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Biological  Structure  and  Function 
Volume  II 


Biological  Structure 
and  Function 


Proceedings  of  the  First  lUBjIUBS  International 
Symposium  Held  in  Stockholm,  September  12-17,  1960 


Edited  hv 


T.  W.  GOODWIN  O.  UNDBERG 

Department  of  Agricultural  Biochemistry  The  Wenner-Gren  Institute  for 

Institute  of  Rural  Science  Experimental  Biology.  University 

Penglais.  IVales  of  Stockholm,  Sweden 


Volume  II 


1961 
ACADEMIC  PRESS  •  LONDON  •  NEW  YORK 


ACADEMIC   PRESS    INC.    (LONDON)    LTD. 

17    OLD   QUEEN    STREET 

LONDON,    S.W.I 


U.S.  edition  published  by 

ACADEMIC   PRESS   INC. 

Ill    FIFTH   AVENUE 

NEW   YORK    3,    NEW   YORK 


Copyright  ©  1961,  by  Academic  Press  Inc.  (London)  Ltd. 


Librory  of  Congress  Catalog  Card  Number:   61-17329 


PRINTED    IN    GREAT    BRITAIN    BY 

SPOTTISWOODE,    BALLANTYNE    &    CO.    LTD., 

COLCHESTER    AND    LONDON 


Contributors  to  Volume  II 

BjORN  A.  Afzelius,  TJie  Wenner-Gyen  Institute  for  Experimental  Biuloi^y, 
University  of  Stock/iolin,  Stveden. 

Robert  D.  Allen,  Department  of  Bio/o»v,  Princeton  University,  New  Jersey, 
U.S.A. 

Daniel  I.  Arnon,  Laboratory  of  Cell  Physiology,  University  of  California, 
Berkeley,  California,  U.S.A. 

Giovanni  Felice  Azzone,  The  Wenner-Gren  Institute  for  Experimental 
Biology,  University  of  Stockholm,  Sweden. 

M.  J.  Bailie,  Laboratory  of  Physiological  Chemistry,  University  of  Amster- 
dam, Netherlands. 

Herrick  Baltscheffsky,  The  Wenner-Gren  Institute  for  Experimental 
Biology,  University  of  Stockholm,  Sweden. 

J.  A.  Bergeron,  Biology  Department,  Brookhaven  National  Laboratory, 
Upton,  Long  Island,  N.Y.,  U.S.A. 

].  BoUMAN,  Laboratory  of  Physiological  Chemistry,  University  of  Amsterdam, 
Netherlands. 

Britton  Chance,  The  Eldridge  Reeves  Johnson  Eoundation  for  Medical 
Physics,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania, 
U.S.A. 

J.  B.  Chappell,  Department  of  Biochemistry,  University  of  Cambridge, 
England. 

Thomas  E.  Conover,  Public  Health  Research  Institute  of  the  City  of  New 
York,  NewYork,  N.Y.,  U.S.A. 

H.  E.  Davenport,  University  of  Bristol,  Research  Station,  Long  Ashton, 
Bristol,  England. 

Bernard  D.  Davis,  Department  of  Bacteriology  and  Immunology,  Harvard 
Medical  School,  Boston,  Massachusetts,  U.S.A. 

Lars  Ernster,  The  Wenner-Gren  Institute  for  Experimental  Biology, 
University  of  Stockholm,  Szceden. 

Albert  \V.  Frenkel,  Department  of  Botany,  University  of  Minnesota, 
Minneapolis,  Minnesota,  U.S.A. 

R.  C.  Fuller,  Dartmouth  Medical  School,  Hanover,  Nezv Hampshire,  U.S.A. 

V 


VI  CONTRIBUTORS  TO  VOLUME  II 

R.  J.  GoLDACRE,  Chester  Beattv  Research  Institute,  Institute  of  Cancer 
Research,  Royal  Cancer  Hospital,  London,  England. 

T.  W.  Goodwin,  Department  of  Agricultural  Biochemistry,  Institute  of 
Rural  Science,  Penglais,  Aberystzoyth,  Wales. 

T.    GusTAFSON,    The    Wenner-Gren    Institute  for   Experimental   Biology, 

University  of  Stockholm,  Sweden . 
Donald  D.  Hickman,  Department  of  Botany,   University  of  Minnesota, 

Minneapolis,  Minnesota,  U.S.A. 

F.  A.  HoLTON,  Royal  Veterinary  College,  London,  England. 

F.  Edmund  Hunter,  Jr.,  The  Edward  Mallinckrodt  Department  of  Phar- 
macology, Washington  University  School  of  Medicine,  St.  Louis, 
Missouri,  U.S.A. 

Andre  T.  Jagendorf,  Biology  Department  and  McCollum-Pratt  histitute. 
The  Johns  Hopkins  University,  Baltimore,  Maryland,  U.S.A. 

Joseph  S.  Kahn,  Department  of  Botany,  North  Carolina  State  College, 

North  Carolina,  U.S.A. 
Martin  D.  Kamen,  Brandeis  University,  Waltham,  Massachusetts,  U.S.A. 

Martin  Klingenberg,  Physiologisch-Chemisches  Institut  der  Universitdt, 
Marburg,  Germany. 

Henry  Lardy,  Institute  for  Enzyme  Research,    University  of  Wisconsin, 

Madison,  Wisconsin,  U.S.A. 
Albert  L.  Lehninger,  Department  of  P/iysiological  Chemistry,  The  Johns 

Hopkins  School  of  Medicine,  Baltimore,  Maryland,  U.S.A. 
Olov   Lindberg,    The    Wenner-Gren  Institute  for  Experimental  Biology, 

University  of  Stockholm,  Szveden. 
W.  F.  LooMis,  The  Loomis  Laboratory,  Greenzvich,  Connecticut,  U.S.A. 

Hans  Low,  The  Wenner-Gren  Institute  for  Experimejital  Biology,  University 
of  Stockholm,  Szceden. 

J.  ]\L  Marshall,  Jr.,  Department  of  Anatomy,  School  of  Medicine,  Univer- 
sity of  Pennsylvania,  Philadelphia,  Pennsylvania,  U.S.A. 

Daniel  Mazia,  Department  of  Zoology,  University  of  California,  Berkeley, 
California,  U.S.A. 

Peter  Mitchell,  Chemical  Biology  Unit,  Department  of  Zoology,  University 
of  Edinburgh,  Scotland. 

V.  T.  Nachmias,  Department  of  Anatomy,  School  of  Medicine,  University 
of  Pemisylvania,  Philadelphia,  Pennsylvania,  U.S.A. 

Lester  Packer,  Department  of  Microbiology,  University  of  Texas  South- 
western Medical  School,  Dallas,  Texas,  U.S.A. 


CONTRIBUTORS  TO  VOLUME  II  VU 

Harvey  S.  Pexefsky,  Division  of  Xutrition  and  Physiology,  The  Public 
Health  Research  Institute  of  the  Citv  of  Xezc  York,  Inc.,  New  York, 
N.Y.,  U.S.A. 

Douglas  C.  Pratt,  Department  of  Botany,  University  of  Minnesota, 
Minneapolis,  Minnesota,  U.S.A. 

D.  M.  Prescott,  Biology  Division,  Oak  Ridge  National  Laboratory,  Oak 

Ridge,  Tennessee,  U.S.A. 

^NIaynard  E.  Pullman,  Division  of  Xutrition  and  Physiology,  The  Public 
Health  Research  Institute  of  the  City  of  Xew  York,  Inc.,  Xezv  York, 
X.Y.,  U.S.A. 

E.  Packer,    Division   of  Xutrition   and  Physiology,    The   Public   Health 

Research  Institute  of  the  City  of  Xew  York,  Inc.,  Xezv  York,  AM'., 
U.S.A. 

E.  R.  Redfearn,  Department  of  Biochemistry,  The  University  of  Liverpool, 
England. 

J.  RuNNSTROM,  The  Wenner-Gren  Institute  for  Experimental  Biology, 
University  of  Stockholm,  Szceden. 

E.  ScHOFFENiELS,  Listitut  Leon  Fredericq,  Laboratoires  de  Biochimie, 
Universite  de  Liege,  Belgium. 

Thomas  P.  Singer,  Edsel  B.  Ford  Institute  for  Medical  Research,  Henry 
Ford  Hospital,  Detroit,  Michigan,  U.S.A. 

E.  C.  Slater,  Laboratory  of  Physiological  Chemistry,  University  of  Amster- 
dam, Xetherlands. 

James  H.  C.  Smith,  Carnegie  Institution  of  Washington,  Department  of  Plant 
Biology,  Stanford,  California,  U.S.A. 

D.  D.  Tyler,  Medical  Research  Council,  Experimental  Radiopathology 
Research  L  nit.  Hammersmith  Hospital,  London,  England. 

BiRGiT  Vennesland,  Department  of  Biochemistry,  University  of  Chicago, 
Chicago,  Illinois,  U.S.A. 

J.  S.  C.  \\'essels.  Philips  Research  Laboratories,  X.J'.  Philips'  (Jloeilampen- 
Jabrieken,  Eindhoven,  Xetherlands. 

Erik  Zeuthen,  The  Biological  Institute  of  the  Carlsberg  Foundation, 
Copenhagen,  Denmark. 

Daniel  AL  Ziegler,  Institute  for  Enzyme  Research,  University  of  Wisconsin, 
Wisconsin,  U.S.A. 


Preface 

In  191^6  The  International  Union  of  Biological  Sciences  (lUBS)  decided 
to  set  up  a  Biochemistry  Section  Committee,  which  would  be  a  Co- 
ordinating Committee  between  lUBS  and  the  International  Union  of 
Biochemistry  (lUB)  and,  through  a  Co-ordinating  Committee  of  lUB  and 
the  International  Union  of  Pure  and  Applied  Chemistry  (lUPAC),  would 
also  have  contact  with  lUPAC.  It  was  considered  that  the  Committee  would 
be  specifically  concerned  with  chemical  biology  within  the  framework  of 
the  Unions  federated  to  the  Councils  of  Scientific  Unions  (ICSU).  The 
members  of  the  Biochemistry  Section  Committee  are  at  present :  R, 
Brunei  (Toulouse)  and  O.  Lindberg  (Stockholm)  (appointed  by  lUBS), 
W.  Florkin  (Liege)  and  T.  W.  Goodwin  (Aberystwyth)  (appointed  by 
lUB),  and  P.  Boyer  (Minneapolis)  and  F.  Lynen  (Munich)  (co-opted 
members).  Florkin  and  Goodwin  were  elected  Chairman  and  Secretary 
respectively. 

The  first  Committee  meeting  was  held  in  1958  during  the  4th  Inter- 
national Congress  of  Biochemistry  in  Vienna.  It  had  been  visualized 
throughout  the  discussions  that  an  important  function  of  the  Committee 
would  be  to  make  suggestions  for  various  International  Symposia  to  both 
I  UBS  and  lUB.  It  was  agreed  that  subjects  would  be  appropriate  only  if 
both  biochemistry  and  the  biological  sciences  were  combining  to  produce 
a  rapidly  expanding  sphere  of  knowledge.  A  number  of  possibilities  were 
considered  at  Vienna  and  it  was  eventually  decided  that  "Biological 
Structure  and  Function"  was  most  appropriate  at  this  time.  This  idea 
was  accepted  by  the  two  International  Unions  and  plans  began  to  be 
formulated.  It  was  readily  agreed  that  the  most  suitable  centre  in 
Europe  for  such  a  symposium  was  the  Wenner-Gren  Institute,  with  its  well- 
established,  international  reputation  in  this  field  and,  furthermore,  the 
project  had  the  blessing  and  support  of  Dr.  Axel  \\'enner-Gren  himself, 
who  honoured  the  Symposium  by  agreeing  to  act  as  Patron  of  Honour 
and  by  attending  the  Inaugural  Session  to  deliver  the  opening  address. 

The  lUB  and  lUBS  have  supported  this  Symposium  financially  but 
the  realization  of  the  Symposium  would  not  have  been  possible  without 
the  generous  aid  of  the  ^^'enner-Gren  Foundation,  and  of  the  various 
bodies  in  difterent  countries  which  support  the  attendance  of  scientists  at 
important    international    meetings.    It    was    extremely    satisfying   to    the 


X  PREFACE 

organizers  to  know  that  these  official  bodies  considered  this  First  lUB/ 
lUBS  Joint  Symposium  worthy  of  support,  and  mention  should  be  made 
of  the  National  Science  Foundation  which  supported  so  many  of  our 
US.  participants;  furthermore,  in  this  connection  the  work  done  on  our 
behalf  by  Dr.  Elmer  Stotz,  the  treasurer  of  lUB,  should  not  be  forgotten. 

The  organizers  hope  that  this  Symposium  will  be  the  forerunner  of  a 
long  line  of  similar  international  symposia  based  on  fruitful  co-operation 
between  biochemists  and  biologists  from  all  nations. 

The  organizers  are  most  grateful  to  the  Institute  of  Physics,  University 
of  Stockholm,  for  their  generosity  in  putting  their  attractive  new  lecture 
theatre  at  the  disposal  of  the  Symposium. 

In  preparing  the  proceedings  for  the  press  the  organizers  have  been 
greatly  helped  by  Miss  J.  T.  Peel,  who  transcribed  the  recorded  dis- 
cussions, and  by  Mr.  D.  J.  Howells,  who  prepared  the  subject  index. 

April,  1 96 1  T.  W.  Goodwin 

O.    LiNDBERG 


Contents  of  Volume  II 


Contributors  to  Volume  II 

Preface 

Contents  of  Volume  I  . 


PAGE 
V 

ix 

XV 


MlTOCHONDRL\L    STRUCTURE    AND    FUNCTION 

Etfects  of  Thyroxine  and  Related  Compounds  on  Liver  Mito- 
chondria in  Mtro.  By  Olov  Lindberg,  Hans  Low,  Thomas  E. 
Conover,  and  Lars  Ernster  ......  3 

Components  of  the  Energy-Couphng  Mechanism  and  Mitochon- 
drial Structure.  By  Albert  L.  Lehninger       .  .  .  .  31 

Ascorbate-Induced  Lysis  of  Isolated  Mitochondria — A  Pheno- 
menon Different  from  Swelling  Induced  by  Phosphate  and 
Other  Agents.  By  F.  Edmund  Hunter,  Jr.    .  .  .  .  53 

Integrated  Oxidations  in  Isolated  Mitochondria.  By  J.  B.  Chappell         71 

Metabolic  Control  of  Structural  States  of  Mitochondria.  By  Lester 

Packer '  .  .85 

Stable  Structural  States  of  Rat  Heart  Mitochondria.  Bv  F.  A.  Holton 

and  D.  D.  Tyler        .  .  .  .  .  .  .  .9^ 

Solubilization  and  Properties  of  the  DPXH  Dehydrogenase  of  the 

Respiratorv  Chain.  Bv  Thomas  P.  Singer     .  .  .  .        ic^ 

Reversal  of  Electron  Transfer  in  the  Respiratory  Chain.  By  Britton 

Chance   .  .  .  .  .  .  .  .  .  .119 

Function  of  Flavoenzymes  in  Electron  Transport  and  Oxidative 

Phosphorylation.  By  Lars  Ernster        .  .  .  .  -139 

Coupling  of  Reduced  Pyridine  Nucleotide  Oxidation  to  the  Ter- 
minal Respiratory  Chain.  Bv  T.  E.  Conover  .  .  .169 

Mitochondrial  Lipids  and  their  Functions  in  the  Respiratorv  Chain. 

By  E.  R.  Redfearn "  .  .        iSi 

The  Functional  Link  of  Succinic  Dehydrogenase  with  the  Terminal 

Respiratory  Chain.  By  Giovanni  Felice  Azzone      .  .  .193 

Pyridine  Nucleotides  in  Mitochondria.  Bv  E.  C.  Slater,  M.  J.  Bailie 

and  J.  Bouman  ........        207 


80166 


XU  CONTENTS  OF  VOLUME  II 

Nucleotides  and  Mitochondrial  Function :  Influence  of  Adenosine- 
triphosphate  on  the  Respiratory  Chain.  By  Martin  KUngenberg       227 

The  Role  of  ATPase  in  Oxidative  Phosphorylation.  By  Maynard  E. 

Pullman,  Harvey  S.  Penefsky  and  E.  Racker  .  .  .        241 

The  Mechanism  of  Coenzyme  Q  Reduction  in  Heart  Mitochondria. 

By  Daniel  M.  Ziegler         .......       253 

Reactions  Involved  in  Oxidative  Phosphorylation  as  Disclosed  by 

Studies  with  Antibiotics.  By  Henry  Lardy   ....        265 

Structure  and  Function  of  Chloroplasts  and  Chromatophores 

Chairman's  Opening  Remarks.  By  T.  W.  Goodwin      .  .  .        271 

Haem  Protein  Content  and  Function  in  Relation  to  Structure  and 
Early  Photochemical  Processes  in  Bacterial  Chromatophores, 
By  Martin  D.  Kamen        .......        277 

Observations  on  the  Formation  of  the  Photosynthetic  Apparatus  in 
RhodospiriUum  riibrum  and  Some  Comments  on  Light- 
Induced  Chromatophore  Reactions.  By  Douglas  C.  Pratt, 
Albert  W.  Frenkel,  and  Donald  D.  Hickman  .  .  .        295 

The    Photosynthetic   Macromolecules   of  Chlorobiiim    TJiiosidfato- 

philum.  By  J.  A.  Bergeron  and  R.  C.  Fuller     ....        307 

Some  Physical  and  Chemical  Properties  of  the  Protochlorophyll 

Holochrome.  By  James  H.  C.  Smith   .  .  .  .  .325 

Photosynthetic     Phosphorylation     and    the     Energy     Conversion 

Process  in  Photosynthesis.  By  Daniel  I.  Arnon      .  .  .        339 

The   Mechanism   of  the   Hill   Reaction   and   Its   Relationship   to 

Photophosphorylation.  By  Birgit  Vennesland         .  .  .411 

Electron  Transport  and  Phosphorylation  in  Light-Induced  Phos- 
phorylation. By  Herrick  Baltscheft'sky  .  .  .  .431 

Reduction  of  Dinitrophenol  by  Chloroplasts.  By  J.  S.  C.  Wessels       443 

The  Relationship  between  "  Methaemoglobin  Reducing  Factor" 
and  "Photosynthetic  Pyridine  Nucleotide  Reductase".  By 
H.  E.  Davenport        ........       449 

ATP  Formation  by  Spinach  Chloroplasts.  By  Andre  T.  Jagendorf 

and  Joseph  S.  Kahn  .......       455 

Intact  Cellular  Structure  and  Function 

Chairman's  Introduction:  Remarks  on  Control  of  Structure  and 

Difl^erentiation  in  Cells  and  Cell  Systems.  By  J.  Runnstrom     .       465 

The  Central  Problems  of  the  Biochemistry  of  Cell  Division.    By 

Daniel  Mazia  ........       475 


CONTEXTS  OF  VOLUME  II  XIU 

Studies  on  the  Cellular  Basis  of  Morphogenesis  in  the  Sea  Urchin. 

By  T.  Gustafson       ........       497 

Cell  Differentiation :  A  Problem  in  Selective  Gene  Activation 
Through  Self-Produced  Micro-Environmental  Differences  of 
Carbon  Dioxide  Tension.  By  W.  F.  Loomis  .  .  .        509 

RXA  Synthesis  in  the  Nucleus  and  RNA  Transfer  to  the  Cyto- 
plasm in  Tetrahymena pyriformis.  By  D.  M.  Prescott         .  ,        527 

Cell  Division  and  Protein  Synthesis.  By  Erik  Zeuthen  .  .        537 

Structure  and  Function  in  Amoeboid  Movement.    By  Robert  D. 

Allen       ..........        549 

Some  Problems   of  Ciliary   Structure   and   Ciliary   Function.   By 

Bjorn  A.  Afzelius       ........       557 

Specific  Membrane  Transport  and  its  Adaptation 
Chairman's  Introduction.  By  Bernard  D,  Davis  .  .  .        571 

Approaches  to  the  Analysis  of  Specific  Membrane  Transport.  By 

Peter  Mitchell 581 

Protein  Uptake  by  Pinocytosis  in  Amoebae :  Studies  on  Ferritin  and 

Methylated  Ferritin.  By  V.  T.  Nachmias  and  J.  M.  ?vlarshall,  Jr.  605 
Comparative  Study  of  Membrane  Permeability.  By  E.  Schoft'eniels  621 
Active  Transport  and  Membrane  Expansion-Contraction  Cycles. 

By  R.  J.  Goldacre      ........       633 

Author  Index       .........       645 

Subject  Index       .........       657 


Contents  of  Volume  I 

Macromolecular  Structure  and  Function 
Introduction.  By  A.  Tiselius 

The  Structure  of  Globular  Proteins.  By  J.  C.  Kendrew 
Molecular  Configuration  of  Xucleic  Acids.  By  \l.  H.  F.  Wilkins 
Partition  of  Alacromolecules  in  Aqueous  Two-Phase  Systems.  By  Per- 

Ake  Albertsson 
The  Reactivity  of  Certain  Functional  Groups  in  Ribonuclease  A  towards 
Substitution  by  i-Fluoro-2,4-dinitrobenzene.  Inactivation  of  the 
Enzyme  by  Substitution  at  the  Lysine  Residue  in  Position  41.  By 
C.  H.  W.  Hirs,  jMirjam  Halmann  and  Jadwiga  PI.  Kycia 
The  Relation  of  the  Secondary  Structure  of  Pepsin  to  Its  Biological 
Activity.  By  Gertrude  E.  Perlmann 

The  Problem  of  Nucleotide   Sequence  in   Deoxyribonucleic  Acids.   By 

Erwin  Chargaff 
Problems  in  Polynucleotide  Biosynthesis.  By  J.  N.  Davidson 
Enzymic  Formation  of  Deoxyribonucleic  Acid  from  Ribonucleotides.  By 

Peter  Reichard 
Studies  on  the  Mechanism  of  Synthesis  of  Soluble  Ribonucleic  Acid.  Bv 

E.  S.  Canellakis  and  Edward  Herbert 

Microsomes  and  Protein  Synthesis 

The  P^ndoplasmic  Reticulum:  Some  Current  Interpretation  of  Its  Form 

and  Functions.  By  Keith  R.  Porter 
Pinocytosis.  By  H.  Holter 
The  Ergastoplasm  in  the  Mammary  Cjland  and  Its  Tumours:  An  Electron 

Microscope    Study    with    Special    Reference    to    Caspersson's    and 

Santesson's  A  and  B  Cells.   By  F.  Haguenau  and  K.  H.  Hollmann 
The  External  Secretion  of  the  Pancreas  as  a  Whole  and  the  Communication 

between   the   Endoplasmic   Reticulum   and   the    Golgi   Bodies.    By 

Gottwalt  Christian  Hirsch 
Immunological  Studies  of  Microsomal  Structure  and  Function.  By  Peter 

Perlmann  and  Winfield  S.  Morgan 


XVI  CONTENTS  OF  VOLUME  I 

Amino  Acid  Incorporation  by  Liver  Microsomes  and  Ribonucleoprotein 

Particles.  By  Tore  Hultin,  Alexandra  von  der  Decken,  Erik  Arrhenivis 

and  Winlield  S.  Morgan 
The  Effects  of  Spermine  on  the  Ribonucleoprotein  Particles  of  Giiinea- 

Pig  Pancreas.  By  Philip  Siekevitz 
The  Correlation  between  Morphological  Structure  and  the  Synthesis  of 

Serum  Albumin  by  the  Microsome  Fraction  of  the  Rat  Liver  Cell. 

By  P.  N.  Campbell 
Amino  Acid  Transport  and  Early  Stages  in  Protein  Synthesis  in  Isolated 

Cell  Nuclei.  By  Vincent  G.  AUfrey 
Effects  of  8-Azaguanine  on  the  Specificity  of  Protein  Synthesis  in  Bacillus 

cereus.  By  H.  Chantrenne 
Purine  and  Pyrimidine  Analogues  and  the  Mucopeptide  Biosynthesis  in 

Staphylococci.  By  H.  J.  Rogers  and  H.  R.  Perkins 
Studies  on  the  Incorporation  of  Arginine  into  Acceptor  RNA  o{  Escherichia 

coli.  By  H.  G.  Boman,  I,  A.  Boman  and  W.  K.  Maas 

Polysaccharides 
Introduction.  By  Gunnar  Blix 

The  Growth  of  Saccharide  Macromolecules.  By  Shlomo  Hestrin 
Mucopolysaccharides  of  Connective  Tissue.  By  Albert  Dorfman  and  Sara 

Schiller 
Separation  of  Oligosaccharides  with  Gel  Filtration.  By  Per  Flodin  and 

Kare  Aspberg 


MITOCHONDRIAL  STRUCTURE  AND  FUNCTION 


VOL.  II — n 


Effects  of  Thyroxine  and  Related  Compounds  on  Liver 
Mitochondria  in  Vitro*t 

Olov  Lindberg,  Hans  Low,  Thomas  E.  Conover,|  and  Lars  Ernster 

The  Wenner-Greu  Institute  for  Experimental  Biology, 
University  of  Sfockliohn,  Sweden 

I.  Historical  Survey 

A  first  although  not  successful  attempt  to  demonstrate  an  effect  of 
thyroxine  on  oxidative  phosphorylation  in  mitochondria  was  reported  by 
Judah  and  Williams- Ashman  [i]  in  195 1.  Later  the  same  year  Judah  [2] 
demonstrated  a  slight  effect  on  the  P/0  ratio  of  liver  mitochondria  isolated 
from  thyroxine-treated  rats.  He  also  compared  the  effect  of  thyroxine 
with  that  of  2,4-dinitrophenol,  and  pointed  out  that  no  similarity  existed 
between  the  modes  of  action  of  the  two  compounds.  At  about  the  same 
time  Martins  and  Hess  [3]  briefly  reported  that  thyroxine,  either  adminis- 
tered in  vivo  or  added  in  vitro,  lowered  the  phosphorylation  of  isolated  rat 
liver  mitochondria.  Niemeyer  et  al.  [4],  however,  found  no  effect  on  the 
P/0  ratio  of  liver  mitochondria  from  rats  treated  with  thyroxine  in  vivo, 
but  were  able  to  demonstrate  a  significant  decrease  of  the  respiratory 
control  by  phosphate  acceptor  in  these  mitochondria. 

In  the  first  comprehensive  work  on  thyroxine  eflect  in  vitro.  Lardy  and 
Feldott  [5]  demonstrated  in  195 1  that  this  compound  at  a  concentration 
of  io~'^  M  inhibited  the  oxidation  of  glutamate  and  of  certain  other  DPN- 
linked  substrates  by  a  washed  residue  of  homogenized  rat  kidney.  The 
inhibition  could  be  partly  relieved  by  added  DPN.  LTsing  particulate 
preparations  of  rat  liver,  a  certain  extent  of  decrease  of  the  P/0  ratio  was 
also  noticed,  both  when  thyroxine  was  added  in  vitro,  and  in  preparations 
from  hyperthyroid  animals.  The  following  year  Lardy  [6]  advanced  the 
hypothesis  that  the  hormonal  effect  of  thyroxine  resides  in  its  capacity  to 
uncouple  one,  rate-limiting,  phosphorylation  from  the  respiration,  thus 
enhancing  both  respiratory  rate  and  net  output  of  high-energy  phosphate 

*  This  work  has  been  supported  by  grants  from  the  Swedish  Medical  Research 
Council  and  the  Swedish  Cancer  Society. 

t  Abbreviations:  ADP,  adenosine  diphosphate;  ATP,  adenosine  triphosphate; 
DPN,  diphosphopyridine  nucleotide;  DPNH,  reduced  diphosphopyridine 
nucleotide;  TPXH,  reduced  triphosphopyridine  nucleotide. 

\  Fellow  of  the  National  Foundation.  Present  address :  Public  Health  Research 
Institute  of  the  City  of  Netv  York,  Netv  York,  N.Y.,  U.S.A. 


4  OLOV    LINDBHRG    et    al. 

at  the  expense  of  thermodynamic  efficiency.  A  somewhat  similar  hypo- 
thesis, based  on  a  chemical  relationship  between  thyroxine  and  dinitro- 
phenol  (both  being  substituted  phenols),  was  also  put  forward  by  Martius 
[7].  In  the  many  attempts  [812]  to  prove  these  hypotheses  experimentally, 
it  has  been  possible,  in  some  instances,  to  demonstrate  a  partial  uncoupling 
of  respiration  from  phosphorylation  in  isolated  mitochondria  due  to  thy- 
roxine treatment,  both  ///  vitro  and  in  vivo.  However,  these  effects  did  not 
appear  in  a  consistent  manner,  nor  could  a  preferential  uncoupling  of  one 
of  the  three  respiratory  chain  phosphorylations  be  established. 

In  1954  Hoch  and  Lipmann  [13]  reported  that  a  consistent  decrease 
of  the  P/0  ratio  in  isolated  rat  liver  mitochondria  by  thyroxine  could  be 
obtained,  if  the  mitochondria  were  preincubated  with  thyroxine  for  a 
period  of  time  before  the  addition  of  substrate.  Hamster  liver  mitochondria, 
on  the  other  hand,  required  no  preincubation.  However,  even  in  this  case, 
the  results  were  rather  inconsistent  from  one  experiment  to  another.  The 
significance  of  the  loss  of  respiratory  control  without  an  actual  loss  of 
phosphorylating  capacity,  found  in  earlier  work  [4],  was  re-emphasized.  In 
parallel  papers  by  Bain  [14]  and  by  Mudd  et  al.  [15]  it  was  shown  that  the 
effect  of  thyroxine  on  the  P/0  ratio  could  be  prevented  by  magnesium  ions. 

By  this  time,  attention  became  directed  towards  the  effect  of  thyroxine 
on  mitochondrial  structure.  In  1953  Aebi  and  Abelin  [16]  reported  that 
liver  mitochondria  from  thyrotoxic  rats  exhibited  an  increased  tendency 
to  spontaneous  swelling  ///  vitro.  Subsequently  Klemperer  [17]  found  an 
increased  water-content  in  thyroxine-treated  mitochondria.  Tapley  et  al. 
[18]  demonstrated  in  1955  that  thyroxine  added  ///  vitro  enhances  the 
swelling  of  KCl-suspended  normal  rat  liver  mitochondria.  A  similar  effect 
was  obtained  with  kidney  mitochondria,  while  the  swelling  was  much 
weaker  with  mitochondria  from  muscle,  brain  and  testes  [19].  It  was  also 
shown  [20]  that  the  P/0  ratio  of  phosphorylating  mitochondrial  fragments, 
prepared  with  digitonin  from  liver  mitochondria,  was  not  affected 
by  thyroxine  whereas  it  was  still  sensitive  to  dinitrophenol.  From  these 
findings  it  was  concluded  (cf.  also  [21,  22])  that  thyroxine,  in  contrast  to 
dinitrophenol,  exhibits  its  effect  on  oxidative  phosphorylation  by  a 
secondary  mechanism  which  is  somehow  correlated  with  the  mitochondrial 
structure. 

The  swelling  effect  of  thyroxine  ///  vitro  has  been  subsequently  studied 
in  great  detail  in  a  series  of  papers  by  Lehninger  and  associates  ([23-28] ; 
for  review,  cf.  [29]).  It  emerged  from  these  studies  that  this  effect  is  similar 
to  that  obtained  when  mitochondria  are  incubated  for  a  period  of  time 
("aged")  in  a  phosphate-containing  medium  ([30-46]  for  review,  cf.  [47]). 
In  both  cases,  the  swelling  seems  to  be  the  result  of  an  active  process, 
which  is  typically  temperature-  and  time-dependent.  It  requires  the 
presence    of  an    oxidizable    substrate,    and    is    prevented    by    respiratory 


EFFECTS  OF  THYROXINE  AND   RELATED   COMPOUNDS  ON   LIVER  MITOCHONDRIA  5 

inhibitors.  Moreover,  it  is  prevented  by  dinitrophenol,  adenine  nucleotides, 
and  in  general  apparently  by  conditions  that  pre\ent  the  accumulation  of 
high-energy  intermediates  within  the  mitochondria.  Significantly,  active 
swelling  is  reversed  by  ATP,  and  this  "contraction"  of  the  mitochondrial 
structure  has  been  shown  to  be  reflected  in  an  extrusion  of  water  from  the 
mitochondria,  paralleled  by  a  splitting  of  ATP.  It  has  also  been  demon- 
strated [29,  48]  that  the  ATP-induced  contraction  is  dependent  on  the 
presence  in  the  mitochondria  of  a  specific  protein  fraction ;  the  symbol, 
"M  factor",  has  been  used  to  denote  this  fraction. 

Parallel  to  the  swelling,  the  mitochondria  lose  their  endogenous  content 
of  DPN.  When  DPX  is  added  to  such  mitochondria  in  the  presence  of 
ATP,  a  rebinding  of  the  DPN  to  the  mitochondrial  structure  takes  place. 
Whether  the  loss  of  DPN  is  a  cause  or  a  consequence  of  the  swelling  is  not 
quite  clear,  although  recent  investigations  by  Kaufmann  and  Kaplan  [49] 
would  seem  to  indicate  that  the  latter  is  the  case. 

A  further  characteristic  feature  of  the  process  of  active  swelling  is  that 
it  is  not  immediately  accompanied  by  an  uncoupling  of  phosphorylation 
from  respiration.  Thus,  mitochondria  which  have  reached  a  state  of  swollen 
structure  following  exposure  to  ageing  in  the  presence  of  phosphate  or 
thyroxine  are  still  capable  of  exhibiting  an  electron  transport-coupled 
phosphorvlation  when  DPN  is  added  to  restore  respiration  (with  succinate 
as  substrate  the  situation  seems  to  be  somewhat  more  complicated  [35], 
owing  probably  to  the  recently  disco\ered  requirement  of  high-energy 
phosphate  for  the  oxidation  of  this  substrate  [50  54]).  However,  simul- 
taneously with  the  swelling  and  the  loss  of  DPN,  or  even  preceding  these 
effects,  the  mitochondria  lose  the  tight  coupling  between  respiration  and 
phosphorylation,  the  former  becoming  independent  of  the  presence  of 
orthophosphate  and  ADP.  In  such  mitochondria,  thus,  coupled  phos- 
phorylation can  take  place,  when  phosphate  and  phosphate  acceptor  are 
present,  but  respiration  can  proceed  at  maximal  rate  even  in  the  absence 
of  these  additions.  It  has  been  shown  by  Lehninger  and  associates  [55,  56] 
that  this  state  of  "loose-coupling"  can  be  induced  in  intact  mitochondria 
not  only  by  the  above  treatments  but  also  by  the  addition  of  a  protein 
factor,  called  "R  factor",  which  can  be  obtained  from  mitochondria 
after  disruption  with  sonic  waves ;  intact  mitochondria  thus  seem  to  contain 
this  factor  in  an  inactive  state.  "Loose-coupling"  efi^ects  can  be  induced  in 
mitochondria  also  by  a  number  of  common  uncoupling  agents  if  these  are 
added  in  low  concentrations  [57,  58]. 

Although  thyroxine  is  not  the  only  agent  capable  of  enhancing  active 
swelling  and  related  svmptoms  in  mitochondria  besides  inorganic 
phosphate,  calcium  ions  [t^^,  37,  59]  and  more  recently  phloridzine  [60] 
have  been  shown  to  exhibit  similar  eftects — several  attempts  have  been 
made  to  explain  the  primary  mode  of  action  of  thyroxine  in  terms  of  these 


6  OLOV    LINDBERG    et   al. 

effects  (cf.  [29]).  According  to  one  of  the  visualized  mechanisms,  the 
mitochondrial  DPN  might  constitute  the  target  molecule  for  thyroxine 
action,  the  latter  causing  a  displacement  of  the  bound  DPN  and  thereby  a 
disorganization  of  the  structure.  The  possibility  has  also  been  considered 
that  thyroxine  might  act  primarily  by  activating  the  "R  factor",  thus 
inducing  a  loose-coupling  of  phosphorylation  from  respiration,  or  alterna- 
tively by  inhibiting  the  activation  of  the  "M-factor"  and  thus  interfering 
with  the  contractile  mechanism  responsible  for  the  maintenance  of  a 
tight  mitochondrial  structure. 

A  marked  swelling  of  liver  mitochondria  m  vivo  following  treatment 
of  rats  with  large  doses  of  thyroxine  has  been  described  in  electron 
microscopic  studies  by  Schulz  et  al.  [61].  However,  when  these  mito- 
chondria were  isolated  they  exhibited  normal  respiration  and  P/O  ratio, 
and  differed  from  normal  mitochondria  only  with  regard  to  an  increased 
susceptibility  to  agents  eliciting  swelling  such  as  calcium  ions  [62]. 

A  state  of  loose-coupling  of  the  oxidative  phosphorylation,  of  the  type 
earlier  described  by  Hoch  and  Lipmann  [13]  in  liver  mitochondria  from 
thyrotoxic  hamsters,  was  recently  reported  by  Ernster  et  al.  [63]  to  occur 
in  skeletal  muscle  mitochondria  from  patients  with  thyrotoxicosis.  These 
mitochondria  revealed  a  markedly  lowered  respiratory  control  as  compared 
with  those  from  normal  subjects,  whereas  the  P/O  ratio  obtained  in  the 
presence  of  phosphate  and  phosphate  acceptor,  as  well  as  the  rate  of 
oxidation  of  DPN-linked  substrates,  were  virtually  normal.  Interestingly, 
the  same  findings  were  also  made  with  skeletal  muscle  mitochondria 
from  a  patient  exhibiting  an  extremely  severe  hypermetabolism  (BMR 
around  +  200%)  of  non-thyroid  origin  which  is  now  being  considered 
to  be  related  to  an  inborn  defect  of  the  mitochondrial  structure. 

In  summarizing  this  brief  historical  survey,  it  may  be  said  that  there 
exists  today  a  well-established  symptomatology  of  the  action  of  thyroxine 
on  isolated  mitochondria  in  vitro,  and  that  some  of  the  symptoms,  though 
not  all,  can  also  be  seen  in  mitochondria  exposed  to  toxic  levels  of  thyroid 
hormone  in  vivo.  However,  some  of  these  effects,  such  as  a  decreased 
P/O  ratio,  are  inconsistent  from  one  case  to  another,  and  those  which 
are  consistent,  such  as  the  enhanced  swelling,  the  loss  of  bound  DPN 
and  the  loose-coupling  of  phosphorylation  from  respiration,  are  in  their 
nature  connected  with  a  time  factor,  thus  giving  the  impression  of  being 
consequences  of  some  other  primary  event. 

2.  Some  instantaneous  effects  of  thyroxine  and  related  compounds 
on  partial  reactions  of  oxidative  phosphorylation 

In  the  present  paper,  certain  effects  of  thyroxine  and  some  related 
compounds  on  various  enzyme  activities  in  mitochondria  in  vitro  will 


EFFECTS  OF  THYROXINE  AND   RELATED  COMPOUNDS  ON   LIVER  MITOCHONDRIA  7 

be  described,  which  differ  from  those  outhned  above  in  being  both 
consistent  and  instantaneous.  Furthermore,  some  of  these  effects  can  also 
be  demonstrated  in  mitochondrial  fragments  and  even  at  the  level  of  the 
purified  enzvme. 

The  reactions  studied  can  be  divided  into  two  categories.  The  first 
category  involves  the  mitochondrial  ATPase  reactions  (both  the  dinitro- 
phenol-  and  the  Mg  +  ^-activated  ATPases),  the  P-ATP  exchange 
reaction,  and  an  ATP-ADP  exchange  reaction  catalyzed  by  certain 
mitochondrial  subfractions.  In  the  second  category  belong  certain  flavin- 
catalyzed  electron-transfer  reactions,  such  as  the  DPNH  diaphorase, 
DPNH-cytochrome  c  reductase,  and  the  DPNH  oxidase  reactions,  as  well 
as  a  second  diaphorase  reaction,  which  is  non-specific  with  respect  to 
pyridine  nucleotides.  From  the  data  presented  the  conclusion  is  derived 
that  thyroxine  and  related  compounds  inhibit,  in  a  consistent  and  in- 
stantaneous manner,  reactions  which  involve  a  part  or  the  whole  of  the 
fiavin-linked  respiratory  chain  phosphorylation.  Some  implications  of 
these  results  as  to  the  mode  of  action  of  thyroxine  analogues  on  mito- 
chondria in  vitro  will  be  discussed. 

(a)  ATPase  reactions 

Agents  which  uncouple  oxidative  phosphorylation  in  li\er  mito- 
chondria usually  also  evoke  an  increased  ATPase  activity  [64-68].  Two 
types  of  ATPase  reactions  may  be  distinguished.  One  is  elicited  by 
dinitrophenol  and  related  uncoupling  agents.  This  ATPase  reaction 
occurs  in  structurally  intact  mitochondria  and  requires  no  addition  of 
Mg  ^  ^  to  exhibit  maximal  activity.  Another  type  of  liver-mitochondrial 
ATPase  appears  when  the  structure  of  the  mitochondria  is  damaged  by 
physical  or  chemical  means,  so  as  to  disrupt  the  obligatorv  coupling 
between  respiration  and  phosphorylation.  This  ATPase  reaction  is 
strictly  dependent  on  added  Mg  ^  .  According  to  a  widely  held  opinion 
[65,  67,  69-78]  one  or  both  of  the  ATPase  reactions  reflect,  in  a  modified 
form,  a  part  of  the  reaction  sequence  involved  in  oxidative  phosphorylation. 

Early  considerations  that  thyroxine  and  related  compounds  may  act  as 
uncouplers  of  oxidative  phosphorylation  were  paralleled  by  the  assump- 
tion that  these  agents  would  also  evoke  a  high  mitochondrial  ATPase 
activity.  Data  presented  by  Lardy  and  Maley  [10]  and  by  Malev  [79] 
showed  that  this  was  the  case,  even  though  the  ATPase  activity  appearing 
in  rat  liver  mitochondria  in  the  presence  of  thyroxine  was  relatively  low 
as  compared  to  that  induced  by  dinitrophenol.  While  recently  confirm- 
ing these  data  in  our  laboratory,  the  rather  unexpected  finding  was  made 
that  certain  thyroxine  analogues  markedly  inhibited  the  ATPase  activities 
of  rat  liver  mitochondria,  both  that  induced  by  dinitrophenol  and  that 
elicited  by  destruction  of  the  mitochondrial  structure. 


8  OLOV   LINDBERG    et   al. 

Inhibition    of  dinitrophenol-  and  Mg  +  ^-activated  A  TPases   by  thyroxine 

and  rehited  compounds 

In  Fig.  I,  the  effects  of  thyroxine,  triiodothyronine  and  desamino- 
thyroxine  on  the  dinitrophenol  induced  ATPase  of  rat  Hver  mitochondria 
are  illustrated.  Of  the  three  compounds,  desaminothyroxine  exhibited 
the  strongest  inhibition,  giving  half-inhibition  at  a  concentration  of 
about  0-02  mM.  The  effect  of  the  same  compounds  on  the  Mg  +  +-acti- 
vated  ATPase  is  shown  in  Fig.  2.  For  the  study  of  this  reaction  a 
preparation  of  mitochondrial  fragments,  obtained  after  disruption  of 
mitochondria  with  a  rapidly  rotating  Super-Thurrax  blender  was  used. 


mM 

Fig.  I .  Effect  of  L-thyroxine,  DL-triiodothyronine  and  desaminothyroxine  on 
the  dinitrophenol-induced  ATPase  activity  of  rat  liver  mitochondria.  For 
experimental  details  see  [76]. 

The  procedure  was  adapted  from  Kielley  and  Kielley  [80]  who  devised  it 
for  enriching  mitochondrial  ATPase  free  from  adenylate  kinase.  As  can 
be  seen  in  Fig.  2,  the  ATPase  activity  of  the  Kielley  and  Kielley  prepara- 
tion was  also  inhibited  by  the  three  compounds  tested,  and  again,  desami- 
nothyroxine exhibited  the  strongest  inhibition.  The  half  inhibitory  con- 
centration of  desaminothyroxine  was  roughly  the  same  as  in  the  case  of 
the  dinitrophenol-induced  ATPase.  However,  in  contrast  to  this  latter 
reaction,  the  inhibitions  given  by  the  triodothyronine  and  thyroxine  were 
not  progressive  with  concentration,  but  levelled  off  at  about  0  •  i  mM  to 


EFFECTS  OF  THYROXINE  AND   RELATED   COMPOUNDS  ON   EIVKR   MITOCHONDRIA 


m  M 

Fig.  2.  Effect  of  L-thyroxine,  DL-triiodothyronine  and  desaminothyroxine  on 
the  ATPase  activity  of  mitochondrial  fragments  prepared  according  to  Kielley 
and  Kielley  [80].  For  experimental  details  see  [84]. 


Desaminothyroxine         o 
Thyroxine         a 


noMg**     Mg'^^^mM 


'0  01 

m  M 

Fig.  3.  Effect  of  added  Mg  "  ^  on  the  sensitivity  of  the  dinitrophenol-induced 
ATPase  activity  of  rat  liver  mitochondria  to  DL-thyroxine  and  desaminothyroxine. 
When  indicated,  Mg  +  +  was  added  in  a  final  concentration  of  4  mM.  Other  experi- 
mental conditions  as  in  Fig.  i. 


10  OLOV   LINDBERG    et    al. 

give  a  maximal  inhibition  of  about  30  and  60"  q,  respectively.  A  similar 
pattern  of  inhibition  could  be  obtained  also  in  the  case  of  the  dinitrophenol- 
induced  ATPase  if  this  reaction  was  measured  in  the  presence  of  added 
Mg  +  +  (Fig.  3).  Thus,  whereas  added  Mg  +  +  did  not  alter  the  inhibition 
of  the  dinitrophenol-induced  ATPase  by  desaminothyroxine,  it  rendered 
the  inhibition  with  thyroxine  less  efficient  and  with  a  maximal  inhibition 
of  about  only  30",,.  It  would  seem  that  this  effect  of  Mg  +  +  was  not  due 


None 


Desamino 
thyroxine 


mi  n 


Fig.  4.  Time-course  of  inhibition  of  the  dinitrophenol-induced  ATPase 
reaction  by  desaminothyroxine.  When  indicated  desaminothyroxine  was  added 
in  a  final  concentration  of  0-2  mM.  Other  experimental  conditions  as  in  Fig.  i. 


primarily  to  a  binding  of  thyroxine  (in  which  case  the  protection  by  Mg  +  + 
should  have  been  overcome  with  higher  concentrations  of  thyroxine), 
but  rather  to  an  ability  of  Mg  +  +  to  restrict  the  number  of  active  sites  in 
the  preparation  accessible  to  thyroxine.  For  this  reason  the  investigations 
to  follow  were  performed  with  desaminothyroxine. 

The  inhibitory  effect  of  desaminothyroxine  on  the  dinitrophenol- 
induced  ATPase  reaction  was  instantaneous,  as  shown  in  Fig.  4.  Pre- 
incubation with  desaminothyroxine  prior  to  the  addition  of  ATP  did  not 
influence  the  extent  of  inhibition,  neither  in  the  case  of  this  reaction,  nor 
in  the  case  of  the  Mg  +  +-activated  ATPase  reaction  catalyzed  by  mito- 
chondrial fragments  (Fig.  5). 


EFFECTS  OF  THYROXINE  AND   RELATED  COMPOUNDS  ON   LIVER  .MITOCHONDRIA  11 

preincubation 
•   =      0   min 
o   =    1  5       .. 
D    =    30       .. 
X   =   60       .. 


0    10 


10""  lO"-"  10' 

M     Desam  i  not  hyroxine 


Fig.  5.  Lacking  effect  (jf  preincubation  on  the  desaminothyroxine  sensitivity 
of  the  ATPase  activity  of  mitochondrial  fragments  prepared  according  to  Kielley 
and  Kielley  [80].  Experimental  conditions  as  in  Fig.  2. 


Comparison  7cit/i  other  A  TPasc  inhibitors 

A  further  characterization  of  the  effect  of  desaminothyroxine  on  the 
mitochondrial  ATPase  reactions  was  considered  possible  by  comparing  it 
with  the  effects  of  known  ATPase  inhibitors. 

It  has  been  known  for  some  time  that  the  liver  mitochondrial  ATPase 
reactions  are  inhibited  by  azide  [75,  81  83]  and  by  a  number  of  flavin 
antagonists,  including  atebrin  [76]  and  chlorpromazine  [77].  The  effect 
of  the  flavin  antagonists  on  the  dinitrophenol-induced  ATPase  is  diphasic, 
consisting  of  a  stimulation  at  low  concentrations  and  an  inhibition  at  high 
concentrations  [76,  77].  Figure  6  compares  the  effects  of  desaminothy- 
roxine, azide,  atebrin  and  chlorpromazine  on  the  dinitrophenol-induced 
ATPase  activitv  on  the  basis  of  concentration.  Desaminothyroxine  was  the 
most  potent  of  the  four  inhibitors,  and  its  effect  lacked  the  dual  character 
shown  by  the  flavin  antagonists. 


12  OLOV    LINDBERG    et    ul. 

It  was  shown  previously  that  amytal  inhibits  shghtly  the  dinitro- 
phenol-induced  ATPase  [75],  and  that  this  inhibition  can  be  greatly 
potentiated  if  a  stimulating  concentration  of  atebrin  [76]  or  chlorpromazine 
[77]  is  added.  Fig.  7  illustrates  this  effect  and  shows  that  a  similar  poten- 
tiation did  not  occur  with  azide  or  desaminothyroxine.  In  fact,  des- 
aminothyroxine  seemed  even  to  eliminate  the  slight  inhibition  given  by 
amytal. 


log  M 


Fig.  6.  Comparison  of  effects  of  azide,  atebrine,  chlorpromazine  and  desamino- 
thyroxine on  the  dinitrophenol-induced  ATPase  activity  of  rat  liver  mitochondria. 
Experimental  conditions  as  in  Fig.  i . 

An  interesting  effect  of  atebrin  was  discovered  by  observing  that  this 
compound  in  a  concentration  of  o  •  5  mM  was  able  to  relieve  almost  com- 
pletely the  inhibitory  effect  of  desaminothyroxine  on  the  dinitrophenol- 
induced  ATPase  reaction  (Table  I).  Peculiarly  enough,  this  effect  of 
atebrin  was  not  shared  by  chlorpromazine.  Similarly,  no  atebrin-like 
effect  was  found  with  flavin  nucleotides. 

It  appeared  from  the  above  findings  that  the  effect  of  desaminothy- 
roxine on  the  dinitrophenol-induced  ATPase  clearly  differed  from  those 
of  the  flavin  antagonists,  whereas  the  difference  from  that  of  azide  was  less 
obvious.  However,  a  clear-cut  distinction  was  found  also  between  des- 
aminothyroxine and  azide  in  the  effects  of  the  inhibitors  on  the  Mg  +  +- 
activated  ATPase  of  the  Kielley  and  Kielley  preparation.  As  was  reported 
previously  [76],  the  Mg  "^  -activated  ATPase  is  characterized  by  a  stimula- 
tion, up  to  about  50*)^,  by  0"5-i  niM  sodium  dithionite.  This  compound, 
however,  not  only  stimulates  the  Mg  +  +-activated  ATPase  reaction  but 


EFFECTS  OF  THYROXINE  AND  RELATED  COMPOUNDS  ON  LIVER  MITOCHONDRIA  1 3 

TABLE  I 

Abolition   of  the   Desaminothyroxine-inhibition   of   the   Dinitrophenol- 
Induced  ATPase  Activity  of  Liver  Mitochondria  by  Atebrin 
Experimental  conditions  as  in  Fi^-  i. 


moles  Pj  20  min. 

Desamino- 
thyroxine, 

M 

None 

Atebrin, 

5    ■     10    '    M 

Chlorpro- 
mazine, 

5    >    10    ■''    M 

Chlorpro- 
mazine, 

ID    *   M 

FAD, 

5  X   10    "*  M 

0 

2x10^ 
2  X  10  J 

4-2 
0  ■  2 

5  9 

4-7 

4-0 

6-4 

1-8 

0-5 

3-7 

I  •  I 

0-<) 

40 

1-6 

0  ■  2 

is  also  able  to  counteract  to  a  remarkable  extent  the  inhibitorv  effects  of 
azide,  atebrin  and  chlorpromazine  in  this  reaction.  This  latter  effect  of 
dithionite,  which  is  illustrated  in  fig.  S,  was  strongest  in  the  case  of  azide, 


Nons 

Desaminotnyroxine,  10     M 

+  Azide,  25xlO"^M 

Chlorpromazine  75x10      M 

Atebrin,  75xlO'^M 

1  2  3 

Amytal ,  mM 

Fig.  7.  Influence  of  azide,  atebrin,  chlorpromazine  and  desaminothyroxine 
on  the  amytal  sensitivity  of  the  dinitrophenol-induced  ATPase  activity  of  rat  liver 
mitochondria.  Experimental  conditions  as  in  Fig.  i. 

followed  by  atebrin  and  chlorpromazine.  In  the  case  of  the  inhibition  by 
desaminothyroxine  no  counteraction  bv  dithionite  could  be  observed.  The 
only  other  inhibitor  of  the  Alg "'  -activated  ATPase  which  was  found  not 
to  respond  to  dithionite  was  pentachlorophenol. 

Finally,  as  briefly  reported  elsewhere  ['^4],  desaminothyroxine  exhibited 


14 


OLOV   LINDBERG    et   al. 

Az  f  de 


0.05 


0.15 


0.25mM 


At  ebrin 


3     mM 


C  hlorpromazine 


0.5mM 


0.5  mM 


1  mM 


Fig.  8.  Influence  of  sodium  dithionite  on  the  inhibition  of  the  ATPase 
activity  of  mitochondrial  fragments  by  various  agents.  Experimental  conditions 
as  in  Fig.  2.  The  ATPase  activity  of  the  sample  without  inhibitors  was  5-11  and 
7-03 /xmoles  Pj/2omin.  in  the  absence  :— C  and  presence  •—•of  o -5  mM  dithionite, 
respectively. 


EFFECTS  OF  THYROXINE  AND  RELATED  COMPOUNDS  ON  LIVER  MITOCHONDRIA         1 5 

no  photosensitizing  effect  on  the  Mg  +  ^-activated  ATPase,  in  contrast  to 
the  flavin  antagonists,  atebrin  and  chloropromazine. 

From  the  resuhs  presented  above  it  would  thus  appear  that  the  effect 
of  desaminothvroxine,  and  probably  of  thyroxine  analogues  in  general, 
differs  in  its  mechanism  from  those  of  other  known  inhibitors  of  the  two 
types  of  liver  mitochondrial  ATPase  reactions. 

(b)    exchange    REACTIONS 

P-A  TP  exchange 

The  effect  of  desaminothyroxine  on  the  P—ATP  exchange  reaction  of 
liver  mitochondria  was  investigated  using  the  conditions  previously 
established  in  this  laboratory  [85].  Under  these  conditions  the  ATP  and 
P;  concentration  are  virtually  constant  during  the  measurement,  and  the 
exchange  rate  is  about  0-35  /xmole/min.  per  mitochondria  from  200  mg. 
wet-weight  liver.  As  shown  in  Table  II,  the  P— ATP  exchange  reaction 

TABLE  II 

Inhibition  of  the  Pj-ATP  Exchange  Reaction  of  Rat  Liver  Mitochondria 

BY  Desaminothyroxine 

Assay  conditions  as  in  [85],  except  that  50,  rather  than  20,  /xmoles  P,  were 
added  per  sample. 

Desamino-                                                   Per  cent  ^-P  in  ATP  after 
thyroxine  — 


M  3  min.  13  mins. 

o  3-91  12-05 

ID**  3-92  11-30 

10-^  1-89  .         2-74 

IO-*  1-25  0-72 

was  strongly  inhibited  by  desaminothyroxine,  10^^  m  giving  an  almost 
complete  inhibition.  Again,  the  inhibition  was  present  from  the  onset  of 
the  incubation,  the  preincubation  with  desaminothyroxine  prior  to  the 
addition  of  Pj  and  ATP  had  no  influence  on  its  extent. 

ATP~ADP  exchange 

Wadkins  and  Lehninger  [86]  described  recently  the  occurrence  of  an 
exchange  reaction  between  ATP  and  ADP  in  phosphorylating  digitonin- 
preparations.  This  reaction  was  characterized  by  a  sensitivity  to  dinitro- 
phenol  which  was  lost,  without  loss  of  the  exchange  activity,  when  the 
preparations  were  damaged  so  as  to  lose  their  phosphorylating  capacity. 
Azide,  also,  although  not  inhibitory  to  the  ATP-ADP  exchange  reaction. 


1 6  OLOV    LINDBERG    et   al. 

was  able  to  render  the  reaction  insensitive  to  dinitrophenol.  Hence, 
Wadkins  and  Lehninger  [86]  proposed  that  the  dinitrophenol-insensitive 
ATP-ADP  exchange  reaction  represents  the  terminal  step  of  phosphate 
transfer  in  respiratory  chain  phosphorylation.  Since  a  similar  conclusion 
concerning  the  Mg  +  '  -activated  ATPase  was  previously  reached  in  our 
laboratory  [75-78]  it  was  of  interest  to  investigate  whether  the  ATPase 
activity  of  the  preparation  of  mitochondrial  fragments  was  paralleled  by 
an  ATP  ADP  exchange.  Such  a  connection  between  the  two  reactions 
has  recently  been  postulated  by  Bronk  and  Kielley  [87]  from  data  obtained 
with  phosphorylating  fragments  of  sonicated  mitochondria.  If  such  a 
connection  existed,  it  was  of  interest  to  investigate  whether  the  ATP-ADP 
exchange  reaction  was  also  sensitive  to  desaminothyroxine. 

TABLE  III 

Influence  of  Some  Agents  on  the  ATPase  and  ATP-ADP  Exchange  Reactions 
IN  Mitochondrial  Fragments  Prepared  According  to  Kielley  and  Kielley 

[80]. 

Conditions :  for  ATP-ADP  exchange  see  [87]  and  forATPase  see  exp.  in  Fig.  2. 
Incubation  for  4  min.  at  30  . 


/xmole 

P  transferred 

Additions 

ATPase 

ADP  exchange 

^i)  none 

0-79 

o-6o 

io~*  M  azide 

0-17 

0-63 

10^*  M  atebrin 

0-42 

0-64 

10-=^  M  AMP 

0-73 

0-31 

(2)   none 

o-6i 

0-45 

2  X  io~-  M  NaF 

o-o8 

0-34 

2  X  10  =*  M  AMP 

0-57 

0-14 

2X10    •■'  M  AMP  +  2  X   TO" 

-M  N 

aF 

o-o8 

0-07 

The  ATP-ADP  exchange  was  measured  by  using  terminally  labelled 
^^P-ADP  following  the  procedure  described  bv  Bronk  and  Kielley  [87]. 
Table  III  summarizes  some  properties  of  the  mitochondrial  fragment 
preparation  regarding  ATPase  and  ATP  ADP  exchange  activities.  In 
accordance  with  the  findings  of  Wadkins  and  Lehninger  [86]  the  exchange 
reaction  was  not  inhibited  by  a  concentration  of  azide  which  strongly 
inhibited  the  ATPase.  A  similar  effect  was  obtained  with  sodium  fluoride. 
Conversely,  however,  AMP  at  a  concentration  of  2  x  10^^  m  strongly 
inhibited  the  exchange  reaction  but  left  the  ATPase  activity  practically 
unaffected. 

In  Table  IV  the  effect  of  desaminothyroxine  on  the  ATP-ADP 
exchange  reaction  is  shown.  The  exchange  was  inhibited  almost  completely 


EFFECTS  OF  THYROXINE  AND  RELATED   COMPOUNDS  ON  LUTR  MITOCHONDRIA  17 

by  2  X  10'^  M  desaminothyroxine.  The  extent  of  inhibition  was  thus 
comparable  to  that  of  the  ATPase  activity.  It  was  found  on  the  other  hand 
(Table  IV)  that  an  ATP-ADP  exchange  reaction  occurred  also  in  the 
supernatant  obtained  in  this  preparation,  and  that  also  this  reaction  was 
strongly  inhibited  by  desaminothyroxine.  This  fraction  was  free  of  ATPase 
activity,  in  agreement  with  Kielley  and  Kiellev  [80].  The  exchange 
activity  found  in  the  supernatant  was  actually  higher  than  that  of  the 
sediment.  It  was  distinct  from  the  latter  as  indicated  by  the  fact  that  the 
residual  activity  could  not  be  removed  from  the  pellet  by  washing.  The 
possibility  was  considered  that  the  ATP-ADP  exchange  activity  of  the 
supernatant  might  be  a  reflection  of  the  adenylate  kinase  reaction,  which 
is  recovered  in  this  fraction  in  the  present  procedure.  However,  the  follow- 
ing findings  indicated  that  the  two  acti\ities  were  not  correlated:  (i)  The 

TABLE   IV 

Effect  of  Desaminothyroxine  on  the  ATP-ADP  Exchange  and  ATPase 
Reactions  of  Submitochondrial  Fractions  Prepared  According  to  Kielley 

AND  Kielley  [80]. 
Conditions  as  in  Table  III,  except  that  time  of  incubation  was  2  min.  Sediment 
and  supernatant  assayed  in  equivalent  amounts  in  terms  of  wet  weight  liver. 


Preparation  Additions 


ATPase  activity         Exchange  activity 
/xmoles  P  hydrolyzed  /Lmioles  P  exchanged 


Sediment 

none 
desaminothy- 

0 

33 

roxme,  02  mM 

0 

1 1 

Supernatant 

none 
desaminothv- 

0 

00 

roxme,  0-2  mM 

0 

00 

0-56 

o*o6 
I  -46 


adenylate  kinase  reaction  (as  measured  with  ADP  as  substrate  and  hexo- 
kinase  and  glucose  as  trapping  agent  for  the  ATP)  was  unafl:"ecced  bv 
2x10  ^.M  desaminothyroxine  whereas  the  ATP-ADP  exchange  was  almost 
completely  inhibited  (cf.  Table  IV).  (2)  The  adenylate  kinase  reaction  is 
inhibited  by  2  x  10  -  m  sodium  fluoride  [88],  whereas  the  ATP-ADP 
exchange  was  virtually  unaftected  (cf.  Table  III).  (3)  The  ATP-ADP 
exchange  activity  of  the  supernatant  compared  with  the  net  adenvlate 
kinase  activity  of  the  same  fraction  was  considerablv  higher  than  the 
corresponding  ratio  of  the  two  activities  in  a  purified  preparation  of  muscle 
myokinase. 

It  would  seem  from  these  data  that  a  desaminothyroxine-sensitive 
ADP-ATP  exchange  reaction  is  present  in  subfractions  of  rat  liver  mito- 
chondria; however,  the  relation  of  this  reaction  to  the  ATPase  is  not 
clear,  since  it  is  present  both  in  the  fraction  in  which  the  ATPase  is 
concentrated  and  in  the  fraction  devoid  of  ATPase  activitv. 


1 8  OLOV   LINDBERG    et   al. 

(c)   ATP-SPLITTING   REACTIONS   OF   NON-MITOCHONDRIAL   ORIGIN 

At  this  Stage  is  was  of  interest  to  test  the  effect  of  desaminothyroxine 
on  ATP-spHtting  reactions  of  non-mitochondrial  origin.  Myosin  ATPase, 
muscle  myokinase,  potato  apyrase,  and  yeast  hexokinase,  were  all  un- 
affected by  a  concentration  of  desaminothyroxine  of  io~^  m  (Table  V), 
indicating  that  desaminothyroxine  is  not  a  general  inhibitor  of  ATP- 
splitting  enzymes.  A  similar  correlation  was  previously  [89]  reached 
concerning  atebrin  and  chlorpromazine.  It  may  be  of  interest  on  the  other 
hand  that  a  liver  microsomal  ATPase  recently  studied  in  our  laboratory 
[90]  seems  to  be  sensitive  both  to  atebrin  and  chlorpromazine  and  to 
thyroxine  analogues. 

TABLE  V 
Effect  of   Desaminothyroxine  on  a   Number  of  ATP-Splitting   Enzymes 

The  mitochondrial  ATPase,  myosin,  potato  apyrase,  and  hexokinase  were 
assayed  in  the  manner  described  by  Low  [89].  Myokinase  was  assayed  by  measuring 
the  decrease  in  7  min.-P  in  the  presence  of  enzyme,  hexokinase  and  glucose. 


/^moles  ATP  split 

Enzyme 

0 

io-« 

10  5 

10-* 

M  desaminothyroxine 

Submitochondrial  ATPase 

Myosin-ATPase 

Potato  Apyrase 

Hexokinase 

Myokinase 

5-1 

5-8 
6-1 
4-1 

5-0 

5-0 
5-7 
6-9 
4-9 

3-2 

5-0 
5-7 
5-9 
4-7 

09 

4-5 
5-2 
4-6 
4-8 

(d)    DIAPHORASE    REACTIONS 

Previous  work  in  this  laboratory  [76-78,  85]  has  given  rise  to  the  con- 
cept that  the  mitochondrial  ATPase  reactions,  both  that  induced  by 
dinitrophenol  in  intact  phosphorylating  mitochondria,  and  the  Mg^  ^- 
activated  ATPase  reaction  appearing  in  structurally  damaged  mito- 
chondrial preparations,  involve  the  diaphorase  flavoprotein  as  intermediate 
phosphate  carrier.  A  possible  explanation  for  the  sensitivity  of  these 
reactions  to  thyroxine  and  related  compounds  would  seem  therefore  to  be 
that  these  compounds  interfere  in  some  way  with  the  mitochondrial 
diaphorase.  The  Kielley  and  Kielley  preparation  proved  to  be  a  suitable 
system  for  investigating  this  question,  since  it  was  found  [53]  that  this 
preparation  exhibited  besides  a  high  Mg  +  +-activated  ATPase  activity 
a  DPNH  diaphorase  reaction  also.  It  was  found,  moreover,  that  the  dia- 
phorase present  in  this  preparation  was  an  integral  part  of  a  mitochondrial 
DPNH  oxidase  system,  as  indicated  by  its  sensitivity  to  both  amytal  and 


EFFECTS  OF  THYROXINE  AND  RELATED  COMPOUNDS  ON  LIVER  MITOCHONDRIA  1 9 

antimycin  A.  A  further  valuable  property  of  this  preparation  was  that  it 
also  contained  the  external  type  of  DPNH-cytochrome  c  reductase,  known 
to  occur  in  liver  mitochondria  and  characterized  by  an  insensitivity  to 
amytal  and  antimycin  A  [38,  39,  47,  91-95]. 

IOOL 


02(+cytc) 


DCPIP 


0.05 

D-?sanrtmof hyroxine  ,mM 


0.1 


Fig.  9.  Effect  of  desaminothyroxine  on  the  oxidation  of  DPNH  by  various 
electron  acceptors  in  mitochondrial  fragments  prepared  according  to  Kielley  and 
Kielley  [Ho].  All  assay  systems  contained  0-02  M  phosphate  buffer,  pH  7-5,  and 
Q- 1  m.M  DPNH,  in  a  final  volume  of  3  ml.  In  the  case  of  O2  as  acceptor,  either  no 
further  additions  were  made  (line  marked  "O.,"),  or  0-005  iri.M  cytochrome  c  was 
added  ("  O.,  ( +  cyt.  c.) "),  and  the  oxidation  of  DPXH  was  followed  at  340  m/x.  In 
the  case  of  2,6-dichlorophenolindophenol  ("DCPIP")  as  terminal  electron 
acceptor,  the  dyestuff  was  added  in  a  final  concentration  of  0-04  mM,  and  its  reduc- 
tion was  followed  at  600  m/t ;  in  the  case  of  cytochrome  c  ("  cyt.  c  ")  this  was  added 
in  a  final  concentration  of  0-05  mM,  and  its  reduction  was  followed  at  550  m/x. 
In  both  latter  cases,  0-33  mM  KCN  was  included  in  the  test.  "  loo^^o  activity" 
was  (in  terms  of  /xmoles  DPNH  oxidized /min.  per  g.  liver):  0-146  with  O.., 
0-218  with  0._,  (+  cyt.  r),  0-620  with  cyt.  r,  and  0-487  with  DCPIP  as  electron 
acceptor. 


As  can  be  seen  in  Fig.  9,  desaminothyroxine  greatly  inhibited  the 
DPNH  oxidase  activity  of  the  Kielley  and  Kielley  preparation  as  measured 
without  added  cytochrome  r,  as  well  as  the  diaphorase  activity  as  measured 


20  OLOV    LINDBERG    et    al. 

with  2,6-dichlorophenoHndophenol  as  the  terminal  electron  acceptor.  At 
the  same  time  desaminothyroxine  only  shghtly  inhibited  the  DPNH 
oxidase  activity  obtained  in  the  presence  of  a  catalytic  amount  of  cyto- 
chrome c  and  the  DPNH-cytochrome  c  reductase  activity  as  measured 
with  cytochrome  c  as  terminal  electron  acceptor.  As  shown  in  Table  VI, 
the  sensitivity  to  desaminothyroxine  of  the  diaphorase  reaction  was 
roughly  equal  to  that  of  the  Mg  +  +-activated  ATPase  and  that  the  des- 
aminothyroxine sensitivity  of  the  latter  reaction  was  not  influenced  by  the 
presence  of  DPNH  and  cytochrome  c.  Conversely,  addition  of  ATP  and 
Mg^  +  to  the  DPNH-cytochrome  c  reductase  system  did  not  increase  the 
sensitivity  of  this  system  to  desaminothyroxine. 

TABLE  VI 

Comparison  of  Effects  of  Desaminothyroxine  on  DPNH  Diaphorase,  DPNH- 
Cytochrome  c  Reductase  and  ATPase  Activities  of  Mitochondrial  Frag- 
ments Prepared  According  to  Kiellev  and  Kielley'  [8o]. 

For  experimental  conditions  see  Figs.  2  and  9. 

",,  inhibition 
Reaction  by  10  *  M 

desaminothyroxine 

DPNH  diaphorase  (in  presence  of  ATP  and  Mg  +  +)  81 

DPNH-cyt.  c  red.  (in  absence  of  ATP  and  Mg  +  +)  18 

DPNH-cyt.  c  red.  (in  presence  of  ATP  and  Mg  +  +)  24 

ATPase  (in  absence  of  DPNH  and  cyt.  c)  84 

ATPase  (in  presence  of  DPNH  and  cyt.  c)  76 


It  would  appear  to  follow  from  these  data  that  the  DPNH  diaphorase 
component  of  the  amytal-  and  antimycin  A-sensitive  mitochondrial  DPNH 
oxidase,  which  probably  represents  the  main  phosphorylative  pathway  of 
terminal  electron  transport  in  the  intact  liver  mitochondria,  is  inhibited 
by  desaminothyroxine  to  the  same  extent  as  the  mitochondrial  ATPase 
reactions.  In  contrast,  the  non-phosphorylating  amytal-  and  antimycin 
A-insensitive  DPNH-cytochrome  c  reductase  appears  to  be  much  less 
sensitive  to  this  agent. 

Another  pyridine  nucleotide  oxidizing  flavoprotein  which  shows  a 
relatively  high  sensitivity  to  thyroxine  analogues  is  the  so-called  DT 
diaphorase.  This  enzyme,  the  detection  [96,  97]  and  purification  [98]  of 
which  was  reported  some  time  ago,  and  which  now  [53]  appears  to  be 
identical  with  the  vitamin  K  reductase  of  Martins  and  collaborators 
[99-102],  catalyzes  the  oxidation  of  both  DPNH  and  TPNH  by  various 
dyestuffs  and  quinones.  The  enzyme  occurs  mainly  in  the  soluble  cytoplasm 
but  is  present  to  a  small  extent  also  in  mitochondria  and  microsomes 


EFFECTS   OF   THYROXINE  AND   RELATED   COMPOUNDS   ON    LIVER   MITOCHONDRIA      21 

[98,  103].  The  inhibition  of  this  enzyme  by  thyroxine  and  related  com- 
pounds is  illustrated  by  Fig.  lo.  The  half  inhibitory  concentration  lies  at 
around  2  x  iq-^  m  in  the  case  of  desaminothyroxine  and  6  x  iq-^  m  in 
the  cases  of  thyroxine  and  triiodothyronine. 


o   desaminothyroxine 
D    L-lhyroxine 
d   L- triiodothyronine 
100. 


z     50 


7  6  5 

concentration. 


log  M 


Fig.  10.  Effect  of  L-thyro.xine,  L-triiodothyronine  and  desaminothyroxine  on 
DT-diaphorase.  The  450-fold  purified  enzyme  was  prepared  and  assayed  as 
described  in  [98]. 


(e)    RESPIRATION    AND    PHOSPHORYLATION 

All  the  effects  of  thyroxine  and  related  compounds  described  up  to 
now  in  this  paper  were  consistent  in  their  occurrence  and  magnitude  from 
one  experiment  to  another.  They  were  also  all  instantaneous  effects,  the 
extent  of  inhibition  being  independent  of  the  time  of  measurement.  In 
sharp  contrast,  no  consistency  was  obtained  when  the  effects  of  the  same 
compounds  on  the  respiration  and  phosphorylation  were  investigated. 
Furthermore  the  effects  were  often  progressive  with  the  time  of  incubation. 
To  illustrate  this  inconsistency,  some  of  the  experiments  performed  are 
summarized  in  Table  VII.  It  can  be  seen  that  both  thyroxine  and  des- 
aminothyroxine were  able  to  inhibit  respiration  (measured  with  glutamate 
as  substrate)  in  some  experiments  (Expts.  2a,  4a,  6a,  ib,  2b,  3b,  4b,  5b), 
whereas  in  others,  virtually  no  respiratory  inhibition  was  obtained  at  a 
concentration  as  high  as  10^'*  m  for  both  compounds  (Expts.  la,  6b).  In 
general  desaminothyroxine  was  more  inhibitory  than  thyroxine,  although 
reservation  must  be  made  here  for  the  presence  of  magnesium  ions  in  the 
system.  In  no  case  was  there  a  clear-cut  uncoupling  effect  obser\ed,  the  P/0 


22  OLOV   LINDBERG    et   al. 

TABLE  VII 

Effect  of  l-Thyroxine  and  Desaminothyroxine  on  Respiration  and  Phos- 
phorylation OF  Rat  Liver  Mitochondria  in  Absence  and  Presence  of  added 

DPN. 
Each  Warburg-flask  contained:  lo  mM  L-glutamate,  25  mM  potassium  phos- 
phate, pH  75,  I  mM  ATP,  4  mM  MgCl.j,  125  mM  sucrose,  30  mM  glucose,  an 
excess  of  yeast  hexokinase,  and,  when  indicated,  0-05  mM  DPN,  in  a  final  volume 
of  2  ml.  Gas  phase,  air.  Centre  well:  02  ml  2  M  KOH.  Temp.,  30  .  Time  of 
incubation,  20  min. 

(«)  h-T/iyroxine 


Amount  of 
mito- 
chondria 
per  flask 
(mg.  eq. 
liver) 

L-Thy- 
roxine 
mM 

Without  DPN 

„              Phos- 

^■^>'^""'  phate,       P/O 
(tatoms           , 

/tmoles 

With  DPN 

Expt. 

No. 

Oxygen, 

/^t  atoms 

Phos- 
phate 
/umoles 

P/O 

la 

200 

0 

9-7 

25-3 

2-62 

III 

24-0 

216 

o- 1 

95 

24-0 

2-50 

9-8 

3-3 

0-33 

2a 

200 

0 

5-8 

15-5 

2-67 

60 

15 -6 

2-58 

005 

06 

I  -7 

(2-91) 

5  '  5 

I  -2 

0-2I 

300 

0 

9-2 

22-4 

2-43 

92 

23-7 

2-50 

0-05 

60 

14-3 

2  40 

8-9 

2-8 

0-31 

400 

0 

134 

32-9 

2-40 

14-5 

33-0 

2-28 

0-05 

II    9 

29-8 

2-50 

132 

6-5 

049 

3a 

300 

0 

13-0 

34-4 

265 

13-1 

34-7 

265 

005 

100 

23-3 

2-33 

IO-8 

25-8 

2    40 

4a 

200 

0 

95 

23 -2 

2-44 

II  •  I 

22-8 

2 -06 

0-04 

7-9 

i6-3 

2 -06 

7-2 

i8-o 

2-52 

005 

4-4 

9-8 

2-22 

7-6 

6-9 

091 

5a 

200 

0 

8-0 

21  -3 

2-66 

8-7 

20 -3 

2-33 

005 

7-3 

19-6 

2  -69 

91 

18 -9 

2 -08 

005 

7-8 

168 

215 

92 

II-4 

1:24 

6a 

400 

0 

12-5 

35   4 

2    83 

12-8 

31-3 

2-45 

005 

8-8 

24   5 

2-79 

III 

99 

0-89 

o- 1 

4-6 

10 -8 

2-35 

12-8 

3-6 

0-28 

7a 

200 

0 

9-6 

27-2 

2-83 

IO-3 

27-0 

2-62 

005 

7-5 

21  -6 

2-86 

9-5 

I  I  -o 

116 

300 

0 

14-3 

40-3 

2-82 

14-6 

40  5 

2-78 

0-05 

12-7 

35-3 

2-78 

132 

34-9 

2-64 

EFFECTS  OF  THYROXINE  AND  RELATED  COMPOUNDS  ON   LIVER  MITOCHONDRIA      23 

TABLE  YU— continued 

(b)  Desaminot/iyroxirie 


Amount  of 

mito- 
chondria 
per  flask 
(mg.  eq. 

liver) 

Desa- 
mino- 
thy- 
roxine, 
mM 

Wi 

Oxygen 
/tatoms 

thout  DPN 

With  DPN 

Expt. 
No. 

Phos- 
phate 
/j,moles 

P  0 

Oxygen 

/natoms 

Phos- 
phate 
/xmoles 

P/O 

lb 

200 

0 

9-7 

25-3 

2-62 

1 1  ■  I 

24-0 

216 

o- 1 

0 

1-4 

— 

5-4 

2-5 

0-46 

2b 

300 

0 

i8-3 

45-7 

2  50 

i8-5 

45 -o 

2-44 

003 

i6-o 

43-6 

2-72 

i6-7 

42-1 

2-52 

o- 1 

95 

14-4 

152 

i6-6 

40-6 

245 

3b 

200 

0 

7 '  5 

ig-Q 

2-64 

6-3 

i6-2 

2-56 

0-05 

0-9 

I  -4 

(1-49) 

5-6 

3-7 

0-66 

/ 

0  •  I 

0-2 

— 

— 

2-7 

— 

— 

4b 

200 

0 

II-3 

31-6 

2-8i 

12-3 

32-3 

2  63 

0-02 

101 

26-9 

2-66 

IO-2 

27-0 

2-66 

005 

4-6 

8-4 

1-85 

5-3 

6-6 

1-25 

5b 

200 

0 

7  '7 

i8-i 

2-36 

9-3 

19  -6 

211 

003 

6-3 

i6-2 

2-57 

9-5 

20-9 

2 -20 

0  •  I 

2  -2 

I  -o 

0-45 

7-8 

1-6 

0-2I 

6b 

300 

0 

13-0 

38-0 

292 

13-8 

34-0 

2-46 

0-03 

II  -s 

31  -9 

2-70 

12-3 

33 -o 

2-68 

o- 1 

IO-2 

24-8 

2-43 

II-7 

28-9 

2-47 

ratios  being  roughly  normal  even  in  those  cases  where  respiration  was 
partially  inhibited.  Added  DPX  was  able  to  restore  the  inhibited  respiration 
and  the  restoration  was  as  a  rule  complete  in  the  case  of  thvroxine  but  most 
often  only  partial  in  the  case  of  desaminothyroxine.  In  the  case  of  des- 
aminothyroxine  inhibition,  the  stimulation  of  the  respiratory  rate  by  DPN 
was  accompanied  either  by  no  change  or  by  an  increase  in  the  rate  of  phos- 
phate uptake.  Most  peculiarly,  a  somewhat  different  effect  of  DPN  on 
phosphorylation  was  obtained  in  the  presence  of  thyroxine.  In  this  case  the 
increased  respiratory  rate  was  never  followed  by  an  increase  in  phosphate 
uptake  and  often  it  even  resulted  in  a  serious  decrease  of  the  latter,  thus 
giving  the  impression  of  a  true  uncoupling  effect  (Expts.  la,  2a,  4a,  5a, 
6a).  Despite  great  efforts  it  has  not  yet  been  possible  to  obtain  this  effect  in 


24  OLOV   LINDBERG   et   al. 

a  consistent  manner  and  it  would  appear  that  it  occurs  only  in  a  very 
narrow  range  of  thyroxine /mitochondrial  protein  ratio  (cf.  Expt.  2a). 

Thus  the  situation  especially  as  far  as  thyroxine  is  concerned  seems  to 
be  very  complicated  indeed.  This  is  further  emphasized  by  the  recent 
findings  of  Bronk  [104]  and  of  Dallam  et  al.  [105,  106]  that  in  their  systems 
thyroxine  was  even  able  to  cause  an  increase  of  the  phosphate  uptake 
coupled  to  the  oxidation  of  jS-hydroxybutyrate. 


3.  Concluding  remarks 

Evidence  has  been  presented  above  that  thyroxine  analogues  inhibit 
in  a  consistent  and  instantaneous  manner  the  P^-ATP  exchange  and 
dinitrophenol-induced  ATPase  reactions  taking  place  in  intact  liver  mito- 
chondria, as  well  as  the  Mg  ^  -activated  ATPase  and  ATP-ADP  exchange 
reactions  observed  in  mitochondrial  fragments.  A  common  denominator 
of  all  these  reactions  is  that  they  are  considered  to  include  one  or  several 
steps  of  the  reaction  sequence  involved  in  phosphorylation  coupled  to 
electron  transport.  The  succinate-linked  reduction  of  mitochondrial  DPN, 
a  process  which  is  also  considered  to  involve  a  partial  reaction  of  electron- 
transport-coupled  phosphorylation,  has  recently  been  reported  by  Chance 
and  Hollunger  [107]  to  be  highly  sensitive  to  thyroxine. 

It  has  been  concluded  from  previous  work  in  this  laboratory  [76-78, 
85]  that  the  mitochondrial  P--ATP  exchange  and  ATPase  reactions 
reflect  predominantly  only  one  of  the  three  phosphorylations  occurring 
along  the  respiratory  chain,  that  located  in  the  DPN-flavin  region.  Also 
the  succinate-linked  reduction  of  mitochondrial  DPN  is  thought  to  involve 
primarily  a  partial  reversal  of  this  phosphorylation  [51,  53,  107,  108].  It 
would  therefore  seem  that  the  observed  instantaneous  effects  of  thyroxine 
and  related  compounds  concern  primarily  the  DPN-flavin-coupled  phos- 
phorylation. The  finding  that  these  compounds  inhibited  the  diaphorase 
component  of  the  amytal-  and  antimycin  A-sensitive  DPNH  oxidase 
system,  is  consistent  with  this  conclusion,  and  may  indicate  that  the 
effect  of  thyroxine  and  related  compounds  on  the  flavin-linked  phos- 
phorylation consists  of  a  direct  action  on  this  enzyme.  The  DPNH- 
cytochrome  c  reductase,  which  is  insensitive  to  amytal  and  antimycin  A, 
and  which  probably  represents  a  non-phosphorylating  pathway  of  electron 
transport  [38,  39,  47,  91-95],  was  only  marginally  inhibited  by  the  com- 
pounds studied.  The  significance  of  the  observed  inhibition  of  the  DT 
diaphorase  cannot  be  understood  as  yet,  since  the  role  of  this  enzyme  in 
mitochondria  is  unclear  (cf.  [109-112]). 

The  effect  of  thyroxine  and  related  compounds  was  clearly  less  re- 
producible on  the  integrated  processes  of  respiration  and  phosphorylation 
than  it  was  when  studied  with  the  above  component  reactions  as  test 


EFFECTS  OF   THYROXINE   AND   RELATED   COMPOUNDS   ON    LIVER   MITOCHONDRIA      25 

systems.  This  lack  of  reproducibility  may  perhaps  be  explained  bv  assuming 
that  the  fla\in-linked  electron  transport  and  phosphorylation  reactions 
usually  occur  in  the  mitochondria  at  an  excess  capacity  in  comparison  to 
the  overall  rates  of  respiration  and  phosphorylation.  Such  an  assumption 
would  be  in  line  with  the  repeated  findings  [69,  83,  113-115]  that  the  rate 
of  P -ATP  exchange  considerably  exceeds  the  rate  of  phosphate  uptake 
in  mitochondria  under  conditions  of  maximal  respiration  and  phosphorv- 
lation.  The  possibility  that  the  inconsistent  and  gradual  character  of  the 
effects  of  thyroxine  and  related  compounds  on  respiration  and  phosphory- 
lation could  be  due  to  a  poor  penetration  of  these  compounds  through  the 
intact  mitochondrial  membrane  seems  improbable,  since  consistent  and 
instantaneous  effects  ensued  in  the  case  of  the  P—ATP  exchange  and 
dinitrophenol-induced  ATPase  reactions,  both  of  which  were  measured 
in  intact  mitochondria.  Moreover,  as  has  been  demonstrated  recentlv  by 
Tapley  and  Basso  [116],  the  uptake  of  thyroxine  and  related  compounds 
by  mitochondria  occurs  in  an  instantaneous  manner. 

As  outlined  in  the  introduction,  mitochondrial  swelling  and  related 
symptoms  seem  to  be  dependent  on  an  active  oxidative  phosphorvlation ; 
this  is  indicated  bv  the  findings  that  swelling  does  not  occur  in  the  absence 
of  oxidizable  substrate,  and  is  prevented  by  respiratory  inhibitors  and  bv 
dinitrophenol.  This  state  of  affairs  raises  the  question  as  to  how  mito- 
chondria in  a  state  of  acti\e  phosphorylation  are  able  to  maintain  their 
structural  integrity.  It  has  been  pointed  out  [29]  that  the  ATP-induced 
contraction  of  the  mitochondria  cannot  be  due  to  a  simple  reversal  of  the 
process  underlying  the  swelling,  since  the  contraction  is  not  inhibited  by 
dinitrophenol.  However,  there  are  now  indications  [50-53,  85,  117,  118] 
that  dinitrophenol  interferes  onlv  with  the  forward  reaction,  and  not  the 
reversal,  of  electron  transport-coupled  phosphorylation.  It  has  also  been 
shown  [26]  that  amytal  inhibits  the  ATP-induced  contraction  of  mito- 
chondria and  that  this  effect  is  not  shared  by  antiniycin  A  and  cyanide. 
These  facts  point  thus  to  the  possibilitv  that  a  reversal  of  the  fla\in-linked 
phosphorylation  may  play  a  part  in  the  contraction  of  the  mitochondria.  It 
would  not  seem  inconceivable,  therefore,  that  the  great  excess  capacity  of 
this  phosphorvlation  in  the  mitochondria  as  compared  with  the  overall 
rate  of  respiration  and  phosphorvlation  might  be  endowed  with  the 
important  function  of  maintaining  the  actively  phosphorylating  mito- 
chondrion in  a  structurallv  and  functionally  intact  shape.  It  would  be 
understandable,  then,  that  exposure  of  mitochondria  to  toxic  concentra- 
tions of  thvroid  hormone,  therebv  depriving  them  of  this  excess  capacity 
of  the  fla\"in-linked  phosphorvlation,  mav  lead  to  a  gradual  loss  of  their 
integrated  properties. 

In  summarv,  then,  the  present  data  seem  to  pro\'ide  a  first  information 
about  a  direct  effect  of  thvroxine  and  related  compounds  on  the  mito- 


26  OLOV   LINDBERG    et   al. 

chondrial  oxidative  phosphorylation  system,  with  the  flavoenzyme  com- 
ponent of  the  respiratory  chain  as  the  probable  site  of  action.  The  relation 
of  this  effect  to  those  established  in  previous  literature,  such  as  loss  of 
respiratory  control,  release  of  bound  DPN,  enhanced  swelling  of  the  mito- 
chondrial structure,  and  general  uncoupling  of  phosphorylation  from 
respiration,  remains  for  the  moment  unclear.  It  is  tempting  to  speculate, 
however,  that,  since  the  latter  effects  are  all  time-dependent  whereas  those 
described  in  the  present  paper  are  instantaneous,  there  might  exist  a 
cause-effect  relationship  between  them. 


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EFFECTS   OF   THYROXINE  AND  RELATED    COMPOUNDS   ON    LIVER    MITOCHONDRIA      29 

112.  Conover,  T.  E.,  this  volume. 

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Discussion 

Hess  :  I  would  like  to  mention  earlier  experiments  regarding  the  protecting 
activity  of  thyroxine  on  mitochondrial  function.  Martius  and  I  found  that  with 
small  concentrations  of  thyroxine  (lo"'^  m)  the  phosphorylating  activity  of  rat  liver 
mitochondria  was  conserved  over  a  period  of  16  hr.  in  the  cold  in  comparison  to  the 
control  which  lost  an  appreciable  amount  of  activity.  This  effect  could  be  due  to  an 
inhibition  of  a  latent  ATP-ase  activity,  which  is  activated  by  ageing  or  chemical 
actions  like  DXP  or  Mg"^  (Biocheni.  Z.  326,  191  (1955)).  On  the  other  hand,  I 
am  wondering  about  the  physiological  significance  of  the  action  of  thyroxine  or 
triiodothyronine  on  the  mitochondrial  ATP-ase,  because  we  found  that  triiodo- 
thyronine acts  on  the  cytochrome  region  of  oxidative  phosphorylation  in  digitonin 
particles  of  rat  liver  mitochondria.  The  particles  were  prepared  free  from  ATP-ase 
and  myokinase  and  no  ATP-ase  activity  was  released  by  the  addition  of  the 
hormone.  The  synthesis  of  ^'-P-ATP  in  the  presence  of  ascorbate,  cytochrome  c, 
ADP  and  inorganic  phosphate,  being  activated  (max.  35  "o)  by  triiodothyronine  in 
the  concentration  range  of  5  x  io~^  M,  responds  with  half  maximal  inhibition  to  the 
presence  of  5  x  io~®  M  triiodothyronine  (unpublished  experiments).  Perhaps,  in  the 
light  of  the  data  of  Prof.  Lindberg  and  ourselves  it  seems  that  there  can  be  a 
common  site  of  hormonal  action  upon  various  components  of  the  respiratory  chain, 
whose  elucidation  depends  upon  the  experimental  conditions.  I  am  wondering 
whether  you  have  data  which  shed  light  on  the  vertebral  level  of  the  hormonal 
action.  Is  there  a  phosphate  requirement  in  your  diaphorase  experiments  ? 

Lindberg:  No.  There  is  no  phosphate  requirement. 

Ch.ance  :  I  was  very  glad  to  hear  Prof.  Lindberg  mention  that  the  effect  of 
succinate  on  DPN  reduction  might  be  involved  in  thyroxin  action.  Hollunger  and 
I  did  investigate  how  much  thyroxine  would  be  required  to  inhibit  half  maximally 
the  effect  of  succinate  on  DPN  reduction  and  found  that  in  the  absence  of  magne- 
sium and  without  an  incubation  period  of  over  a  minute,  that  less  than  10  •*  M 
thyroxine  gave  half  maximal  inhibition  with  a  protein  concentration  of  about  one 
mg.  per  ml.,  so  it  is  extremely  sensitive  to  thyroxine.  I  don't  know  whether  this  is 
the  primary  site  of  thyroxine  action. 

Lindberg  :  It  is  our  feeling  that  it  is  difficult  to  reproduce  experiments  with 
thyroid  compounds  on  respiration  and  phosphorylation,  but  the  closer  you  come  to 
a  certain  region,  the  diaphorase  region,  in  the  respiratory  chain  the  easier  it  is  to 
get  a  good,  consistent  result. 


Components  of  the  Energy-Coupling  Mechanism  and 
Mitochondrial  Structure* 

Albert  L.  Lehxixger 

Department   of  Physiological   Chemistry,    The  Johns   Hopkins    ScJiool   of 
Medicine,  Baltimore,  Md.,  U.S.A. 

In  this  paper  approaches  taken  in  our  laboratory  to  the  isolation  and 
identification  of  active  mitochondrial  proteins  involved  in  the  mechanism 
of  respiratory  energy-coupling  and  in  the  mitochondrial  swelling  and 
contraction  cycle  will  be  described.  This  information  is  still  only  frag- 
mentary, but  it  gives  increasing  hope  that  the  mechanism  of  oxidative 
phosphorylation,  the  structure  of  the  mitochondrial  membranes,  and  the 
physical  nature  of  swelling  and  contraction  may  be  studied  in  molecular 
terms  and  that  these  entities  may  ultimately  be  at  least  partlv  reconstructed 
in  vitro  from  the  isolated  catalysts. 

Although  considerable  progress  has  been  made  bv  biochemists  in  the 
study  of  the  mitochondrion  actually  much  of  this  work  has  involved 
study  of  the  more  organized  physiology  of  the  mitochondrion,  its  control 
mechanisms,  and  the  action  of  hormones  and  drugs.  The  major  reason, 
without  question,  for  our  lack  of  specific  molecular  knowledge  of  mito- 
chondrial chemistry  is  the  fact  that  the  most  interesting  of  the  mito- 
chondrial reactions  take  place  in  what  I  once  designated  as  a  "solid-state 
enzyme  system"  [i],  namely  the  insoluble  complex  lipoprotein  structure 
of  the  mitochondrial  membranes,  which  contain  the  assemblies  of  res- 
piratory enzymes.  Since  the  first  efforts  of  Warburg  and  Keilin  many 
years  ago,  the  respiratory  enzymes  in  these  membranes  have  been  found 
to  be  remarkably  refractory  to  isolation  in  soluble,  homogeneous  form. 
The  relatively  limited  information  we  now  have  available  on  the  respiratorv 
carriers  and  coupling  enzymes,  as  isolated  molecular  entities,  has  figura- 
tively had  to  be  carved  out  of  solid  rock.  The  direct  approach  to  chemical 
study  of  the  molecular  components  of  the  mitochondrion  has  thus  been 
forbidding  and  frustrating,  and  has  given  rise  to  development  of  indirect 
methods  and  sometimes  less  than  direct  study  objectives. 

*  Original  work  in  the  author's  laboratory  was  supported  by  grants  from  the 
National  Institutes  of  Health,  the  National  Science  Foundation,  the  Nutrition 
Foundation,  Inc.,  and  the  Whitehall  Foundation. 


32  ALBERT   L.    LEHNINGER 

Respiratory  chain  phosphorylation,  the  most  prominent  mitochondrial 
activity,  is  not  only  a  very  complex  enzyme  system,  but  one  which  differs 
from  many  other  enzyme  systems  in  that  it  may  never  be  understood  in 
mechanism  until  the  active  sites  of  the  individual  enzymes  are  identified, 
since  there  are  apparently  no  low-molecular  weight  diffusible  inter- 
mediates. The  active  sites  of  the  enzymes  involved  are  thus  the  "inter- 
mediates". No  amount  of  indirect  experimentation  or  descriptive,  physio- 
logical study  of  the  mitochondrion  can  thus  replace  direct  isolation  and 
chemical  study  of  the  catalysts  of  energy-coupling  and  the  catalysts  activa- 
ting the  swelling-contraction  cycle. 

The  three  most  conspicuous  properties  of  mitochondria  are  {a)  the 
catalysis  of  respiration  and  energy  coupling,  {b)  the  occurrence  of  reversible 
swelling  and  contraction,  leading  to  water  movements,  which  are  geared 
to  respiration  and  (r)  ion  transport,  also  geared  to  the  respiratory  chain. 
After  considering  the  mitochondrial  membranes,  in  which  these  functions 
apparently  reside,  this  paper  will  deal  with  recent  work  on  some  isolated 
components  involved  in  these  functions. 

Molecular  organization  of  the  mitochondrial  membranes 

It  is  now  clear  from  many  items  of  evidence  that  the  enzymes  of 
respiration  and  coupled  phosphorylation  are  more  or  less  firmly  embedded 
in  or  on  the  mitochondrial  membranes ;  indeed  circumstantial  evidence 
suggests  that  the  inner  membrane  which  presumably  forms  the  cristae 
is  the  site  of  these  enzymic  activities.  When  the  mitochondria  are 
subjected  to  disruption  by  either  digitonin  or  sonic  oscillation,  they  shatter 
into  fragments  having  a  wide  spectrum  of  particle  weights.  We  have 
examined  the  enzymic  properties  of  a  series  of  such  fragments  differing 
in  sedimentation  rate  and  have  found  that  they  have  a  fairly  constant 
content  per  mg.  protein  N  of  cytochrome  oxidase,  ^-hydroxybutyric 
dehydrogenase,  succinoxidase,  and  ATP-ase,  regardless  of  particle  size 
[2],  suggesting  that  the  membranes  are  made  up  of  a  large  number  of 
recurring  structural  units,  each  of  which  may  contain  a  complete  assembly 
of  respiratory  carriers  in  finite  ratio,  as  determined  by  difference  spectra. 

Calculations  suggest  that  an  individual  liver  mitochondrion  may 
contain  5000-10  000  or  more,  of  such  respiratory  assemblies,  which  are 
more  or  less  evenly  distributed  on  the  membrane.  Extension  of  such 
calculations,  with  certain  assumptions,  indicates  that  a  large  fraction  of 
the  total  mass  of  the  membrane  is  made  up  of  these  assemblies  of  catalyti- 
cally  active  molecules — perhaps  as  much  as  40%  by  weight  [2].  The 
membranes  also  contain  considerable  "  phosphoprotein  "  and  the  phosphate 
groups  undergo  replacement  at  a  high  rate.  The  exact  disposition  of 
protein  and  lipid  molecules  in  the  membranes  is  not  yet  clear.  The  original 


COMPONENTS   OF   THE   ENERGY-COUPLING   MECHANISM  33 

concept  of  the  structure  proposed  by  Sjostrand  is  now  under  some  modi- 
fication and  refinement  by  other  workers,  notably  Robertson  [3].  In  any 
case,  alternation  of  oriented  lipid  and  protein  molecules  in  unimolecular 
layers  appears  to  be  the  basic  structural  plan. 

In  the  light  of  these  considerations  it  is  clear  that  the  permeability 
and  physical  state  of  the  mitochondrial  membranes  could  logically  be 
expected  to  be  functions  of  the  activity  or  state  of  the  catalytically  active 
proteins  which  apparently  make  up  such  a  large  part  of  the  structural 
mass  of  the  membranes.  Thus  the  swelling-contraction  cycle  of  the  mem- 
branes and  their  characteristic  selective  permeability  may  be  attributed  to 
mechano-chemical  changes  of  the  respiratory  and  coupling  enzymes, 
analogous  to  the  mechano-chemical  activities  of  the  actomyosin  complex. 
Furthermore  work  of  Gamble  in  our  laboratory  [4]  has  demonstrated  that 
the  membranes  are  also  the  site  of  perhaps  the  most  prominent  reaction 
of  mitochondrial  active  transport,  namely  the  active  binding  of  K+. 
Isolated  digitonin  fragments  of  the  membranes  bind  K  +  specifically 
during  coupled  phosphorylation  to  an  extent  which  can  account  nearly 
completely  for  the  entire  activity  of  intact  mitochondria. 

Finally  it  should  be  pointed  out  that  the  selective  permeability  of  the 
mitochondrial  membranes  may  be  an  element  in  physiological  control 
mechanisms.  For  example,  it  has  been  assumed  in  some  recent  specula- 
tions on  the  mechanism  of  the  Pasteur  reaction  [5]  that  ATP  generated 
by  mitochondria  is  segregated  or  compartmented  in  the  mitochondria,  so 
that  it  does  not  "mix"  with  glycolytically  generated  ATP. 

"Partial  reactions"  and  the  mechanism  of  oxidative 
phosphorylation 

No  attempt  will  be  made  to  review  in  any  detail  thedexelopment  of  ideas 
and  the  experimentation  which  have  led  to  current  outlines  of  knowledge ; 
recent  reviews  by  Slater  [6],  Lehninger  [i,  2,  7,  9,  10],  and  Chance  [8] 
may  be  referred  to.  However,  some  of  the  most  valuable  information  has 
come  from  study  of  the  so-called  "partial  reactions"  of  oxidative  phos- 
phorylation which  are  reflections  of  the  fact  that  at  least  some  if  not  all 
the  intermediate  reactions  are  reversible.  The  most  fundamental  dis- 
covery was  probably  the  finding  that  the  uncoupling  agent  dinitrophenol 
stimulates  hydrolysis  of  ATP,  indicating  that  a  "leak"  in  the  coupling 
mechanism  occurs  in  the  presence  of  this  reagent  (cf.  [11]).  Since  DNP  can 
release  respiration  from  its  dependence  on  ADP  in  the  absence  of  in- 
organic phosphate  (cf.  [6]),  the  site  of  action  of  DNP  appears  to  be  at  a 
point  prior  to  the  uptake  of  phosphate. 

A  second  "partial  reaction"  of  great  significance  is  the  ATP-P,^- 
exchange.  In  the  absence  of  net  electron  transport  the  terminal  phosphate 


34  ALBERT   L.    LEHNINGER 

group  of  ATP  exchanges  very  rapidly,  a  reaction  which  is  completely 
inhibited  by  dinitrophenol  [12]. 

Both  the  ATP-ase  and  ATP-F;^'-  exchange  have  been  studied  to 
greatest  advantage  in  so-called  digitonin  fragments  of  the  membranes  of 
rat  liver  mitochondria  [2,  13],  which  contain  complete  respiratory  chains 
and  coupling  mechanisms  but  do  not  show  Krebs  cycle  activity.  These 
fragments  are  relatively  free  of  enzymes  not  relevant  to  oxidative  phos- 
phorylation and  are  not  so  subject  to  compartmentation  phenomena  as 
are  intact  mitochondria.  With  these  fragments  it  was  found  that  the 
partial  reactions  are  specific  for  nucleotides  of  adenine.  Further,  the  require- 
ments and  kinetics  of  the  ATP  exchange  reaction  could  be  examined  more 
closely.  It  was  found  that  ADP  was  a  necessary  componerit  in  the  ATP-Pj^^ 
exchange  [14]  and  also  that  this  exchange  was  most  rapid  when  the 
respiratory  carriers  were  in  the  fully  oxidized  state  [15]. 

During  further  examination  of  the  mechanism  of  the  ATP-P^^^  ex- 
change, it  was  found  that  digitonin  preparations  also  catalyze  an  exchange 
of  labelled  ADP  into  ATP  which  was  inhibited  by  DNP  [14,  16].  This 
exchange,  which  is  specific  for  adenine  nucleotides,  does  not  require  in- 
organic phosphate  and  was  found  not  to  be  caused  by  other  phosphate- 
transferring  enzymes  known  to  catalyze  ATP-ADP  exchanges,  such  as 
adenylate  kinase  and  protein  phosphokinase.  The  exchange  activity  is 
stable  but  on  ageing  loses  its  sensitivity  to  DNP.  This  striking  finding  was 
corroborated  by  independent  experiments  with  azide;  this  agent  does  not 
afiect  the  rate  of  the  ATP-ADP  exchange  but  prevents  it  from  being 
inhibited  by  DNP.  The  tentative  conclusion  was  drawn  that  the  ATP- 
ADP  exchange  reaction  is  a  reflection  of  the  action  of  the  terminal  enzyme 
of  oxidative  phosphorylation,  but  that  this  enzyme  is  not  itself  sensitive 
to  DNP.  However,  it  was  postulated  that  its  sensitivity  to  DNP  was 
conferred  on  it  because  it  is  in  equilibrium  with  a  preceding  reaction  in 
the  coupling  sequence  which  has  a  DNP-sensitive  component. 

The  information  on  the  ATP-ase  activity  and  the  phosphate  and  ADP 
exchange  reactions  therefore  suggested  that  the  general  form  of  the 
energy-coupling  reactions  could  be  expressed  bv  the  following  equations 
[2,  14.  15]: 

Carrier r^ J  +  X        — ^  Carrier^^ '--' X  (i) 

Carrier^^j.'--' X  +  P^^     ^Carrier^^  +  P^—X        (2) 
P--X  +  ADP        TZlATP  +  X  (3) 

Reaction  (3)  thus  accounts  for  the  ATP-ADP  exchange,  reactions  (2) 
+  (3)  for  the  ATP-Pj-'^-  exchange,  and  the  sequence  of  reactions  3  +  2 
plus  the  following  reaction  (4)  for  DNP-stimulated  ATP-ase  activity: 

Carrier  '-^  X  — ^->  Carrier  +  X  (4) 


COMPONENTS   OF   THE   ENERGY-COUPLING    MECHANISM  35 

While  this  represents  the  simplest  statement,  as  will  be  seen  it  is  possible 
that  one  or  more  additional  intermediate  reactions  may  also  occur.  The 
above  sequence  accounts  for  the  finding  that  ADP  is  necessary  for  m- 
corporation  of  P,  into  ATP  and  that  P,  is  not  necessary  for  incorporation 
of  ADP  into  ATP  [2].  It  is  suggested  that  this  basic  mechanism  occurs  at 
all  three  phosphorylation  sites  of  the  respiratory  chain,  but  it  is  not  vet 
known  to  what  extent  each  of  the  three  sites  contributes  to  the  overall 
rates  of  the  partial  reactions.  The  outline  of  the  reaction  pattern  described 
here  is  in  general  consistent  with  most  experimental  observations,  but 
there  have  been  some  difi^erences  in  interpretation  which  are  fuUv  out- 
lined by  Slater  [6].  The  value  of  any  hypothesis  is  the  fruitfulness  of 
experimentation  which  it  may  suggest. 

Separation  of  the  ATP-ADP  exchange  enzyme 

It  was  found  that  the  relatively  stable  enzvme  catalyzing  the  ATP- 
ADP  exchange  could  be  extracted  from  acetone  powders  of  digitonin 
fragments  and  of  mitochondria  in  soluble,  highly  active  form  and  in 
nearly  complete  yield  [16].  It  has  now  been  purified  approximatelv  i  Re- 
fold by  Dr.  Charles  L.  Wadkins,  using  ammonium  sulphate  fractionation 
and  chromatography  on  cellulose  columns.  While  a  minor  component  is 
still  present,  preliminary  examination  indicates  that  the  protein  is  of 
relatively  small  molecular  weight  and  that  it  is  free  of  lipid.  The  highly 
purified  enzyme  requires  Alg^^  or  AIn~^  for  activity,  is  quite  stable  to 
dialysis  and  storage,  and  is  reversibly  inhibited  by  /i-chloromercuri- 
benzene  sulphonate  (PCMB).  It  has  been  assayed  for  activity  in  promoting 
other  phosphate-transferring  reactions  which  are  known  to  bring  about 
ATP-ADP  exchanges,  such  as  myokinase  and  protein  phosphokinase,  but 
such  activities  are  absent.  In  addition  the  enzyme  does  not  show  ATP-P^^'- 
exchange  activity  or  ATP-ase  activity,  in  the  presence  or  absence  of  DNP. 
The  enzyme  is  not  identical  with  that  described  by  Chiga  and  Plant  [17] 
who  have  obtained  a  highly  purified  enzyme  from  heart  mitochondria 
catalyzing  both  the  ATP-P,^-  exchange  and  ATP  ADP  exchange  and 
which  is  most  active  with  Mn  "^ '^. 

Recombination    of   soluble    ATP  ADP    exchange    enzyme    with 

digitonin  fragments 

The  soluble  form  of  the  ATP  ADP  exchange  enzyme  is  completely 
insensitive  to  dinitrophenol.  In  this  form  it  therefore  possesses  no  dis- 
tinctive characteristics  which  identify  it  as  a  portion  of  the  energy-coupling 
machinery  of  oxidative  phosphorylation.  Experimental  approaches  were 
therefore  taken  to  establish  more  firmly  the  relevance  of  this  enzyme  in 


36  ALBERT   L.   LEHNINGER 

its  soluble  form  to  the  mechanism  of  respiratory  energy  coupling.  In  intact 
mitochondria  [18]  or  membrane  fragments  [16]  the  ATP-ADP  exchange 
is  sensitive  to  DNP,  but  only  indirectly,  as  pointed  out  above.  If  the 
soluble  ATP-ADP  exchange  enzyme  could  be  "reconnected"  with  the 
DNP-sensitive  reaction,  the  soluble  enzyme  might  regain  its  DNP- 
sensitivity.  We  have  now  found  it  possible  to  reconfer  DNP-sensitivity  on 
the  soluble  form  of  the  ATP-ADP  exchange  enzyme  quite  simply  by 
adding  it  to  fresh  preparations  of  digitonin  fragments  [19].  The  typical 
experiment  illustrated  in  Fig.  i  shows  that  addition  of  the  DNP-insensitive, 
soluble  exchange  enzyme  to  fresh  rat  liver  digitonin  fragments  in  which  the 


500 


E 
< 


200 


i 


DP  SOL  COMBINED     EXPECTED 

ENZ  FOUND  IF    NO 

INTERACTION 


Fig.  I.  "Recombination"  of  soluble,  DNP-insensitive  ATP-ADP  exchange 
enzyme  with  digitonin  particles  to  restore  DNP-sensitivity.  System  contained 
o-oi  M  ATP,  o-oo6  M  ["C]-ADP,  and  5  x  10-^  m  DNPwhere  shown.  Black  portion 
of  bars  indicates  fraction  of  activity  sensitive  to  DNP. 


inherent  nucleotide  exchange  activity  is  inhibited  significantly  by  dinitro- 
phenol,  causes,  in  the  combined  system,  a  summation  of  the  total  exchange 
activities  in  the  absence  of  DNP.  However,  it  is  evident  that  a  very  large 
fraction  of  the  combined  ATP-ADP  exchange  activity  is  now  sensitive  to 
dinitrophenol,  to  a  far  greater  extent  than  would  be  expected  by  simple 
addition  of  the  two  reactions  measured  separately.  Many  experiments  of 
this  kind  thus  demonstrate  the  conferral  of  DNP  sensitivity  on  the  soluble 
nucleotide  exchange  reaction  by  adding  it  to  fresh  digitonin  particles. 
Dinitrophenol  sensitivity  is  not  conferred  on  the  soluble  enzyme  by  aged 
digitonin  particles,  which  are  incapable  of  oxidative  phosphorylation 
(Fig.  2).  Furthermore  the  DNP-sensitivity  of  the  recombined  system  is 
abolished  in  the  presence  of  azide,  which,  as  mentioned  above,  can  dis- 
sociate the  particulate  ATP-ADP  exchange  from  the  DNP-sensitive  site 
(Fig.  3).  The  conferral  of  DNP-sensitivity  on  the  soluble  ATP-ADP 
exchange  enzyme  is  thus  specific  and  this  finding  establishes  the  identity 


COMPONENTS   OF   THE   ENERGY-COUPLING   MECHANISM  37 

of  the  latter  with  the  exchange  activity  observed  in  intact  mitochondria. 
Addition  of  adenylate  kinase,  which  also  catalyzes  an  ATP-ADP  exchange, 
to  fresh  digitonin  particles  does  not  confer  DNP-sensitivity  on  this  re- 
action, for  example. 


800 


|=DNP-SENSITIVE    PORTION 


<j  a 
X  t- 

UJ    < 


FRESH  _ 

PARTICLES 


AGED 


u 


DP        SOL 


COMB- 
INED 


DP        SOL 


COMB- 
INED 


Fig.  2.  Failure  of  aged  digitonin  particles  (48  hr.  at  2  )  to  confer  DNP- 
sensitivity  on  soluble  ATP-ADP  exchange  enz\Tne. 

We  have  concluded  that  the  digitonin  particles  have  lost,  during  the 
course  of  preparation,  a  significant  fraction  of  the  molecules  of  the  ATP- 
ADP  exchange  enzyme  present  in  mitochondria.  However,  the  binding 
sites  to  which  these  molecules  are  normallv  attached  are  still  functional 


400 


■|=DNP   SENSITIVE    PORTION 
CONTROL 


+  0.002  M 
AZIDE 


DP        SOL      COMB- 
INED 


DP 


SOL      COMB- 
INED 


Fig.   3.   Elffect  of  0002  m  azide  on  "recombination  "  of  ATP-ADP  exchange 
enzyme  with  digitonin  particles. 


and  capable  of  "  rebinding"  the  soluble  form  of  the  exchange  enzyme  in  a 
specific  manner  so  as  to  bring  the  nucleotide  exchange  reaction  it  catalyzes 
into  equilibrium  with  a  DNP-sensitive  reaction.  In  consonance  with  this 
conclusion  we  have  found  that  there  is  an  upper  limit  to  the  capacity  of 


38  ALBERT    L.    LEHNINGER 

any  sample  of  digitonin  particles  to  "  rebind  "  soluble  ATP-ADP  exchange 
enzyme;  this  upper  limit  in  molar  terms  is  approximately  equal  to  the 
total  potential  ability  of  the  preparations  to  catalyze  phosphate  uptake  at 
a  P  :  O  ratio  of  3.  The  specific  rebinding  to  phosphorylating  assemblies 
indicates  that  the  soluble  ATP  ADP  exchange  enzyme  is  a  part  of  the 
coupling  machinery.  It  also  indicates  that  this  exchange  enzyme  has 
another  functional  site  which  is  reactive  with  an  as  yet  unknown  "sub- 
strate "  molecule,  presumably  a  preceding  enzyme  of  the  coupling  sequence, 
with  which  ATP  and  ADP  must  come  into  equilibrium. 

In  preliminary  experiments  it  has  been  found  that  the  soluble  ATP- 
ADP  exchange  enzyme,  when  added  to  digitonin  fragments  giving 
suboptimal  phosphorylation,  will  significantly  increase  the  P:0  ratio 
[9,20].  While  the  effect  requires  further  investigation,  it  gives  further 
evidence  for  participation  of  this  enzyme  in  the  mechanism  of  oxidative 
phosphorylation. 

M-factor 

With  the  availability  of  highly  purified  preparations  of  the  ATP-ADP 
exchange  enzyme,  efforts  were  begun  to  establish  the  nature  of  the  binding 
of  this  enzyme  to  preceding  components  of  the  coupling  mechanism,  in 
the  hope  that  the  chemical  nature  of  the  intermediate  reactions  catalyzed 
in  the  energy-coupling  sequence  could  thus  be  approached.  An  important 
lead  into  the  enzymic  aspects  of  the  recombination  phenomena  was 
afforded  by  the  finding  that  extracts  of  mitochondria  contain  a  soluble 
heat-labile  substance  of  protein  nature  (designated  as  M-factor)  which 
when  added  to  normal  digitonin  particles  greatly  increases  the  sensitivity 
of  the  inherent  ATP-ADP  exchange  reaction  to  dinitrophenol  [21]. 

Most  preparations  of  digitonin  particles  are  substantially  but  not 
completely  inhibited  by  dinitrophenol  [16].  The  degree  of  inhibition, 
which  varies  from  20-90*^,' (,  among  different  preparations  cannot  be  in- 
creased simply  by  increasing  dinitrophenol  concentrations  above  the  level 
of  approximately  5  x  io~^  m  (which  produces  essentially  complete  un- 
coupling of  oxidative  phosphorylation).  This  finding  suggests  that  the 
total  ATP-ADP  exchange  activity  of  any  given  sample  of  digitonin 
particle  consists  of  two  components:  a  "coupled"  component,  sensitive 
to  DNP,  and  a  "dissociated"  or  "uncoupled"  ATP-ADP  exchange 
activity,  which  may  be  a  portion  of  the  coupling  machinery  but  which 
has  been  "  dislocated  "  from  the  DNP-sensitive  reaction  during  preparation 
of  the  particles. 

Data  in  Fig.  4  show  that  addition  of  soluble  partly  purified  protein 
fractions  from  mitochondrial  extracts  can  greatly  increase  the  fraction  of 
the  total  ATP-ADP  exchange  activity  which  is  sensitive  to  dinitrophenol. 


COMPONENTS    OF    THE    ENERGY-COUPLING    MECHANISM 


39 


This  activity,  called  M-factor,  can  be  assayed  semi-quantitatively  with 
the  system  shown  and  it  has  been  purified  over  fortyfold.  The  starting 
material  is  either  a  phosphate  extract  of  acetone-powdered  mitochondria, 
from  which  M-factor  can  be  precipitated  by  relatively  low  concentrations 
of  ammonium  sulphate  or,  curiously,  simple  extracts  of  whole  fresh  rat 
liver  mitochondria  made  with  0-3  M  ammonium  sulphate.  Al-factor 
activitv  from  such  extracts  can  then  be  recovered  by  further  treatment 
with  ammonium  sulphate.  The  M-factor  preparations  contain  essentially 
no  ATP-ase  activitv,  ATP-P/'^-  exchange  activity  or  ATP-ADP  exchange 
acti\itv.  Thev  are  also  free  of  adenylate  kinase  and  protein  phosphokinase. 


100 


DNP-SENSITIVE    PORTION 


0  5^q.         lO^g         20;ig 

M-FACTOR 


Fig.  4.  Increase  of  DXP-sensitivity  of  .\TP-ADP  exchange  in  digitonin 
particles  by  Al-factor. 

Two  possibilities  are  open  for  the  mechanism  of  action  of  M-factor. 
The  first  is  that  M-factor  is  a  specific  "cementing"  protein  capable  of 
binding  with  the  ATP~ADP  exchange  enzyme  molecule  in  such  a  manner 
as  to  hold  it  in  the  appropriate  geometry  on  the  digitonin  particle  so  that 
it  may  become  reactive  with  the  preceding,  DPX-sensitive  reaction.  On 
the  other  hand,  Al-factor  may  itself  be  an  intermediate  enzyme  of  the 
energy-coupling  mechanism.  A  possible  mode  of  action  is  given  in  the 
following  equations: 


c 

arrier 

^  M 

c 

arrier 

-M  + 

P 

P~M  +  E 

P^ 

-E  + 

ADP 

electron 


Carrier-'^M 

Carrier +  P'^ 
P-^E  +  M 
ATP  +  E 


A I 


(7) 

(S) 


in  which  E  represents  the  ATP-ADP  exchange  enzyme  and  Carrier-^-M 
the  high-energv  complex  of  the  carrier  generated  during  electron  transfer. 
M  thus  could  be  visualized  as  replacing  the  X  of  the  earlier  formulation 


40  ALBERT   L.    LEHNINGER 

given  above.  M-factor  therefore  could  serve  as  an  intermediate  enzyme  in 
the  respiratory  energy  coupUng  sequence,  which  is  capable  of  transferring 
high  energy  groups  from  the  coupled  carrier  to  the  terminal  enzyme  E 
catalyzing  the  ATP-ADP  exchange  reaction.  This  possibility  is  being 
examined  directly  (a)  by  studying  the  participation  of  M-factor  in  the 
binding  of  external  soluble  ATP-ADP  exchange  enzyme  to  the  presumably 
empty  sites  in  digitonin  particles,  to  determine  the  sequence  and  stoi- 
chiometry  of  rebinding,  and  (b)  by  examination  of  complex  formation 
between  M-factor  protein  and  the  ATP-ADP  exchange  enzyme  by 
physical  methods  and  by  kinetic  approaches.  Recently  we  have  found  that 
when  the  soluble  ATP-ADP  exchange  enzyme  is  more  highly  purified, 
it  no  longer  can  "recombine"  with  digitonin  fragments  to  restore  DNP- 
sensitivity.  It  appears  possible  that  purification  has  removed  a  factor 
necessary  for  "recombination"  and  work  is  in  progress  to  determine 
whether  M-factor  is  involved  in  binding  soluble  ATP-ADP  exchange 
enzyme. 

Relationship  to  other  soluble  factors  supporting  oxidative 
phosphorylation 

While  many  investigators  have  observed  that  soluble  protein  fractions, 
particularly  from  bacterial  extracts,  can  increase  the  P  :  O  ratio  of  res- 
piratory chain  preparations  (cf.  [22,  23]),  in  general,  little  is  known  of 
the  enzymic  capabilities  of  such  soluble  fractions  and  the  contribution 
they  make  to  the  overall  coupling  mechanism.  Similarly  the  protein 
fraction  isolated  by  Titchener  and  Linnane  from  beef  heart  mitochondria 
[24],  which  increases  the  P  :  O  ratio  of  pretreated  beef  heart  particles,  is 
of  relatively  unknown  enzymic  competence.  However,  the  important 
work  of  Pullman,  Penefsky,  and  Packer  [25]  has  shown  that  a  highly 
purified  soluble  enzyme  catalyzing  DNP-stimulated  ATP-ase  in  the 
presence  of  Mg  +  +,  increases  the  P  :  O  ratio  of  mechanically  disrupted 
beef  heart  mitochondria.  This  factor,  which  shows  extraordinary  lability 
to  cold,  does  not  catalyze  the  ATP-Pj^^  exchange  reaction  or  an  ATP-ADP 
exchange  reaction.  While  this  soluble  ATP-ase  seems  not  to  be  identical 
with  either  our  ATP-ADP  exchange  enzyme  or  the  M-factor  described 
above,  at  least  on  superficial  comparison  of  properties,  yet  it  cannot  be 
excluded  that  there  are  elements  of  identity.  It  is  possible  for  example 
that  a  complex  of  the  ATP-ADP  exchange  enzyme  and  M-factor  may  be 
equivalent  to  the  Pullman  ATP-ase,  at  least  in  some  respects.  In  any  case 
further  development  of  both  lines  of  work  and  comparison  of  the  findings 
should  be  of  great  importance.  It  must  be  recalled  that  there  are  three 
phosphorylation  sites  in  the  respiratory  chain.  While  it  is  comforting  to 
think  that  all  three  operate  by  the  same  mechanism,  this  need  not  be  the 


COMPONENTS   OF   THE   ENERGY-COUPLING   MECHANISM  41 

case  and  it  is  therefore  possible  that  the  two  laboratories  are  studying 
reconstruction  of  different  phosphorylation  sites  in  the  chain. 

Of  greatest  importance,  however,  is  the  fact  that  it  now  seems  possible 
to  obtain  from  mitochondria  in  soluble  and  fairly  stable  condition,  specific 
protein  factors  which  appear  to  be  concerned  in  the  mechanism  of  res- 
piratory energy  coupling.  Identification  of  the  specific  enzymatic  capabili- 
ties of  these  reactions  may  represent  a  "breakthrough"  to  real  under- 
standing of  the  mechanism  of  oxidative  phosphorylation.  It  is  of  course 
quite  possible  that  current  hypotheses  on  the  mechanism  of  phosphoryla- 
tion and  the  postulated  role  of  these  factors  are  wrong.  However,  the 
important  thing  is  that  these  soluble  factors  are  now  at  hand  and  that  they 
can  be  examined  more  carefully  at  the  molecular  level  in  reconstituting 
oxidative  phosphorylation. 

The  swelling-contraction  cycle  of  mitochondria 

An  independent  approach  to  the  mechanism  of  oxidative  phosphorvla- 
tion  comes  from  work  on  the  contraction  of  mitochondria.  Abundant 
evidence  now  exists  that  both  the  swelling  of  mitochondria  and  their 
active  contraction,  leading  to  uptake  and  extrusion  of  water  respectivelv, 
are  phenomena  which  are  geared  to  the  activity  or  state  of  the  respiratory 
carriers  and  or  the  energy  coupling  mechanism  bv  which  ATP  is  formed 
in  mitochondria  (cf.  [26]).  The  enzymes  of  respiration  and  phosphorylation 
are  located  in  the  membranes  and  are  thus  in  a  strategic  position  to  provide 
mechano-chemical  control  over  membrane  properties,  such  as  their  mole- 
cular geometry  and  their  permeability.  In  the  following  discussion, 
swelling  and  contraction  will  refer  to  those  changes  in  membrane  properties 
specifically  associated  with  the  respiratory  chain  and  the  coupling 
mechanisms  which  can  lead  to  changes  in  the  mitochondrial  volume.  The 
transitory  and  purely  osmotic  changes  which  can  be  effected  in  mito- 
chondrial volume  for  some  seconds  on  altering  merely  the  osmotic  pressure 
of  the  medium  with  solutes  of  varying  degrees  of  penetrabilitv  [27]  will 
not  be  discussed  here.  Such  properties  are  of  course  common  to  all 
structures  bounded  by  semipermeable  membranes. 

That  mitochondrial  swelling  is  a  function  of  the  activitv  of  the  res- 
piratory chain  is  shown  most  strikingly  by  the  finding  that  swelling  is 
inhibited  by  respiratory  inhibitors  such  as  amytal,  antimvcin  A  and  cvanide 
or  by  simple  anaerobiosis  [2S-30].  These  factors  inhibit  swelling  induced 
by  a  variety  of  agents  (cf.  [26,  31])  such  as  phosphate,  thyroxine,  calcium, 
phlorizin,  and  many  others.  At  first  it  was  concluded  that  this  inhibition 
was  due  to  the  maintenance  of  the  carriers  in  the  reduced  state,  particularly 
DPNH  [28,  29].  However,  more  extensive  work  by  Chappell  and  Greville 
[32]  has  shown  that  it  is  more  likely  that  mitochondrial  swelling  requires 


42  ALBERT   L.    LEHNINGER 

occurrence  of  active  electron  transport  which  may  be  atforded  by  endo- 
genous substrates.  The  susceptibihty  to  swelHng  agents  can  be  conferred 
by  electron  transfer  in  different  segments  of  the  respiratory  chain.  On  the 
other  hand,  data  of  Birt  and  Bartley  among  others,  suggest  [33]  that  both 
the  oxidation-reduction  state  of  the  carriers  and  the  net  electron  flux  may 
be  elements  in  susceptibility  of  mitochondria  to  swelling. 

Another  piece  of  evidence  implicating  the  coupled  respiratory  chain  in 
the  swelling  process  is  that  sucrose  and  other  polyhydroxylic  compounds 
inhibit  swelling  [29,  34].  These  compounds  also  inhibit  respiration  and 
uncouple  phosphorylation  in  the  osmotically  insensitive  digitonin  fragments 
of  the  mitochondrial  membrane,  suggesting  they  act  as  enzyme  inhibitors 
rather  than  in  an  osmotic  sense  [35].  In  addition,  dinitrophenol  has  been 
found  to  inhibit  swelling  when  added  to  fresh  mitochondria  [36],  whereas 
on  delayed  addition  it  becomes  an  activator  of  swelling  [37]. 

Mitochondrial  swelling  /;/  vitro  induced  by  thyroxine  or  phosphate 
leads  to  an  increase  of  volume  of  between  100  to  200  per  cent  over  a 
period  of  10-15  min.  at  20.  Small-amplitude  swelling  of  tightly-coupled 
mitochondria  has  also  been  found  to  be  dependent  on  respiration  or 
respiratory  state  by  Holton  [38]  and  Packer  [39]. 

In  large  amplitude  mitochondrial  swelling  taking  place  over  longer 
periods,  a  large  part  of  the  respiratory  control  by  ADP  is  lost,  as  well  as 
ability  to  phosphorylate,  possibly  as  a  consequence  of  the  "stretching"  of 
respiratory  assemblies  in  the  membranes.  However,  as  is  shown  below, 
such  drastic  mitochondrial  swelling  is  still  reversible  by  ATP  [34,  40]. 

Mitochondrial  contraction 

Price  et  al.  first  established  in  their  thorough  study  [41]  that  re- 
institution  of  phosphorylating  respiration  in  swollen  mitochondria  by 
appropriate  supplements  to  the  test  medium  would  cause  a  contraction 
with  gravimetrically  measurable  extrusion  of  water.  Similar  observations 
were  reported  by  Beyer  et  al.  [42].  Since  the  mechanism  of  oxidative 
phosphorylation,  at  least  in  its  terminal  stages,  has  been  thought  to  be 
reversible,  it  would  have  appeared  likely  that  ATP  alone  in  the  absence 
of  respiration  might  be  able  to  effect  mitochondrial  contraction.  However, 
with  the  exception  of  a  very  limited  contraction  observed  by  Chappell 
and  Perry  in  pigeon  breast  muscle  mitochondria  by  the  addition  of  ATP 
[43],  no  significant  success  was  reported  in  effecting  contraction  by  mere 
addition  of  ATP  to  swollen  mitochondria.  In  1959  we  found  that  the 
failure  of  ATP  to  effect  contraction  could  be  traced  to  the  presence  of 
sucrose  in  the  test  media  ordinarily  used  in  such  experiments  [29,  44]. 
Sucrose  in  approximately  isotonic  concentrations  completely  inhibits  con- 
traction of  swollen  liver  mitochondria  by  ATP,  whereas  mitochondria 


COMPONENTS    OF    THE    ENERGY-COUPLING    MECHANISM  43 

contract  quite  well  in  a  butfered  KCl  medium  on  addition  of  ATP.  The 
addition  of  ATP  +  Mg  ^  +  +  serum  albumin  was  found  to  cause  immediate 
contraction  of  mitochondria  swollen  by  a  wide  variety  of  agents,  including 
thyroxine,  oleate,  phlorizin,  calcium,  PCAIB,  phosphate,  and  many  others 
[44]  and,  curiously,  mitochondria  swollen  by  digitonin  and  by  carbon 
tetrachloride.  Actual  extrusion  of  water  was  demonstrated  by  gravimetric 
methods  to  accompany  the  optical  changes.  It  was  shown  that  several 
hundred  moles  of  water  could  be  extruded  per  mole  of  ATP  split  [34]. 

The  molecular  mechanism  of  the  ATP-induced  contraction  can  be 
shown  to  be  completely  independent  of  respiration  and  net  phosphoryla- 
tion, since  it  proceeds  perfectly  well  in  a  medium  containing  sufficient 


P:0  =  2- 

K^  =  i2m/^M/MG 


P:O  =  00 
K^=OOI 


Q    03 


[PO^OO 
iK*  =002 


Time  (rnin) 

Fig.  5.  Independence  of  ATP-induced  contraction  from  oxidative  phosphoryla- 
tion and  K  ~  binding.  .Allowing  mitochondria  to  stand  in  swollen  state  at  25  for 
extended  periods  abolishes  phosphorylation  and  K  ^-binding,  without  affecting 
abilitv  to  contract. 


cyanide  or  other  respiratory  inhibitors  to  block  respiration  or  in  the 
presence  of  sufficient  dinitrophenol  to  completely  uncouple  oxidative 
phosphorylation,  as  long  as  ATP  is  in  excess  [34].  On  the  other  hand,  it 
is  clear  that  the  ATP-induced  contraction  must  employ  at  least  a  portion 
of  the  energy  coupling  machinery,  since  this  contraction  is  blocked  by 
inhibitors  such  as  azide,  which  disconnects  the  ATP-ADP  exchange 
reaction  from  the  dinitrophenol  sensitive  site,  and  is  also  inhibited 
characteristically  by  sucrose  and  many  other  sugars  and  polvhvdroxvlic 
alcohols,  which  are  also  known  to  inhibit  oxidative  phosphorylation  and 
the  ATP-P^'-  exchange  reaction  as  well  as  ATP-ase  [3^].  Furthermore, 
contraction  of  mitochondria  by  ATP  is  not  dependent  on  any  specific 
ionic  environment  and  can  occur  in  mitochondria  whose  abilitv  for  K  + 
transport  is  completely  inactivated  [40]  (Fig.  5). 

It   appears   likely   that   mitochondrial    contraction    induced   bv   ATP 


44  ALBERT   L.    LEHNINGER 

causes  extrusion  of  small  solute  molecules  along  with  water.  On  the  other 
hand,  since  swollen  mitochondria  are  still  relatively  impermeable  to  large 
molecular  weight  compounds  such  as  serum  albumin  and  polyvinylpyrroli- 
done [29],  the  soluble  proteins  and  other  high  molecular  weight  substances 
in  the  intramitochondrial  space  probably  do  not  leave  during  contraction. 
If  this  is  the  case,  then  osmotic  work  is  carried  out  during  ATP-induced 
contraction,  because  it  leads  to  a  more  concentrated  solution  of  the  high 
molecular  weight  solutes  inside  the  mitochondria. 

Preliminary  examination  of  thyroxine-swoUen  and  ATP-contracted 
mitochondria  with  the  electron  microscope  [45]  shows  the  swollen  mito- 
chondria to  be  very  large  and  spherical,  containing  large  optically  clear 
vesicles  and  few  or  no  recognizable  cristae.  After  contraction,  they  are 
much  smaller,  optically  dense,  contain  no  vesicles,  and  show  nearly  normal 
cristae. 

Swelling  and  contraction  of  mitochondria  therefore  clearly  involve  the 
respiratory  chain  and  the  associated  energy  coupling  mechanisms,  but  the 
two  phases  employ  or  are  activated  by  different  segments  or  portions  of 
this  complex  enzymic  machinery.  Swelling  requires  the  action  of  the 
respiratory  chain,  but  the  contraction  does  not;  however,  terminal  stages 
of  energy  coupling  appear  to  be  involved  in  the  latter  phase.  The  swelling 
and  contraction  therefore  appear  not  to  be  reversible  in  the  sense  that  they 
employ  reversibly  the  same  controlling  catalysts.  Furthermore,  because 
of  the  occurrence  of  two  mitochondrial  membranes  it  is  possible  that 
swelling  may  be  a  function  of  the  properties  of  the  inner  membrane,  for 
example,  and  contraction  a  function  of  the  outer  membrane,  since  all 
kinds  of  mitochondrial  swelling  can  be  contracted  again  by  ATP  [40]. 

Because  sucrose  and  other  polyhydroxylic  compounds  such  as  glucose, 
raffinose,  fructose,  dextran,  xylose,  mannitol,  and  sorbitol  in  concentrations 
of  o-i  M  to  0-6  M  inhibit  both  swelling  and  contraction  (the  latter  more 
strongly),  as  well  as  ATP-ase  and  the  ATP-P^^-  exchange  in  osmotically 
insensitive  digitonin  particles,  we  have  suggested  that  these  compounds 
are  efficacious  in  preserving  mitochondrial  morphology  during  isolation 
more  for  their  ability  to  act  as  inhibitors  of  an  intermediate  enzymic 
reaction  involved  in  the  swelling-contraction  cycle  than  for  their  relative 
slowness  of  penetration  [29,  35].  Simple  alcohols  or  compounds  like 
ethylene  glycol  and  glycerol  do  not  inhibit.  It  has  been  suggested  that  the 
polyhydroxylic  alcohols  act  as  artificial  acceptors  in  "transferase "  reactions, 
displacing  the  normal  group  acceptor. 

Biochemistry  of  the  contractile  process 

It  now  seems  possible  to  approach  chemical  analysis  of  the  mechanism 
of  contraction.  A  guiding  principle  for  such  approaches  is  the  hypothesis 


COMPONENTS   OF   THE   ENERGY-COUPLING   MECHANISM  45 

that  the  swelling  and  contraction  are  reflections  of  the  action  of  "  mechano- 
enzyme"  systems  similar  to  the  actomyosin  of  muscle,  in  which  inter- 
mediate enzymes  of  the  energy-coupling  mechanism  may  act  as  "  mechano- 
enzymes"  and  undergo  change  of  shape  or  charge  distribution.  If  the 
ATP  ADP  exchange  enzyme  can  exist  in  phosphorylated  form,  this  might 
difi^er  in  configuration  or  in  geometrical  arrangement  from  the  unphos- 
phorylated  form  and  account  for  changes  in  the  geometry  or  properties  of 
the  membrane  [26,  34].  There  is  in  fact  a  striking  resemblance  between  the 
ATP-ase  activity  of  the  actomyosin  system  and  that  of  the  phosphory- 
lation mechanism. 

Mechanisms  of  membrane  changes 

Y 

I      Electron  transport  »-  carrier~X 

Carrier  ~X  +  P,     <         "    carrier  +  P~X 


P~X  +  (E 


—(E)    +  X 
.P 
'ADP 


^.  ADP  ^=^   (!i'^=^  di' 
\-  k  ^ADP  i    ^ 


ATP 


■■mechanoproteins" 


n    ATP  + 


Membrane 
protein 


Protein 
phospho 
kinase 


Fig.  6.  Two  possible  mechanisms  for  alteration  of  membrane  state  through 
ATP-driven  changes  of  shape  or  conformation  of  protein  molecules.  In  the  first, 
the  mechano-enzyme  may  be  an  intermediate  enzyme  of  energy  coupling,  such  as 
\Ej  whose  shape  may  change  as  a  function  of  binding  of  P  or  ADP  or  ATP.  In  the 
second,  an  independent  membrane  protein  (possibly  the  "phosphoprotein "  of 
mitochondria)  may  be  activated  by  ATP  to  yield  mechanical  changes. 

Figure  6  indicates  two  possible  ways  in  which  contraction  might  be 
visualized.  In  the  upper  half  is  shown  a  representation  in  which  an  inter- 
mediate enzyme  of  the  energy-coupling  sequence  is  the  "mechano- 
enzyme"  activating  the  contractile  changes.  It  is  postulated  to  change 
shape  or  charge  distribution  when  it  is  phosphorylated. 

On  the  other  hand,  it  is  possible  that  the  contractile  protein  is  not  a 
member  of  the  coupling  sequence  itself  but  perhaps  is  in  equilibrium  with 
it.  We  have  suggested  that  the  "phosphoprotein"  of  mitochondria  is  a 
possible  candidate,  since  earlier  work  with  Friedkin  had  shown  that  the 


46  ALBERT   L.   LEHNINGER 

phosphorus  of  this  fraction  has  a  significantly  high  rate  of  turnover  [46]. 
Although  preliminary  work  by  Dr.  Ishikawa  as  a  test  of  this  possibility 
appeared  very  promising,  because  the  mitochondria  contain  a  protein 
phosphokinase  [47],  analytical  difficulties  of  an  unexpected  nature  still 
prevent  a  clear-cut  evaluation. 

However,  a  rather  different  development  provided  an  important 
approach  to  the  chemistry  of  contraction.  This  was  the  finding  that 
mitochondrial  swelling  induced  by  reduced  glutathione  is  different  from 
swelling  caused  by  other  agents  such  as  phosphate  or  thyroxine  in  its 
kinetics  and  in  its  control  [48].  Furthermore,  swelling  induced  by  gluta- 
thione is  not  reversed  by  ATP  under  the  same  conditions  which  can  reverse 
swelling  caused  by  other  agents.  It  was  soon  found  that  this  failure  of 
contraction  was  due  to  the  detachment  from  the  mitochondria  of  a  necessary 


Fig.  7.  Requirement  of  C-factor  for  contraction  of  glutathione-swollen 
mitochondria. 

protein  factor  (designated  C-factor)  on  exposure  to  glutathione  [49].  This 
factor  leaked  out  into  the  medium  and  could  be  recovered.  Only  when  this 
factor  was  added  back  in  appropriate  concentrations  to  the  test  medium 
could  contraction  of  the  mitochondria  be  observed  in  the  presence  of 
ATP  +  Mg  +  +  +  BSxA..  The  C-factor  can  be  assayed,  as  is  shown  in 
Fig.  7,  by  the  level  of  contraction  achieved  as  a  function  of  the  concentra- 
tion of  the  factor  in  the  medium.  With  this  simple  bioassay  it  was  found 
possible  to  demonstrate  the  occurrence  of  C-factor  in  sonic  extracts  of 
mitochondria  and  in  extracts  of  digitonin  particles.  It  was  found  not  be  to 
dialyzable,  it  is  labile  to  heat,  and  survives  acetone  drying  or  lyophilization. 
It  has  now  been  purified  over  fifty-fold  by  Dr.  Diether  Neubert. 

Recently  we  have  carried  out  an  examination  of  C-factor  activity  in 
different  tissues  and  in  different  tissue  fractions  [50]  with  the  surprising 
finding  that  this  factor  is  found  not  only  in  mitochondria  but  also  in  extra- 
mitochondrial  cytoplasm.  C-factor  activity  has  been  found  in  the  mito- 
chondria and  extra-mitochondrial  cytoplasm  of  a  number  of  tissues  of  the 


COMPONENTS    OF   THE   ENERGY-COUPLING    MECHANISM  47 

rat,  in  Ehrlich  ascites  tumour  cells,  and  also  in  erythrocytes  and  extracts 
of  Escherichia  coli.  Of  great  significance  is  the  finding  that  C-factor 
activity  is  especially  rich  in  contractile  tissues  like  skeletal  muscle  and  car- 
diac muscle.  We  have  earlier  called  attention  to  the  possibility  that  C- 
factor  may  bear  a  relation  to  the  mitochondrial  membrane  analogous  to 
that  born  by  actin  to  myosin.  While  this  analogy  is  only  suggestive  at  this 
stage,  it  is  of  interest  to  point  out  that  erythrocyte  membranes  can  be 
induced  to  change  shape  in  the  presence  of  ATP  [51]  and  it  is  now  well 
known  from  studies  of  Abrams  and  others  that  bacterial  protoplast  mem- 
branes also  undergo  swelling-contraction  cycles  which  are  metabolism- 
dependent  [52]. 

It  is  also  significant  that  the  partlv  purified  specimens  of  C-factor 
contain  some  ATP-ase  activity,  which  suggests  that  they  may  be  related 
to  the  factor  described  by  Pullman  et  al.  [2^^  which  is  capable  of  restoring 
oxidative  phosphorylation  in  heart  preparations. 

Other  factors  in  mitochondrial  contraction 

There  is  evidence  that  mitochondrial  substances  other  than  C-factor 
are  necessary  in  contraction.  It  has  been  found  bv  measuring  the  light- 
scattering  envelope  of  intact  mitochondria  [^^t^]  that  the  ratio  of  light 
scattered  at  135  to  that  scattered  at  45'  to  the  incident  beam  measures  a 
change  in  mitochondrial  configuration  induced  bv  ATP  which  is  not 
measureable  by  simple  light  absorption  or  bv  light  scattered  at  90  .  This 
change  is  promoted  by  substance(s)  "leaking"  from  mitochondria  stored 
simply  at  o    in  sucrose  which  are  apparently  not  identical  with  C-factor. 

Lastly,  the  rather  puzzling  effect  of  L-thyroxine  in  stimulating  mito- 
chondrial contraction  by  ATP  [54]  must  be  mentioned.  L-thvroxine  is 
thus  not  only  a  swelling  agent,  but  can  also  stimulate  contraction. 

Concluding  remarks 

A  number  of  soluble  mitochondrial  factors  having  significant  action  of 
an  apparently  enzymic  nature  on  oxidative  phosphorylation  and  mito- 
chondrial swelling  and  contraction  have  now  been  recognized.  These 
include  (i)  the  soluble  ATP-ADP  exchange  enzyme,  (2)  M-factor,  (3) 
C-factor,  (4)  sucrose-extracted  contraction  factor,  as  well  as  earlier  des- 
cribed entities  such  as  (5)  U-factor  [^^^  (presumablv  an  uncoupling  fatty 
acid)  and  (6)  R-factor,  a  protein  fraction  which  releases  respiration  from 
its  dependence  on  ADP  but  which  does  not  uncouple  phosphorvlation 
[56].  With  the  protein  factors  from  beef  heart  mitochondria  separated  by 
Titchener  and  Linnane  [24]  and  by  Pullman  et  al.  [25],  as  well  as  the  in- 
creasing successes  in  dissociation  and  recognition  of  the  respiratorv  carriers, 
a  significantly  large  number  of  elements  of  the  mitochondrial  membrane 


48  ALBERT   L.   LEHNINGER 

are  recognizable  and  are  susceptible  to  assay,  purification,  and  use  in 
reconstruction  experiments.  Furthermore,  applications  of  physical 
methods  to  isolated  proteins  of  the  membrane,  particularly  the  coupling 
enzymes,  may  provide  direct  approaches  to  study  of  the  "mechano- 
enzyme"  nature  which  we  have  postulated  to  account  for  the  swelling- 
contraction  phenomena. 

While  our  knowledge  of  oxidative  phosphorylation  and  of  the  mech- 
anism of  mitochondrial  swelling  and  contraction  is  still  fragmentary  and 
there  are  many  loose  ends  still  to  be  accounted  for  satisfactorily,  it  is 
evident  that  these  complex  chemical  and  mechano-chemical  activities  of 
the  mitochondrial  membrane  are  approachable  on  the  molecular  level  and 
can  be  at  least  partly  reconstructed  or  reconstituted.  It  is  of  course  im- 
portant to  examine  these  phenomena  as  they  occur  in  intact  mitochondria 
because  of  the  extraordinary  possibilities  they  afford  for  physiological 
control  mechanisms,  however  it  is  clear  that  the  greatest  challenge  and  the 
most  significant  developments  toward  full  knowledge  of  the  molecular 
biology  of  the  mitochondrion  can  be  expected  to  come  from  examination 
of  the  separate  molecular  entities  participating  in  these  complex  reactions. 


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50  ALBERT   L.    LEHNINGER 

Discussion 

Estabrook:  Dr.  Fugmann  and  I  have  studied  the  reactions  of  the  endogen- 
ous pyridine  nucleotides  of  the  digitonin  particles  and  we  find  that  the  extent  of 
reduction  of  the  pyridine  nucleotide  during  the  steady  state  is  largely  dependent 
on  the  presence  of  versene.  In  other  words  one  cannot  get  a  cyclic  response  of  the 
pyridine  nucleotide  with  ADP  without  versene  being  there.  Have  you  tried  any  of 
these  metal  chelators  or  do  you  have  any  indication  that  the  action  of  the  M-factor 
has  any  relation  to  chelating  properties  ?  My  second  question  is :  according  to  your 
second  mechanism,  phosphate  itself  should  serve  as  an  uncoupler  because  it  will 
liberate  high  concentrations  of  free  carrier.  I  wonder  if  you  have  any  explanation 
for  this  point. 

Lehninger  :  With  regard  to  your  first  question  I  should  have  mentioned  that 
for  the  action  of  the  M-factor  we  need  Mg-+  so  possibly  some  function  of  the  metal 
is  required  here,  but  the  effect  is  not  given  by  EDTA.  With  regard  to  your  second 
question,  I  don't  think  that  the  mechanism  I  had  on  the  board  is  the  last  word  on  the 
mechanism  of  phosphorylation  but  you  have  to  start  somewhere  with  a  working 
hypothesis.  The  value  of  any  of  these  hypotheses  is,  I  think,  just  a  matter  of  how 
many  crucial  experiments  you  can  design  based  on  them.  In  the  case  of  the 
second  mechanism,  phosphate  could  act  as  an  uncoupling  agent  only  if  P~M  is 
split  by  either  hydrolysis  or  reaction  with  E,  to  regenerate  free  M,  which  is 
required  for  respiration  as  well  as  the  carrier.  Since  the  molar  amount  of  E  is 
limited,  free  M  could  be  regenerated  only  if  free  E  could  be  regenerated.  Arsenate 
can  uncouple  because  the  intermediate  arsenylated  compounds  are  presumably 
unstable. 

Chance:  I  was  very  interested  in  Dr.  Lehninger's  discussion  of  the  mechano- 
protein  which  I  think  is  a  very  important  concept,  especially  in  view  of  the  work  of 
Pullman  and  Racker.  However,  being  part  physical  chemist,  there  is  a  critical 
question  which  I  can  ask,  that  is  whether  the  time-course  of  contraction  of  the 
hypothetical  mechano-protein  in  mitochondria  is  one  that  is  compatible  with  its 
function  in  oxidative  phosphorylation.  It  is  true  that  the  ADP-induced  light 
scattering  increase  on  contraction,  observed  by  Packer  and  myself,  is  a  rapid  one 
and  is  possibly  compatible  with  the  mechano-protein  idea,  but  it  seems  to  me  that 
it  is  the  opposite  sense  to  the  ATP-induced  contraction,  which  also  seems  to  me 
to  be  on  a  slower  time  scale.  Are  there  two  kinds  of  mechano-proteins,  one  studied 
in  the  presence  of  ATP  and  on  a  fairly  long  time  scale,  and  one  observed  on  ADP? 

Lehninger  :  In  our  earlier  publications  it  appeared  that  ATP-induced  contrac- 
tion was  quite  slow;  this  was  because  we  did  not  understand  the  optimum  condi- 
tions. Now  on  more  refined  investigation  I  am  not  sure  that  there  is  a  great  differ- 
ence in  the  speed  of  ATP-  and  ADP-induced  contraction.  I  don't  think  there  is  any 
great  disparity  there,  but  there  is  no  doubt  that  the  changes  that  we  and  Packer 
observed  lag  behind  the  changes  in  the  steady  state  of  the  carriers.  However,  I 
don't  think  this  is  completely  mcompatible  with  the  picture  I  have  drawn  for  the 
following  reason :  the  morphology  of  the  mitochondrion  is  pretty  complicated,  and 
it  is  possible  that  in  ATP-  or  ADP-induced  contraction  there  is  a  sequence  of 
events.  First,  in  the  primary  event  a  given  molecule  changes  shape  or  contracts  in 
the  presence  of  ATP,  possibly  synchronous  with  change  in  oxidation-reduction 


COMPONENTS   OF   THE   ENERGY-COUPLING    MECHANISM  5 1 

State.  However  after  the  primary  event  there  are  certainly  secondary  events,  which 
follow  the  ATP-induced  change,  but  it  may  take  a  certain  finite  lag  period  before 
the  rest  of  the  very  complicated  mitochondrial  structure  undergoes  those  changes 
which  register  as  total  light  scatter.  Does  that  make  sense  ? 

Ch.\nce  :  The  facts  are  that  we  don't  have  fast  ADP  changes  and  the  ATP  ones 
are  slower. 

Jagendorf  :  When  you  restore  DXP  sensitivity  to  ATP  :  ADP  exchanges  by 
mixing  the  isolated  enzyme  with  fresh  particles  do  you  have  to  deplete  these  parti- 
cles first  ? 

Lehninger:  Xo.  They  are  already  partly  depleted.  We  can  deplete  them 
further  by  exposing  them  to  high  salt  concentrations  or  to  glutathione. 

Jagendorf:  Could  this  M-factor  be  a  polynucleotide  as  in  Pinchot's  experi- 
ments ? 

Lehninger:  .Although  M-factor  is  a  heat-labile,  non-dialysable  substance,  it  is 
possible  that  it  carries  a  bound  polynucleotide.  We  have  tried  polynucleotides  and 
at  least  with  the  polyadenylic  acid  and  polyuridylic  acid  specimens  we  had  they  did 
not  work. 

Mitchell:  I  was  very  interested  in  Dr.  Lehninger's  concept  of  a  mechano- 
protein,  but  I  would  suggest  that  we  need  to  take  more  care  not  to  be  too  romantic 
about  this  concept.  We  heard  in  the  first  discussion  of  this  s>Tnposium  (Dr. 
Kendrew)  how,  during  the  uptake  of  oxygen,  the  haemoglobin  molecule  can  be- 
come reorientated  and  change  shape  ;  and  I  suggest  that  in  thinking  about  mechano- 
proteins  we  should  bear  this  example  in  mind  as  it  illustrates  the  principle  of  change 
of  orientation  implicit  in  the  mechano-protein  conception  and  is  free  of  the 
potentially  misleading  associations  of  the  usual  elastic  catapult  kind  of  model. 
Perhaps  it  would  be  better  to  use  the  phrase  "mechano-protein  complex",  because 
we  do  not  want  to  give  the  feeling  that  a  change  of  shape  and  the  contraction  which 
results  is  necessarily  due  to  the  contraction  of  individual  protein  molecules  as  we 
used  to  think  in  the  case  of  muscle. 

Lehninger:  I  completely  agree  that  we  should  not  be  too  precise  about  speci- 
fying the  mechanism.  You  are  quite  right,  I  would  regard  haemoglobin  as  an 
example  of  a  mechano-protein  in  this  very  general  way.  Obviously  changes  in 
charge  distribution  or  a  dissociation  are  also  possible  molecular  mechanisms  under 
this  generic  name. 

Azzone  :  You  have  tried  to  calculate  the  stoicheiometry  between  the  moles  of 
HiO  extruded  from  the  mitochondria  and  the  moles  of  ATP  split.  I  wonder 
whether  it  is  necessary  for  ATP  to  be  hydrolyzed  or  merely  to  be  bound  to  the  mito- 
chondrial membrane.  Do  you  think  it  is  possible  to  inhibit  the  ATP-ase  activity 
and  still  maintain  the  ATP-induced  contraction  of  the  mitochondria  ? 

Lehninger  :  As  I  said  we  are  not  prepared  to  state  that  ATP-ase  activity  is 
necessary  for  the  contraction ;  contraction  may  require  only  binding  of  ATP,  just  as 
in  Morales'  theory  of  contraction  of  actomyosin.  Secondly,  we  have  not  found  any 
inhibitor  or  circumstances  where  we  can  inhibit  ATP-ase  without  also  inhibiting 
contraction. 


Ascorbate-Induced  Lysis  of  Isolated  Mitochondria — 

A  Phenomenon  Different  from  Swelling  Induced 

by  Phosphate  and  Other  Agents* 

F.  Edmund  Hunter,  jR.f 

The    Edzcard    Mallinckrodt    Department    of    Pharmacology,     Washington 
University  School  of  Medicine,  St.  Louis,  Mo.,  U.S.A. 

Many  substances  induce  swelling  in  isolated  liver  mitochondria. 
Relatively  few  substances  other  than  surface  active  agents  such  as  the 
detergents  produce  a  lytic  type  of  effect.  In  the  course  of  earlier  work  [i] 
we  observed  that  low  concentrations  of  ascorbate  have  a  characteristic 
lytic  effect.  This  phenomenon  has  been  studied  to  establish  the  nature  of 
the  reaction,  to  obtain  clues  on  the  key  groups  in  the  mitochondrial 
membrane,  and  to  explore  the  possibility  of  preparing  submitochondrial 
particles  or  units  and  soluble  proteins  in  this  way. 

Glutathione  and  cysteine  produce  effects  which  appear  to  be  similar 
to  but  not  identical  with  those  seen  with  ascorbate.  Feldott,  Johnson,  and 
Lardy  [2]  mention  a  lytic  effect  of  cysteine,  and  Lehninger  [3]  has  ex- 
tensively studied  swelling  induced  by  10  mM  glutathione. 

In  the  present  work  the  swelling  of  isolated  liver  mitochondria  [i]  was 
followed  by  light  scattering  or  absorbancy  changes  of  dilute  suspensions 
in  0-33  M  sucrose  containing  0-025  ^^  ^"s  buffer,  pH  7-4.  Routinelv  the 
temperature  was  22-25"  ^^^  5^0  m/x  light  was  used.  To  minimize  inter- 
ference by  certain  additions  such  as  dyes,  775  m^u,  w^as  used  in  some 
experiments.  Protein  was  measured  by  the  method  of  Lowry  et  al.  [4]. 

Figure  i  illustrates  the  striking  differences  between  the  absorbancv 
changes  occurring  with  low^  concentrations  of  ascorbate  and  those  ac- 
companying swelling  induced  by  phosphate  or  /S-hydroxybutyrate.  Charac- 
teristically ascorbate  induced  swelling  or  lysis  has  a  lag  period  averaging 
20  min.  This  is  followed  by  a  rapid  fall  of  the  optical  density  to  very  low 

*  Abbreviations  used  in  this  report  are :  DNP  for  2,4  dinitrophenol,  EDTA 
for  ethylenediaminetetraacetate,  P-P  for  inorganic  pyrophosphate,  P-P-P  for 
inorganic  triphosphate,  DEDTC  for  diethyldithiosemicarbazide,  /)CMB  for 
/)-chloromercuribenzoate,  DHF  for  dihydrox^-fumarate,  DHM  for  dihydroxy- 
maleate,  and  DHA  for  dehydroascorbate. 

t  The  work  in  this  communication  was  carried  out  with  the  collaboration  and 
assistance  of  Francisco  Guerra,  Beverly  Schutz,  Joan  Fink,  Lillian  Ford,  Audrey 
Scott,  and  Ellen  Smith. 


54 


F.    EDMUND    HUNTER,    JR. 


values,  much  lower  than  the  plateau  seen  with  phosphate  and  substrates. 
An  important  characteristic  is  that  ascorbate  lysis  occurs  only  with  low 
concentrations,  o-2-i  mM  being  optimal.  Higher  concentrations  lengthen 


0,5 


0.4 


0,3 


0.2 


0.1 


CONTROL 


-•-■-♦—*      "--a. 


•^ 


ASCORBATE 
5mM 
^.    lOmM 


i 
5m  M    PO4 

2mM^-0H-BUTYRATE    \ 

A 

0.2-  ImM  ASCORBATE        ^ 


20  30  40 

MINUTES 


50 


60 


Fig.  I.  Absorbancy  changes  at  520  m/it  when  dilute  suspensions  of  liver 
mitochondria  are  treated  with  phosphate,  /3-hydroxybutyrate,  or  ascorbate.  All 
additions  made  at  zero  time.  Mitochondrial  protein  was  150  /^g./ml. 


0.5 


0.4 


o  0.3 
in 


0.2  - 


0.1    - 


-»                • 

^. 

CONTROL 

v^»\r^ 

• 

■ « 

'\v\   \ 

\  \\        \ 

\«     \  ^ 

5mM  GSSG 

gsh\^    *^ 

\^  2mM-''\ 

^  • 

4 

\  \ 

\    \\ 
\    1  ^ 

\  5mM       \ 

VlOmM      \ 
yK    l5mM     \ 

'  \r 

i          \    5mM 

\ 

V 

\^      VCYSTEINE 

x^^ 

■• 

1                1 

• 

• 

L 

— ""    ■  '   " * 

10 


20 


30  40 

Ml  NUTES 


50 


60 


Fig.   2.   Absorbancy  changes  due  to  swelling  or  lysis  of  mitochondria  induced 
by  cysteine  or  glutathione. 

the  lag  period  and  inhibit  swelling,  until  15  mM  prevents  swelling  com- 
pletely for  I  to  2  hr.  This  has  special  significance,  for  high  concentrations 
of  ascorbate  do  not  block  phosphate  or  substrate-induced  swelling. 

Figure  2  demonstrates  that  reduced  glutathione  (GSH)  and  cysteine 
produce  an  absorbancy  change  like  that  seen  with  low  concentrations  of 


ASCORBATE-INDUCED    LYSIS   OF    ISOLATED    MITOCHONDRIA 


55 


ascorbate,  but  the  concentrations  required  are  considerably  higher.  In 
addition,  raising  the  concentration  shortens  the  lag  period.  If  there  is  a 
high  concentration  which  inhibits  swelling  it  was  not  reached  in  these 
experiments.  Oxidized  glutathione  (GSSG)  produces  a  slow  steady  swel- 
ling, possibly  the  result  of  partial  reduction.  High  concentrations  of  as- 
corbate do  prevent  glutathione  induced  swelling. 

Ascorbate  Ivsis  is  more  difficult  to  produce  as  the  concentration  of  the 
mitochondrial  suspension  is  increased,  and  unlike  phosphate-induced 
swelling,  it  is  not  seen  at  all  in  concentrated  suspensions.  While  this  could 
result  from  rapid  exhaustion  of  ascorbate  or  Oo,  increasing  ascorbate  in 
proportion  to  the  mass  of  mitochondria  and  thorough  oxygenation  have 

0,5 


0.4 


0.3 


CONTROLS 


30      "     60 
MINUTES 

Fig.  3.  Absorbancy  changes  due  to  ascorbate  lysis  of  mitochondria  are  pre- 
vented as  long  as  strict  anaerobiosis  is  maintained.  Admitting  air  after  50  min. 
results  in  a  typical  effect  of  ascorbate. 

been  only  partly  successful  in  more  concentrated  suspensions.  Perhaps 
trace  metal  binding  by  the  greater  mass  of  protein  is  involved. 

Ascorbate  induced  lysis  of  the  mitochondria  requires  the  presence  of 
some  oxygen  (Fig.  3).  Strict  anaerobiosis  will  prevent  lysis  for  at  least 
2  hr.  If  air  is  admitted  after  i  hr.,  lysis  occurs  in  the  usual  characteristic 
fashion,  with  the  possible  exception  that  the  lag  period  may  be  a  little 
shorter.  In  this  requirement  for  oxygen,  ascorbate  lysis  resembles  swelling 
induced  by  phosphate  and  many  other  agents.  While  swelling  with  these 
other  agents  has  been  demonstrated  to  be  dependent  on  endogenous  or 
added  substrate  in  nearly  every  case,  this  is  not  true  for  ascorbate.  Ageing 
or  other  treatments  of  mitochondria  which  deplete  endogenous  substrate 
do  not  alter  the  response  to  ascorbate.  Long-term  ageing  at  o^  tends  to 
shorten  the  lag  period. 

Because  of  the  oxygen  requirement,  the  effect  of  electron  transport 


56  F.   EDMUND   HUNTER,   JR. 

chain  inhibitors  on  ascorbate-induced  lysis  was  investigated.  These  in- 
hibitors have  been  shown  to  block  the  oxygen  and  substrate  requiring 
swelling  induced  by  phosphate,  etc.  [i,  5,  6].  Figure  4  illustrates  that 
amytal  does  not  block  ascorbate  lysis,  while  2  mM  NaCN,  4-6  /xM  anti- 
mycin  A,  or  4-6  [xM  SN  5949  inhibit  completely.  These  observations 
suggest  that  inhibition  of  electron  transport  from  cytochrome  b  and  above 
prevents  the  ascorbate  effect.  Somewhat  puzzling  is  the  fact  that  slightly 
higher  cyanide  and  antimycin  concentrations  are  required  to  block 
ascorbate  than  to  block  the  phosphate  type  of  swelling.  Moreover,  10  mM 
NaNg,  which  is  moderately  effective  against  phosphate,  produces  only 
slight  inhibition  of  ascorbate  and  GSH  induced  swelling.  Possibly  these 


0.5 


A  +  6«M  ANTIMYCIN^     A  +  6z^M   SN  5949 


A 


l^^^4^^::zz:         ^  \   ' 


20  30 

Ml  NUTES 

Fig.  4.  Effect  of  electron  transport  chain  inhibitors  on  the  absorbancy  change 
associated  with  ascorbate  lysis  of  mitochondria. 


reducing  agents  interfere  with  the  action  of  azide.  Perhaps  an  explanation 
for  some  of  the  other  differences  will  evolve  from  the  work  of  Chappell 
and  Greville  [7]. 

Ascorbate  has  long  been  known  to  feed  electrons  into  the  electron 
transport  chain  via  added  cytochrome  c  [8,  9].  A  much  lower,  but  not 
insignificant  oxygen  consumption  occurs  without  added  cytochrome  c. 
Just  how  much  of  this  represents  electron  transfer  via  the  cytochromes  is 
uncertain  at  the  moment.  If  ascorbate  lysis  depends  on  ascorbate  oxidation 
(electron  transfer  from  ascorbate),  inhibition  by  antimycin  A  suggests 
electrons  entering  the  electron  transport  chain  at  cytochrome  b,  ubiquinone, 
or  lower,  rather  than  cytochrome  c.  Figure  5  shows  an  experiment  to  test 
whether  added  cytochrome  c  would  change  the  effect  of  ascorbate.  It  does 
not,  but  the  experiment  is  inconclusive,  as  the  concentration  differences 
make  it  impossible  for  all  the  ascorbate  to  be  oxidized  instantaneously  by 


ASCORBATE-IXDUCED    LYSIS   OF    ISOLATED    MITOCHONDRIA  57 

the  cytochrome  c.  Some  persists  for  a  period  of  time,  possibly  feeding 
electrons  to  cytochrome  b.  It  is  clear  that  rapid  transfer  of  electrons  from 
ascorbate  to  added  cytochrome  c  to  O2  via  the  electron  transport  chain, 
as  must  be  occurring,  is  not  capable  of  producing  swelling  in  the  presence 
of  15  niM  ascorbate.  If  electron  transfer  through  some  carrier  like  cyto- 
chrome b  is  of  special  importance,  one  possible  mechanism  for  the  blocking 
action  of  high  concentrations  of  ascorbate  would  be  that  cytochrome  c 
is  kept  so  completely  reduced  that  electron  transfer  through  cytochrome  b 
is  not  possible. 

DNP  at  ID"'*  M,  which  blocks  phosphate  +  substrate  induced  swelling 
under  the  conditions  used  here,  does  not  influence  the  action  of  ascorbate 


0.5 


0.4 


o  0,3  - 


0,2  - 


^  lOi^M  CYT0CHR0MEC+15mM  ASCORBATE 


0.3m M  ASCORBATE       \^\ 
+  CYT0CHR0ME  C   -^X    \ 


l5mM  ASCORBATE 


10 


20  30  40 

MINUTES 


Fig.   5.   Failure  of  added  cytochrome  c  to  modify  the  effects  of  either  low  or 
high  concentrations  of  ascorbate  on  mitochondrial  suspensions. 


(Fig.  6).  Lower  concentrations  (lO"''  m),  which  hasten  substrate-induced 
swelling,  do  not  shorten  the  lag  period  with  ascorbate. 

The  chelating  agent  EDTA  (10  '  to  io~^  m)  blocks  ascorbate  induced 
lysis  at  least  as  easily  as  it  blocks  virtually  all  other  swelling  inducing 
agents  (Fig.  6).  Other  chelating  compounds  are  \ery  effective  in  preventing 
ascorbate  lysis,  even  though  they  may  be  much  less  effective  or  ineffective 
in  blocking  phosphate  type  swelling.  Complete  inhibition  of  the  ascorbate 
effect  was  seen  with  i  mM  penicillamine,  2  mM  o-phenanthroline,  o  •  i  mM 
8-hydroxyquinoline,  2  mM  citrate,  i  mM  inorganic  pyrophosphate,  i  mM 
inorganic  triphosphate,  10  m.M  oxalate,  and  0-2  mM  diethyldithiocar- 
bamate  (Fig.  6).  In  each  case  the  concentration  is  roughly  the  minimum 
for  complete  inhibition  lasting  an  hour  or  more.  Lower  concentrations 
cause  partial  block  or  markedly  prolong  the  lag  period. 

Oxaloacetate,  pyruvate,  and  phenvlpyruvate  block  ascorbate  (Fig.  7) 


58  F.   EDMUND    HUNTER,    JR. 

just  as  they  do  other  swelhng  agents.  Possibly  they  should  be  grouped  with 
EDTA.  As  has  been  observed  in  other  work  Mn  +  +  is  considerably  more 
effective  than  Mg  +  +  in  preserving  mitochondrial  structure.  Because  of 


0.5 


20  30  40 

MINUTES 

Fig.  6.   Inhibition  of  ascorbate-induced  lysis  of  mitochondria  by  metal  com- 
plexing  agents. 


0.5 


0.4- 


0.3 


o 

CO 
ID 
< 


0.2  - 


0.1  - 


-• — •—  • — • « 

•v^        ASCORBATE  +    / 
ImM    PYRUVATE 
\or  0.1  mM  Mn*"^ 
■^  or2mMOXALACETATE 

A 

0.3mMASC0RBATE\ 

\  +  5mM       \ 
\~ ^*^ 

1                1 

V^-r^^^^-r . 

10 


20 


30  40 

MINUTES 


50 


60 


Fig.  7.   Inhibition   of  ascorbate-induced    lysis    of  mitochondria   by   Mg  +  +, 
Mn  +  +,  pyruvate,  and  oxaloacetate. 


the  inhibitory  effect  of  15  mM  ascorbate,  several  possible  reducing  sub- 
stances were  tested.  Two  mM  nitrite  has  only  a  slight  effect,  but  20  mM 
frequently  blocks  completely  for  some  time  (Fig.  8).  Hydroquinone  and 
catechol  completely  prevent  ascorbate-induced  lysis  in  concentrations 
which  have  no  effect  or  smaller  effects  on  phosphate  swelling.  This  may 


ASCORBATE-INDUCED    LYSIS    OF    ISOLATED    MITOCHONDRIA  59 

well  be  the  result  of  complexing  with  metals  rather  than  reducing  action, 
for  quinone  is  even  more  effective  than  hydroquinone.  At  5  and  10  mM 
hvdroquinone  or  quinone  alone  cause  a  small  amount  of  swelling  of  the 
phosphate  type. 

The  question  whether  ascorbate  induced  lysis  of  mitochondria  is 
dependent  on  entry  of  electrons  into  the  electron  transport  chain  cannot 
be  answered  completely  just  now.  It  appears  to  be  dependent  on  some 
trace  metal  effect.  For  the  moment  we  must  keep  in  mind  the  fact  that 
CN,  antimycin  A,  and  SN  5949  may  act  as  metal  complexing  agents  as 
well  as  electron  transport  chain  inhibitors. 


0.5 


0.4 


0.3 


0.2 


0.1 


0.3mM    ASCORBATE 
(A 


10  20  30 

MINUTES 

Fig.  8.  Comparison  of  nitrite,  hydroquinone,  and  quinone  with  15  mM 
ascorbate  as  inhibitors  of  the  lysis  of  mitochondria  induced  by  0-3  mM 
ascorbate. 


The  lytic  action  of  low  ascorbate  concentrations  is  clearly  established 
as  a  different  phenomenon  by  experiments  in  which  ascorbate  is  added 
after  phosphate  +  substrate  swelling  is  more  or  less  complete.  In  Fig.  9  it 
may  be  seen  that  the  typical  ascorbate  type  of  optical  density  change  curve 
occurs  after  phosphate  swelling.  The  lag  is  similar  and  the  absorbancy 
falls  to  very  low  values.  This  figure  also  illustrates  the  fact  that  the  typical 
lag  period  (usually  shortened  a  little)  occurs  after  the  mitochondria  have 
been  at  25"  for  30  min.  Similar  results  are  obtained  after  60  min.  This 
clearly  indicates  that  ascorbate  lysis  is  basically  unchanged  by  ageing  and 
is  not  dependent  on  endogenous  substrate.  In  Fig.  10  it  may  be  seen  that 
the  same  inhibitors  block  ascorbate-induced  lysis  after  phosphate  swelling 
has  occurred  as  with  fresh  mitochondria. 

Because  of  the  possibility  that  HoOo  production  was  involved  in  the 
metal-ascorbate  induced  lysis,  the  effects  of  catalase  and  of  HoO.^  were 


6o 


F.    EDMUND    HUNTER,    JR. 


tested.  Figure  ii  illustrates  that  single  or  multiple  additions  of  catalase 
did  not  significantly  alter  the  effect  of  ascorbate.  Likewise,  single  or 
multiple  additions  of  HoOo  as  such,  or  generation  of  H2O2  by  the  glucose 


0.5 


20 


30        40  50 

MINUTES 


60 


70        80 


Fig.  9.  Demonstration  that  ascorbate-induced  lysis  of  mitochondria  occurs 
after  depletion  of  endogenous  substrates  (ascorbate  added  after  the  mitochon- 
dria were  at  24°  for  33  min.)  and  after  phosphate-induced  swelling  has  occurred 
(ascorbate  added  at  33  min.). 


5mM  PO4+  0.5mMyg-0H-B 


NHIBITORS 
,       '^   AT  32' 


ASCORBATE  + 
ImM    EDTA 
or  AaU  ANTIMYCIN  A 
or    ImM  PYRUVATE 
"or    ZmMNaCNn, 


0.3mM  ASCORBATE 
AT  37' 


\\*n\,  control 

\^    v\       ASCORBATE + 
mM  AZIDE 
or  4mM 

AMYTAL 


ASCORBATE  +  O.ImM 
DNP 


40 


50         60        70 
MINUTES 


80 


90 


Fig.  10.  Effect  of  various  inhibitors  on  ascorbate-induced  lysis  of  mito- 
chondria after  phosphate  +  ^-hydroxybutyrate-induced  swelling  has  already 
occurred. 

oxidase  system,  did  not  produce  an  ascorbate-like  effect.  High  concen- 
trations of  glucose  oxidase  tend  to  inhibit  ascorbate  lysis.  This  is  probably 
due  to  destruction  of  ascorbate  and  to  a  non-specific  protein  effect  (metal 
binding  }).  All  evidence  on  a  role  for  H.2O2  is  at  present  negative. 


ASCORBATE-INDUCED    LYSIS    OF    ISOLATED    MITOCHONDRIA 


6i 


Because  the  experimental  data  could  suggest  an  ascorbate-metal 
catalyzed  oxidative  change  in  some  labile  key  group  in  the  membrane, 
possibly  not  really  involving  the  electron  transport  chain,  we  investigated 


0.5 


0.4 


ImM  HpOp   REPEATED 
■  ^  *  i 


30  40 

MINUTES 

Fig.    II.   Failure  of  addtd  H.^Oj,  H.,Oo-forming  enzyme  systems,  or  catalase 
to  modify  significantly  ascorbate-induced  lysis  of  mitochondria. 


CONTROL--^ 

U  b 
0,4 

— • — • •-  _^ « 

O.ImM            ~~~r-« 
N-ETHYLMALEIMIDE-^ 
V                  03mM 
yr-i^-ASOORBATE 

0.3 

-  5mM  LIPQATE  /       ) 

■"r--L"^p-OH-MERCURI- 
\_^V^  BENZOATE 

0.2 

5lJ^*x^ 

ImM  ARSENITE 

\       — ~~~~--^  * 

5mM  lODOACETAMlDE 

\ 

0.1 

1                1                1 

1 

1       1 

20  30 

MINUTES 


40 


50 


60 


Fig.  12.  Comparison  of  the  absorbancy  changes  due  to  ascorbate  lysis  of 
mitochondria  with  those  produced  by  lipoate  and  reagents  which  react  with  thiol 
and  dithiol  groups. 

additional  sulphvdrvl  compounds  and  reagents  which  react  with  thiol  and 
dithiol  groups.  The  swelling  inducing  effect  of  arsenite  [i]  and  p-ch\or- 
mercuribenzoate  have  been  reported  [lo].  Carefully  investigated  over  a 
wide  range  of  concentrations,  none  of  these  compounds  produced  a 
typical  ascorbate-like  effect  (Fig.  12).  Oxidized  lipoic  acid,  arsenite,  and 


62 


F.    EDMUND    HUNTER,    JR. 


iodoacetamide  produce  swelling,  but  it  resembles  that  seen  with  phosphate 
more  than  that  due  to  ascorbate.  An  interesting  observation  from  these 
experiments  is  that  very  low  ^-hydroxymercuribenzoate  concentrations 
produce  swelling  after  a  lag  period,  while  60-100  /xM  levels  have  a  three- 


0.5 


0.4 


0.3 


0.2 


0.1 


CONTROL 


p-OH-MERCURIBENZOATE 


10 


20 


30  40 

MINUTES 


50 


60 


Fig.    13.   Absorbancy  changes  due  to  different   concentrations   of  /)-hydroxy- 
mercuribenzoate.  High  concentrations  show  a  three-phase  curve. 


^      •_-_  .                              CONTROLv, 

^\'«>            ""V^ 

U.b 

-     \\^                     \^            0.3mM  ASCORBATE 

U.4 

^        •--.:.--rX-.,..„, 

0 

0.3 

0.  ImM              /\\        \         *"""■-, 

CVJ 

in 
< 

p-OH-MERCURlBENZOATE\\          ^                 "-.^ 
+  ASCORBATE   AT  oO^\<^\ 

0,2 

-        +  ASCORBATE  AT  20' -Av           \ 
♦              * 

0.1 

— *~  —  ~( 
1                 1                 1                1                1 

20  30  40 

MINUTES 


50 


60 


Fig.    14.   Ascorbate  added  with  or  after  p-hydroxymercuribenzoate  produces 
fairly  typical  lysis. 

phase  effect — first  a  rapid  fall  in  absorbancy,  then  a  definite  plateau  for 
about  10  min.,  then  a  further  fall  (Fig.  13).  This  may  indicate  two  sites  of 
action  or  an  immediate  and  a  delayed  effect  from  a  single  site  of  action. 
Pyruvate  seems  to  inhibit  the  initial  phase  with  little  effect  on  the  second 
fall. 


ASCORBATE-IN'DUCED    LYSIS    OF    ISOLATED    MITOCHONDRIA  63 

Although  reagents  reacting  with  sulphhydryl  groups  did  not  mimic 
ascorbate,  it  was  of  considerable  importance  to  determine  whether  re- 
action of  these  substances  with  the  mitochondrial  membrane  prevented 
the  action  of  ascorbate.  In  Fig.  14  it  may  be  seen  clearly  that  ascorbate 
lysis  seems  to  be  independent  of  the  action  of  /)-hydroxymercuribenzoate. 
Lysis  occurs  in  a  typical  fashion  whether  ascorbate  is  added  simultaneously 
with  or  20  min.  after  the  inhibitor.  Similar  data  have  been  obtained  with 
arsenite.  These  data  suggest  that  thiol  or  dithiol  groups  may  not  be  critical 
for  the  lytic  action  of  ascorbate. 

We  have  investigated  the  specificity  of  the  ascorbate  type  of  effect  by 
testing  substances  structurally  related  to  ascorbate.  The  ones  of  primary 


0 

c 

0 

0 

c 

C 

HO-C-H 

HO-C 

0-C 

HO-C-H 

HO-C 

0-C 

H-C 0 

H-C C 

H-C -0 

HO-C-H 

HO-C-H 

HO-  C-H 

CH2OH 

CHjOH 

CHjOH 

GULONOLACTONE 

ASCORBIC 

DEHYDROASCORBIC 
0 

' 

c 

COOH 

COOH 

HO-C 

C-OH 

C-OH 

HO-C 

C-OH 

HO-C 

H-C 6 

COOH 

COOH 

H-C-OH 
CHjOH 

Dl-OH-FUMARiC 

DI-OH-  MftLEiC 

IS0A5C0RBIC 

Fig.  15.  Formulae  for  ascorbic  acid  and  some  compounds  with  structures 
related  to  part  of  the  ascorbate  molecule. 

interest  are  shown  in  Fig.  15.  Isoascorbate  gives  an  effect  identical  with 
ascorbate  (Fig.  16).  In  experiments  followed  for  just  60  min.,  dehydro- 
ascorbate,  the  oxidation  product  of  ascorbate  had  no  effect,  but  longer 
experiments  revealed  that  it  may  produce  an  effect  after  very  long  lag 
periods  (60-90  min.).  How  much  reduction  occurs  is  unknown.  The 
precursor  of  ascorbate,  gulonolactone,  does  not  induce  lysis  at  all.  Two 
compounds  containing  groups  similar  to  the  active  oxido-reduction  centre 
of  ascorbate,  dihydroxyfumarate  and  dihydroxymaleate,  produce  lysis 
which  appears  identical  to  that  with  ascorbate,  with  one  important 
difference — the  lag  period  is  40-50  min.  instead  of  15-25.  The  active 
concentrations  are  identical  with  ascorbate,  and  other  concentrations  do 
not  give  a  shorter  lag  period. 

Not  only  do  low  concentrations  of  dihvdroxvfumarate  and  dihy- 
droxymaleate act  like  ascorbate,  high  concentrations,  like  high  ascorbate, 
do  not  cause  lysis.  Moreover,  high  concentrations  of  the  dihydroxy  acids 
block  low  concentrations  of  ascorbate  and  high  concentrations  of  ascorbate 


64  F.   EDMUND   HUNTER,   JR. 

block  low  concentrations  of  dihydroxy  acids  (Fig.  17).  It  is  also  of  some 
interest  that  dehydroascorbate  shows  steadily  increasing  antagonism  of 
ascorbate  action  as  the  concentration  is  raised  to  10  mM. 


0.5 


40  60 

Ml N  UTES 


Fig.    16.  Ascorbate-like  lysis  of  mitochondria  produced  by  certain  substances 
with  related  structures.   Note  especially  the  marked  differences  in  the  lag  period. 


U.D 

0.4 

—♦::—•-    -T^ 

\        0.3mMA+lOmMDHF'l    i 

\or  a3mMA+lOmMDHA   S/ 

\or0.3mMDHF  +  l5mMAj 

0.3 

0.3mM/  \ 
ASCORBATE   \ 
-        (A)              \ 

\ 

\ 

0.2 

\ 

\ 
\ 
\ 

• 

0.1 

0.3  m  M     -'^'V 
DI-OH-FUMARATE 
(OHF) 

1 

—                   \ 

1                  ,   1                   -J 

20 


40  60 

MINUTES 


80 


Fig.  17.  Inhibition  of  ascorbate-induced  lysis  of  mitochondria  by  high  con- 
centrations of  dihydroxyfumarate  (DHF)  and  dehydroascorbate  (DHA). 
Inhibition  of  DHF-induced  lysis  by  high  concentrations  of  ascorbate. 

More  knowledge  concerning  what  is  happening  during  the  lag  period 
undoubtedly  would  tell  us  something  about  the  mechanism  of  action  of 
ascorbate.  We  have  very  little  information  on  this  point.  In  fact  some 
titrations  with  indophenol  raise  a  question  as  to  whether  ascorbate  is 
disappearing.  More  experiments  are  needed.  However,  in  the  course  of 


ASCORBATE-INDUCED   LYSIS   OF    ISOLATED   MITOCHONDRIA  65 

testing  various  substances  several  striking  alterations  in  the  effect  of 
ascorbate  have  been  observed.  Two  of  these  are  shown  in  Fig.  i8.  Five  niM 
a-ketoglutarate  usually  causes  a  little  swelling.  In  this  experiment  it  caused 
almost  none,  but  it  drastically  shortened  the  lag  period  for  ascorbate 
lysis.  Even  more  remarkable  is  the  effect  of  lo  mi\i  gulonolactone.  Alone 
it  never  causes  swelling,  but  it  converted  ascorbate-lysis  into  typical 
phosphate  type  swelling.  Only  further  work  can  add  information  on  these 
effects. 

We  do  not  know  much  about  the  mechanism  of  the  ascorbate  lysis 
phenomenon,  but  we  can  describe  it  in  terms  other  than  just  light  scattering 


0,5 


CONTROL 


20  30 

MINUTES 


Fig.    18.  Effect  of  a-ketoglutarate  and  gulonolactone  on  the  lag  period  and 
absorbancy  curve  associated  with  ascorbate-induced  lysis  of  mitochondria. 


changes.  The  experiments  in  Fig.  19  show  the  distribution  of  protein 
recoveries  on  differential  centrifugation  after  dilution  of  mitochondrial 
suspensions  and  various  experimental  treatments.  Control  suspensions, 
whether  held  at  o  or  25*^,  yield  about  8o"o  of  the  protein  in  the  regular 
mitochondrial  pellet  at  8000  x  g  and  15-20%  in  the  supernatant  after 
I  hr.  at  100  000  X  g.  Phosphate-  and  succinate-produced  swelling  alter 
this  distribution  remarkably  little.  However,  after  treatment  with  ascorbate 
75°,j  of  the  protein  appears  in  the  "soluble"  fraction,  3  to  7%  in  the 
submitochondrial  particle  fraction  (100  000  x  g  pellet).  The  20  000  x  g 
pellet  contains  drastically  swollen  and  damaged  mitochondria.  The  sub- 
mitochondrial particle  fraction  (100  000  x  g  pellet)  is  greatly  increased  by 
ascorbate  treatment. 

Electron  microscopy  has  been  used  to  examine  the  nature  of  the 
morphological  changes.  After  phosphate-induced  swelling  swollen  and 
unchanged  mitochondria  are  clearly  seen.  With  ascorbate  treatment  most 


66 


F.    EDMUND    HUNTER,    JR. 


of  the  mitochondria  disappear.  The  differential  centrifugation  yields 
pellets  which  demonstrate  the  disintegration  of  most  of  the  mitochondria 
into  smaller  particles  and  soluble  protein.  Essentially  all  of  the  pyridine 
nucleotide  is  released  into  the  soluble  fraction  during  ascorbate  lysis. 

Protein  Distribution  after  Mitochondrial  Swelling 


Percentage  reco\ 

ered  protein  in 

Treatment 

8  000  g 
Pellet 

20  000  g 
Pellet 

100  000  g 
Pellet 

Super- 
natant 

o°  Control 
25"  Control 
Ascorbate  25° 

83-0 
78-4 
IO-8 

2-6 
3-0 

lO-O 

0-5 
09 

7-3 

13-7 
i8-o 
72-0 

Ascorbate  25" 
PO4  25^ 
Succinate  25'' 

12-9 
83-0 
78-0 

8-9 
1-6 

2-5 

6-4 
0-7 

0-5 

72 

14-8 

19-0 

Ascorbate  25'' 
PO,  25" 
Succinate  25 

12  •  I 
68-8 

71-8 

8-8 
6-3 
8-0 

2-9 
1-6 

0-7 

76-5 
23-6 
19-4 

Mitochondria  diluted  i  :25  for  treatment  with  0-5  mM  ascorbate,  or  5  mM 
PO4,  or  3  mM  succinate,  centrifuged  8  000  x  g  for  10  min.,  20  000  x  g  for  20  min., 
100  000   X   g  for  60  min. 

Fig.    19.  Protein  distribution  after  mitochondrial  swelling. 

Figure  20  outlines  some  possible  interrelationships  between  swelling 
inducing  agents,  inhibitors,  and  the  electron  transport  chain.  While  the 
evidence  for  active  electron  transfer  or  a  closely  associated  high  energy 
intermediate  conditioning  the  membrane  response  is  strong  in  the  case  of 
a  great  many  swelling-inducing  substances,  this  question  cannot  be 
answered  completely  jusi  now  for  ascorbate-lysis.  We  must  determine 
whether  inhibitors  like  cyanide,  antimycin  A,  and  SN  5949  act  by  preven- 
ting electron  transport  or  by  chelation  of  metal  ions.  There  is,  of  course, 
the  possibility  that  ascorbate-lysis  is  dependent  on  two  conditions :  (a)  some 
electron  transfer,  and  (b)  an  action  of  an  ascorbate-metal  complex. 

The  failure  of  dehydroascorbate  (DHA)  to  act  more  like  ascorbate 
suggests  that  ascorbate  is  not  acting  as  a  simple  oxidation-reduction 
couple  with  DHA.  Conceivably  a  half-oxidized  radical  [11]  (rather  than 
DHA)  might  be  involved.  Another  possibility  which  must  be  given 
serious  consideration  is  that  ascorbate  catalyzed  formation  of  lipid  per- 
oxides [12,  13]  may  be  responsible  for  disintegration  of  the  mitochondrial 
membranes.  Additional  possibilities  include  alterations  in  lipoproteins, 
activation  of  some  lytic  enzyme  like  lecithinase,  release  of  lysolecithin, 
lysoplasmalogen,  or  a  fatty  acid. 


ASCORBATE-INDUCED    LYSIS    OF    ISOLATED    jMITOCHONDKIA  67 

An  important  question  not  fully  answered  is  whether  ascorbate  causes 
rupture  of  the  membrane  at  one  or  at  many  points.  The  latter  appears  to 
be  more  likely,  for  many  submitochondrial  particles  of  small  size  are 
formed.  Since  ascorbate  lysis  occurs  after  swelling  induced  by  phosphate 
and  other  agents  has  stopped,  when  the  membrane  permeability  has 
greatly  increased  and  sucrose  has  probably  come  to  equilibrium  internally 


Ascorbate +^  /A 


t 
Cyto.  A  +  Aj CN,  azide 

added  cyto.  C 

Cyto.C 

t 
Cyto.C, 

..A Antimycin  A 

7  '  SN  5949 

Ascorbate    ■ »-  Cyto.  B  or  UQ 

Flavin  Flavin 

Malonate ]-•-  ---T Amytal 

Succinate  DPN 

t 
/3-OH-Butyrate 

Fig.  20.  Possible   interrelationships    between   swelling   inducing   agents,    in- 
hibitors, and  the  electron  transport  chain. 

and  externally,  the  ascorbate  effect  would  seem  to  involve  rupture  of 
links  in  the  membrane  structure  and  not  further  osmotic  swelling.  The 
action  of  ascorbate  may  give  clues  to  key  groups  or  links  in  the  membrane 
structure.  It  seems  unlikely  that  this  effect  is  related  to  the  vitamin  role 
of  ascorbate,  but  this  cannot  be  ruled  out  completely. 

References 

1.  Hunter,  F.  Edmund,  Jr.,  Levy,  J.  F.,  Fink,  J.,  Schutz,  B.,  Guerra,  F.,  and 
Hurwitz,  A.,  J.  biol.  Cheni.  234,  2176  (1959). 

2.  Alaley,  G.  F.,  and  Johnson,  D.,  Biochim.  biophys.  Acta  26,  522  (1957). 

3.  Lehninger,  A.  L.,  and  Schneider,  M.,^.  biophys.  biocheni.  Cytol.  5,  109  (1959). 

4.  Lowry,  O.  H.,  Rosebrough,  N.  J.,  Farr,  A.  L.,  and  Randall,  R.  J.,  J.  biul. 
Chem.  193,  265  (1951). 

5.  Lehninger,  A.  L.,  and  Schneider,  M.,  Z.  physiol.  Cluni.  313,  138  (1958). 

6.  Chappell,  J.  B.,  and  Greville,  G.  D.,  Nature,  Loud.  183,  1525  (1959). 

7.  Chappell,  J.  B.,  and  Greville,  G.  D.,  Nature,  Land.  183,  1737  (1959). 

8.  Lehninger,  A.  L.,  ul  Hussan,  AI.,  and  Sudduth,  H.  C,  J.  biol.  C/iern.  210, 
911  (1954). 

9.  Maley,  G.  F.,  and  Lardy,  H.,jf.  biol.  Chetn.  210,  903  (1954). 

10.  Lehninger,  A.  L.,  "Proceedings  of  the  International  Symposium  on  Enzyme 
Chemistry,  Tokyo,  1957".  Alaruzen,  Tokyo,  297  (1958). 

11.  Knox,  W.   E.,   "Proceedings  of  the   Fourth   International   Congress  of  Bio- 
chemistry, Vienna  1958",  Vol.  XI.  Pergamon  Press,  London,  307  (i960). 

12.  Ottolenghi,  A.,  Arch.  Biochem.  Biophys.  79,  355,  (1959). 

13.  Thiele,  E.  H.,  and  HufT,  J.  W.,  Arch.  Biochem.  Biophys.  88,  203  (i960). 


68  F.    EDMUND    HUNTER,    JR. 


Discussion 

Williams  :  May  I  make  one  general  comment  ?  I  am  interested  in  the  kinetics 
of  the  swelling  that  you  get  and  I  have  a  feeling  that  we  in  the  mitochondrial  field 
should  be  thinking  about  this  type  of  kinetics  which  is  more  familiar  in  the  erythro- 
cyte field  where  you  regularly  find  S-shaped  progress  curves  which  represent  the 
integral  form  of  the  Gaussian  distribution  of  red  cell  fragilities.  If  we  had  better 
methods  than  just  optical  density  measurement  for  following  lysis  then  we  might 
observe  this  more  often.  We  have  observed  such  curves  in  measuring  the  onset  of 
choline  oxidation  in  mitochondria  which  is  related  to  the  integrity  of  the  mito- 
chondrial membrane.  It  might  even  be  useful  to  use  them  to  measure  the  homo- 
geneity of  a  population  of  mitochondria. 

DiscHE :  What  was  the  technique  of  homogenization  which  you  used  in  studying 
the  distribution  of  different  particles  after  ascorbate  treatment,  and  in  what 
medium  were  the  mitochondria  suspended  when  you  treated  them  with  ascorbate  ? 

Hunter:  We  used  0-33  m  sucrose  plus  0-025  m  tris  buffer  pH  74  in  all  these 
experiments.  In  the  protein  distribution  experiments  the  mitochondrial  lysis 
experiment  was  carried  out  in  this  medium  in  the  usual  fashion  and  then  the 
suspension  was  subjected  to  differential  centrifugation  and  separated  into  four 
fractions,  8000  x  g,  20  000  x  g  and  100  000  x  g  pellets  plus  the  supernatant. 

Dische:  The  distribution  of  fractions  in  the  non-treated  mitochondria  was 
simply  determined  by  centrifugation  of  your  suspension  of  mitochondria  ? 

Hunter:  We  have  centrifuged  the  suspensions  at  o  and  at  25"  under  exactly 
the  same  conditions  but  in  the  absence  of  ascorbate. 

Dische:  Would  you  not  suspect  that  under  these  conditions  your  population 
of  mitochondria  was  very  inhomogeneous,  because  you  have  already  got  a  certain 
distribution  on  your  original  suspension  ? 

Hunter:  Not  entirely,  although  it  undergoes  some  change.  Actually  we  think 
that  the  largest  change  is  due  to  dilution.  Mitochondria  when  diluted  out  as  they 
are  for  experiments  like  this  do  begin  to  undergo  changes.  We  have  electron 
microscope  evidence  for  this.  If  you  take  a  mitochondrial  suspension  and  recentri- 
fuge  it  without  much  dilution  you  will  get  far  more  than  So",,  of  the  protein  in  the 
so-called  mitochondrial  pellet.  If  you  dilute  it  out  even  at  o  you  get  some  change 
just  by  that  dilution,  and  you  get  only  80%  of  the  protein  in  the  mitochondrial 
fraction  and  an  appreciable  portion  in  the  soluble  fraction,  which  I  am  sure  is  not 
all  a  contaminating  soluble  protein. 

Weinbach  :  Was  the  ascorbate  effect  you  noted  preceded  by  the  loss  of  pyridine 
nucleotide,  in  other  words  did  you  examine  what  happened  during  the  lag  phase  ? 

Hunter:  Within  the  limits  of  methods  we  use  we  could  not  say  that  it  preceded 
this,  but  it  occurs  simultaneously  with  it. 

Hess  :  Do  you  know  what  the  fate  of  ascorbate  is  and  how  the  kinetics  are  ? 
How  does  it  compare  to  the  swelling  action  ? 

Hunter:  I  can't  answer  that  question  exactly.  We  were  interested  in  what 
happened  to  the  ascorbate  during  the  lag  period.  We  were  somewhat  amazed  when 
the  preliminary  experiments  indicated  that  it  was  not  disappearing,  yet  later 
experiments  with  an  oxygen  electrode  indicated  an  oxygen  consumption.  That 


ASCORBATE-INDUCED    LYSIS    OF    ISOLATED    MITOCHONDRIA  69 

might  be  due  to  stimulation  of  an  endogenous  substrate  or  something.  These 
experiments  do  bring  to  mind  that  Ottolenghi  in  his  experiments  on  lipid  per- 
oxidation by  mitochondria  found  that  ascorbate  was  oxidized  only  until  the  lipid 
was  used  up.  Then  oxidation  stopped  and  ascorbate  was  not  used.  It  probably 
depends  on  the  ratio  of  ascorbate  to  mitochondria ;  if  you  use  an  excess  of  ascorbate 
most  of  it  will  be  there  when  your  experinient  is  finished. 


Integrated  Oxidations  in  Isolated  Mitochondria 

J.  B.  Chappell* 
Department  of  Biochemistry,  Uuirersity  of  Cambridge,  England 

It  appears  that,  in  intact  mitochondria,  a  straightforward  reduction 
of  pyridine  nucleotide  by  substrate,  followed  by  reoxidation  of  the  reduced 
nucleotide  by  the  cytochrome  system  does  not  occur  without  complications 


Ag-AgCI 


0-5M  KCI 


Pt  electrode 


Perspex  disc 


Tlea' 


O'  ring  supporting 
membrane 


-Support 
for  vessel 


1cm 

Fig.    I.  The    Clark    oxygen    electrode    adapted    for    following    mitochondrial 
respiration. 

arising  because  of  subsequent  events  at  the  substrate  level.  This  paper 
represents  an  attempt  to  justify  this  statement.  All  the  investigations 
reported  have  been  performed  w-ith  the  Clark  oxygen  electrode  set  up  as 

*  Present    address :     TJie  Johnsnii    Foundation,     University    of    Pennsylvania, 
Philadelphia,  U.S.A. 


72 


J.    B.    CHAPPELL 


shown  in  Fig.  i  [i].  Most  experiments  were  performed  with  rat  liver 
mitochondria  isolated  in  0-25  M-sucrose  containing  5  mM-2-amino- 
hydroxymethylpropane-i  :3-diol-hydrochloride  (tris)  buffer,  pH  7-4,  but 
some  results  obtained  with  kidney  mitochondria  prepared  in  the  same 
medium  are  presented. 

Isocitrate  oxidation 

When  the  oxidation  of  isocitrate  by  liver  mitochondria  was  followed 
in  a  medium  containing  80  mM  KCl,  6  mM  MgClg,  15  mM  phosphate  and 
ID  mM  substrate  and  respiration  was  stimulated  by  addition  of  small 
quantities  of  adenosine-diphosphate  (ADP)  an  unusual  pattern  resulted 


100 

Me            Mc 

T    ADP    ;    ADP 

— \         '      \       1 

90 

\             \    232 

80 

\273      \ 

/socitrate 

70 

P/0  2-9   \                \ 

86 

60 

\       ADP 
37A 

37     ADP         P/O  2-8 

50 

^230 

40 

Glutamate                   \ 

\^I29 

30 

\        ADP 

V 

ADP 

— . T 

20 

\  235 

10 

- 

\ 

\ 

0 

\. 

\| 

0      12      3     4      5 
Time  (mm) 

Fig.  2.  Stimulation  by  ADP  of  glutamate  and  isocitrate  oxidation  in  a  medium 
containing  15  mM-phosphate.  The  numbers  juxtaposed  to  the  curves  in  this  and 
subsequent  figures  represent  Q,,.,  (n)  values  (/d.  O.i/mg.  N/hr.). 


(Fig.  2).  When  L-glutamate,  a-ketoglutarate,  /3-hydroxybutyrate,  proline 
or  succinate  served  as  substrates  the  State  3  rate  [2]  was  linear  until  nearly 
all  the  ADP  had  been  converted  into  adenosine  triphosphate  (ATP), 
when  the  rate  characteristic  of  State  4  ensued.  With  Lg(  +  )-isocitrate  as 
substrate  after  a  short  period  in  State  3  the  rate  of  oxidation  declined  and 
this  lower  rate  persisted  until  the  added  ADP  was  exhausted.  A  subsequent 
addition  of  ADP  after  a  short  period  in  State  4  led  to  a  further  rapid  rate 
of  oxidation  followed  by  a  slower  rate.  However,  the  longer  the  period  of 


INTEGRATED    OXIDATIONS    IN    ISOLATED    MITOCHONDRIA  73 

observation  the  less  obvious  was  the  second  slower  rate.  When  io~^  to 
10"*  M-2  :4-dinitrophenol  (DNP)  was  used  to  stimulate  isocitrate  oxidation 
an  initial  rapid  rate  was  observed  followed  by  a  very  much  slower  rate, 
which  persisted  for  at  least  15  min. 

When  the  phosphate  concentration  in  the  medium  was  reduced  below 
5  niM,  linear  rates  of  isocitrate  oxidation  occurred,  both  when  ADP  and 


DNIP 


0       12       3      4       5 
Time  (min) 

Fig.  3.  The  stimulation  of  isocitrate  oxidation  by  malate.  Three  separate 
experiments  are  shown.  In  each  case  10^  M  DNP  was  added  followed  4  min. 
later  (*)  either  by  i  -o  mM-L-malate  or  10  niM  -L,(  +  )— isocitrate,  or  isocitrate  and 
then  malate. 


DXP  wert  used  to  stimulate  respiration.  However,  even  in  a  medium 
containing  a  low  concentration  of  phosphate,  if  the  mitochondria  were 
depleted  partly  of  their  endogenous  substrates  by  preincubating  them 
with  DNP  or  ADP  for  5  min.,  added  isocitrate  was  not  oxidized  at  ap- 
preciable rates  for  many  minutes.  The  addition  of  low  concentrations  of 
malate,  fumarate  or  higher  concentrations  of  oxaloacetate  led  to  a  marked 
increase  in  oxygen  consumption.  In  the  absence  of  isocitrate,  and  under 
these  conditions,  malate,  fumarate  or  oxaloacetate  did  not  produce  any 
marked  oxygen  uptake  (Fig.  3). 


/socitrate- 

Y 

-Oxidized- 
TPN 

Y 

»■  Reduced- 
DPN, 

Y 

-Oxalo 

C02+«-KG- 
r 

(1) 

•-  Reduced  - 

(2) 

-Oxidized' 

(3) 

-Ma 

74  J.    B.    CHAPPELL 

Either  5  mM-malonate  or  ^-chlorovinylarsenious  oxide  (0-5  /xg./ml.) 
severely  inhibited  isocitrate  oxidation  even  in  the  low  phosphate  medium. 
These  inhibitory  effects  were  largely  reversed  by  the  addition  of  low 
concentrations  of  the  dicarboxylic  acids  mentioned  above.  Half  maximal 
effect  was  obtained  with  2  x  io~*  M-malate.  With  mitochondria  which 
had  been  depleted  of  their  endogenous  substrates,  or  in  the  presence  of 
malonate  or  ^-chlorovinylarsenious  oxide,  neither  acetoacetate  nor 
pyruvate  together  with  bicarbonate  were  able  to  restore  isocitrate  oxidation. 

These  results  suggest  that  the  pyridine  nucleotide  reduced  by  the 
isocitrate  dehydrogenase  is  reoxidized  by  coupling  with  the  malate 
dehydrogenase.  Neither  the  ^-hydroxybutyrate  dehydrogenase  nor  the 
malic  enzyme  appears  to  be  able  to  do  this.  The  scheme  outlined  in  Fig.  4 
is  thought  to  represent  the  sequence  of  events  occurring  in  the  oxidation 
of  isocitrate  by  liver  mitochondria.  This  scheme,  besides  accounting  for 

tate-*^  ^^-Reduced— ^  /-^^Oi 

: ^^j^Oxidized^^j^HjO 

I 

TCA  cycle 

(1)  /socitrate  dehydrogenase  (3)  Malate  dehydrogenase 

(2)  Pyridine  nucleotide  transhydrogenase  (4)  Cytochrome  system 

Fig.  4.  The  proposed  pathway  of  isocitrate  oxidation. 

the  experimental  observations  which  have  been  given  above,  also  takes 
into  account  the  following  facts:  (i)  there  exists  in  liver  mitochondria  a 
triphosphopyridine  nucleotide  (TPN)-linked  and  not  a  diphosphopyridine 
nucleotide  (DPN)-linked  dehydrogenase  [3,  4],  (2)  the  DPN  specificity  of 
the  mitochondrial  malate  dehydrogenase  [5]. 

Similar  results  to  those  given  above  have  been  obtained  with  kidney 
mitochondria. 


COMPARISON    OF    GLUTAMATE    AND    ISOCITRATE    OXIDATION 

In  liver  mitochondria  two  possible  routes  of  glutamate  oxidation  are 
available,  one  involving  glutamate-aspartate  transaminase,  the  other 
utilizing  glutamate  dehydrogenase  [6,  7].  The  transaminase  pathway  is 
analogous  to  the  pathway  which  appears  to  exist  for  isocitrate  oxidation; 
both  are  coupled  oxido-reductions  and  both  involve  the  utilization  cf 
oxaloacetate.  The  former  uses  pyridoxal  phosphate  as  a  cofactor,  the 
latter  pyridine  nucleotide.  The  pathway  for  glutamate  oxidation  involving 
the  use  of  the  dehydrogenase,  leads  to  the  reduction  of  pyridine  nucleotide, 


INTEGRATED    OXIDATIONS    IN    ISOLATED    MITOCHONDRIA 


75 


which  in  this  case  is  directly  oxidized  by  the  cytochrome  system,  since, 
even  in  mitochondria  depleted  of  their  endogenous  substrates,  glutamate 
was  oxidized  immediately  and  rapidly.  This  was  also  the  case  when 
^-hydroxybutyrate,  proline  and  malate  served  as  substrates. 

However,  even  when  glutamate  is  oxidized  by  the  dehydrogenase 
pathway,  the  rate  of  this  reaction  is  intimately  dependent  upon  the  rate  at 
which  a-ketoglutarate  is  removed.  This  can  be  shown  clearly  by  a  study  of 
the  effects  of /S-chlorovinylarsenious  oxide  on  glutamate  oxidation  (Fig.  5). 


100 
90 
80 

B  70 

I    60 

:    50 
> 

°    40 
<v 

30|- 
20I-' 
10 


-»- 


0        OS      1-0      1-5      20    25 
^/S-chlorovinylarsenious  oxide 
(/^g/4ml,) 

Fig.  5.  The  eflFect  of  ^-chlorovinylarsenious  oxide  on  ADP-stimulated  oxida- 
tion of  glutamate  (x  —  x  ),   a-ketoglutarate    (D — D),    succinate     (H h)    and 

proline(c — c). 


This  arsenical  inhibited  oxidation  of  glutamate  and  a-ketoglutarate  to  the 
same  extent  at  the  same  concentrations.  Under  identical  conditions 
j8-hydroxybutyrate,  malate,  proline,  and  succinate  oxidation  and  the 
associated  phosphorylation  were  unaffected.  The  inhibitory  effect  was 
readily  reversed  by  2  :3-dimercaptopropanol  (Fig.  6).  The  arsenical,  at 
these  concentrations,  does  not  inhibit  the  glutamate  dehydrogenase  itself, 
since  in  disrupted  mitochondria,  when  DPN  and  cytochrome  c  were  added, 
/S-chlorovinylarsenious  oxide  did  not  inhibit  glutamate  oxidation.  In  intact 
mitochondria  it  appears  that  glutamate  oxidation  cannot  occur  when 
a-ketoglutarate  accumulates.  Alternatively  it  may  be  concluded  that  the 
glutamate  dehydrogenase  is  not  functional  in  intact  liver  mitochondria  and 
only  serves  to  "spark"  the  oxidation  of  glutamate  by  the  transaminase 


76  J.    B.    CHAPPELL 

pathway.  The  possibiHty  must  not  be  overlooked  that  glutamate  dehydro- 
genase serves  a  synthetic  rather  than  a  degradative  function  in  hver  mito- 
chondria. 

The  phosphate  requirement  for  DPN-stimulated  glutamate  oxidation 
[8,  9]  and,  when  oligomycin  [10]  is  present,  the  requirement  for  ADP  [11] 
are  presumably  reflections  of  the  demands  of  the  substrate  level  phos- 
phorylation associated  with  a-ketoglutarate  oxidation.  These  requirements 
have  also  been  observed  when  ferricyanide  acted  as  terminal  electron 
acceptor. 


100  r 


90 


80 


S    70 

U 

-S    60 
o 

I    50 

40 

30 
20 


Lewisite 


0       12      3      4      5 

Time  (min) 


Fig.  6.  The  effect  of  2  :3-dimercaptopropanol  (BAL)  on  the  inhibition  of 
glutamate  oxidation  by  ^-chlorovinylarsenious  oxide.  /3-chlorovinylarsenious  oxide, 
0"5  Mg-/4  rnl.;  BAL,  i  ng./^.  ml. 

Similarly,  isocitrate  oxidation  showed  the  same  requirement  for 
phosphate,  and  in  the  presence  of  oligomycin,  for  ADP,  when  respiration 
was  stimulated  by  DNP.  In  this  case  it  is  not  the  accumulation  of  a-keto- 
glutarate, but  the  failure  to  produce  sufiicient  quantity  of  oxaloacetate, 
required  for  the  coupling  process,  which  is  responsible  for  the  low  rates 
of  oxygen  consumption.  The  addition  of  malate,  in  catalytic  quantities, 
abolishes  the  requirement  for  phosphate  and  ADP,  for  isocitrate  oxidation. 


Succinate  oxidation 

In  confirmation  of  the  findings  of  Azzone  and  Ernster  [12]  liver  mito- 
chondria which  had  been  pre-incubated  with  2  mM  arsenate  and  io~*  m 


INTEGRATED    OXIDATIONS    IN    ISOLATED    MITOCHONDRIA  77 

DNP  did  not  oxidize  succinate  at  significant  rates  until  ATP  was  added 
(Fig.  7).  I  mM  ATP,  ADP  or  inosine  triphosphate  were  all  equally  effective. 
However,  lower  concentrations  of  ATP  were  less  effective,  unless  oligomy- 
cin  (2  •  5  fig. /ml.)  and  amytal  were  added.  Under  these  conditions  3  ni/xmoles 
of  ATP  served  to  catalyze  the  oxidation  of  2  ftmoles  of  succinate,  and  it  is 
apparent  therefore  that  ATP  is  not  required  in  stoicheiometric  amounts. 
Furthermore,  since  oligomycin  inhibits  the  enzymes  involved  in  oxidative 


Amytal 


12        3       4 
Time  (mm) 
Fig.   7.   Effect  on  succinate  oxidation  of  preincubating  liver  mitochondria  with 
DNP  and  arsenate  (cf.  Azzone  and  Ernster,  [12]).  DXP,  lo^^  m;  amytal,  i  -8  mM ; 
succinate,  10  mM;  ATP,  i  mM. 

phosphorylation  [10]  it  is  unlikely  that  AIT  acts  by  reversing  this  process 
as  suggested  by  Azzone  and  Ernster  [12].  Indeed  oligomycin  accentuated 
the  effect  of  ATP,  especially  at  low  nucleotide  concentrations,  presumablv 
because  this  antibiotic  inhibits  the  mitochondrial  DXP-stimulated  ATPase 
[10]. 

When  I  -8  niM  amytal,  as  well  as  arsenate  and  DNP,  was  present  during 
the  preincubation  period,  ATP  was  not  required  for  succinate  oxidation. 
The  amytal  almost  entirely  abolished  the  endogenous  respiration  of  the 
liver   mitochondria.    With   kidney   mitochondria,   which   had   an   almost 


78  J.    B.    CHAPPELL 

immeasurably  small  endogenous  respiration,  preincubation  with  arsenate 
and  DNP  did  not  induce  a  requirement  for  ATP  for  succinate  oxidation. 
However,  when  0-5  mM  malate  was  present  during  the  preincubation 
period,  the  situation  was  exactly  the  same  as  it  was  with  liver  mitochondria, 
namely  ATP  was  required  before  succinate  was  oxidized  at  significant 
rates  and  the  addition  of  amytal  at  zero  time,  which  of  course  prevented  the 
oxidation  of  malate,  abolished  the  requirement  for  ATP.  It  is  a  reasonable 
hypothesis  therefore  that  when  liver  mitochondria  are  preincubated  with 


Kidney 
Mitochondria 


0     1      2     3     4     5 
Time  (mm) 

Fig.  8.  Effect  of  arsenate  and  oxaloacetate  on  succinate  oxidation  by  kidney 
mitochondria.  The  oxaloacetate  concentration  was  i  mM,  other  conditions  as  for 
Fig.  7.  The  early  parts  of  the  traces  and  the  additions  made,  were  the  same  in  all 
cases,  but  are  not  shown  for  the  two  traces  on  the  right. 


arsenate  and  DNP  the  endogenous  substrates  give  rise  to  oxaloacetate 
which  is  responsible  for  the  inhibition  of  succinate  oxidation.  This  effect 
can  be  demonstrated  directly  with  kidney  mitochondria  (Fig.  8).  In  this 
case,  when  DNP,  arsenate  and  oxaloacetate  were  added  before  the  succinate, 
ATP  was  required  for  maximal  rates  of  oxidation.  Oxaloacetate  had  no 
efi"ect  in  the  absence  of  arsenate.  The  amount  of  oxaloacetate  required  to 
produce  this  effect  was  about  i  mM,  which  was  40-100  times  greater  than 
the  amount  of  oxaloacetate  which  can  be  calculated  to  have  arisen  from 
added  malate  in  the  experiment  described  above.  It  is  possible  that 
enzymically  generated  oxaloacetate  is  more  effective  because  it  is  produced 


INTEGRATED    OXIDATIONS    IN    ISOLATED    MITOCHONDRIA  79 

in  the  vicinity  of  the  succinate  dehydrogenase  and  that  intact  mitochondria 
are  relatively  impermeable  to  oxaloacetate. 

With  particulate  preparations  derived  from  liver  mitochondria  by 
lysis  with  phosphate  and  washing  with  KCl  [13]  and  from  kidney  by  the 
method  of  Slater  [14],  concentrations  of  oxaloacetate  of  the  order  of  10  juM 
had  a  profound  inhibitory  action  on  succinate  oxidation.  With  these 
preparations  permeability  effects  would  be  expected  to  be  far  less  pro- 
nounced. However  the  order  of  addition  of  substrate  and  inhibitor  had  a 


Kp     Kp  OAA 
100  n-7        -t        :. 


^P         i^P    Malonate 

T  T 


0      12      3     4     5 
Time  (mm) 

Fig.  9.  Effect  of  oxaloacetate  (12-5  /^m)  and  malonate  (125  /<m)  on  the  oxida- 
tion of  succinate  (10  niM)  by  a  Slater  kidney  preparation.  The  medium  contained 
20  mM  tris,  pH  7  45. 


marked  effect  on  the  inhibition  of  oxygen  uptake.  This  is  illustrated  for  a 
kidney  preparation  in  Fig.  9.  If  10  to  50  /xM  oxaloacetate  were  added 
before  the  succinate,  no  oxygen  uptake  occurred  for  approximately  i  min., 
after  which  the  steady-state  of  inhibited  respiration  was  observed.  On  the 
other  hand,  when  the  oxaloacetate  was  added  after  the  succinate  several 
minutes  elapsed  before  the  steady-state  was  established.  In  the  latter  case, 
the  lower  the  oxaloacetate  concentration  the  longer  was  the  time  taken 
before  the  final  rate  was  observed.  In  contrast  the  order  of  addition  of  sub- 
strate and  inhibitor  was  unimportant  when  malonate  and  pyrophosphate 
were  used. 


8o 


J.    B.    CHAPPELL 


The  same  dependence  of  order  of  addition  on  the  immediate  effect 
of  oxaloacetate  on  succinate  oxidation  has  been  observed  with  intact 
liver  and  kidney  mitochondria.  Mitochondria  which  had  been  preincu- 
bated  with  arsenate,  DPN  and  amytal,  oxidized  succinate  at  rapid  rates. 
However,  if  i  mM  oxaloacetate  were  added  as  little  as  2  sec.  before  the 
succinate,  oxygen  uptake  was  severely  and  sometimes  completely  inhibited, 
whereas  if  the  inhibitor  were  added  after  the  succinate  no  significant  effect 
was  observed  (Fig.  10). 


100 

-r 

c 

\ 



Succ. 

Mc 

OAA 

T 

T\ 

90 

\              1 

80 

- 

DNP 

\      DNP 

Succ. 

> 

ATP; 

.              no  ATP 

\                   N 

70 

- 

\  750 

\ 

£   60 

U 

oaa\ 

\ 

0 

\ 

0 
^    40 

LU 

- 

\ 

30 

- 

Liver 

\ 

Mitoch 

Dndria 

\ 

20 

- 

\ 

10 

- 

noOAA'^    \ 

1 

I 

\ 

0 

1 

1 

I 

1 

1         1         1     \ 

2       3       4 
Time  (mm) 


Fig.  10.  Effect  of  adding  oxaloacetate  (i  mM),  both  before  and  after  succinate, 
on  the  rate  of  oxygen  uptake  of  liver  mitochondria.  2  mM  arsenate  and  i  •  8  mM 
amytal  were  present  at  zero  time.  Other  conditions  as  in  Fig.  7. 


These  observations  enable  an  explanation  of  the  effect  of  Azzone  and 
Ernster  [12]  to  be  given.  Preincubation  with  DNP  and  arsenate  leads  to 
the  accumulation  of  oxaloacetate  from  endogenous  substrates,  the  keto- 
acid  then  forms  a  stable  complex  with  the  succinate  dehydrogenase,  which 
dissociates  with  difficulty.  Very  little  can  be  said  of  how  ATP  reverses  this 
inhibition ;  it  may  be  that  in  some  way  ATP  dissociates  the  dehydrogenase- 
oxaloacetate  complex,  but  this  is  unlikely  since  ATP  had  no  effect  on  the 
oxaloacetate   inhibition   of  succinate   oxidation   by  kidney   preparations 


INTEGRATED    OXIDATIONS    IN    ISOLATED    MITOCHONDRIA  01 

and  saline-phosphate  treated  Hver  mitochondria.  An  alternative  hypothesis 
is  that  the  ATP  is  required  for  removal  of  the  oxaloacetate  by  the  phos- 
phoenolpyruvate  carboxylase  reaction. 

Malate  oxidation 

EFFECT    OF    FLUOROMALATE 

DL-3-fluoromalate  is  a  competitive  inhibitor  of  purified  mitochondrial 
malate  dehydrogenase;  2  mM  fluoromalate  causes  a  99" u  inhibition  of 
DPN  reduction  with  i  mM  L-malate  as  substrate  [5].  Malate  oxidation 
occurs  at  relatively  low  rates  in  liver  mitochondria,  especially  when  DNP 
is  used  to  stimulate  respiration.  However,  when  glutamate  and  /3-chloro- 
vinyl  arsenious  oxide  (0-5  /xg./ml.)  were  used  the  oxaloacetate  produced 
by  the  malate  dehydrogenase  was  removed  by  transamination  and  a  rapid 
rate  of  oxygen  uptake  resulted.  The  arsenical  inhibited  the  oxidation  of 
glutamate,  as  was  described  previously,  and  aspartate  was  shown  to 
accumulate  in  the  medium.  In  this  system  5  niM  fluoromalate  inhibited 
the  oxidation  of  10  mM  malate  by  more  than  90'^*  o ;  at  lower  malate  con- 
centrations the  inhibition  was  even  more  marked. 

However,  fluoromalate,  even  at  a  concentration  of  10  mM,  had  no 
observable  effect  on  two  systems  which  are  thought  to  involve  the  oxidation 
of  malate,  namely  the  isocitrate  system  (Fig.  4)  and  the  inhibition  of 
succinate  oxidation  which  occurs  when  mitochondria  are  preincubated 
with  arsenate  and  DNP.  Both  these  latter  systems  may  be  thought  of  as 
"  internal "  and  it  may  be  that  fluoromalate  is  unable  to  penetrate  to  them. 
iVIitochondria  behave  as  though  they  are  partly  impermeable  to  oxalo- 
acetate and  it  is  not  inconceivable  that  they  are  also  impermeable  to  fluro- 
malate. 

Figure  11  is  a  summarv  of  some  of  the  findings  which  have  been  dis- 
cussed and  an  attempt  to  correlate  the  structural  relationships  of  the 
enzymes  within  the  mitochondrion  with  their  function. 

It  is  apparent  that  the  level  of  oxaloacetate  in  mitochondria  can  under 
certain  conditions  control  the  rates  of  glutamate,  isocitrate,  succinate  and 
malate  oxidation.  Under  conditions  in  which  the  rate  of  oxaloacetate 
production  is  inhibited  (lewisite,  malonate)  glutamate  and  isocitrate  oxida- 
tion occur  at  markedly  reduced  rates.  On  the  other  hand  succinate  and 
malate  oxidation  are  inhibited  by  oxaloacetate  accumulation.  If  these 
factors  are  suitably  controlled,  e.g.  by  providing  sufiicient  oxaloacetate  for 
isocitrate  oxidation  or  by  preventing  the  accumulation  of  this  keto-acid  in 
the  case  of  malate  and  succinate  oxidation,  the  rates  of  glutamate,  isocitrate 
and  malate  (in  the  presence  of  lewisite  and  glutamate)  occur  at  the  same 
rate  (360  400  /d.  02/mg.  N/hr.)  when  ADP  is  used  to  stimulate  respiration. 
Succinate   oxidation    occurs   at    50  60",,    greater   rates.    However   in   the 

VOL.  n. — (; 


82 


J,    B.    CHAPPELL 


presence  of  io~^  m  DNP  only  glutamate  and  isocitrate  oxidation  occur 
at  these  rates;  both  malate  (600-800  fxl.  Oo/mg.  N/hr.)  and  succinate  (up 
to  1600  ^1.  Oo/mg.  N/hr.)  oxidation  occur  at  considerably  greater  rates. 
These  results  indicate  that  many  mitochondrial  oxidations  are  controlled 
by  the  activity  of  the  enzymes  directly  involved  in  the  synthesis  of  ATP 
and  subsequent  to  the  site  of  action  of  DNP.  On  the  other  hand  ^-hydroxy- 
butyrate  was  oxidized  at  only  60'^,,   of  the  ADP-rate   for  other   DPN 


Outside 


Inside 


/socitrate 
dehydrogenase 


Oxaloacetate 

Oxaloacetate 

|-«-- DPN,  arsenate 

Pyruvate 

Phosphoenol- 
pyruvate 

Inhibition  -•- 

Fig.    II.  Postulated  spatial  relationship  of  some  mitochondrial  enz^Tnes. 

linked  substrates;  in  this  case  some  other  factor,  presumably  the  activity 
of  the  dehydrogenase,  controls  the  rate  of  oxidation. 

Acknowledgment 

I  wish  to  thank  Miss  Freda  Johnson  for  her  expert  technical  assistance. 


References 

1.  Chappell,  J.  B.  (ig6i).  To  be  published. 

2.  Chance,  B.,  and  Williams,  G.  R.,  Advanc.  Enzy7noL  l.'J.i  65  (1956). 

3.  Purvis,  J.  L.,  BiocJu'm.  hiopliys.  Acta  30,  440  (1958). 

4.  Stein,  A.  M.,  Kaplan,  N.  O.,  and  Ciotti,  M.  M.,y.  biol.  Chem.  234,  979  (1959). 

5.  Thorne,  C.  J.  R.,  personal  communication  (i960). 

6.  Borst,  P.,  and  Slater,  E.  C,  Biochim.  hiophys.  Acta  41,  170  (i960). 

7.  Krebs,  H.  A.,  and  Bellamy,  D.,  Biochcm.J.  75,  523  (i960). 

8.  Lardy,  H.  \.,  and  Wellman,  H.,7-  biol.  Chem.  195,  215  (1952). 


INTEGRATED    OXIDATIONS    IN    ISOLATED    MITOCHONDRIA  83 

9.  Borst,  P.,  and  Slater,  E.  C,  Xatiire,  Loud.  184,  1396  (1959). 

10.  Lardv,  H.  A.,  Johnson,  D.,  and  McMurrav,  W.  C,  Arch.  Biochein.  Biophys. 
78,587(1958). 

11.  Chappell,  J.  B.,  and  Greville,  G.  D.,  Nature,  Loud.  190,  502  (1961). 

12.  Azzone,  G.  F.,  and  Ernster,  L.,  Xatiire,  Land.  187,  65  (i960). 

13.  Estabrook,  ^.,J.  biol.  Chem.  230,  755  (1957). 

14.  Slater,  E.  C,  Biochern.J.  45,  i  (1949). 


Discussion 

Lowenstein:  Dr.  Chappell's  first  slide  showed  that  isocitrate  was  oxidized  as 
rapidly  as  glutamate.  Some  years  ago  Dr.  Lardy  showed  that  isocitrate  is  oxidized 
much  more  slowly  than  glutamate.  Is  this  a  question  of  the  conditions  that  you  use  ? 

Chappell  :  Dr.  Lardy  was  using  Warburg  manometers  and  I  am  using  an 
oxygen  electrode. 

Lowenstein  :  We  used  an  oxygen  electrode  and  obtained  the  same  results. 

Ch.^ppell:  What  was  your  level  of  phosphate,  M  25  phosphate  ? 

Lowenstein:  We  used  somewhat  lower  concentration,  plus  hexokinase  and 
glucose. 

Chappell:  M  25  phosphate  is  inhibitory  unless  you  add  catalytic  amounts  of 
malate.  I  think  the  point  of  action  of  the  phosphate  is  on  fumarase  because  at  low 
fumarate  concentrations  phosphate  does  inhibit  fumarase  quite  markedly,  thus  it 
is  acting  as  malonate  would,  or  Lewisite,  and  preventing  the  catalyst  from  getting 
into  its  position  to  catalyse  more  citrate  oxidation. 

Lowenstein:  You  think  it  is  the  concentration  of  orthophosphate  which  is 
critical  ? 

Ch.appell  :  I  am  sure  it  is,  you  can  show  it  very  easily. 

Lardy  :  I  should  like  to  point  out  that  De  Luca  and  Steenbock  have  found  very 
striking  differences  between  the  rate  of  oxidation  of  isocitrate  in  rats  with  and 
without  vitamin  D  and  they  think  that  this  is  a  controlling  factor  determining  the 
levels  of  citrate  in  tissues ;  there  is  a  considerable  increase  in  the  citrate  content  of 
tissues  in  the  animals  getting  the  normal  supplement  of  vitamin  D.  I  wonder 
whether  your  experiments  could  not  be  investigated  using  vitamin  D-deficient 
rats  to  see  what  effects  vou  would  get. 


Metabolic  Control  of  Structural  States  of  Mitochondria 

Lester  Packer 

Departtnent  of  Microbiology, 

University  of  Texas  Southwestern  Medical  School, 

Dallas,  Tex.,  U.S.A. 

I  would  like  to  discuss  briefly  certain  properties  of  the  swelling-shrink- 
ing phenomenon  as  it  occurs  ///  vitro  and  perhaps  in  the  living  cell. 
Although  many  of  the  questions  surrounding  this  phenomenon  are  un- 
answered, some  experiments  suggest  directions  in  which  explanations 
may  lie. 


Sub- 
strate 


Z 
PN 


FP 


Cr 


^  Oxygen  I 


A.  '  >■ 

-n    Carrier~I    ■- 


I 
X~I 

X~P 


ATP 


Fig.   I.  Scheme  for  oxidative  phosphorylation.  The  components  of  the  mito- 
chondrial membrane  are  enclosed  bv  the  dashed  line. 


In  particular  there  are  two  points  I  would  like  to  raise,  both  are  still 
speculative,  but  it  appears  that  their  clarification  may  contribute  to  our 
understanding  of  the  phenomenon  at  the  macromolecular  and  cellular 
levels.  The  first  is  that  it  seems  certain  that  changes  in  mitochondrial 
volume  can  be  directed  in  vitro  by  the  activity  of  the  enzymes  of  the 
respiratory  chain  and  oxidative  phosphorylation.  The  system  is  schematized 
in  Fig.  I.  The  reactions  enclosed  by  the  dashed  lines  represent  the  res- 
piratory chain  and  associated  enzymes  of  the  coupling  mechanism  located 
in  the  membrane.  This  outline  embodies  current  concepts  of  several 
laboratories  [i,  2]  in  which  it  is  thought  that  following  electron  transport 


86 


LESTER    PACKER 


an  energy-linked  form  of  the  oxidation-reduction  carriers  arises,  and  that 
this  component  is  capable  of  giving  rise  to  further  intermediates  which 
interact  with  inorganic  phosphate  and  adenosine  diphosphate  (ADP) 
leading  to  adenosine  triphosphate  (ATP)  synthesis.  The  reactants  of  the 
system  are  clearly  substrate,  which  may  interact  at  different  sites,  and 
oxygen  for  electron  transport,  and  phosphate  and  ADP  required  for  the 
synthesis  of  ATP.  It  happens  that  these  reactants  are  capable  of  inducing 
characteristic  changes  in  mitochondrial  volume.  The  product  of  the 
process,  ATP,  also  can  control  mitochondrial  volume  but  it  appears  to 
have  a  rather  special  role.  Very  early  in  the  study  of  the  swelling-shrinking 
phenomenon  Raaflaub  [3]  and  Brenner-Holzach  and  Raaflaub  [4]  reported 
that  swelling  of  rat  liver  mitochondria  was  retarded  by  ATP  and  also  that 
the  state  of  swelling  was  correlated  with  the  intramitochondrial  content 
of  ATP.  Dr.  Lehninger  and  his  associates  [5,  6]  have,  of  course,  clearly 


Glutamate  (5mM 


O,  =  0 


7  9 

Time  (mm) 


15 


Fig.   2.   Effect  of  the  reactants  of  respiratory  chain  phosphorylation  in  mito- 
chondrial swelling  and  shrinking. 


shown  that  ATP  acts  as  a  potent  agent  to  reverse  swelling  induced  by 
treating  mitochondria  with  a  wide  variety  of  the  reagents,  but  that  reversal 
of  swelling  appears  to  be  most  effective  after  some  treatment  of  the  mito- 
chondria occurs  which  renders  the  membrane  more  permeable  to  this 
substance.  Thus  reversal  of  swelling  by  ATP  was  found  to  be  more  effective 
when  mitochondria  were  suspended  in  potassium  chloride  rather  than 
sucrose  solutions.  Reversal  of  light-scattering  changes  in  fragmented 
mitochondrial  membranes  is  also  readily  brought  about  by  ATP  [7].  The 
fact  that  the  reactants  and  product  of  this  system  under  appropriate  cir- 
cumstances can  interact  with  the  swelling-shrinking  phenomenon  suggests 
that  the  phenomenon  is  controlled  by  some  common  intermediate.  An 
example  of  rapid  metabolically-driven  volume  changes  in  rabbit  cardiac 
muscle  mitochondria  is  shown  in  Fig.  2.  The  apparatus  employed  was  a 
Brice-Phoenix  light-scattering  photometer  adapted  for  recording  with  the 
photomultiplier  positioned  at  90'  to  the  incident  beam  at  546  m/.t.  In  some 


METABOLIC    CONTROL    OF    STRUCTURAL    STATES    OF    MITOCHONDRL\  87 

instances,  kinetics  of  scattering  changes  were  measured  simultaneously 
with  the  utihzation  of  oxygen  by  means  of  the  vibrating  platinum  electrode. 
The  electrode  was  placed  in  the  cuvette  and  employed  in  a  manner  similar 
to  that  described  bv  Chance  and  Williams  [8].  Mitochondria  were  isolated 
in  sucrose-Versene  by  the  technique  of  Cleland  and  Slater  [9]  and  sus- 
pended in  a  medium  of  o-i  m  sucrose  fortified  with  0-025  ^^  "tris" 
buffer  at  pH  7-5.  An  increase  in  light-scattering  indicates  shrinking. 
Addition  of  reducing  equivalents  in  the  form  of  glutamate  gave  a  rapid 
shrinkage  which  terminated  in  a  steady  state  after  a  few  seconds.  As 
electron  transport  bv  tightly  coupled  mitochondria  requires  phosphate, 
very  little  respiration  of  glutamate  was  recorded  at  this  time;  this  is 
denoted  bv  the  figure  o-oi  which  refers  to  the  calculated  rate  of  oxygen 
utilized  in  /xM  sec.  Adding  phosphate  augmented  respiration  sevenfold, 
and  also  initiated  swelling  which  continued  over  several  minutes  before 
reaching  a  steadv  state.  The  reverse  experiment  of  adding  phosphate  in  the 
absence  of  substrate  does  not  result  in  swelling.  Thus  phosphate  is  unable 
to  induce  swelling  in  the  absence  of  reducing  equivalents  interacting 
with  the  electron  transport  chain.  The  dependence  of  swelling  on  electron 
transport  has  been  widely  reported  on  by  Chappell  and  Greville  [10] 
and  Hunter  et  al.  [11].  Initiation  of  phosphorylation  in  the  mitochondrial 
suspension  bv  the  addition  of  ADP  results  in  acceleration  of  respiration 
and  a  rapid  shrinkage.  When  ADP  is  converted  into  x-lTF,  respiration 
declines,  and  the  scattering  state  returns  to  that  found  prior  to  the  brief 
cycle  of  phosphorylation.  Synthesis  of  ATP  under  these  conditions 
apparentlv  has  not  given  rise  to  a  net  change  in  mitochondrial  volume. 
When  Dr.  Chance  and  I  [12]  first  observed  this  rapid  reversal  of  swelling 
by  ADP,  and  the  relative  lack  of  eff'ectiveness  of  ATP  under  these  con- 
ditions, we  suggested  that  energy-linked  intermediates  may  be  more 
efi^ective  than  ATP  itself.  Implications  for  the  role  of  intermediates  in 
the  control  of  this  phenomenon  have  been  reported  by  others,  for  example, 
Ernster  [13]  and  Lehninger  and  associates  [5],  employing  diflPerent  systems. 
Again  returning  to  the  experiment,  after  some  lapse  of  time,  the  dissolved 
O2  of  the  system  is  exhausted  and  at  this  point  a  reversal  of  swelling  occurs 
which,  if  allowed  to  continue  in  this  record,  would  have  reached  the  level 
seen  in  the  presence  of  ADP.  This  effect  has  been  termed  autonomic 
reversible  swelling  by  Beechey  and  Holton  [14].  The  experiment  suggests 
how  fluctuations  in  the  concentration  of  the  reactants  of  the  respiratory 
system  are  capable  of  controlling  the  state  of  mitochondrial  volume 
[i:^,  16],  as  the  ability  of  these  substances  to  elicit  changes  in  volume 
follows  closely  their  effects  upon  the  respiratory  chain  on  a  concentration 
basis.  Thus  the  half-maximal  value  for  activation  of  respiration  by  ADP 
and  phosphate  is  about  50  |UM  and  i  mM  respectively  [17]  and  they  have  a 
half-maximal  effect  on  shrinkage  or  swelling  at  the  same  concentrations. 


88 


LESTER    PACKER 


Therefore  it  may  be  anticipated  that  a  small  fluctuation  in  the  concentration 
of  ADP  would  have  a  more  extensive  effect  upon  mitochondrial  volume 
than  a  similar  change  in  phosphate.  How  about  the  extent  of  the  reversible 
volume  changes  ?  When  the  metabolically  induced  changes  are  calibrated 
by  comparing  them  with  an  osmotically  induced  change  in  volume,  then 
the  calculated  magnitude  of  the  metabolic  changes  is  of  the  order  of  20% 
of  their  total  volume  [15].  It  must,  of  course,  be  asked — can  such  changes 
in  mitochondrial  volume  be  induced  through  fluctuations  in  the  con- 
centrations of  the  reactants  of  the  respiratory  chain  in  vivo  ?  If  this  is  so, 
what  effect  would  this  have  upon  metabolic  reactions  ?  Perhaps  in  swollen 
or  shrunken  states  the  ability  of  metabolites  to  cross  the  membranes  is 


0-22        '          ' 

1           1 

^<           0-68 

^   130 

i N. 

002 

Ol 

c 

"Mannose  (SmM) 

^  r 

S   120 

4_j 

- 

0 

^0^ 

u 

^^ 

:.  110 

/ 

-C 

t        / 

CD 

§  100 

1           1           1 

- 

240 


120    5. 

6 
60 

0 


0 


2         3         4         5 
Time  (min) 

Fig.  3.  Respiration  and  light-scattering  changes  in  Ehrlich  ascites  tumour 
cells  observed  after  mannose  addition.  The  numbers  above  the  upper  trace  refer 
to  the  calculated  rates  of  oxygen  consumption  in  /xM/l./sec. 


changed.  This  would  affect  the  competition  which  exists  between  cyto- 
plasmic and  mitochondrial  compartments  in  the  cell  for  the  same  meta- 
bolite. Indeed,  the  membrane  has  been  implicated  as  a  possible  site  for 
the  regulation  of  certain  effects  in  the  intact  cell  such  as  the  Pasteur  and 
Crabtree  phenomena,  which  involve  complex  interactions  between 
different  compartments  in  the  cell.  This  subject  was  authoritatively  dis- 
cussed at  the  recent  symposium  on  regulation  of  cell  metabolism  [18].  For 
this  reason  it  seemed  promising  to  Dr.  Colder  and  me  [19]  to  attempt  to 
demonstrate  light-scattering  changes  in  ascites  tumour  cells  following  the 
addition  of  certain  carbohydrates.  The  rapid  changes  in  metabolism  which 
characterize  the  early  phases  of  the  Crabtree  effect  have  been  probed  in 
detail  by  Chance  and  Hess  [20].  Figure  3  demonstrates  a  simultaneous  re- 
cording of  O2  consumption  and  light-scattering  in  a  suspension  of  these 
cells  in  Krebs-Ringer  phosphate  medium.  Addition  of  mannose  initiates  an 
acceleration   of  respiration  lasting  for  several   minutes,  which    is   then 


METABOLIC    CONTROL    OF    STRUCTURAL    STATES    OF    MITOCHONDRIA  89 

followed  by  a  strong  inhibition.  An  increase  in  light-scattering  also  begins 
after  mannose  addition.  The  respiration  and  scattering  changes  are  com- 
pleted at  almost  the  same  time.  Other  hexoses  such  as  2-deoxyglucose  and 
glucose  give  similar  results.  Although  scattering  changes  have  been  ob- 
served which  are  due  to  tonicity  changes  of  the  cells  themselves  (Lucke 
and  Parpart,  [21]),  rapidly  penetrating  carbohydrates  such  as  mannose 
cause  no  measurable  changes  in  cell  volume  as  judged  by  direct  determina- 
tions in  control  experiments.  It  was  therefore  proposed  that  the  changes 
were  of  intracellular  origin.  Similar  light-scattering  or  shrinkage  changes 
are  observed  by  ADP  addition  to  isolated  mitochondria  from  many  sources 
including  those  of  ascites  tumour  cells.  Mitochondrial  shrinkage  and 
acceleration  of  respiration  would  be  the  result  of  the  carbohydrate-induced 
hexokinase  reaction  which  increases  the  intramitochondrial  ADP  level. 
The  extensive  inhibition  of  metabolism  is  believed  to  result  from  the  un- 
availability of  ATP  in  the  cytoplasmic  system.  Chance  and  Hess  [20]  and 
Racker  [22]  imply  that  the  cause  is  an  alteration  in  the  structure  of  the 
mitochondrial  membrane.  In  this  experiment  such  a  change  would  seem 
disclosed  by  the  light-scattering  effect.  As  the  production  of  ADP  would 
be  expected  to  be  quite  high  under  the  conditions  where  scattering  was 
increasing,  these  results  may  indicate  that  shrunken  mitochondria  in 
vivo  can  retard  the  escape  of  ATP  synthesized  by  oxidative  phosphorylation. 

In  this  regard  it  is  interesting  to  recall  the  experiments  of  Gamble  [23] 
who  reported  increased  retention  of  bound  potassium  ions  by  intact 
mitochondria  under  conditions  of  phosphorylation  and,  presumably,  high 
shrinkage.  Certain  structural  states  of  the  membrane  may  favour  potassium 
binding. 

Attempts  are  being  made  to  design  other  experiments  to  test  the  avail- 
ability of  ATP  synthesized  by  oxidative  phosphorylation  for  extramito- 
chondrial  processes.  In  one  series  of  experiments  Dr.  Watanabe  and  I 
have  made  a  crude  reconstruction  of  a  living  muscle  fibre  [24].  The  ATP 
synthesized  by  oxidati\e  phosphorylation  of  cardiac  muscle  mitochondria 
from  ADP  and  phosphate  is  made  available  for  the  isometric  development 
of  tension  by  a  glycerinated  muscle  fibre.  Tension  development  evoked  by 
ATP  alone  and  by  ATP  produced  by  mitochondrial  phosphorylation  of 
ADP  were  compared  and  the  results  are  recorded  in  Fig.  4.  Respiration 
of  the  mitochondrial  suspension  was  traced  polarographically  and  tension 
development  was  simultaneously  recorded  by  use  of  a  strain  gauge  trans- 
ducer. It  was  found  that  tension  development  runs  very  closely  with 
oxidative  phosphorylation  over  a  range  of  ADP  concentrations  varying 
between  10^^  and  lo^^  m.  In  an  experiment  in  the  presence  of  ADP  and 
mitochondria  half-maximum  tension  was  developed  when  the  con- 
centration of  ADP  was  2-5  X  10  '^  M  (curve  A).  In  the  absence  of 
mitochondria    with    ATP    only,    half-maximum    tension   was   developed 


90  LESTER    PACKER 

with  1-25  X  io~^  M  ATP  (curve  B),  The  results  show  that  the  tension 
response  is  faster  than  ATP  production  by  mitochondrial  phosphorylation. 
Escape  of  ATP  from  the  mitochondria  may  be  the  rate-limiting  react'on, 
and  this  process  may  be  connected  with  the  extensive  mitochondrial 
shrinkage  states  present. 

In  summary,  it  was  shown  that  changes  in  mitochondrial  volume  or 
membrane  structure  are  brought  about  by  changes  in  the  activity  of  the 
respiratory  chain.  Certain  evidence  suggests  that  these  structural  changes 
may  lead  to  altered  reaction  rates  of  ATP-requiring  systems  which  react 
at  or  near  the  membrane  surface. 

The  second  point  which  I  would  like  to  consider  is  the  locus  of  the 
coupling  mechanism  involved  in  the  swelling-shrinking  phenomenon, 
which  has  been  raised  by  some  experiments  we  have  done  with  p-chloro- 
mercuribenzoate    (PCMB).    Certain    striking    similarities    between    this 


o  0-5 


200  400  600  800 

Concentration  of  ADP  or  ATP  (//M) 

Fig.  4.  Contraction  of  glj'cerol-treated  fibres  of  rabbit  psoas  muscle  with  ATP 
synthesized  by  rabbit  cardiac  muscle  mitochondria  (a)  or  ATP  only  (b).  (Courtesy 
of  the  Journal  of  Biological  Chemistry.) 

system  and  the  contractile  protein  of  muscle,  myosin  B,  are  apparent. 
Tapley  [25]  and  Lehninger  and  Ray  [26]  have  studied  the  action  of  PCMB 
on  mitochondrial  volume  and  report  that  it  enhanced  swelling.  Significantly 
Lehninger  and  Ray  [26]  found  that  swelling  was  more  rapid  under  aerobic 
than  anaerobic  conditions.  Tapley  [25]  suggested  that  sulphydryl  groups 
may  be  important  in  determining  mitochondrial  structure.  In  Fig.  5  the 
time  course  is  shown  of  the  effect  of  83  /xM  PCMB  on  shrinking  and  swelling 
of  a  suspension  of  cardiac  mitochondria  respiring  different  substrates.  In 
the  absence  of  substrate,  PCMB  exerts  no  appreciable  effect  upon  mito- 
chondrial shrinkage  for  over  2  min.,  and  then  extensive  swelling  occurs. 
When,  however,  PCMB  is  added  when  substrates  are  present  it  immediately 
initiates  an  enormous  shrinkage.  Later  a  reversal  occurs  and  swelling  ensues, 
resulting  in  a  decrease  in  light-scattering  similar  to  that  in  absence  of 
substrates.  PCMB-induced  shrinkage  is  dependent  on  the  presence  of 
substrate   and   on  the   concentration   of  the   SH-binding  reagent.   With 


METABOLIC    CONTROL    OF    STRUCTURAL    STATES    OF    MITOCHONDRIA  9 1 

high  concentrations  of  PCMB  the  initial  shrinkage  period  is  only 
transient,  but  at  low  concentrations  the  high  shrinkage  state  is  maintained 
for  considerable  time  and  reversal  only  occurs  after  long  incubation  times. 
Similar  findings  have  been  observed  with  uncoupling  agents. 

It  is  suggested  that  the  period  of  shrinkage  induced  by  PCMB  and 
uncoupling  agents  prior  to  swelling  is  the  result  of  their  ability  to  interact 
with  an  enzyme  of  the  coupling  mechanism  to  augment  adenosine  triphos- 
phatase activity  recently  reported  by  Cooper  [27]  through  interaction  with 
inhibitory  sulphydryl  groups.  However,  after  PCMB  has  been  able  to 
bind  sulphydryl  groups  more  extensively  a  deformation  of  the  macro- 
molecular  structure  occurs  which  leads  to  severe  swelling.  Based  upon  the 


PCBM  (83//M) 


Clutamate 
3-hydroxybutyrate 


(/-ketoglutarate 


0        1         2        3        4        5        6        7 
Time  (min) 

Fig.   5.   Effect  of /)-chloromt'rcuribenzoate  on  the  shrinking-swelling  phenome- 
non in  cardiac  muscle  mitochondria. 

current  concepts  of  myosin  B-ATPase  action,  it  is  postulated  that  a  protein 
very  similar  to  but  difi^erent  from  the  contractile  muscle  proteins  will  be 
isolated  from  mitochondrial  systems  as  was  suggested  4-5  years  ago  by 
Chappell  and  Perry  [28]. 

References 

1.  Chance,  B.,  and  Williams,  G.  R.,  Advaiic.  Enzyniol.  17,  65  (1956). 

2.  Slater,  ¥..  C,  Aiist.J.  exp.  Biol.  med.  Set.  36,  51  (1958). 

3.  Raaflaub,  J.,  Helv.  physiol.  acta  1 1,  242  (1953). 

4.  Brenner-Holzach,  O.,  and  Raaflaub,  J.,  Helv.  physiol.  acta  12,  242  (1954). 

5.  Lehninger,  A.  L.,_7.  hiol.  Clioii.  234,  2465  (1959). 

6.  Lehninger,  A.  L.,  Ray,  B.  L.,  and  .Schneider,  M.,^.  biophys.  hiochem.  Cytol.  5, 

97  (1959)- 

7.  Packer,  L.,  and  Tappel,  A.  L,.,jf.  biol.  Client.  235,  525  (i960). 

8.  Chance,  B.,  and  Williams,  G.  R.,^.  biol.  Cbem.  217,  383  (1955). 

9.  Cleland,  K.  W.  and  Slater,  E.  C,  Biochem.  J.  53,  547  (1953). 

10.  Chappell,  J.  B.,  and  Greville,  G.  D.,  Nature,  Lottd.  182,  813  (1958). 
ri.   Hunter,  F.  E.,  Jr.,  Levy,  J.  F.,  Fink,  J.,  Schutz,  B.,  Guerra,  F.,  and  Hurwitz, 
A.,y.  biol.  Ghent.  234,  2176  (1959). 


92  LESTER    PACKER 

12.  Chance,  B.,  and  Packer,  L.,  Biochem.  J.  68,  295  (1958). 

13.  Ernster,  L.,  Ann.  Rev.  Physiol.  20,  13  (1958). 

14.  Beechey,  R.  B.,  and  Holton,  F.  A.,  Proc.  biochem.  Soc.  73,  29P  (1959). 

15.  Packer,  L.,^.  biol.  Cheni.  235,  242  (i960). 

16.  Packer,  L,.,jf.  biol.  Chern.  236,  214  (1961). 

17.  Chance,  B.,  ?'«  "  Ciba  Foundation  Symposium  on  Regulation  of  Cell  Metabo- 
lism". Little,  Brown,  and  Co.,  Boston,  Mass.,  91  (1959). 

18.  Wolstenholme,  G.  E.  W.,  and  O'Connor,  C.  M.,  "Ciba  Foundation  Sym- 
posium on  Regulation  of  Cell  Metabolism".  Little,  Brown,  and  Co.,  Boston, 
Mass.  (1959). 

19.  Packer,  L.,  and  Golder,  R.  H.,y.  biol.  Chem.  235,  1234  (i960). 

20.  Chance,  B.,  and  Hess,  B.,  Science  129,  700  (1959). 

21.  Lucke,  B.,  and  Parpart,  A.  K.,  Cancer  Res.  14,  75  (1954). 

22.  Packer,  E.,  and  Wu,  R.,  in  "Ciba  Foundation  S>Tnposium  on  Regulation  of 
Cell  Metabolism".  Little,  Brown,  and  Co.,  Boston,  Mass.  205  (1959). 

23.  Gamble,  J.  L.,  ]r.,y.  biol.  Chem.  228,  955  (1957). 

24.  Watanabe,  S.,  and  Packer,  L.,  _7.  biol.  Chem.  236,  1201  (1961). 

25.  Tapley,  D.  F.,_7.  biol.  Che?n.  222,  325  (1956). 

26.  Lehninger,  A.  L.,  and  Ray,  B.  L.,  Biochim.  biophys.  Acta  26,  643  (1957). 

27.  Cooper,  C.,jf.  biol.  Chem.  235,  18 15  (i960). 

28.  Perry,  S.  V.,  "Proc.  3rd  Intern.  Congress  Biochem.,  Brussels",  364  (1956). 

Discussion 

Lehninger  :  These  experiments  carried  out  by  Dr.  Packer  bring  into  focus  one 
of  the  apparent  discrepancies  which  can  be  seen  in  our  respective  approaches. 
Dr.  Packer  is  interested  in  the  physiological  description  of  reversible,  low- 
amplitude  swelling  and  shrinking  in  tightly  coupled  mitochondria.  Our  own 
interest  has  been  in  dissociating  the  high  amplitude  swelling  and  contraction 
processes  from  all  the  other  enzymic  machinery  in  mitochondria  which  are  not 
necessarily  tightly  coupled.  Actually  we  can  make  mitochondria  contract  with 
ATP  after  respiration  and  phosphorylation  are  irreversibly  lost.  We  have  a  different 
approach  and  the  interesting  thing  is  that  our  drastically  swollen  mitochondria 
contract  with  ATP  but  not  ADP  and  Dr.  Packer's  slightly  swollen  mitochondria 
contract  on  the  addition  of  ADP  but  not  ATP.  I  think  that  his  experiments  point 
to  the  reason  for  this  apparent  discrepancy  if  I  understand  him  right.  It  is  now  the 
current  conception  that  the  mitochondrial  membrane  in  vivo  is  relatively  imper- 
meable to  ATP.  The  internal  and  external  nucleotides  have  different  turnover 
rates.  Such  compartmentation  has  been  invoked  in  explaining  the  Pasteur  reaction. 
I  think  it  is  possible  then  under  conditions  of  very  drastic  swelling  such  as  we  use 
that  the  permeability  of  the  mitochondrial  membrane  has  in  fact  changed,  though 
I  can  hardly  agree  with  Dr.  Packer  that  exposure  with  KCl  can  be  regarded  as 
unphysiological.  It  is  a  more  physiological  substance  than  sucrose.  In  any  case  it 
is  possible  that  for  shrinking  to  occur  ATP  must  penetrate  inside,  or  that  it  must 
be  generated  inside  from  ADP.  Do  you  believe  that  ADP  can  penetrate  inside  and 
there  generate  ATP  which  can  drive  the  contraction  ? 

Packer:  I  think  the  reconstruction  experiment  shows  that  it  is  very  difficult 
in  isolated  mitochondria  to  retain  the  ATP  synthesized  by  oxidative  phosphoryla- 
tion ;  it  almost  all  comes  out,  bvit  when  a  change  in  permeability  or  when  a  large 


METABOLIC    CONTROL    OF    STRUCTURAL    STATES    OF    MITOCHOXDRLA  93 

swelling  occurs  apparently  the  ATP  has  the  ability  to  bind  on  the  membrane  or 
penetrate  whereas  before  it  was  ineffective.  I  think  that  ATP  and  ADP  act  through 
some  common  mechanism  by  influencing  energy-linked  intermediates.  With  regard 
to  the  KCl  comment  Jackson  and  Pace  have  shown  that  the  half  time  for  penetra- 
tion of  KCl  is  seconds  whereas  for  sucrose  it  is  hours. 

Lehninger  :  If  this  is  the  case  then  ATP  leaks  very  readily. 

Packer:  Yes.  However,  this  may  not  be  so  in  vivo. 

Lehninger:  In  view  of  what  you  say  I  am  therefore  a  little  puzzled  over  those 
explanations  of  the  Pasteur  reaction  which  are  based  on  "  compartmentation " 
of  ATP. 

P.\CKER :  Well  the  reconstruction  experiments  indicate  that  the  leakage  of  ATP 
through  the  membrane  is  rate-limiting.  If  we  assume  that  in  the  course  of  isolation 
of  mitochondria  from  the  tissue  some  swelling  has  taken  place  it  is  reasonable  that 
this  property  of  the  escape  of  .ATP  is  now  retained  to  a  lesser  extent,  but  still 
sufficiently  to  detect  it. 

Hess:  If  one  isolates  the  mitochondria  from  glucose  ascites  cells  then  it  is 
apparent  that  they  take  in  quite  a  lot  of  ATP  in  comparison  with  mitochondria 
isolated  from  ascites  tumour  cells  which  do  not  contain  glucose.  Now  the  shrinkage 
of  mitochondria  seems  to  be  associated  with  the  retention  of  .ATP  as  far  as  the 
experimental  data  are  concerned.  It  is  worthwhile  to  point  out  that  cytologists  have 
evidence  about  mechano-proteins  in  living  cells  and  have  found  in  a  number  of 
cells  contractile  proteins  in  the  cytoplasm  which  can  be  readily  activated  by  the 
addition  of  ATP.  My  question  is:  there  is  a  certain  discrepancy  in  the  interpreta- 
tion in  the  Crabtree  effect ;  what  is  the  rate-limiting  material  which  controls  rate 
of  respiration  whether  phosphate  or  .ADP  ?  .As  far  as  I  see  from  your  data  if  you 
have  low  inorganic  phosphate  concentration,  mitochondria  are  swollen  and  if  you 
have  a  low  ADP  concentration  then  the  mitochondria  are  shrunk.  Xow  could  you 
draw  any  conclusions  from  your  experiments  on  this  point  ? 

Packer  :  We  have  been  interested  in  trying  to  examine  the  effect  of  phosphate 
on  the  ascites  cell  with  respect  to  the  scattering  problem.  In  this  connection  we 
have  tried  to  prepare  phosphate-free  ascites  cells,  but  apparently  we  have  not  yet 
been  able  to  remove  sufficient  phosphate  to  lower  the  endogenous  respiration,  so 
I  don't  feel  that  we  have  been  able  to  put  the  effect  of  phosphate  on  the  shrinkage 
phenomenon  to  the  test. 

Siekevitz:  1  wish  to  recall  the  experiments  Dr.  Potter  and  I  did  in  relation  to 
the  amount  of  ATP  available  in  mitochondria  under  conditions  of  oxidative 
phosphorylation.  When  we  added  hexokinase  we  obtained  the  phosphate  of  the 
ATP  as  glucose  6-phosphate  in  the  medium.  Under  these  conditions,  we  found 
that  the  hexokinase  does  not  attack  the  ATP  inside  the  mitochondria  but  the  ATP 
coming  out.  So  under  these  conditions  of  active  phosphorylation,  I  do  not  know 
whether  the  mitochondria  are  shrunken  or  swollen,  but  the  .ATP  can  come  out 
very  fast  indeed. 

Ch.'\nce:  Just  a  very  short  comment  on  the  ability  of  .ATP  rapidly  to  enter  the 
mitochondria.  With  tightly  coupled  mitochondria  ATP  can  enter  rapidly  and  at 
the  same  time  cause  them  to  swell  (unpublished  observations). 

EsTABROOK :  On  the  point  of  the  comparison  of  rates  of  ATP  getting  out  of  the 
mitochondria  and  ADP  going  back  in,  one  can  show  using  the  coupled  hexokinase 


94  LESTER    PACKER 

system  that  the  rate-limiting  step  with  isolated  mitochondria  is  ATP  going  out  to 
hexokinase,  rather  than  ADP  going  back  in  to  the  mitochondrial  phosphorylating 
system. 

Packer:  Of  course  very  early  in  this  study  of  the  swelling-shrinking  phe- 
nomenon Raaflaub  observed  that  the  level  of  swelling  in  the  mitochondria  was 
related  to  the  intramitochondrial  ATP  level,  but  we  may  have  to  distinguish 
between  ATP  which  is  bound  and  unavailable  and  that  which  is  free,  and  this  may 
be  the  area  where  some  of  these  discrepancies  lie. 

AzzoNE :  As  far  as  I  can  understand  the  main  discrepancy  between  your  results 
and  those  of  Dr.  Lehninger  is  that  you  get  shrinking  when  mitochondria  are  in 
State  3,  that  is  when  there  is  ADP  in  the  medium  and  no  high  energy  phosphate 
intermediate(s)  can  accumulate  in  the  mitochondria;  when  all  the  ADP  in  the 
medium  is  transformed  into  ATP  the  mitochondria  begin  to  accumulate  high 
energy  phosphate  intermediate(s)  at  the  same  time  as  they  begin  to  swell.  On  the 
contrary,  in  Dr.  Lehninger's  experiments,  the  mitochondria  are  swollen  without 
any  addition  to  the  incubation  medium,  and  after  addition  of  ATP  they  begin  to 
shrink.  One  possible  explanation  of  this  discrepancy  is  that  in  Dr.  Lehninger's 
experiments  the  mitochondria  are  completely  uncoupled.  Thus  it  would  appear 
that  in  uncoupled  mitochondria  external  ATP  is  reqviired  for  shrinking  whereas  in 
coupled  mitochondria  it  is  the  presence  of  ATP  and  of  high  energy  phosphate 
intermediate(s)  inside  the  mitochondria  which  causes  swelling. 


Stable  Structural  States  of  Rat  Heart  Mitochondria 

F.  A.  HoLTON*  AND  D.  D.  Tyler 

Medical  Research   Council,   Experimental  Radiopathology   Research    Unit, 
Hammersmith  Hospital,  London,  England 

Recent  work  of  Packer  [1,2]  has  shown  that  isolated  mitochondria  of 
both  hver  and  heart  in  dilute  suspension  may  be  induced  to  undergo 
rapidly  reversible  changes  in  light-scattering  properties  and  that  the 
direction  of  these  changes  is  related  to  the  relative  concentrations  of  ADP 
and  ATP  in  the  reaction  medium.  Changes  in  the  light-scattering  properties 
of  suspensions  of  mitochondria  are  commonly  ascribed  to  alterations  in  the 
structure  of  the  individual  particles.  This  hypothesis  has  been  adopted  by 
Packer  and  is  also  assumed  in  what  follows  here. 

Rapidlv  reversible  changes  in  mitochondrial  structure  may  also  be 
demonstrated  in  the  absence  of  external  adenine  nucleotide  [3],  but  lately 
we  have  followed  Packer  in  the  use  of  externally  added  adenine  nucleotide, 
and  in  experiments  with  heart  mitochondria  we  have  confirmed  many  of 

TABLE  I 

Ratios  of  Rates  of  Chax(;e  of  Extinction  Observed  in  a  Suspension  of  Heart 
Mitochondria  at  434  m/t  and  477  •  5  mfj.  in  Response  to  Additions  of  Phosphate 

and  Succinate 

Data  calculated  from  Fig.  i.  The  values  given  in  the  last  column  should  be 
compared  with  the  theoretical  ratio  calculated  from  Rayleigh's  law.  The  theoretical 
ratio  for  measurements  at  434  m^tt  and  477  •  5  m/x  where  the  changes  of  extinction 
are  due  solely  to  changes  in  the  light-scattering  properties  of  the  material  is  i  -47. 


Experiniental 
time 
(sec.) 

Rate  of  ch 

( 

an 
m 

ge 
e. 

of 

u.,' 

extinction  {z-) 
5ec.) 

Ratio  of 
rates 

434  m/' 

477-5  miL 

^.434 

^.477.5 

190 
230 

270 

1-39 

2-21 

I-4S 

0-89 
I  -50 
1-03 

1-56 

1-47 
I  -44 

*   Present  address:   Royal   J^tterinarv  College,   Royal  College  Street,   London, 

X.IV.T. 


96  F.    A.    HOLTON    AND    D.    D.    TYLER 

his  findings.  In  particular,  we  have  repeated  Packer's  important  observa- 
tion that  the  phosphorylating,  close-coupled  heart  mitochondrion  may  be 
made  to  alternate  between  a  stable  expanded  condition  and  a  stable  con- 
tracted condition  according  to  the  state  of  the  respiratory  chain  (expanded 


477-5  m/t 


Time,  sec 

Fig.  I.  Establishment  of  a  stable  expanded  condition  of  mitochondria  in 
response  to  phosphate  and  succinate. 

Extinction  changes  at  434  m/^t  and  at  477  •  5  m/x  were  measured  simultaneously 
by  the  method  of  Holton  [10]  using  a  rectangular  glass  cell  with  clear  walls,  sur- 
rounded on  three  sides  with  polished  metal  plates  and  maintained  at  a  constant 
temperature  of  18 -8'.  Rat  heart  mitochondria  (sarcosomes)  were  isolated  as 
described  previously  [11]  and  were  diluted  from  time  o  into  a  medium  containing 
0-28  M  sucrose,  o-oi  M  disodium  potassium  ethylenediaminetetraacetate,  pH  7-4, 
to  give  50  ml.  of  a  reaction  mixture  containing  0-51  mg.  biuret  protein  per  ml.  At 
time  177  sec.  the  optical  recording  was  interrupted  and  0-5  ml.  of  0-5  M  potassium 
phosphate  buffer,  pH  7  -4,  was  rapidly  stirred  into  the  suspension.  At  time  210  sec. 
0-5  ml.  of  0-4  M  potassium  succinate,  pH  7-4,  was  added  in  a  similar  way.  Final 
concentrations  were:  phosphate,  4-9  mvi ;  succinate,  3-9  mM. 

Measurements  of  the  rates  of  change  of  extinction  observed  at  the  two  wave- 
lengths studied  yielded  the  ratios  of  rates  given  in  Table  I.  These  values  show 
reasonable  agreement  with  the  ratio  calculated  from  Rayleigh's  law  of  light-scatter- 
ing. This  constitutes  evidence  that  the  progressive  changes  of  extinction  illustrated 
above  were  caused  by  changes  in  the  light-scattering  properties  of  the  mitochondria. 

condition  with  external  nucleotide  as  ATP,  respiratory  chain  in  state  4; 
contracted  condition  with  external  nucleotide  as  ADP,  respiratory  chain 
in  state  3,  following  the  nomenclature  of  Chance  and  Williams  [4]). 

More  recently  we  have  attempted  to  answer  by  experiment  two 
questions  concerning  the  establishment  of  stable  structural  states  in  these 


STABLE   STRUCTUR.\L   STATES    OF   RAT    HEART    MITOCHONDRIA 


97 


mitochondria.  The  first  is  as  follows,  i .  Are  there  only  tzco  stable  structural 
states  or  is  it  possible  to  demonstrate  a  series  of  intermediate  stable  states 
lying  betzceen  the  fully  expanded  and  tJie  fully  contracted  conditions  ? 

The  second  of  these  alternatives  appears  to  be  correct.  Rat  heart  mito- 
chondria may  be  brought  to  a  stable  expanded  configuration  by  a  short 


0 

!                                    1                                    1 

■    ■—  ■■  1 

-450 

__ 

+  0  04 

1 

t 

t 

t 

t 

Mg 

ADP 

ADP 

ADP 

\ 

i 

i 

i 

0 

-477  5 

*v- 

i 

*>■>- 

-H003 

\— 

1                               II 

( 

50 

Time. 

100 
sec 

150 

Fig.  2.  Data  illustrating  {a)  the  absence  of  a  discernible  effect  on  mitochondrial 
structure  when  magnesium  chloride  is  added  to  a  suspension  of  heart  mitochondria 
which  have  been  brought  to  a  stable  expanded  condition  with  phosphate  and 
succinate;  and  {b)  part  of  a  "shrinkage  titration"  showing  the  establishment  of  a 
series  of  steadv  structural  states  of  mitochondria  with  increasing  concentrations  of 
ADP. 

Extinction  changes  were  recorded  and  rat  heart  mitochondria  were  isolated  as 
described  in  the  legend  to  Fig.  i .  Only  two  of  the  three  simultaneous  recordings 
are  shown.  Downward  deflection  of  the  traces  indicates  increase  of  extinction. 
Horizontal  lines  have  been  drawn  at  intervals  of  o-oi  extinction  units.  Temperature 
20•9^ 

9  min.  before  the  beginning  of  the  above  record  a  concentrated  suspension  of 
freshly  isolated  mitochondria  was  diluted  into  0-32  M  sucrose,  pH  7-4,  to  give  48 
ml.  of  a  reaction  mixture  containing  0-21  mg.  biuret  protein  per  ml.  Phosphate 
buffer,  pH  7-4  and  potassium  succinate,  pH  7-4  were  then  added  to  give  final 
concentrations:  phosphate,  5-1  mM ;  succinate,  4-1  mM.  Extinction  changes 
similar  to  those  shown  in  Fig.  i  were  then  observed,  indicating  swelling  of  the 
mitochondria.  A  stable  expanded  condition  was  finally  established  and  is  indicated 
above  by  the  steady  level  of  the  traces  at  the  beginning  of  the  record.  \x  the  point 
marked  Mg  the  recording  was  interrupted  and  0-5  ml.  of  0-09  m  magnesium 
chloride  was  rapidly  stirred  into  the  suspension  (final  concentration  0-92  mM). 
The  small  decrease  of  extinction  which  followed  was  attributable  to  the  effect 
of  dilution.  At  the  points  marked  ADP  00s  ml.  of  0-02  M  ADP  in  0-32  m  sucrose 
was  added  in  the  same  way.  Three  further  additions  of  .A.DP  were  made  at 
intervals  immediately  following  the  above  record.  The  optical  effects  of  these  later 
additions  are  not  shown  above  but  are  included  in  Fig.  3  together  with  the  above 
data. 


98  F.   A.    HOLTON   AND    D.    D.    TYLER 

incubation  with  phosphate  and  succinate  (Fig.  i).  Addition  of  magnesium 
chloride  does  not  alter  this  configuration  (Fig.  2),  but  it  activates  a  power- 
ful ATPase  [5]  which  can  hydrolyze  newly  synthesized  ATP  at  least  as 


►  002 


+  001 


50  100  150 

External   ADP  concentration 
/i.  Molar 

Fig.  3.  Relationship  between  the  concentration  of  externally  added  ADP  and 
the  resultant  extinction  changes  measured  simultaneously  at  three  separate  wave- 
lengths in  a  suspension  of  heart  mitochondria  in  the  presence  of  magnesium  ions. 
Data  of  Fig.  2  graphed,  with  addition  of  results  relating  to  later  additions  of  ADP 
and  also  from  simultaneous  observations  at  434  m/x. 

The  changes  of  extinction  caused  by  ADP  clearly  did  not  obey  Rayleigh's  law, 
since  for  any  given  concentration  of  ADP  they  did  not  decrease  regularly  with 
increasing  wavelength  of  observation.  The  data  suggests  that  a  minor  part  of  the 
extinction  changes  observed  was  due  to  an  alteration  in  the  extinction  due  to  pig- 
ments as  ADP  was  added.  They  are  consistent  with  the  hypothesis  that  the  states 
of  oxidation  of  both  cytochrome  b  and  flavoprotein  were  moved  in  the  direction  of 
oxidation  by  addition  of  ADP,  an  eifect  to  be  expected  from  the  work  of  Chance  and 
Baltscheffsky  [12].  If  it  is  assumed  that  there  was  no  contribution  of  pigments  to 
the  extinction  changes  recorded  at  450  m/x,  it  is  possible  to  calculate  for  the  other 
two  wavelengths  of  observation  values  of  the  ratio 

extinction  change  due  to  light  scattering 
extinction  change  due  to  pigment 
For  observations  at  both  434  m^i  and  477  ■  5  m/Lt  the  above  data  give  a  value  of 
3  -4  for  the  above  ratio.  This  value  may  be  compared  with  that  from  the  work  of 
Chance  and  Packer  [13],  who  added  ADP  to  a  suspension  of  rat  heart  sarcosomes 
and  deduced  a  value  of  about  4  for  the  same  ratio.  (In  their  work  the  extinction 
change  due  to  scattering  was  equated  to  that  observed  at  443  m/x  and  the  extinction 
change  due  to  pigment  was  equated  to  the  difference  between  the  changes  observed 
at  430  m/t  and  at  443  m/i.) 

fast  as  the  respiratory  chain,  oxidizing  succinate,  can  produce  it.  Under 
these  conditions  added  ADP  is  maintained  at  the  concentration  at  which 
it  has  been  added  while  the  processes  of  oxidative  phosphorylation  con- 
tinue normally.  Figures  2  and  3  illustrate  the  optical  effects  of  successive 


STABLE    STRUCTURAL    STATES    OF    RAT    HEART    MITOCHONDRL^  99 

additions  of  small  amounts  of  ADP  to  expanded  mitochondria  with  mag- 
nesium present.  A  succession  of  stable  structural  states  is  established  with 
increasing  concentrations  of  ADP  until  an  equilibrium  contracted  con- 
dition is  reached. 

Parallel  measurements  of  respiration  rates  carried  out  at  the  same  time 
as  the  spectrophotometric  observations  with  the  same  preparation  of 
mitochondria  under  identical  experimental  conditions  showed  that  the 
mitochondria  in  their  expanded  condition  exhibited  respiratory  control  [6] 
with  ADP  temporarily  accelerating  their  respiration  in  a  characteristic  way. 
This  fact  suggested  a  second  question.  2.  Is  the  influence  of  ADP  concen- 
tration on  structural  change  quantitatively  similar  to  its  effect  on  respiration 
rate  ? 

We  have  found  that  the  two  relationships  are  quantitatively  very 
similar.  The  results  given  in  Fig.  2  are  repeated  in  a  different  form  in 
Fig.  3  in  order  to  show  the  relation  between  the  total  concentration  of 
added  ADP  and  the  total  change  of  extinction  caused  by  its  addition. 
Half-maximal  shrinkage  was  effected  at  a  concentration  of  18  fiM  ADP. 
Respiration  measurements  show  that  half-maximal  acceleration  of  respira- 
tion is  brought  about  at  approximately  the  same  concentration  of  ADP 
(e.g.  26  juM  for  pigeon  breast  muscle  mitochondria  [7]). 

The  hypothesis  that  the  respiration  rate  of  mitochondria  in  vivo  is 
controlled  by  the  concentration  of  ADP  in  the  cytoplasm  has  been  well 
ventilated  recently  [8].  Both  the  work  of  Packer  and  his  associates  and  the 
above  experiments  emphasize  that  reversible  alterations  in  mitochondrial 
structure  probablv  represent  a  feature  of  the  same  control  mechanism. 
They  provide  evidence  supporting  the  suggestion  of  Ernster  [9]  that  "a 
reversible  labilization  of  the  mitochondrial  structure  may  constitute  a 
physiological  principle  of  metabolic  control." 


References 

1.  Packer,  L.  Ann.  N.Y.  Acad.  Sci.  72,  518  (1959)- 

2.  Packer,  L.  7-  biol.  Chem.  235,  242  (i960). 

3.  Beechey,  R.  B.,  and  Holton,  F.  A.,  Biochein.J.  73,  29P  (i959)- 

4.  Chance,  B.,  and  Williams,  G.  R.,  J.  biol.  Chem.  217,  409  (i955)- 

5.  Holton,  F.  A.,  Hiilsmann,  W.  C,  Myers,  D.  K.,  and  Slater,  E.  C,  Biuchem.J. 

67.  579  (1957)- 

6.  Chance,  B.,  in  "  Ciba  Foundation  Symposium  on  the  Regulation  of  Cell 
Metabolism",  ed.  G.  E.  W.  Wolstenholme,  C.  M.  O'Connor,  London, 
Churchill,  91  (1959). 

7.  Chance,  B.  (i960),  personal  communication. 

8.  Wolstenholme,  G.  E.  W.,  and  O'Connor,  C.  M.,  "  Ciba  Foundation  Symposium 
on  the  Regulation  of  Cell  Metabolism".  London,  Churchill  (i959)- 

9.  Ernster,  L.,  Exp.  Cell.  Res.  lO,  721  (1956). 
10.  Holton,  F.  A.,  Biochem.J.  66,  37P  (1957)- 


lOO  F.    A.    HOLTON   AND    D.    D.    TYLER 

11.  Holton,  F.  A.,  Biochetn.J.  6l,  46  (1955). 

12.  Chance,  B.,  and  Baltscheffsky,  M.,  Biochetn.J.  68,  283  (1958). 

13.  Chance,  B.,  and  Packer,  L.,  Biochem.J.  68,  295  (1958). 


Discussion 

Klingenberg  :  May  I  mention  that  the  phenomenon  of  reversible  swelling  was 
also  reported  some  years  ago  by  us  for  flight  muscle  mitochondria.  These  show, 
with  glycerolphosphate  as  substrate,  a  very  pronounced  reversible  swelling  which 
can  be  related  to  respiratory  control  and  to  changes  in  the  redox  state  of  respiratory 
components;  in  further  unpublished  studies  we  noted  that  with  pyruvate  plus 
malate  which  are  very  effective  respiratory  substrates  for  mitochondria  practically 
no  reversible  swelling  could  be  observed.  Sometimes  even  contraction  of  the  mito- 
chrondria  can  be  observed.  Studies  on  the  internal  adenine  nucleotide  content 
showed  that  the  ATP  to  ADP  ratio  in  mitochondria  is  the  same  whether  one  has 
glycerolphosphate  or  pyruvate  plus  malate  there.  The  difference  in  other  nucleo- 
tides can  only  be  seen  in  the  reduction  of  DPN  or  in  the  reduction  of  flavin  nucleo- 
tide, the  glycerolphosphate  reduces  the  DPN  to  a  higher  extent  and  the  flavin  also, 
whereas  pyruvate  plus  malate  do  not  reduce  the  DPN  or  the  flavin  nucleotide  to  an 
appreciable  extent. 

Holton  :  Am  I  right  in  interpreting  your  feeling  that  the  reactions  important 
in  controlling  structural  state  are  at  the  flavin  end  and  that  they  are  not  related 
directly  to  the  ATP-ADP  equilibria  inside  the  mitochondria. 

Klingenberg  :  Yes. 

Mitchell:  You  say.  Dr.  Holton,  that  this  kind  of  swelling  and  shrinking,  the 
reversible  kind,  is  quite  diflFerent  from  the  kind  studied  by  Dr.  Lehninger.  Are  you 
in  fact  studying  the  same  membrane  ?  There  are  two  membranes  here.  Are  you 
quite  sure  that  both  these  cases  of  swelling  and  shrinking  are  due  to  effects  on  the 
same  membrane  ? 

Holton  :  I  would  say  that  the  fact  that  the  mitochondria  that  Dr.  Lehninger 
studies  are  a  good  deal  further  from  the  native  state  than  ours  is  consistent  with 
your  idea,  since  the  reversible  phenomena  that  both  we  and  Dr.  Packer  study  don't 
last  long.  I  suppose  it  might  well  be  a  more  intact  particle  which  shows  the  rever- 
sible phenomena  and  one  which  has  got  holes  in  it  which  shows  the  other. 

Lehninger:  I  would  just  like  to  add  that  occurrence  of  two  different  morpho- 
logical types  of  swelling  could  be  a  plausible  resolution  of  the  apparent  differences 
in  the  properties.  It  should  be  made  clear  that  there  is  no  disagreement  as  to  the 
observations.  We  have  confirmed  the  effect  of  ADP  on  a  tightly  coupled  system. 
On  the  other  hand  Dr.  Holton  has  confirmed  our  findings  that  ATP  is  specific  for 
shrinking  drastically  swollen  mitochondria.  As  I  pointed  out  in  one  of  my  slides 
there  are  at  least  two  diflferent  ways  in  which  mitochondria  can  swell  and  we  have 
suggested  in  fact  that  it  may  be  the  outer  membrane  which  is  the  ATP-activated 
structure.  On  the  other  hand  damage  to  the  membranes  on  drastic  swelling  could 
produce  a  diflference  in  access  of  ATP  and  ADP,  but  it  seems  unlikely  that  a  strict 
ADP  specificity  will  change  to  a  strict  ATP  specificity  on  damaging  a  structure. 
I  would  like  to  ask  Dr.  Holton  a  question  which  I  think  will  contrast  the  different 


STABLE   STRUCTURAL    STATES   OF   RAT   HEART   MITOCHONDRL\  lOI 

kinds  of  systems  we  are  studying.  In  the  more  drastic  swelling  we  study,  the  ampli- 
tude of  the  cycle  is  large.  There  is  a  two  or  three-fold  increase  in  volume.  I  would 
like  to  know  whether  Dr.  Holton  can  give  an  estimate  of  the  per  cent  difference  in 
mitochondrial  volume  between  his  two  stable  states  ? 

HoLTON :  No,  I  am  afraid  that  we  have  no  data  on  which  to  base  such  an  esti- 
mate, but  perhaps  a  rough  comparison  of  the  magnitudes  of  the  extinction  changes 
we  are  observing  in  our  respective  systems  would  be  helpful.  I  suspect  that  you  use 
a  lo-mm.  cell  and  a  concentration  of  mitochondria  of  perhaps  0-5  mg./a  ml.  of 
protein.  Is  that  reasonable  ?  With  this  arrangement  you  get  changes  of  about  one 
extinction  unit.  Our  reversible  changes  would  not  be  discernible  at  this  optical 
thickness  and  concentration.  We  use  0-2  mg./ml.  and  a  60  mm.  cell  where  the  sides 
are  mirrored,  and  then  we  observe  a  total  change  for  reversible  swelling  of  about 
0-02  extinction  units.  You  see  that  there  are  several  degrees  of  magnitude  between 
the  amount  of  extinction  change  which  are  seen  under  your  conditions  and  ours. 


Solubilization  and  Properties  of  the  DPNH 
Dehydrogenase  of  the  Respiratory  Chain* 

Thomas  P.  Singer 

Edsel  B.  Ford  Institute  for  Medical  Research,  Henry  Ford  Hospital,  Detroit, 

Mich.,  U.S.A. 

Although  the  work  of  our  laboratory  for  the  past  few  years  has  been, 
in  the  main,  concerned  with  the  systematic  isolation  and  detailed  study  of 
the  various  dehydrogenases  which  are  structural  and  functional  com- 
ponents of  the  respiratory  chain,  until  recently  we  have  not  attempted  to 
isolate  one  of  the  most  interesting  enzymes  in  this  group,  the  respiratory 
chain-linked  DPNH  dehydrogenase.  One  reason  for  this  was  the  large 
number  of  preparations  of  mitochondrial  origin  described  in  the  literature 
which  are  capable  of  oxidizing  DPNH  under  suitable  conditions.  A  closer 
study  of  the  relevant  literature  reveals,  however,  that  few  of  these  prepara- 
tions have  been  well  characterized ;  even  fewer  could  be  ascribed  a  definite 
function  in  cellular  metabolism,  and  no  soluble,  purified  preparations 
could  be  assuredly  identified  with  the  enzyme  which  links  the  oxidation 
of  DPNH  to  the  respiratory  chain. 

Among  animal  tissues  heart  mitochondria  appear  to  have  been  most 
intensively  studied  with  respect  to  DPNH  oxidation.  Limiting  this  dis- 
cussion to  soluble  enzymes,  free  from  respiratory  chain  components,  there 
have  been  two  relatively  well-defined  preparations  isolated  from  heart 
mitochondria:  Straub's  diaphorase  [i]  and  Mahler's  DPNH  cytochrome 
reductase  [2].  Both  of  these  enzymes  have  been  thought,  one  time  or 
another,  to  be  artifacts  of  isolation,  a  view  based  on  the  harsh  methods 
employed  in  their  isolation.  As  to  Mahler's  enzyme,  the  group  at  the  Enzyme 
Institute  still  believes  that  it  is  an  artifact  [3],  since  the  peculiar  properties 
of  its  flavin  group  may  be  reproduced  by  applying  the  alcohol  treatment 
used  in  its  isolation  to  other  preparations,  although  JMassev  has  produced 
considerable  evidence  to  indicate  that  the  enzyme  in  fact  pre-exists  in 
mitochondria  [4].  In  either  event  there  is  little  in  its  properties  that  would 
suggest  that  it  is  the  flavoprotein  component  of  the  DPNH  oxidase  chain. 

*  Supported  by  grants  from  the  National  Heart  Institute,  National  Institutes 
of  Health  and  the  American  Heart  Association,  Inc.,  and  by  contract  No.  Nonr 
1656  (00)  between  the  Office  of  Naval  Research  and  this  Institute. 


I04  THOMAS    P.    SINGER 

As  to  diaphorase,  Massey's  work  [5,  6]  has  clearly  demonstrated  that  it  is 
part  of  the  a-ketoglutarate  oxidase  complex  and,  as  such,  it  is  concerned 
with  the  reduction  of  DPN,  not  the  oxidation  of  DPNH.  More  recently, 
Ziegler  aiid  colleagues  [3]  have  reported  the  isolation  of  a  lipid-bound 
DPNH  dehydrogenase  of  very  high  molecular  weight  and  lipid  content 
and  with  very  interesting  properties,  and  King  and  Howard  reported  the 
extraction  of  DPNH  dehydrogenase  from  heart  muscle  mince  by  treatment 
with  phospholipase  [7].  Regarding  the  former  preparation,  it  remains  to 
be  seen  whether  its  high  lipid  content  represents  a  fimctional  component 
or  an  impurity.  In  either  case,  we  felt  that  it  was  desirable  to  isolate  the 
dehydrogenase  without  recourse  to  organic  solvents,  bile  salts,  or  other 
harsh  treatments  and  in  a  lipid-free  form,  so  that,  as  in  the  case  of  succinic 
dehydrogenase,  the  properties  of  the  protein  could  be  adequately  charac- 
terized. The  possible  relation  of  King  and  Howard's  soluble  preparation 
to  the  enzyme  I  shall  describe  will  be  discussed  later  on  in  this  paper. 

Our  initial  work  on  this  problem  w^as  concerned  wuth  the  linkage  of 
DPNH  dehydrogenase  to  the  respiratory  chain.  It  has  been  known  for  over 
five  years  that,  although  succinic  and  DPNH  dehydrogenases  operate  via 
a  common  respiratory  chain  and  are  interlinked  at  the  oxidation  level  of 
cytochrome  h,  methods  which  solubilize  succinic  dehydrogenase  from 
respiratory  chain  preparations  [8]  fail  to  extract  DPNH  dehydrogenase. 
Thus,  superficially,  the  bonds  holding  these  two  closely  related  enzymes 
to  the  electron  transport  system  appear  to  be  quite  different.  During  the 
isolation  of  a-glycerophosphate  dehydrogenase  from  brain  mitochondria 
a  few  years  ago  [9],  we  found  that  the  incubation  of  brain  mitochondria 
with  phospholipase  A  resulted  not  only  in  the  extraction  of  a-glycero- 
phosphate dehydrogenase  in  soluble  form  but  also  of  considerable  DPNH 
dehvdrogenase  activity.  Following  this  initial  lead,  with  Drs.  Minakami 
and  Ringler,  w-e  decided  to  undertake  its  isolation  and  characterization  [10]. 

Two  problems  faced  us  at  the  outset :  the  choice  of  starting  material 
and  the  assav.  In  our  work  on  other  mitochondrial  dehydrogenases  it  has 
been  shown  that  these  are  the  paramount  factors  deciding  the  success  or 
failure  of  the  isolation.  As  to  starting  material,  we  did  not  wish  to  use 
heart  muscle  mince  or  even  mitochondria,  since,  besides  the  enzyme  we 
were  after,  they  were  bound  to  contain  other  DPNH  dehydrogenases, 
such  as  the  Straub  diaphorase,  possibly  Mahler's  reductase,  and  any 
DPNH  dehydrogenases  arising  from  microsomal  contamination.  We 
decided,  therefore,  to  use  the  particulate  DPNH  oxidase  (ETP)  prepara- 
tion of  Crane,  Glenn,  and  Green  [11],  since  this  has  been  reported  to  be  a 
purified  form  of  the  DPNH  dehydrogenase  linked  to  the  cytochrome  chain, 
free  from  numerous  contaminating  enzymes,  particularly  diaphorase,  in 
which  the  oxidation  of  DPNH  is  completely  antimycin  and  amytal- 
sensitive   [11].   Thus  any  soluble  preparation  isolated  from  it  would  be 


SOLUBILIZATION   AND    PROPERTIES    OF   THE   DPNH    DEHYDROGENASE         IO5 

reasonably  certain  to  represent  the  flavoprotein  responsible  for  DPNH 
oxidation  in  mitochondria. 

Considerable  effort  was  expended  on  elaborating  a  reliable  assay 
method  for  the  enzyme.  Early  in  this  work  it  became  apparent  that, 
contrary  to  general  impression,  the  assay  of  DPNH  dehydrogenase  in 
mitochondria  or  in  particles  such  as  ETP  is  a  relatively  difficult  task. 
Among  electron  acceptors  to  be  employed  phenazine  methosulphate  was 
eliminated  because  of  its  rapid  non-enzymic  reaction  with  DPNH, 
quinones,  such  as  menadione,  because  of  their  failure  to  react  with  the 
isolated  dehydrogenase  at  significant  rates,  and  2,6-dichlorophenol- 
indophenol  because  of  relatively  high  blanks  and  the  great  dependence  of 
the  measured  activity  on  dye  concentration  characteristic  of  this  oxidant. 


15 

, 

^    10 

.y^^^^ 

-'"'ri       .^^^ 

'-'''/^^'I--— ••'•^* 

^^-^'^'S 

■"'''W*' 

,,,'''' 

/ 

^''' 

1 

2  4  6  8 

l/ml,  FelCN)^**" 

Fig.  I.  Ferricyanide  assay  of  particulate  DPNH  dehydrogenase  in  the  presence 
of  6  X  lo"^  M  DPXH.  The  assays  were  performed  in  the  presence  of  120  /^/moles 
phosphate,  pH  7-4,  i-8  /xmoles  DPXH,  DPXH  oxidase,  and  quantities  of  o-oi 
M  ferricyanide  as  indicated,  in  a  total  volume  of  3  ml.  The  determinations  were 
made  at  30  using  a  recording  spectrophotometer  and  a  30  to  60  sec.  total  reaction 
time.  The  reduction  of  ferricyanide  was  followed  at  various  wave  lengths  and 
corrected  to  £400-  X — X,  assay  without  further  additions,  O— o,  in  the  presence 
of  2  X    io~^  M  antimj'cin  A,  • — •,  in  the  presence  of  io~^  m  cyanide. 


A  suitable  method  was  eventually  elaborated  which  is  based  on  the 
spectrophotometric  measurement  of  the  initial  rate  (15  or  3c  sec.)  of  re- 
duction of  ferricyanide  [12].  The  application  of  this  method  to  heart  mito- 
chondria or  to  particles  deri^■ed  therefrom,  such  as  ETP,  entails  several 
problems,  some  of  which  are  illustrated  in  Fig.  i.  This  figure  is  a  Line- 
weaver-Burk  plot  of  the  variation  of  measured  activity  with  ferricyanide 
concentration  in  an  ETP  preparation  at  moderately  high  (6  x  iq-^  m) 
initial  substrate  concentration.  In  the  absence  of  inhibitors  (crosses)  a 
definite  break  is  seen  in  the  curve  relating  reciprocal  activity  to  reciprocal 
ferricyanide  concentration.  The  reason  for  this  is  that  ferricyanide  has  two 


io6 


THOMAS    P.    SINGER 


reaction  sites  in  the  DPNH-oxidase  chain  of  heart  mitochondria,  the 
flavoprotein  and  cytochrome  c,  just  as  in  the  succinoxidase  system  [13,  14]. 
The  relatively  flat  part  of  the  curve  represents  primarily  the  reaction  with 
cytochrome  c,  since  the  apparent  Kj^j  of  the  flavoprotein  for  ferricyanide 
is  much  larger  than  that  of  the  cytochrome  c  site  and  thus,  at  low  dye 
concentrations,  the  measured  rate  represents  largely  the  reaction  with 
cytochrome  c.  That  this  is  the  case  may  be  readily  shown  by  the  following 
facts :  the  flatter  curve  is  non-competitively  inhibited  by  amytal  or  antimy- 
cin  A  (open  circles),  as  expected  from  the  fact  that  these  inhibitors  prevent 
the  flux  of  electrons  to  cytochrome  c.  It  is  competitively  activated  by  cyanide 
and  azide  (closed  circles),  because  these  inhibitors  inhibit  the  flux  of 
electrons  via  cytochrome  oxidase  to  Oo  and  thereby  increase  the  flux  of 
electrons  to  ferricyanide.  The  activation  is  competitive  with  respect  to 


Antimycin  C  x)  y^ 

Amytal  (o)   ^^^^ ^■^ 

x/>*     ^         '  Control 

4C*'^  +CN- 


10 


12 


2  4  6  8 

I/ml.  FeCCN)^  ** 

Fig.  2.  Ferricyanide  assay  of  DPNH  oxidation  by  ETP  in  the  presence  of 
2   X    io~*  M  DPNH.  Conditions  as  in  Fig.  i. 


ferricyanide,  since  at  infinite  ferricyanide  concentration  all  the  electrons 
would  flow  to  the  dye.  The  steep  part  of  the  curve  represents  the  sum  of 
both  reaction  sites  of  ferricyanide.  When  the  values  from  the  extrapolation 
of  the  flat  curve  are  subtracted  from  the  experimental  values  obtained  at 
high  ferricyanide  concentrations,  the  inhibition  by  antimycin  and. amytal 
and  the  activation  by  cyanide  or  azide  disappear  in  accord  with  the  fact 
that  these  inhibitors  do  not  afl"ect  the  DPNH-flavoprotein-ferricyanide 
reaction  sequence. 

Attention  should  be  called  to  the  fact  that  the  higher  the  concentration 
of  ferricyanide  employed,  the  greater  the  contribution  of  the  dehydro- 
genase site  to  the  measured  activity.  Thus  it  is  clearly  desirable  to  employ 
as  high  a  concentration  of  the  electron  acceptor  as  compatible  with  the 
optical  arrangement  when  working  at  fixed  concentrations  of  the  oxidant. 
In  heart  particles  high  concentrations  of  ferricyanide  do  not  appear  to  be 


SOLUBILIZATION    AND    PROPERTIES    OF    THE    DPNH    DEHYDROGENASE       IO7 

inhibitory  to  DPNH  dehydrogenase,  ahhough  the  situation  is  quite 
different  in  liver  [15].  Since  at  high  ferricyanide  concentrations  the  curve 
is  very  steep,  however,  for  accurate  and  rehable  assays  it  is  clearly  desirable 
to  determine  the  activity  at  infinite  ferricyanide  concentration. 

The  biphasic  nature  of  the  curve  relating  activity  to  ferricyanide  con- 
centrations at  or  above  6  x  lo^*  m  DPXH  (Fig.  i)  is  not  seen  at  low 
(2  X  10"^  M  or  less)  DPNH  concentrations  (Fig.  2).  The  break  in  the 
curve  also  disappears  on  solubilization  of  the  dehvdrogenase  (Fig.  3),  as 
expected  from  the  fact  that  this  procedure  separates  the  flavoprotein  from 
the  respiratory  chain  and  thus  leaves  only  one  reaction  site  for  ferricvanide. 


8- 


6- 


\       1 

r 

- 

•^ 
y^ 

- 

y 

y      •  I0'*M    A  A.  or 
10    M  dicumarol 
"  2-7mM  amytal 

- 

0  Control 

'               '              ■              t 

0  2  4  6  8  10 

I/ml,  Fe(CN)j^** 

Fig.  3.  Ferricyanide  assay  of  dehydrogenase  after  solubilization  with  phos- 
pholipase  A. 


One  of  the  many  reasons  why  assays  of  this  enzyme  conducted  at  fixed 
ferricyanide  concentrations  tend  to  be  unreliable  is  the  narrow  region  of 
DPNH  concentrations  in  which  apparently  optimal  activity  is  observed. 
As  shown  in  Fig.  4,  both  the  DPNH  oxidase  and  the  DPNH-ferricyanide 
assays  are  seriously  inhibited  by  substrate  concentrations  in  excess  of  i .  5 
to  2  X  iQ-^  M.  "While  inhibition  of  DPNH  dehydrogenases  and  DPNH 
cytochrome  reductases  by  excess  substrate  is  a  fairly  common  finding,  it 
is  interesting  to  note  that  the  inhibition,  at  least  with  the  enzyme  under 
discussion,  is  competitive  with  respect  to  the  electron  acceptor  (Fig.  ^). 
Thus  the  inhibition  by  moderately  high  DPNH  concentration  disappears 
at  infinite  concentration  of  ferricyanide. 

Returning  for  the  moment  to  Fig.  3,  it  may  be  noted  that  the  soluble 
dehydrogenase  employed  here  is  completely  insensitive  to  antimycin  A 
and  to  amytal.  It  may  be  remembered  that  ETP,  the  starting  material 
employed  for  the  extraction  of  the  enzyme,  is  100"  q  inhibited  by  amytal 


io8 


THOMAS   P.   SINGER 


and  antimvcin  A  in  the  DPNH  oxidase  assay  and  that  the  reduction  of 
ferricyanide  by  ETP  is  also  inhibited  by  these  reagents  as  far  as  the  cyto- 
chrome c  site  is  concerned  (Fig.  i).  That  the  sohible  flavoprotein  is 
antimycin-insensitive  is  not  surprising,  since  this  inhibitor  is  thought  to 


4- 


DPNH  oxidase 


Fe(CN)^       assay 


DPNH   (x10''m) 

Fig.  4.  Inhibition  of  DPNH  oxidase  and  of  DPNH  dehydrogenase  activities 
by  excess  substrate.  Left:  DPNH  oxidase  assay  at  30°;  120  /Limoles  phosphate, 
pH  7-4,  o-o6  mg.  protein  (DPNH  oxidase,  Crane,  et  al.  [11]),  and  DPNH  as 
indicated  in  3  ml.  volume.  Right:  Ferricyanide  assay  at  fixed  acceptor  concentra- 
tion. Same  conditions  except  that  0-09  mg.  protein  and  2-5  /xmoles  K3Fe(CN)6 
were  present  in  each  cuvette.  Reaction  time  in  both  experiments  about  30  sec. 


0  2  4  6 

l/ml.  Fe(CN)g'" 

Fig.  5.  Ferricyanide  assay  of  soluble  DPNH  dehydrogenase  at  various  con- 
centrations of  DPNH.  For  assay  conditions  see  Fig.  i.  The  DPNH  concentrations 
were:  1-5    x    ic*  m,  • — •;3-o  x    10"*  m,  o — O;  and6-o  x    10  *  m,  X — X. 


act  between  cytochromes  h  and  c,  but  the  fact  that  it  is  also  amytal- 
insensitive  was  contrary  to  expectations,  since  amytal  had  been  thought 
to  interrupt  the  flow  of  electrons  from  DPNH  to  flavoprotein  [16].  The 
insensitivity  of  the  isolated  dehydrogenase  to  amytal  and  the  insensitivity 


SOLUBILIZATION    AND    PROPERTIES    OF    THE    DPNH    DEHYDROGENASE         lOQ 

of  ETP  or  DPNH  oxidase  preparations  to  this  inhibitor  in  the  flavoprotein- 
ferricyanide  interaction  (cf.  discussion  of  Fig.  i)  suggest  that  amytal 
interrupts  electron  transport  between  flavoprotein  and  the  respiratory 
chain,  as  is  also  the  case  in  the  choline  oxidase  system  of  liver  [17],  and  not 
between  DPNH  and  flavoprotein.  Contrary  conclusions  in  the  earlier 
literature  were  based  on  the  "cross-over  technique"  which  relies  on  the 
measurement  of  the  oxidation  state  of  the  flavoprotein  in  the  450-465  mfj. 
region.  As  will  be  documented  later  in  this  paper,  the  application  of  this 
technique  to  DPNH  dehydrogenase  has  some  major  weaknesses:  the 
diff'erence  spectrum  is  atypical  of  simple  flavoproteins;  the  extinction 
coefficient  of  simple  flavoproteins  at  465  m^u,  is  not  applicable  to  this 
enzyme;  and  it  is  not  even  certain  that  the  flavin  is  fully  reduced  in 
normal  catalysis. 


40 


NAJA-NAJA    1:25 


(mm ) 
Fig.  6.  Progress  of  solubilization  of  DPNH  dehydrogenase  by  phospholipase 
A.  Aliquots  of  a  DPXH  oxidase  preparation  [11]  were  incubated  at  30^,  pH  7-4, 
with  Naja  naja  venom  as  a  source  of  phospholipase.  The  ratios  indicated  define 
the  mg.  weight  of  venom  employed  per  mg.  protein  in  the  particulate  preparation 
(determined  by  biuret  reaction,  using  a  coefficient  of  0-095  for  i  nng.  protein  per 
3  ml.,  I  cm.  light  path,  540  m/w).  At  various  times  aliquots  were  rapidly  cooled  to 
o  ,  centrifuged  at  105  000  x  g  for  30  min.,  and  the  supernatant  solution  was  assayed. 
Activities  are  given  in  arbitrarv  units  on  the  ordinate. 


Turning  now  to  the  isolation  and  characteristics  of  the  dehydrogenase, 
Fig.  6  shows  the  progress  of  solubilization  of  the  enzyme,  starting  with 
ETP,  at  two  levels  of  cobra  venom.  Compared  with  brain  a-glvcerophos- 
phate  dehydrogenase  [9]  and  choline  dehydrogenase  from  liver  [18],  the 
level  of  phospholipase  A  (cobra  venom)  required  for  extensive  solubiliza- 
tion of  DPXH  dehydrogenase  is  quite  high  and  the  progress  of  the  reaction 
under  the  same  conditions  is  rather  slow.  These  observations  are  in  accord 
with  King  and  Howard's  findings  on  the  conditions  necessary  for  the 
extraction  of  the  various  DPXH  oxidizing  activities  from  heart  muscle 
mince  [7].  Table  I  shows  the  balance  of  solubilization.  It  may  be  noted 
that  the  assay  is  reliable  for  both  particles  and  the  soluble  enzyme,  since 
the  recovery  is  satisfactory.  By  repeating  the  incubation  with  a  second 


THOMAS    P.    SINGER 


batch  of  venom  under  the  conditions  of  Table  I,  90^0  or  more  of  the 
activity  may  be  obtained  in  solution. 


TABLE  I 
Balance  of  Solubilization 

^                                                 Activity 

''P                            (^lM  DPNH/min./ml.) 

Per  cent 

DPNH  oxidase                                            333 
Same  after  venom  treatment                      303 
Soluble  enzyme                                             187 
Residue                                                             94 

(100) 
62 
32 

Conditions:  80  min.  incubation  with  i  mg.  A^aja  iiaja  per  25  mg.  protein  at 
30'^.  Solubilization  varied  from  62  to  78%.  Second  incubation  yields  22%  more 
enzyme  in  solution. 

The  fact  that  the  enzyme  is  in  true  solution  has  been  shown  by  the 
usual  criteria :  it  does  not  sediment  in  i  hr.  at  144  000  x  g  even  after  12  hr. 
dialysis  or  repeated  freezing  and  thawing  and  it  may  be  readily  fractionated 
with  (NH4)2S04  in  a  manner  characteristic  of  soluble  proteins. 

The  enzyme  has  been  purified  by  two  cycles  of  (NH4)2S04  fractiona- 
tion at  pH  8-0  and  the  resulting  preparation  has  a  specific  activity  of  about 
200  /xmoles  of  DPNH  oxidized /min. /mg.  protein  (biuret  basis,  coefficient 
=  0-095)  ^^  3°  '  pH  7  •4.  Fractionation  on  calcium  phosphate  gel  or 
hydroxylapatite  has  failed  to  increase  the  purity  further.  The  enzyme  is 
not  held  on  carboxymethylcellulose  at  pH  6-8  and  it  is  excluded  on 
Sephadex  G75.  Fractionation  on  DEAE  cellulose  is  not  feasible,  since  the 
enzyme  is  extremely  strongly  adsorbed  on  this  ion  exchanger.  The  turn- 
over number  per  mole  of  flavin  is  at  least  350  000  at  30°,  pH  7*4  in  the 
ferricyanide  assay. 

Present  knowledge  of  the  properties  of  the  enzyme  may  be  summed 
up  as  follows.  The  enzyme  is  gratifyingly  stable  and  may  be  preserved  for 
prolonged  periods  in  the  frozen  state  with  little  or  no  loss  of  activity. 
Even  after  96  hr.  at  room  temperature  (21  )  at  the  pH  of  optimum  stability 
(pH  7-5)  8o")o  of  the  activity  remained. 

The  dehydrogenase  does  not  act  on  TPNH,  nor  is  TPNH  an  inhibitor. 
DPN,  however,  is  a  powerful  competitive  inhibitor;  the  competition  is 
again  with  respect  to  the  electron  acceptor  (Fig.  7).  This  inhibitory  efl^ect 
of  the  oxidation  product  is  another  reason  why  accurate  assays  must  be 
based  on  the  measurement  of  initial  rates  at  infinite  ferricyanide  con- 
centration. 

The  apparent  Y^j^j  for  DPNH,  based  on  assays  in  which  the  DPNH 
concentration  was  varied  at  infinite  ferricyanide  concentration,  is  i  x  io~^M 


SOLUBILIZATION    AND    PROPERTIES    OF    THE    DPNH    DEHYDROGENASE         III 

at  30°,  pH  7-4  (Fig.  8).  It  should  be  noted  that  this  DPNH  concentration 
is  only  about  one-half  of  that  which  gives  apparent  optimal  activity  (Fig.  5) 
in  the  ferricyanide  assay.  While  an  increase  in  the  initial  DPNH  concentra- 

Effect  of  DPN 


10- 


DPNH  =1  5xI0"''m 
+  9xI0"'m  DPN 


No  DPN 


0  2  4  6  8  10 

l/m"L.  Fe(CN)^"" 

Fig.  7.   Competitive  inhibition  of  soluble,  purified  dehydrogenase  by  9   x    10" 
M  DPN.  Standard  ferricyanide  assay;  DPXH  concentration  =    i  -5    x    10"*  M. 


M  DPNH  X  10^ 


Fig.  8.  Lineweaver-Burk  plot  of  effect  of  DPNH  concentration  on  activity. 
The  values  on  the  ordinate  are  maximal  velocities  of  ferricyanide  reduction 
(^max  with  respect  to  ferricvanide)  corresponding  to  each  concentration  of 
DPNH  at  pH  74,  30  . 

tion  beyond  about  2  x  lo-"*  m  fails  to  increase  the  measured  rate,  even 
under  conditions  where  the  inhibitory  effect  of  excess  substrate  is  elimina- 
ted, it  is  doubtful  if  this  value  represents  a  true  "saturation"  of  the  dehy- 
drogenase. 


112  THOMAS   P.    SINGER 

The  determination  of  the  "pH  optimum"  of  this  enzyme  is  a  particu- 
larly difficult  task.  The  ferricyanide  assay,  as  described,  functions  very 
satisfactorily  in  the  pH  range  of  about  5-5  to  7-8.  In  this  range  double 
reciprocal  plots  of  activity  versus  ferricyanide  concentration  show  a  definite, 

6 


Fig. 


l/ml.  Fe(CN)j"" 
9.  Ferricyanide  assay  of  soluble,  purified  enzyme  at  different  pH  values. 


zlE/min 


pH 

Fig.  10.  Effect  of  pH  on  the  activity  of  purified  DPNH  dehydrogenase. 
Solid  line,  at  fixed  (i  -66  x  io~^  m)  ferricyanide  concentration;  dashed  line,  V^ax 
values.  The  pH  values  given  are  those  of  the  reaction  mixture  at  30°.  Buffers: 
0-04  M  phosphate  (pH  5-5  to  8-5)  or  tris  (above  pH  8-5). 

moderate  slope  and  the  reaction  kinetics  are  of  zero  order,  while  at  pH 
5-0  the  slope  is  negligible  (Fig.  9)  and  the  reaction  assumes  first  order 
characteristics  with  respect  to  the  substrate.  As  the  pH  is  increased  above 
8-0,  the  slope  approaches  infinity  and  at  pH  8-5  to  9  it  intersects  the 
abscissa  and  thus  no  satisfactory  measure  of  F^ax  can  be  obtained.  At 
present  no  satisfactory  explanation  of  this  complex  behaviour  is  evident. 


SOLUBILIZATION'    AND    PROPERTIES    OF    THE    DPXH    DEHYDROGENASE         II3 

Within  the  pH  range  where  assays  based  on  l\^^^  vakies  are  rehable  (5  -5 
to  7  •  8)  no  definite  optimum  is  attained,  but  at  fixed  ferricvanide  concentra- 
tions (i-66  X  io~^  M  or  lower)  the  apparent  optimum  is  around  pH  8 
(Fig.  10). 


0-2- 


With  DPNH 


With  dithionite 


400 


550 


450  500 

Fig.  II.  Absorption  spectrum  of  dehydrogenase  in  soluble  extract,  prior 
to  purification  and  the  effects  of  DPXH  and  of  dithionite  on  the  spectrum.  Protein 
concentration,  3-6  mg.  per  ml.;  pH  =  7-15.  Recorded  with  Cary  Model  11 
spectrophotometer. 


Kinetics  of  bleaching  (at  410) 

0 

-0-02 

-004 

DPNH 

T 

-     L_- 

0        30       60 
(sec) 

Fig.   12.   Kinetics  of  bleaching  by  6-5    x    10^^  m  DPXH.  Conditions  as 
Fig.  II. 


Certain  characteristic  features  of  the  absorption  spectrum  are  readily 
recognizable  in  the  initial  soluble  extract,  prior  to  purification  (Fig.  ii). 
The  main  peak  (upper  curve,  oxidized  enzyme),  which  at  pH  7-4  is 
located  at  410  nijn,  is  not  a  Soret  band  but  is  strongly  reminiscent  of  that 


114 


THOMAS    P.    SINGER 


seen  in  a-glycerophosphate  dehydrogenase  [19]  and  the  succinic  dehydro- 
genase of  I\I.  lactilyticiis  [20],  both  of  them  iron-containing  proteins. 
DPNH  and  hydrosulphite  partially  bleach  the  colour.  As  compared  with 
succinic  dehydrogenase   [21],   decolourization  by  the  substrate  is  quite 


400 


550 


450  500 

Wavelength  (m/z) 

Fig.  13.  Difference  spectrum  (oxidized  minus  DPNH-trealed),  replotted 
from  a  tracing  obtained  with  Cary  recording  spectrophotometer.  Negative  values 
denote  bleaching.  Conditions  as  in  Figs.  11  and  12;  soluble  extract. 


E     0-6- 


400  450  500 

Fig.    14.   Shift  of  absorption  spectrum  with  pH. 


rapid  (Fig.  12).  The  difference  spectrum  resulting  from  bleaching  by  the 
substrate  shows  minima  in  the  410  m^a  as  well  as  in  the  flavin  region  in  the 
initial  extract  (Fig.  13).  In  highly  purified  preparations  a  single  broad 
minimum  centring  around  425  m/x  is  observed  and  the  bleaching  is  more 
extensive  than  could  be  ascribed  to  the  flavin  content. 


SOLUBILIZATION    AND    PROPERTIES    OF    THE    DPNH    DEHYDROGENASE 


115 


Before  leaving  the  subject  it  may  be  worth  mentioning  that  the  absolute 
position  and  the  height  of  the  peak  in  the  near  visible  region  are  strongly 
dependent  on  pH  (Fig.  14). 

The  partly  purified  enzyme  (specific  activity  =  130)  was  found  to  be 
relatively  insensitive  to  inhibition  by  /)-chloromercuribenzoate,  completely 
insensitive  to  dicoumarol  (Fig.  3);  it  did  not  catalyze  the  reduction  of 
coenzyme  Q^,  significantly  (with  or  without  added  mitochondrial  lipid 
and  Triton  X-ioo),  and,  under  the  assay  conditions  recommended  by 
Wosilait  [22],  the  rate  of  reduction  of  menadione  at  V^^^  was  less  than  1% 
of  the  rate  of  reduction  of  ferricyanide.  These  observations  clearly  dis- 

Lipoyl   dehydrogenase  assay 


04  0'8  1-2 

I///1.  Iipoamide 

Fig.  15.  Lipoyl  dehydrogenase  assay  of  DPXH  dehydrogenase.  Conditions 
were  as  recommended  by  Massey  [6].  The  abscissa  denotes  the  reciprocal  volumes 
(in  jA.)  of  0-058  M  Iipoamide  present  in  i  ml.  reaction  mixture. 


tinguish  the  enzyme  from  DT  diaphorase  [23,  24]  and  from  menadione 
reductase  [22]. 

Under  the  conditions  of  the  lipoyl  dehydrogenase  assay  employed  by 
Massey  [6]  the  soluble  extract  obtained  on  treatment  of  FTP  with  phos- 
pholipase  A  shows  only  a  trace  of  activity  on  Iipoamide  (Fig.  15):  ratio  of 
activities  on  ferricyanide  and  Iipoamide,  respectively,  differ  bv  a  factor  of 
about  500  between  this  preparation  and  diaphorase  (Table  II).  This  trace 
of  lipoyl  dehydrogenase  activity  may  well  be  due  to  a  slight  contamination 
with  diaphorase  which  would  be  probably  removed  in  the  purification  pro- 
cedure. The  dehydrogenase  may  be  readily  distinguished  from  DPNH 
cytochrome  c  reductase  [2]  by  its  much  greater  stability  and  bv  the 
extremely  low  rate  of  cytochrome  c  reduction  even  when  assayed  under 
optimal  conditions  for  Mahler's  enzyme  (Table  III).  That  the  residual 


Il6  THOMAS    P.    SINGER 

TABLE  II 

Comparison  of  DPNH  Dehydrogenase  and  Diaphorase 

DPNH  Dehydrogenase  Diaphorase 

Reaction  „  Ratio  Ratio 

,        opntS^/     ■     /     1^     i^ma.  Fe  (CN)e       F^,,  Fe  (CN)6 

(/xM  DPNH /mm. /ml.)    — ^p —     — — — rj- 

t^max  Lipoamide     V ^^^vpodxnxas. 

DPNH  +  Fe  (CN)6  +  +  +  150  56  — 

DPNH  +  Lipoamide  27  — ■  01 

Conditions  of  assay:  as  per  IVIassey  (pH  6-5)  [6].  K,„  for  lipoamide  —  2  mM 
for  DPNH  dehydrogenase,  5    niM  for  diaphorase. 

activity  with  cytochrome  c  may  represent  a  trace  contamination  with 
Mahler's  reductase,  rather  than  a  property  of  DPNH  dehydrogenase,  is 
suggested  by  the  fact  that  the  reactivity  with  cytochrome  c  was  inhibited 
by  the  same  substances  at  the  same  concentrations  as  reported  for  DPNH 
cytochrome  reductase  [25,  26]  (Table  IV).  Such  trace  contamination  would 
not  be  surprising  in  view  of  the  fact  that  these  experiments  were  carried 
out  with  the  initial  soluble  extract  prior  to  fractionation. 

TABLE  III 

Cytochrome  c  Reductase  Activity  of  Partially  Purified  DPNH 
Dehydrogenase 


Assay 

Activity 
(//M  DPNH/min./ml.) 

Ratio 
F„,,  Fe  (CN)6 

f^ma.  Cyt.   C 

DPNH  +  Fe  (CN)6  +  +  + 
DPNH  +  Cyt.  c 

0-53 

I4IO 

Conditions  of  assay:  as  per  Alahler  and  Elowe  [25]. 

Comparison  of  the  properties  of  the  enzyme  described  with  those  of 
the  preparation  of  King  and  Howard  [7]  would  be  interesting  but  is 
rendered  difficult  by  the  fact  that  their  detailed  data,  particularly  the 
assay  conditions  employed,  have  not  yet  been  published.  In  view  of  the 
similarities  in  the  extraction  procedure,  it  seems  very  likely  that  the 
enzyme  here  described  is  one  of  the  DPNH  oxidizing  activities  detected 
by  these  workers  on  chromatographing  their  extracts  on  DEAE  cellulose 
[27].  The  presence  of  several  components  capable  of  oxidizing  DPNH 
in  their  preparation  is  not  surprising  in  view  of  the  heterogeneous  con- 
stitution of  the  starting  material  employed  (Keilin-Hartree  preparation), 
which,  in  turn,  might  make  the  deiinite  identification  of  the  individual 


SOLUBILIZATION    AND    PROPERTIES    OF    THE    DPNH    DEHYDROGENASE       llj 

TABLE  IV 

Effect  of  Known  Inhibitors  of  DPNH-Cytochrome   Reductase  on  Cyto- 
chrome c  Reduction  by  DPXH  Dehydrogenase 

Inhibitor  Inhibition  ("„) 

PO4,  o-oi  M  78 

Pyrophosphate,  o-oi  M  85 

Ca^  ^,  001  M  78 

Mg~  ^,  001  M  76 

Conditions:  as  per  Mahler  and  Elowe  [25];  assay  at  pH  8-5. 

flavoproteins  after  solubilization  rather  difficult.  In  the  present  work  this 
difficulty  was  circumvented  by  the  expedient  of  using  a  starting  material 
known  to  contain  only  one  DPXH  oxidizing  activity,  the  flavoprotein 
attached  to  the  respiratory  chain. 

Assuming  that  the  assay  conditions  are  not  too  dissimilar,  the  best 
preparation  hitherto  obtained  in  the  Detroit  laboratory  is  some  eighty  to 
ninety  times  more  active  than  the  purified  enzyme  described  by  King  and 
Howard  [7].  The  major  differences  between  their  preparation  and  ours 
are  that  while  theirs  is  very  unstable,  ours  is  quite  stable ;  their  preparation 
precipitates  at  a  considerably  higher  range  of  (NH4)2S04  concentrations 
than  does  ours  [7] ;  and,  finally,  that  while  our  enzyme  is  so  thoroughly 
adsorbed  on  DEAE  cellulose  as  to  render  fractionation  on  this  exchanger 
quite  unfeasible,  theirs  is  readily  eluted  from  DEAE  cellulose  [28].  These 
differences  seem  too  great  to  be  readily  accounted  for  by  the  different 
degrees  of  purification  of  the  two  preparations  and,  hence,  at  this  time  it 
is  uncertain  whether  the  two  laboratories  are  indeed  investigating  the 
same  enzyme. 

References 

1.  Stravib,  F.  B.,  Biochem.J.  33,  787  (1939). 

2.  Mahler,  H.  R.,  Sarkar,  N.  K.,  and  Vernon,  L.  P.,  and  Alberty,  R.  A.J.  binl. 
Cheni.  199,  585  (1952). 

3.  Ziegler,  D.  M.,  Green,  D.  E.,  and  Doeg,  K.    A.,  J.   bio!.   Chem.    234,    1916 

(1959)- 

4.  Massey,  \.,  Biochini.  biopJiys.  Acta  37,  310  (i960). 

5.  Massey,  V.,  Biochim.  biophys.  Acta  38,  447  (i960). 

6.  Massey,  V.,  Biochim.  biophys.  Acta  37,  314  (i960). 

7.  King,  T.  E.,  and  Howard,  R.  L.,  Biochini.  biophys.  Acta  37,  557  (i960). 

8.  Singer,  T.  P.,  Kearney,  E.  B.,  and  Bernath,  P.,  J.  biol.  Chcni.  223,  599  (1956). 

9.  Ringler  R.  L.,  and  Singer,  T.  P.,  Biochim.  biophys.  Acta  29,  661  (1958). 

10.  Ringler,  R.  L.,  Minakami,  S.,  and  Singer,  T.  P.,  Biochem.  biophys.  Res.  Comm. 
3,  417  (i960). 

11.  Crane  F.  L.,  Glenn,  J.  L.,  and  Green,  D.  E.,  Biochim.  biophys.  .4cta  22,  475 
(1956). 


Il8  THOMAS    P.    SINGER 

12.  Minakami,  S.,  Ringler,  R.  L.,  and  Singer,  T.  P.,  Biochem.  biophys.  Res.  Comm. 
3>  333  (i960). 

13.  Estabrook,  R.  W.,  Fed.  Proc.  16,  178  (1957). 

14.  Giuditta,  A.,  and  Singer,  T.  P.,X  biol.  Chem.  234,  662,  666  (1959). 

15.  Lusty,  C.  J.,  and  Singer,  T.  P.,  to  be  published. 

16.  Chance,  B.,  in  "Enzymes:  Units  of  Biological  Structure  and  Function",  ed. 
O.  H.  Gaebler.  Academic  Press,  New  York,  447  (1956). 

17.  Packer,  L.,  Estabrook,  R.  W.,  Singer,  T.  P.,  and  Kimura,  T.,  J.  biol.  Chem. 

235.  535  (1960)- 

18.  Rendina,  G.,  and  Singer,  T.  V.,y.  biol.  Chem.  234,  1605  (1959). 

19.  Ringler,  R.  L.,_7.  biol.  Chem.  236,  1192  (1961). 

20.  Warringa  M.  G.  P.  J.,  and  Giuditta,  A.,  J.  biol.  Cheyn.  230,  iii  (1958). 

21.  Kearney,  E.  B.,_7.  biol.  Chem.  229,  363  (1957). 

22.  Wosilait,  W.  D.,7-  biol.  Chem.  235,  1196  (i960). 

23.  Ernster,  L.,  Ljunggren,  M.,  and  Danielson,  L.,  Biochem.  biophys.  Res.  Comm. 
2,  88  (i960). 

24.  Marki,  F.,  and  Martius,  C,  Biochem.  Z.  333,  iii  (i960). 

25.  Mahler,  H.  R.,  and  Elowe,  D.  G.,  J.  biol.  Cheyn.  210,  165  (1954). 

26.  Vernon,  L.  P.,  Mahler,  H.  R.,  and  Sarkar,  N.  F.,  J.  biol.  Chem.  199,  599  (1952). 

27.  King,  T.  E.,  and  Howard,  R.  L.,  Results  presented  at  the  44th  Annual  Meet- 
ing of  the  American  Society  of  Biological  Chemists,  Chicago,  April,  i960. 

28.  Personal  Communication  from  Dr.  T.  E.  King. 


Discussion 

Ernster  :  Does  your  enzyme  react  with  quinones  as  electron  acceptors  ? 

Singer:  We  haven't  had  a  chance  to  test  it  yet. 

Ernster  :  I  should  like  to  recall  an  interesting  observation  which  Dr.  Conover 
and  I  made  some  time  ago  on  a  non-purified  preparation  of  DPNH  oxidase;  we 
found  that  with  vitamin  K3  as  terminal  electron  acceptor  we  obtained  an  appreciable 
amytal-sensitivity,  whereas  with  i  ,4-naphthoquinone  the  am^tal-sensitivity  was  only 
marginal  (cf.  Ernster,  this  volume.  Table  7,  page  150). 

Note  added  in  proof.  The  homogeneous  enzyme  contains  about  16  atoms  of  non- 
haem  iron  and  i  mole  FAD  per  10"  g.  protein.  The  pH  optimum  range,  as  deter- 
mined by  a  transhydrogenase  assay,  is  pH  8  to  9.  In  this  range  the  turnover  number 
per  mole  of  flavin  is  i  •  3  million  per  minute  at  30  . 


Reversal  of  Electron  Transfer  in  the  Respiratory  Chain* 

Brittox  Chanxe 

The  Eldridge  Reeves  Johnson  Foundation  for  Medical  Physics,  University  of 
Pennsylvania,  Philadelphia,  Pa.,  U.S.A. 

I.  Energy-linked  DPN  reduction 

GENERAL    FEATURES    OF    THE    REACTION 

It  was  obsened  some  time  ago  in  collaboration  with  Dr.  G.  R.  Williams 
[i]  that  addition  of  succinate  to  mitochondria  oxidizing  a  DPNH-linked 
substrate  caused  a  significant  increase  of  pyridine-nucleotide  reduction. 
This  phenomenon  was  especially  clear  in  guinea-pig-kidney  and  rat-heart 
mitochondria  studied  with  Dr.  G.  R.  Hollunger  [2, 3]  and  was  most  recently 

DPNH 


Succinate 

"Switch"'  hypothesis 

Fig.  I.  Diagram  illustrating  how  competition  between  DPNH  and  succinate 
for  oxidizing  equivalents  from  cytochrome  chain  could  lead  to  increased  pyridine- 
nucleotide  reduction  ;  the  number  of  arrowheads  indicates  the  proportion  of  electron 
transfer  which  might  flow  in  the  chain  and  its  two  branches.  (MD  102). 

obseryed  in  pigeon-heart  mitochondria  with  Dr.  B.  Hagihara  [4].  The 
result  has  been  confirmed  by  chemical  assays  in  a  number  of  laboratories 
[5-10]. 

Such  a  phenomenon  might  readily  haye  been  ascribed  to  a  competition 
between  succinate  and  DPNH  for  oxidizing  equiyalents  in  the  cytochrome 
portion  of  the  chain  (Fig.  i).-\-  On  this  basis  one  would  haye  expected  that 

*  This  research  was  supported  in  part  by  the  National  Science  Foundation. 

f  The  "switch"  hypothesis  is  discussed  in  some  detail  by  Birt  and  Bartley  [21] 
although  kinetic  studies  were  not  possible  with  their  analytical  methods.  This 
hypothesis  received  only  a  short  discussion  previously  [3]  where  the  general  case 
of  a  "simple  kinetic  explanation"  based  upon  a  greater  speed  of  pyridine-nucleo- 
tide reduction  by  succinate  appeared  to  be  inadequate.  Here  we  elaborate  our 
views  and  present  additional  evidence. 


I20 


BRIXTON    CHANCE 


succinate  would  deprive  DPNH  of  the  oxidizing  capacity  of  the  chain  and 
lead  to  a  greater  degree  of  pyridine-nucleotide  reduction.  However,  many 
features  of  the  reaction  called  our  attention  to  a  need  for  closer  study.  For 
example,  the  rate  at  which  succinate  produced  increased  reduction  of 
pyridine  nucleotide  appeared  to  be  slow  compared  with  that  at  which 


K 50  sec 


-M 


Spectrophotometric  troce 


"^  340-374m//, 

log  Iq/I  =  0  010 


7mM  succinate 
Aerobic   — >•  . — "^ 
mitochondria  ' 

330/xM 

0-5fxW\   / 
PNH/sec 


Platinum  microelectrode  trace 


Pyridine  nucleotide  red 


uction     I 


Fig.  2.  Illustrating  increased  reduction  of  pyridine  nucleotide  in  a  suspension 
of  rat-liver  mitochondria  caused  by  addition  of  7  mM  succinate.  Absorbancy  changes 
measured  spectrophotometrically  by  double-beam  spectrophotometer  and  respira- 
tion by  vibrating  platinum  microelectrode.  Downward  deflection  upon  addition  of 
reagent  indicates  increased  light  absorption  at  340  m/x  relative  to  374  m/x.  Diagram 
indicates  final  concentrations  of  reagents  added,  respiratory  rate  in  /umoles/l./sec. 
and  increment  of  oxygen  taken  up  during  phosphorylation  of  330  [jlm  ADP. 
Rates  of  pyridine-nucleotide  reduction  also  given  in  /xmoles/l./sec.  The  metabolic 
states  of  mitochondria  are  indicated  by  numerals  1-3-4.  Rat-liver  mitochondria 
diluted  in  isotonic  salt  meciium  to  concentration  of  approximately  2  mg.  protein/ 
ml.  at  pH  7-4,  temperature  25'  (Expt.  332-2). 


succinate  could  intercept  oxidizing  equivalents  from  the  respiratory  chain 
and  was  no  more  rapid  than  the  rate  at  which  pyridine  nucleotide  could  be 
reduced  by  DPNH-linked  substrates.  Another  puzzling  feature  of  the  re- 
action was  that  it  w^as  slowed  by  addition  of  very  low  concentrations  of 
uncoupling  agents  and  completely  inhibited  by  larger  concentrations  [3]. 
This  too  seemed  inconsistent  with  a  simple  competitive  reaction  which 
should  also  occur  at  higher  respiratory  rates  caused  by  addition  of  the  un- 
coupling agents.  Further  doubts  as  to  a  simple  explanation  of  the  reaction 


RE\^RSAL   OF   ELECTRON   TRANSFER    IN   THE   RESPIRATORY    CHAIN  121 

were  afforded  by  the  inhibitory  effects  of  amytal  upon  the  rate  of  pyridine- 
nucleotide  reduction  caused  by  succinate.  Since  amytal  inhibits  DPNH 
oxidation  by  the  mitochondrial  respiratory  chain  [ii,  iia],  one  would 
have  expected  that  the  effectiveness  of  succinate  in  intercepting  oxidizing 
equivalents  would  have  been  even  greater  in  the  presence  of  this  inhibitor. 
On  the  other  hand,  the  observation  of  Klingenberg  et  al.  [5]  that  a-gly- 
cerophosphate  causes  enhanced  pyridine-nucleotide  reduction  in  locust 
flight-muscle  mitochondria  was  not  inconsistent  with  the  hypothesis  of  a 
simple  competitive  reaction. 

Our  doubts  about  the  simplicity  of  this  mechanism  led  us  to  carry 
out  an  extensive  study  of  the  nature  of  succinate-linked  pyridine-nucleo- 
tide reduction  in  a  variety  of  mitochondrial  preparations  under  divers" 
conditions  with  emphasis  on  the  kinetics  of  intramitochondrial  reactions. 

An  example  of  the  pyridine-nucleotide  reduction  by  succinate  in 
rat-liver  mitochondria  was  presented  in  1956  [i]  and  illustrated  the 
important  features  of  the  reaction  (Fig.  2).  These  mitochondria  con- 
taining endogenous  substrate  show,  upon  addition  of  succinate,  increased 
pyridine-nucleotide  reduction  indicated  by  the  trace's  downward  deflection 
corresponding  to  increased  absorption  at  340  m^t  measured  with  respect 
to  374  vcijx.  The  reduction  rate  is  not  rapid  and  a  steady  state  is  obtained 
in  about  i  min.  The  increase  of  respiration  is  not  great,  the  initial  rate  of 
0-56  /xM  Oo/sec.  rising  to  0-9  /xM  Oo/sec.  Characteristic  of  the  reduction 
reaction  is  its  reversal  by  ADP,  illustrated  here  by  the  trace's  abrupt 
upward  deflection  upon  addition  of  that  reactant  and  increased  pyridine- 
nucleotide  reduction  upon  exhaustion  of  the  added  ADP.  In  view  of  the 
possible  obscuration  of  DPNH  reduction  by  the  concomitant  reduction  of 
TPNH  in  liver  mitochondria  [5,  12,  13]  we  have  been  studying  heart  and 
kidney  preparations  in  preference  to  those  of  liver  since  1956  because  of 
their  low  TPNH  content  [13].  The  percentage  increase  of  DPN  reduction 
obtained  on  adding  succinate  to  guinea-pig-kidney  mitochondria  runs 
as  high  as  fourfold,  making  such  material  ideal  for  kinetic  and  stoicheio- 
metric  studies  [2]. 

Figure  3  gives  an  example  of  succinate-linked  pyridine-nucleotide 
reduction  in  guinea-pig-kidney  mitochondria  from  work  with  Dr. 
Hollunger  [2].  Mitochondria  are  pretreated  with  4  mM  glutamate  and 
about  25",,  of  the  total  DPN  is  reduced  in  state  4.  At  this  point  addition  of 
succinate  causes  a  striking  increase  in  pyridine-nucleotide  reduction, 
as  indicated  by  the  large  downward  deflection  of  the  trace.  There  appears 
to  be  a  transient  respiratory  acceleration  upon  succinate  addition  to  the 
glutamate-treated  mitochondria.  Thereafter  net  respiratory  acceleration 
on  succinate  addition  to  the  glutamate-treated  material  is  not  extremely 
large.  Note  that  the  rate  at  which  DPN  is  reduced  is  comparable  (on  a  2- 
electron  basis)  to  the  State  4  respiration  rate  in  the  presence  of  succinate 


122  BRITTON    CHANCE 

(0-28  compared  to  0-30).  As  in  the  case  of  liver  mitochondria,  reduced 
pyridine  nucleotide  shows  a  typical  cycle  of  oxidation  and  reduction  upon 
addition  of  ADP.  But  the  initial  rate  of  DPNH  oxidation  is  small  compared 
with  the  steady-state  rate  of  oxygen  utilization  (o  •  56  compared  with  2  -o  in 
2-electron  equivalents).  Thus  the  significant  features  of  this  reaction  are 
the  relatively  slow  changes  in  pyridine-nucleotide  reduction  states,  which 
lead  nevertheless  to  very  large  magnitudes  of  changes  in  steady  state. 


4mM 
succinote 


State  4 
(qlutamate) 


Spectrophotometric  trace 


ccinate) 


Platinum 
microelectrode  trace  "* 


340-374m^-j- 
log  Io/I  =  0- 


h- 50  sec    H 

Fig.  3.  Illustrating  increase  of  pyridine-nucleotide  reduction  caused  by 
adding  succinate  to  glutamate-treated  guinea-pig-kidney  mitochondria.  Down- 
ward deflection  of  trace  indicates  increased  absorbancy  at  340  m/x  measured  with 
respect  to  374  m/it.  Rates  of  pyridine-nucleotide  reduction  and  oxygen  utilization 
indicated  in  jumoles/l./sec.  Mitochondria  diluted  in  sucrose-phosphate  medium  to 
concentration  of  o-6  mg.  protein/ml.,  pH  7-4,  temperature  2$'-  (Expt.  683-1). 
(Reproduced  with  permission  of  The  Journal  of  Biological  Chemistry.) 

While  the  above  experiments  showed  the  important  role  of  succinate 
in  activating  DPN  reduction  in  mitochondria  they  did  not  clearly  rule  out 
the  possibility  that  addition  of  succinate  increased  the  concentration  of  a 
DPN-linked  substrate,  for  example  malate,  according  to  the  sequence 
of  the  citric  acid  cycle  (Fig.  4).  This  hypothesis  is  largely  ruled  out  in 


Succinate — ^  fumarate — ^  malate 
Fig.  4. 


oxalacetate 


experiments  illustrating  the  abrupt  inhibitory  effect  of  malonate  upon 
succinate-linked  DPN  reduction.  For  instance  in  experiments  such  as 
those  recorded  in  Figs.  2  and  3  addition  of  malonate  causes  an  abrupt 
decrease  of  pyridine-nucleotide  reduction  to  the  level  previously  obtained 
in  the  presence  of  the  DPN-linked  substrate  only.  That  this  inhibition 
occurs  with  no  measurable  induction  period,  as  would  have  been  expected 
for  a  mechanism  which  depended  upon  accumulation  of  DPN-linked 
substrate,  rules  against  the  simple  hypothesis  outlined  in  Fig.  4. 


re\t:rsal  of  electron  transfer  in  the  respiratory  chain  123 

Experiments  of  the  type  indicated  by  Figs.  2  and  3  suggest  an  essential 
role  for  succinate  in  particular,  and  for  a  flavin-linked  substrate  in  general, 
in  reduction  of  a  considerable  portion  of  mitochondrial  pyridine  nucleo- 
tide. However,  such  experiments  do  not  plainly  separate  the  electron- 
transfer  and  energy  requirements  for  the  reaction. 

An  energy  requirement  for  this  reaction  is  apparent  from  two  stand- 
points. First,  there  is  at  least  a  300-mv.  unfavourable  potential  difference 
between  the  succinate-fumarate  and  the  DPN-DPNH  couples  and  second, 
the  sensitivity  of  the  reaction  to  uncoupling  agents  suggests  that  the 
energy  requirement  was  met  by  internal  high-energy  intermediates  of 
oxidative  phosphorylation  (Fig.  5).  In  more  recent  experiments  at  the 
Johnson  Foundation,  preparations  of  pigeon  heart  mitochondria  [14]  have 
been  studied  in  which  succinate  causes  no  appreciable  pyridine-nucleotide 
reduction  in  the  absence  of  ATP  and,  more  important,  ATP  causes  no 
appreciable  pyridine-nucleotide  reduction  in  the  absence  of  succinate. 

Succinate  +  X~I   +  DPN  - — -  fumarate  +  DPNH  +  H*  +  X  +1 
Fig.  5. 

Figures  6  and  7  illustrate  the  properties  of  succinate-linked  pvridine- 
nucleotide  reduction  in  pigeon-heart  mitochondria  where  the  electron- 
donor  and  energy-donor  requirements  are  separable.  In  Fig.  6  pigeon- 
heart  mitochondria  are  suspended  in  an  aerobic  medium  containing  o  •  27  M 
mannitol,  0-03  M  sucrose,  0-02  m  "tris"  buffer,  pH  7-4,  free  of  added 
magnesium  and  phosphate.*  Under  these  conditions  succinate  addition 
causes  no  downward  deflection  of  the  spectrophotometric  trace;  there  is 
no  measurable  pyridine-nucleotide  reduction.  However,  upon  addition  of 
100  /LtM  ATP  there  is  an  abrupt  downward  deflection  of  the  trace,  indicating 
reduction  of  DPN.  This  reaction  continues  for  about  2  min.  In  a  similar 
experiment  under  the  same  conditions  mitochondria  are  pretreated  with 
78  /LiM  ATP  (Fig.  7).  There  is  only  a  very  small  downward  deflection  of  the 
trace  amounting  to  about  4-',,  of  the  total  pyridine  nucleotide.  However, 
upon  addition  of  succinate  there  is  an  initial  rapid  reduction  of  DPN 
which  then  proceeds  at  a  slower  rate. 

These  experimental  results  can  now  be  considered  against  the  back- 
ground of  the  various  hypotheses  that  have  been  considered.  It  is  apparent 
that  the  "switch"  hypothesis  (Fig.  i)  is  inapplicable  to  these  experimental 
conditions  since  succinate  alone  causes  no  measurable  pvridine-nucleotide 
reduction. 

An  hypothesis  suggesting  that  onlv  succinate  is  required  for  pyridine- 
nucleotide  reduction  is  apparently  inconsistent  with  these  data,  as  is  one 

*  Thanks  are  due  to  Mr.  K.  Kaminker  and  Miss  H.  Diefenbach  for  pigeon- 
heart  preparations  and  to  Dr.  U.  Fugmann  for  "digitonin"  preparations. 


124  BRIXTON    CHANCE 

suggesting  that  only  ATP  is  required  for  pyridine-nucleotide  reduction. 
These  considerations  eliminate  hypotheses  which  postulate  that  DPN- 
linked  substrates  are  acting  directly  or  through  an  ATP-activated  step, 
since  succinate  or  ATP  alone  should  cause  the  observed  effects. 

A  remarkable  feature  of  the  reaction  and  one  which  seems  to  differ 
considerably  from  the  results  so  far  obtained  by  Klingenberg  and  Azzone 
(this  meeting)  is  the  low  concentration  of  ATP  required  for  pyridine- 
nucleotide  reduction.  Under  favourable  conditions  it  has  been  observed 


Fluorescence  excitation  365nn/i 
Fluorescence  measurement  450m/i 

lOmM  succinate 
PH.  M    +  ==jf_' 


State  1 


Fig.  6. 

Fig.  6.  Illustrating  separation  of 
electron  and  energy-transfer  require- 
ments for  succinate-linked  pyridine- 
nucleotide  reduction.  Addition  of 
succinate  alone  causes  no  fluores- 
cence change,  while  loo  jum  ATP 
causes  a  large  fluorescence  increase 
corresponding  to  pyridine-nucleo- 
tide reduction.  Pigeon-heart  mito- 
chondria suspended  in  sucrose- 
"  tris  "-mannitol  medium,  pH  7-4, 
temperature  26°  (Expt.  133d). 
(Reproduced  with  permission  of 
The  Journal  of  Biological  Chemistry.) 


Fluorescence  excitation  365m/i 
Fluorescence  measurement  450myu 


TS/jM  ATP 


4mM  succinate 


P.H  Mv»_>: 

(state  1) 


Fig.  7. 

Fig.  7.  Illustrating  separate 
energy  and  electron-transfer  require- 
ments for  succinate-linked  pyridine- 
nucleotide  reduction.  Pigeon-heart 
mitochondria  suspended  in  mannitol- 
sucrose-"tris"  medium,  pH  7 '4, 
temperature  25°.  Downward  deflec- 
tion of  fluorescence  trace  indicates 
increase  of  pyridine-nucleotide  re- 
duction (Expt.  133d).  (Reproduced 
with  permission  of  The  Journal  of 
Biological  Chemistry). 


that  as  little  as  two  ATPs  per  DPNH  are  required  [15].  This  result  is 
obtained  under  relatively  poor  conditions  for  optimal  efficiency  of  the 
reaction  since  the  mitochondria  are  capable  of  breaking  down  ATP  with- 
out expending  its  energy  in  the  reduction  of  pyridine  nucleotide.  The  small 
ATP  requirement  for  DPN  reduction  also  sets  a  value  for  any  other  sub- 
stances that  might  react  with  ATP.  First,  the  conditions  of  the  experi- 
ment are  such  that  about  20  /xM  DPN  is  reduced  on  addition  of  about  60 
/xM  ATP.  Thus  readily  detectable  chemical  changes  are  caused  by  the 
ATP  reaction  which  can  easily  be  confirmed  by  extraction  of  the  mito- 
chondria and  subsequent  analysis. 


REVERSAL    OF    ELECTRON    TRANSFER    IN    THE    RESPIRATORY    CHAIN  1 25 

PATHWAY    OF    SUCCINATE-LINKED    DPN    REDUCTION 

In  view  of  the  evidence  in  favour  of  requirements  for  electron  and 
energy  donors  in  this  reaction,  it  is  important  to  consider  the  pathway  by 
which  electron  and  energy  transfer  might  occur.  With  regard  to  electron 
transfer,  the  central  question  is  the  nature  of  the  actual  electron  donor  to 
DPN.  Two  hypotheses  may  be  considered  (Figs.  8,  9).  In  Fig.  8  the  path- 
way of  electron  transfer  from  succinate  to  DPN  involves  carriers  of  the 


t 
Succ fp — -b fp -X--I+DPN 

Fig.  8. 

respiratorv  chain.  In  fact,  electrons  are  depicted  to  travel  part  of  the  way 
toward  the  oxygen  and  then  to  be  bypassed  into  an  energy-requiring  path- 
way involving  pyridine-nucleotide  reduction.  In  this  portion  of  the  path- 
way, electron  transfer  under  the  influence  of  ATP  would  proceed  in  the 
reverse  of  the  usual  direction  in  oxidative  reactions.  Such  a  pathway  would 
be  expected  to  show  inhibitor  sensitivity  typical  of  this  portion  of  the 
electron  pathway— a  sensitivity  to  amytal  and  possibly  to  antimycin-A. 
The  enzvme  system  involved  in  this  reaction  would  presumably  be  tightly 
bound  to  the  mitochondrial  structure. 

Succ  -^I^  succ^-^^^fum  -  DPNH  -  H* 
Fig.  9. 

Figure  9  shows  the  second  hypothesis  by  which  ATP  activates 
succinate  or  some  immediate  oxidation  product  to  an  energetic  form  with 
suitable  thermodvnamic  properties  for  direct  reduction  of  DPN  by  a 
tvpical  dehydrogenase  reaction.  This  hypothesis  differs  from  that  of  Fig.  4 
only  in  that  a  novel  reaction  product  of  succinate  is  postulated  which  has 
so  far  not  been  identified.  This  reaction  mechanism  would  be  expected 
to  be  insensitive  to  inhibitors  of  electron  transfer  through  the  respiratory 
chain,  such  as  amvtal  and  antimycin-A,  and  presumably  would  be 
isolable  in  soluble  form. 

Figure  10  illustrates  that  the  cycle  of  oxidation  and  reduction  of  suc- 
cinate-linked  pvridine  nucleotide  shown  in  Fig.  3  is  greatly  affected  by 
pretreatment  with  o-8  niM  amytal.  In  Fig.  3  we  see  that  oxidation  pro- 
ceeds immediately  upon  addition  of  ADP  and  reduction  occurs  coincident 


126  BRITTON    CHANCE 

with  the  diminution  of  respiratory  rate  following  phosphorylation  of  ADP. 
In  Fig.  ID,  however,  the  amytal-treated  material  shows  pyridine-nucleo- 
tide oxidation  that  does  not  reach  a  steady  state  for  approximately  i  min. 
In  fact,  most  of  the  ADP  has  been  phosphorylated  before  DPNH  oxi- 
dation is  completed.  Subsequently,  upon  exhaustion  of  ADP,  pyridine- 
nucleotide  reduction  proceeds  for  approximately  i  min.  While  it  would  be 
expected  that  oxidation  of  succinate-linked  DPNH  would  be  sensitive  to 
amytal,  in  view  of  the  general  sensitivity  of  DPNH-linked  oxidations  to 
this  inhibitor,  it  is  surprising  that  the  reduction  is  so  severely  inhibited 
unless  the  reaction  is  proceeding  by  reversed  electron  transfer  through 
the  same  carrier  as  that  through  which  the  oxidation  reaction  is  occurring. 

340-374m/x 


ADP 


log  Iq/I  =  0005 


State  4'  /  007/xM 

(succinote    >  —  /     PN/sec 

+  0-8mM  amytal) 


50sec 


Fig.  10.  Effect  of  amytal  upon  cycle  of  oxidation  and  reduction  of  succinate- 
linked  reduced  pyridine  nucleotide  in  presence  of  o  •  8  mM  amytal.  Experimental 
conditions  identical  to  those  in  Fi^.  3  and  comparison  of  rates  of  reaction  in  both 
cases  is  possible  (Expt.  683-3).  (Reproduced  with  permission  of  The  Journal  of 
Biological  Chemistry.) 

We  interpret  this  experiment  as  identifying  an  amytal-sensitive  reaction 
in  not  only  the  oxidation  but  also  the  reduction  of  pyridine  nucleotide. 
This  result  strongly  supports  the  idea  that  energy-linked  reversal  of 
electron  transfer  through  the  respiratory  carriers  is  involved  in  the 
succinate-linked  reductions  of  DPN  (Figs.  5,  8). 

The  question  of  the  level  to  which  electron  transfer  proceeds  toward 
oxygen  before  it  is  bypassed  into  pyridine-nucleotide  reduction  is  sugges- 
ted by  experiments  in  which  reduction  of  DPN  by  succinate  and  ATP  is 
highly  inhibited  by  hydroxyquinoline-N-oxide  (HOQNO)  or  antymycin- 
A.  At  the  present  time  experimental  data  suggest  that  electron  transfer 
proceeds  to  the  antimycin-A-sensitive  point  (Fig.  8). 


REVERSAL    OF    ELECTRON    TRANSFER    IN    THE    RESPIRATORY    CHAIN  1 27 

Also  of  considerable  interest  is  the  pathway  for  energy  transfer  from 
ATP ;  according  to  the  mechanism  of  Fig.  9  this  would  not  necessarily  be 
through  those  transfer  reactions  involved  in  oxidative  phosphorylation 
and  currently  associated  with  iVTPase  and  x'\TP-''^-P  exchange  reactions. 
Figure  1 1  illustrates  the  pathway  of  energy  transfer  following  reversal  of 
oxidative  phosphorylation.  It  is  apparent  that  such  a  sequence  of  reactions 
would  be  sensitive  to  accumulation  of  reaction  products  such  as  ADP. 
This  has  been  demonstrated.  As  mentioned  above,  uncoupling  agents  are 
potent  inhibitors  of  succinate-linked  pyridine-nucleotide  reduction  since 
they  hydrolyze  the  high-energy  intermediates  X~I.  Magnesium  also 
inhibits,  presumably  by  activating  the  breakdown  of  one  of  the  energy- 
rich  intermediates.  Further  experiments  also  show  that  the  reaction  is 
largely  inhibited  by  low  concentrations  of  oligomycin.  On  the  basis  of 
such  data,  it  is  apparent  that  the  pathw^ay  of  energy  transfer  is  essentially 
a  reversal  of  oxidative  phosphorylation.  This  consideration  casts  further 

ATP  +  X  ^—  X~P  +  ADP 
X~P  +  I^—  X~I  +  P, 

X~I   +  rfp  +  DPN^--fp  +  DPNH  +  H*  +  X  +  I 
Fig.  II. 

doubt  upon  the  feasibility  of  the  mechanism  of  Fig.  9,  which  implies  that 
activation  of  succinate  might  follow  a  pathw^ay  other  than  that  employed 
in  oxidative  phosphorylation. 

In  summary  we  can  put  forth  many  experimental  data  indicating  that 
the  pathways  of  electron  and  energy  transfer  in  succinate-linked  pyridine- 
nucleotide  reduction  are  similar  or  identical  to  those  of  oxidative  phos- 
phorylation, the  only  difference  being  that  a  reversal  of  the  process  of 
oxidative  phosphorylation,  in  both  the  energy  and  electron-transfer  steps, 
has  been  revealed  by  these  experiments. 

A  schematic  diagram  of  the  assembly  of  electron  and  energy-transfer 
reactions  by  which  this  reaction  may  be  possible  is  indicated  in  Fig.  12. 
The  important  feature  of  this  mechanism  is  that  it  does  not  require  that  all 
DPNH  reduction  and  oxidation  proceed  through  the  succinate-linked 
pathway,  but  allows  this  to  be  a  side  pathway  which  may  involve  a  small 
fraction  of  the  total  electron  transfer,  as  is  consistent  with  available  kinetic 
data.  In  addition,  an  attempt  has  been  made  to  indicate  the  possible 
participation  of  quinone  in  electron  transfer  between  flavin  and  cyto- 
chrome b  in  the  forward  or  reverse  directions.  Since  the  function  of 
quinone  has  not  been  conclusi\ely  proved  in  either  one  of  these  path- 
ways, the  mechanism  is  arranged  so  that  by-passes  around  the  quinone  are 
feasible.  A  third  feature  of  this  scheme  is  a  mechanism  by  which  a  larger 
amount  of  pyridine  nucleotide  can  be  reduced  in  the  energy-linked  path- 
way than  in  the  usual  dehydrogenase-linked  pathway  of  DPN  reduction. 


128 


BRITTON   CHANCE 


The  succinate-linked  mitochondrial  pyridine  nucleotide  is  postulated  to 
be  separated  from  the  remainder  of  the  DPN  by  a  compartment — possibly 
the  cristae  and  matrices  of  the  mitochondria  are  involved.  This  com- 
partmentation  implies  that  electrons  donated  by  a  DPN-linked  substrate 
cannot  readily  enter  the  succinate-linked  pyridine-nucleotide  pool  while 
those  from  succinate  can. 


Succinate  -^ 


Fumarate 


Malate  -^ 


3X~P+3I 
3ATP+  3X 


dbc 


Fig.  12.  Schematic  diagram  of  electron-transfer  pathways  in  respiratory  chain 
involving  succinate-linked  pyridine-nucleotide  reduction.  This  diagram  is  similar 
to  those  presented  earlier  [i,  i8,  19]  and  includes  the  quinone  component  [20]. 
(Reproduced  with  permission  of  the  Jotirnal  of  Biological  Chemistry.) 

2.  Energy-linked  cytochrome  oxidation 

Since  the  preceding  considerations  demonstrate  reverse  electron  trans- 
fer in  a  branch  of  the  respiratory  chain,  we  have  actively  considered  the 
possibility  that  flavoprotein  may  be  oxidized  in  DPN  reduction  as  indi- 
cated in  Fig.  II,  provided  experimental  conditions  could  be  arranged  so 
that  pyridine  nucleotide  was  oxidized  and  flavoprotein  reduced.  A  suitable 
condition  for  this  can  be  obtained  by  antimycin-A  or  quinoline  oxide 
inhibition  of  the  respiratory  chain,  reinforced  by  hydrosulphide  inhibition 
of  the  oxidase.  The  plan  for  such  an  experiment  is  indicated  by  Fig.  8.  If 
electrons  have  already  been  transferred  up  to  the  level  of  cytochrome  h 
and  flavoprotein,  so  that  the  flavoprotein  involved  in  DPN  reduction  is 
already  reduced,  then  indeed  addition  of  ATP  should  be  all  that  is  needed 
to  cause  pyridine-nucleotide  reduction  with  a  concomitant  oxidation  of 
flavoprotein.  It  has  been  observed  in  pigeon-heart  mitochondria  that 
treatment  of  the  aerobic  suspension  with  4  niM  succinate  and  sufficient 


REVERSAL    OF    ELECTRON    TRANSFER    IX    THE    RESPIRATORY    CHAIN 


129 


hydrosulphide  or  cvanide  to  block  respiration  causes  no  measurable 
pyridine-nucleotide  reduction,  as  observed  fluorometrically  with  365  m^Li 
excitation  and  450  m^t  measurement  [16].  Under  such  conditions  reduction 
of  flavin,  quinone  and  cvtochrome  components  can  be  observed  spectro- 
photometricallv.  Thus  the  respiratory  chain  is  in  a  condition  where  the 
etTect  of  ATP  on  the  sequence  of  reactions  depicted  by  Fig.  8  could  be 
observed  readily.   Figure   13  shows  that  flavoprotein  is  oxidized  at  the 


Aerobic   P.H.  M 


w 


4mM  succinate 

+ 

360 mM  Na2S 

+ 

2y/mt.  HO  Q  NO 


36/^M   ATP 


Flavoprotein  oxidation    I 
465-5l0mu,  * 

_L 
log  Iq/I  =  0005 


0-6mM 
DPNH/sec 


50sec 


Pyridine  nucleotide  reduction    I 
365—  450myu  -»- 

l-l/^M   ,   DPNH 


Fig.  13.  Flavoprotein  oxidation  and  pyridine-nucleotide  reduction  caused  by 
ATP  addition  to  pigeon-heart  mitochondria  inhibited  with  hydroxyquinoline-N- 
oxide  and  sodium  svilphide.  Mitochondria  suspended  in  mannitol-sucrose- 
"tris"  medium,  pH  7-4,  temperature  26  ,  protein  concentration  1-2  mg./ml. 
(Expt.  173).  (Reproduced  with  permission  of  Xature.) 


same  time  that  pvridine  nucleotide  is  reduced  upon  addition  of  ATP  to 
the  HOQXO-  and  XaoS-treated  mitochondria.  This  result  aflFords  strong 
support  for  the  reaction  of  Figs.  8  and  11.  It  is  also  of  interest  that  under 
these  conditions  the  reaction  of  ATP  with  the  rfp-DPN  couple  is  so 
rapid  that  succinate  cannot  maintain  flavoprotein  reduced  against  the 
oxidizing  etfect  of  added  ATP.  However,  when  the  DPN  has  been  reduced, 
the  oxidized  flavoprotein  is  reduced  by  succinate  toward  its  initial  level. 

An  experiment  of  this  tvpe  suggests  the  possibility  of  generalized 
reversal  of  electron  transfer  between  all  couples  of  the  respiratory  chain 
involved  in  oxidative  phosphorylation.  It  is  apparent  that  if  reduced 
flavoprotein  can  be  oxidized  bv  ATP  a  similar  effect  should  be  observed 
at  the  level  of  the  cytochromes  under  appropriate  conditions. 

\\'e  therefore  repeated  an  experiment  similar  to  that  of  Fig.  13  except 


VOL.   II. K 


130  BRITTON    CHANCE 

that  we  observed  at  wavelengths  appropriate  to  cytochrome  c*  and 
omitted  the  quinohne  oxide.  Thus  Fig.  14  represents  an  experiment  in 
which  pigeon-heart  mitochondria  are  pretreated  with  4  niM  succinate  and 
sufficient  hydrosulphide  to  block  respiration.  Under  these  conditions 
cytochrome  c  is  completely  reduced  in  about  i  min.  As  explained  above, 
pyridine  nucleotide  is  not  reduced.  Upon  addition  of  530  /^M  ATP, 
pyridine-nucleotide  reduction  in  agreement  with  Fig.  13  is  observed.  At 
the  same  time  we  recorded  an  abrupt  upward  deflection  of  the  spectro- 
photometric  trace  indicating  an  oxidation  of  cytochrome  c  to  an  extent  of 
71%  of  the  total.  While  pyridine-nucleotide  reduction  proceeds  exponen- 
tially toward  a  highly  reduced  level,  the  response  of  cytochrome  c  to  ATP 

Cytochrome-c  oxidation  t 
550-540mAt 


l-2//.M/sec 

Aerobic  P.  H.  M 

+ 
4mM  succinate 
360mM    N-agS 


t  I 

530 /iM  ATP 


50  sec 


log  Iq/I  -0005 


Pyridine 
nucleotide  reduction  , 
365-450        _L  ^ 

f^M  3-6mM    DPNH 


Fig.  14.  Cytochrome  c  oxidation  and  pyridine-nucleotide  reduction  in 
succinate-  and  sulphide-treated  mitochondria  (concentrations  indicated  on  figure). 
Pigeon-heart  mitochondria  suspended  in  mannitol-sucrose-"  tris "  medium,  pH 
7-4,   temperature   26,   protein   concentration    i-i    mg/ml.    (Expt.    i85b-3). 


addition  is  cyclic  and  reduction  toward  the  initial  level  proceeds  rapidly. 
Other  experiments  show  cytochrome  a  to  be  oxidized  as  well,  cyto- 
chrome b  showing  little  initial  change  and  then  a  slow  reduction*  which 
is  complete  at  about  the  same  time  as  that  of  pyridine  nucleotide.  Studies 

*  Lundegardh  [22]  has  reported  an  effect  of  ATP  (and  fumarate)  upon  the 
interaction  of  cytochromes  b  and  ''dJi"  in  anaerobic  wheat  roots,  cytochrome 
"rf/i"  being  "turned  over  into  a  state  of  strong  reduction  under  the  influence  of 
ATP  (and  fumarate)  simultaneously  with  cytochrome  b  remaining  more  oxidized". 
The  interpretation  of  this  interesting  result  is  rendered  ambiguous  by  the  fact 
that  the  existence  of  cytochrome  " dh"  in  wheat  roots  and  other  plant  or  animal 
tissues  has  not  been  confirmed  by  other  workers  [23]  nor  by  us  using  liquid  nitrogen 
spectroscopy  of  wheat-root  mitochondria  (B.  Chance  and  W.  Bonner,  Jr.,  un- 
published observations). 


REVERSAL    OF    ELECTRON    TRANSFER    IN    THE    RESPIR.ATORY    CHAIN  I31 

of  quinone  under  similar  experimental  conditions  are  difficult  because  of 
the  absorbancy  change  caused  by  addition  of  ATP  at  275  m^a.  However, 
preliminary  studies  show  quinone  to  be  oxidized  and  it  may  be  an  impor- 
tant component  of  the  couple  involved  in  DPN  reduction.  Such  changes 
are  consistent  with  the  idea  that  AIT  is  entering  the  respiratory  chain  in 
the  flavoprotein-pyridine  nucleotide  region  as  well  as  the  cytochrome 
region.  Similar  etfects  ha\  e  been  demonstrated  in  the  presence  of  various 
respiratory  inhibitors,  for  example  cyanide,  and  even  in  the  presence  of 
dithionite.  It  is  found  that  dithionite  does  not  readilv  penetrate  the  mito- 
chondrial membrane  and  thus  mitochondrial  pyridine  nucleotide  is  not 
initially  reduced,  permitting  time  for  studies  similar  to  those  indicated  in 
Fig.  14.  ^'arious  types  of  mitochondria  show  this  reaction,  for  instance  rat- 
liver  mitochondria  and  "  digitonin  "  particles  have  been  tested. 

The  specificity  of  the  reaction  for  various  nucleotides  has  been  investi- 
gated and  found  to  be  highly  specific  for  ATP ;  GTP,  UTP,  CTP,  and  ITP 
show  no  measurable  reduction  of  pyridine  nucleotide  or  oxidation  of 
cytochrome  c  under  conditions  similar  to  those  of  Fig.  14.  These  data 
support  those  already  indicating  that  ATP  is  interacting  with  the  res- 
piratory chain  through  the  pathway  by  which  oxidative  phosphorylation 
occurs. 

Discussion 

As  illustrated  by  Fig.  14  the  interaction  of  ATP  with  the  cytochromes 
appears  to  be  rapid,  but  is  much  slower  than  the  rate  of  ferrocvtochrome 
c  oxidation  obtained  by  the  rapid  flow  apparatus.  The  reaction  is,  however, 
quite  sensiti\e  to  inhibitors  of  the  pathway  illustrated  by  Fig.  11  and 
it  has  been  found  that  ADP,  oligomycin,  and  phosphate  inhibit  the  oxi- 
dation of  cytochrome  c  as  well  as  the  reduction  of  DPX.  Thus  the  pathway 
by  which  ATP  enters  the  respiratory  chain  is  identified  with  the  pathway 
of  oxidative  phosphorylation  by  its  inhibitor  sensiti\itv  and  nucleotide 
specificity.  This  pathway,  which  has  been  identified  with  ATPase  and 
ATP-'^-P  exchange  activities,  is  acting  under  these  conditions  to  transfer 
energy  from  ATP  into  oxidation-reduction  couples  of  the  respiratory  chain 
— an  ATP-electron  transferase  acti\ity.  That  the  activity  of  this  enzyme 
system  can  be  measured  in  the  intact  mitochondria  without  acti\ating 
hydrolysis  of  one  of  the  intermediates  in  the  sequence  of  F'ig.  1 1  presents 
tremendous  advantages  for  two  kinds  of  experiments :  (i)  to  determine  the 
maximal  activity  of  the  ATP-electron  transferase  pathway,  and  (2)  to 
evaluate  the  efiFectiveness  of  reconstituted  phosphorylation  systems  such  as 
those  of  Polls  [17],  Pullman,  and  Lehninger  (this  svmposium). 

The  efficiency  with  which  ATP  can  convert  its  energy  into  electron 
transfer  is  of  considerable  theoretical  and  practical  interest,  particularly  in 
connection  with  theories  of  active  transport  and  photosynthesis.  We  have 


132  BRITTON   CHANCE 

therefore  titrated  the  oxidation  of  cytochrome  c  with  ATP  and  obtained  a 
sigmoid  titration  curve,  presumably  due  to  preferential  expenditure  of 
ATP  at  other  couples  at  low  concentrations  of  ATP.  However,  the 
maximum  slope  of  the  graph  corresponds  to  roughly  4  ATPs  per  electron, 
over  twice  the  observed  value  of  i  •  5  ATPs  per  electron  produced  in  oxida- 
tive phosphorylation.  Our  experimental  value  is  surely  not  a  minimum 
value  since  some  ATP  is  lost  in  the  simultaneous  oxidation  of  other 
carriers  in  addition  to  cytochrome  c  and  in  hydrolysis  of  intermediates 
formed  from  ATP.  Thus  the  efficiency  may  under  appropriate  conditions 
approach  the  higher  values.  It  is  unlikely  that  the  ATP/e  value  for  the 
reversal  of  electron  transfer  would  reach  the  experimentally  observed  value 
for  oxidative  phosphorylation  in  the  forward  direction  since  the  efficiency 
of  the  latter  process  is  probably  less  than  100%.  In  fact  an  estimate  of  the 
efficiency  of  oxidative  phosphorylation  can  be  obtained  by  the  ratio  of  the 
two  values  and  on  the  basis  of  these  preliminary  data  a  value  of  over  50% 
is  obtained. 

In  addition  to  considerable  interest  in  the  stoicheiometric  properties 
of  the  ATP-electron  transfer  interaction,  the  thermodynamic  properties 
are  of  importance  and  preliminary  titrations  of  the  extent  of  oxidation  of 
cytochrome  c  in  "phosphate-potential  buffers"  (ATP/ADP.Pj)  have 
been  made.  We  are  for  the  first  time  able  to  study  the  oxidation  levels  of 
cytochromes  in  the  presence  of  ATP  under  conditions  where  electron 
flow  through  the  respiratory  chain  is  sufficiently  small  to  be  negligible. 
Furthermore,  the  rate  of  ATP  breakdown  due  to  hydrolysis  can  be  so  small 
that  the  initial  concentration  of  ATP  is  practically  constant  during  the 
measurement  of  cytochrome  concentration.  Thus  the  system  can  be 
sufficiently  near  equilibrium  to  consider  the  relationship  between  phos- 
phate potential  and  cytochrome  oxidation.  Experiments  similar  to  those 
of  Fig.  14  but  in  the  presence  of  varying  concentrations  of  ATP,  ADP  and 
Pj  show  that  the  oxidation  of  cytochrome  c  is  very  sensitive  to  small 
amounts  of  phosphate  and  ADP  and  a  considerable  inhibition  of  the  extent 
of  oxidation  can  be  obtained.  Actually  it  is  difficult  to  obtain  50%  oxida- 
tion of  cytochrome  c  under  conditions  where  the  ADP  and  phosphate 
concentrations  are  sufficiently  high  to  insure  that  equilibrium  and  not 
stoicheiometric  factors  are  determining. 

Preliminary  estimates  suggest  that  the  phosphate  potential  necessary 
to  cause  cytochrome  oxidation  does  not  correspond  to  the  complete  free- 
energy  change  from  DPNH  to  oxygen  but  instead  to  a  value  that  would 
be  expected  for  a  single  redox  couple  involved  in  oxidative  phosphorylation. 
This  possibility  is  supported  by  titration  of  the  respiratory  chain  in 
the  absence  of  added  succinate  where  the  [ATP /ADP]/  [PJ  ratio  is  about 
iC*  corresponding  only  to  about  15  kcal.  Since  spectrophotometric  observ- 
ations of  pyridine-nucleotide  reduction  and  the  oxidation  of  cytochrome  c 


REVERSAL    OF    ELECTRON    TRANSFER    IN    THE    RESPIRATORY    CHAIN  1 33 

were  made  under  these  experimental  conditions,  we  have  no  alternative 
but  to  conclude  that  all  the  components  of  the  respiratory  chain  itself 
were  not  in  equilibrium  as  an  electron-transfer  system,  but  the  couples 
of  the  respiratory  chain  were  interacting  individually  with  the  ATP- 
electron  transfer  system,  presumably  because  under  these  particular 
experimental  conditions  the  latter  reaction  is  much  more  rapid  than  that 
of  electron  transfer. 

Such  an  experimental  condition  calls  our  attention  again  to  the  intense 
inhibition  of  electron  transfer  through  the  chain  attributed  to  hypothetical 
"  I  "  compounds.  It  follows  from  Figs.  1 1  and  12  that  a  high  concentration 
of  ATP  would  lead  to  a  concentration  of  the  "I"  compounds  sufficient 
to  bind  the  carriers  tightly  in  their  inhibited  form  [5].  Thus  the  products 
of  the  reaction  of  the  ATP-electron  transfer  activity  are  concluded  to  be 
the  inhibited  and  not  the  free  forms  of  the  carriers,  to  explain  the  low 
values  of  "  phosphate  potential  "  which  can  cause  half-maximal  cytochrome 
oxidation.  However,  this  hypothesis  must  be  considered  a  tentative  one 
because  of  our  limited  experience  with  this  new  phenomenon. 

At  the  present  time  investigations  are  under  wav  to  locate  "crossover 
points"  for  the  ATP-electron  transfer  activity  and  experiments  such  as 
those  of  Fig.  13  suggest  an  interaction  site  between  DPNH  and  flavin. 
The  response  of  cytochrome  h  suggests  that  crossover  points  mav  be  found 
on  either  side  of  this  component. 

Summary 

These  experiments  ha\e  attempted  to  elucidate  two  pathways  by 
which  ATP  may  be  used  in  activating  the  reversal  of  electron  transfer 
through  components  of  the  respiratory  chain.  The  first  pathway  investi- 
gated is  a  succinate-acti\  ated  branch  of  the  respiratory  chain  which  leads 
to  reduction  of  the  majority  of  mitochondrial  pyridine  nucleotide,  pro- 
\iding  that  an  energy  source  such  as  ATP  is  available.  The  specificity  of 
flavin-linked  substrates  such  as  succinate  has  been  studied  as  has  the 
pathway  of  electron  transfer  through  respiratory  carriers.  Similarly,  the 
pathway  of  energy  transfer  from  ATP  to  DPNH  has  been  shown  to  involve 
the  transfer  system  employed  in  oxidative  phosphorylation. 

Of  more  general  concern  is  the  observation  that  ATP  can  cause  oxida- 
tion of  reduced  cytochromes  in  a  magnesium-,  phosphate-,  and  ADP-free 
system,  and  in  a  respiratory  chain  blocked  at  the  oxygen  end  by  a  suitable 
inhibitor  or  by  the  lack  of  oxygen.  This  reaction  may  be  observed  in  spite 
of  the  presence  of  a  reducing  substrate  such  as  succinate.  Three  general 
points  that  must  be  borne  in  mind  in  carrying  out  this  experiment:  (i) 
that  the  ATP-electron  transfer  activity  be  maximal  because  of  the  absence 
of  {a)  reaction  products  such  as  ADP  and  phosphate;  {b)  reagents  hydroly- 
zing  high-energy  intermediates  such  as  magnesium  or  uncoupling  agents ; 


134  BRITTON    CHANCE 

(2)  that  the  cytochrome  should  be  in  a  reduced  state  blocked  from  the 
oxidizing  power  of  molecular  oxygen  either  by  anaerobiosis  or  by  a  suitable 
inhibitor  of  cytochrome  oxidase;  (3)  that  the  respiratory  chain  must 
initially  contain  both  oxidized  and  reduced  components,  since  for  every 
oxidation  there  must  be  a  reduction.  Succinate-linked  pyridine  nucleotide 
can  be  used  as  the  acceptor  of  the  reducing  equivalents  from  cytochromes 
from  other  parts  of  the  chain.  Under  these  conditions  the  efficiency  of  the 
ATP-electron  transfer  reaction  is  high,  the  ATP/f  value  being  3  or  less. 
Thermodynamically  the  phosphate  potential  required  for  cytochrome 
oxidation  suggests  that  ATP  can  interact  with  redox  couples  of  the 
respiratory  chain  as  individuals  without  supplying  a  phosphate  potential 
corresponding  to  the  complete  span  from  pyridine  nucleotide  to  cyto- 
chrome oxidase. 


References 

1.  Chance,  B.,  in  "Enzymes:  Units  of  Biological  Structure  and  Function",  ed. 
O.  H.  Gaebler.  Academic  Press  Inc.,  New  York,  447  (1956). 

2.  Chance,  B.,  and  Hollunger,  G.,  Fed.  Proc.  16,  163  (1957). 

3.  Chance,  B.,  and  Hollunger,  G.,  Nature,  Lond.  185,  666  (i960). 

4.  Chance,  B.,  and  Hagihara,  B.,  Biochem.  biophys.  Res.  Comtn.  3,  6  (i960). 

5.  Biicher,  T.,  and  Klingenberg,  M.,  Angew.  C/ieni.  70,  552  (1958). 

6.  Klingenberg,  M.,  Biochem.  Z.  332,  47  (1959). 

7.  Purvis,  J.  L.,  Abst.,  Anter.  chetn.  Soc.  Boston,  50  (April,  1959). 

8.  Kaufman,   B.  T.,  and  Kaplan,  N.  O.,  Abst.,  Amer.  chern.   Soc.  Boston,  50 
(April,  1959). 

9.  Baessler,  K.  H.,  and  Pressman,  B.  C,  Fed.  Proc.  18,  194  (1959). 

10.  Slater,  E.  C,  this  volume,  p.  207. 

11.  Ernster,  L.,  Jailing,  O.,  Low,  H.,  and  Lindberg,  O.,  Exp.  Cell  Res.  Siippl.  3, 
124  (1955)- 

1 1  a.    Greig,  M.  "E.,  J.  Pharmacol,  exp.  Therap.  87,  185  (1956). 

12.  Glock,  G.  E.,  and  McLean,  P.,  Biochem.  J.  61,  381  (1955). 

13.  Jacobson,  K.  B.,  and  Kaplan,  N.  0.,J.  biol.  Chem.  226,  603  (1957). 

14.  Chance,  B.,  and  Hagihara,  B.,  Biochem.  biophys.  Res.  Comm.  3,  i  (i960). 

15.  Chance,  B.,  Biochem.  biophys.  Res.  Coftim.  3,  10  (i960). 

16.  Chance,  B.,  and  Baltscheffsky,  H.,jf.  biol.  Chem.  233,  736,  (1958). 

17.  Polis,  D.  B.,  and  Schmukler,  H.  W.,  Abst.,  Amer.  chem.  Soc.  128th  Meeting, 
19c  (September,  1955). 

18.  Chance,  B.,^.  biol.  Chem.  234,  1563  (1959). 

19.  Chance,  B.,  in  "International  Symposium  on  Enzyme  Chemistry"  Maruzen 
Co.,  Ltd.,  Tokyo,  295  (1958). 

20.  Chance,  B.,  ///  "  Ciba  Foundation  Symposium  on  Quinones  in  Electron 
Transfer",  ed.  G.  E.  W.  Wolstenholme  and  C.  M.  O'Connor.  J.  and  A. 
Churchill,  Ltd.,  London,  327  (1961). 

21.  Birt,  L.  M.,  and  Bartley,  W.,  BiocJiem.J.  76,  427  (i960). 

22.  Lundegardh,  H.,  Physiol.  Plant.  8,  157  (1955). 

23.  Hartree,  Fl  F.,  Advanc.  Enzytnol.  18,  22  (1957). 


REVERSAL    OF   ELECTRON   TRANSFER    IN    THE    RESPIRATORY    CHAIN  1 35 

Discussion 

Arnon  :  Is  it  a  fair  inference  from  your  talk  that  the  mechanism  of  this  reaction 
(the  reduction  of  DPX  by  succinate  with  the  aid  of  ATP)  offers  a  way  to  study  the 
niechanism  of  electron  transfer  but  that  the  reaction  as  such  is  of  no  physiological 
significance  ?  Do  you  ascribe  any  physiological  significance  to  this  type  of  reaction 
at  the  cellular  level  ? 

Chance  :  I  suppose  that  photosynthesis  may  be  a  physiological  event,  probably 
the  one  to  which  you  were  referring,  and  might  be  of  some  importance  here,  and 
have  some  comments  to  make  on  that  tomorrow,  particularly  on  the  possibility 
that  light-induced  cytochrome  responses  observed  in  anaerobic  photosynthetic 
bacteria  may  be  due  to  photo-produced  ATP.  Professor  Lindberg  referred  this 
morning  to  the  reversal  of  electron  transfer  into  pyridine  nucleotide  that  has  a 
probable  physiological  implication.  Active  transport  by  reversed  electron  transfer 
has  been  considered  for  nearly  a  decade. 

Arnon  :  I  should  add  that  I  was  specifically  excluding  photosynthesis  from  my 
question. 

AzzoNE :  What  type  of  mitochondria  did  you  use  in  your  oligomycin  experi- 
ments ? 

Ch.ance:  Pigeon-heart  mitochondria. 

Azzone:  Why  do  you  think  addition  of  oligomycin  inhibits  the  succinate- 
induced  pyridine  nucleotide  reduction  ?  Is  it  not  possible  to  generate  the  energy 
necessary  for  DPN  reduction  merely  by  succinate  oxidation  ?  My  second  question 
is:  have  you  tested  the  effect  of  dinitrophenol  in  your  system  where  you  get  the 
cytochrome  oxidation  after  addition  of  ATP  in  anaerobiosis  ? 

Chance:  The  answer  to  your  first  cjuestion  is  no.  Our  reaction  involves  the 
oligomycin-sensitive  steps  of  Fig.  1 1.  Suitably  prepared  pigeon-heart  mitochondria 
have  an  absolute  requirement  for  ATP  for  succinate-linked  reduction  of  DPX.  The 
answer  to  the  second  question  is  that  we  have  tested  2,4-dinitrophenol  and  find  it 
to  block  the  ATP-activated  reduction  of  DPN. 

Azzone:  As  Dr.  Ernster  will  report  later  oligomycin,  at  least  in  liver  mitochon- 
dria, does  not  inhibit  the  succinate-induced  pyridine  nucleotide  reduction.  This 
in  our  opinion  means  that,  in  the  presence  of  oligomycin,  liver  mitochondria  can 
still  synthesize  high  energy  intermediate(s)  and  that  these  intermediate(s)  can 
provide  the  energy  required  for  DPX  reduction.  Thus  in  this  system  there  is  no 
requirement  for  externally  added  ATP. 

With  regard  to  the  second  question :  you  know  that  we  have  done  some  experi- 
ments in  collaboration  with  Dr.  Klingenberg  (Xature,  Loud.,  188,  552  (i960))  where 
we  have  found  that  dinitrophenol  does  not  inhibit  the  ATP-induced  pyridine 
nucleotide  reduction. 

Obviously  it  was  more  difficult  to  observe  the  ATP  effect  in  our  system  because 
the  ATP-induced  reduction  was  counter-balanced  by  the  dinitrophenol-induced 
oxidation  of  the  mitochondrial  pyridine  nucleotide.  Thus  your  system  is  perhaps 
more  suited  for  testing  the  effect  of  uncouplers  since  in  anaerobiosis  the  stimulating 
effect  of  dinitrophenol  on  electron  transport  is  abolished. 

Chance:  We  may  use  oligomycin  and  2,4-dinitrophenol  to  block  the  ATP- 


136  BRITTON    CHANCE 

transfer  reactions,  and  to  define  the  path  by  which  energy  is  coupled  to  the  reversal 
of  the  electron  transfer  reactions. 

Singer  :  I  would  like  to  ask  Dr.  Chance  why  he  seems  to  prefer  the  diaphorase 
of  the  ketoglutaric  oxidase  complex  as  being  the  agent  responsible  for  the  reduction 
of  pyridine  nucleotide  rather  than  the  one  of  the  respiratory  chain,  since,  in  the 
first  place,  it  is  commonly  believed  that  there  is  a  spatial  separation  between  these 
enzymes  and,  therefore,  the  mechanism  of  electron  transport  between  these  two 
systems  would  not  be  very  obvious.  In  the  second  place,  the  partial  inhibition  by 
amytal  presents  another  difficulty,  since  the  ketoglutaric  system  is  not  known  to  be 
amytal-sensitive. 

Chance:  I  completely  agree  with  Dr.  Singer,  but  wanted  to  point  out  that 
there  are  flavins  which  had  been  demonstrated  to  reduce  DPN. 

P'renkel  :  Can  you  tell  from  your  difTerence  spectrum  whether  the  DPN  is  free 
or  enzyme-bound  ?  Dr.  Kaplan  has  informed  me  that  the  reduction  of  enzyme- 
bound  DPN  may  require  appreciably  less  free  energy  than  the  reduction  of  free 
DPN. 

Chance  :  That  is  a  very  interesting  observation.  The  intra-mitochondrial  DPNH 
is  bound  but  it  requires  about  two  ATPs  for  each  DPN  reduced  so  considerable 
energy  is  required.  Its  potential  may  indeed  be  higher  than  —300  millivolts  but  its 
surely  not  zero. 

Arnon  :  I  would  still  like  to  come  back  to  Dr.  Chance's  comment  on  the  possible 
significance  of  this  reaction  in  non-photosynthetic  cells. 

Chance:  I  thought  I  had  answered  that  question.  This  morning  Prof.  Lindberg 
referred  to  some  very  interesting  possibilities  where  thyroxine  might  interact  with 
the  reduction  of  DPN  and  I  think  this  is  certainly  an  example  of  how  this  would  be 
a  pathway  of  production  of  reducing  power  in  the  cell,  which  could  be  under 
hormonal  control. 

Slater:  Dr.  Chance's  explanation  of  his  results  on  the  basis  of  reversal  of  the 
respiratory  chain  is  very  feasible.  I  am  not  completely  sure  that  that  is  the  only 
possible  explanation  of  his  results,  but  this  is  something  I  do  not  think  we  can 
possibly  go  into  now.  My  first  question  follows  on  from  what  Dr.  Singer  asked. 
Which  flavoprotein  do  you  think  you  are  studying  when  you  are  following  the 
flavoprotein  spectrum  ?  The  second  question  relates  to  the  very  interesting  anaero- 
bic experiment  with  dithionite  and  ATP  where  you  get  DPN  reduced  and  cyto- 
chrome c  oxidized ;  what  is  the  stoicheiometric  relationship  between  the  amount  of 
DPN  reduced  and  cytochrome  c  oxidized  ? 

Chance:  The  answer  to  the  first  question  is  that  we  don't  know  for  sure, 
because  the  flavins  are  unfortunately  summed  by  measurement  at  465  m/x,  but  the 
amount  of  flavin  which  is  involved  in  this  pathway  is  the  major  portion  of  the 
flavin  which  one  observes  spectroscopically.  Two  types  of  answer  are  available 
to  your  second  question.  We  added  5  -6  /xmoles  ATP  and  we  found  a  total  oxidized 
^3,  a,  c  and  flavin  of  i  -5  one-electron  equivalents  to  be  oxidized.  In  other  experi- 
ments the  DPN  reduction  was  slightly  in  excess  of  the  cytochrome  oxidation,  but 
we  have  not  included  Q  oxidation  because  it  is  hard  to  assay  it  quantitatively  when 
we  add  ATP.  We  find  that  DPN  is  reduced  faster  and  flavin  oxidized  faster  than 
cytochrome  c.  This  is  apparently  the  couple  into  which  energy  can  be  put  most 
easily. 


REVERSAL    OF   ELECTRON   TRANSFER    IN   THE   RESPIRATORY    CHAIN  1 37 

Klingenberg  :  We  are  also  investigating  the  effects  of  ATP  on  the  respiratory 
chain.  This  research  started  with  the  ATP-dependent  DFX  reduction.  The  latest 
results,  which  I  shall  show  later  in  the  afternoon,  have  shown  that  ATP  can  also 
interact  at  the  cytochrome  region  under  aerobic  conditions.  We  can  induce 
respiratory  control  and  by  this  get  a  cross-over  point.  Cytochrome  b  is  further 
reduced  and  cytochrome  c  is  further  oxidized.  We  do  not  see  a  further  oxidation  of 
flavoprotein.  Flavoprotein,  cytochrome  b  and  DFX  are  all  reduced  on  the  addition 
of  ATF,  and  cytochrome  c,  in  a  very  fast  reaction,  is  oxidized.  We  believe  that  this 
signifies  the  interaction  of  ATF  at  the  phosphorylation  step  between  cytochrome  h 
and  cytochrome  c  and  the  skeletal  muscle  mitochondria  are  thus  brought  from  a 
slightly  uncoupled  state  to  a  coupled  state  by  the  addition  of  ATF. 

Chance:  Just  a  question.  Is  oxygen  present  ? 

Klingenberg  :  Yes. 

Ch.'XNCe:  Well  that  is  very  interesting,  because  it  is  much  more  difficult  to 
reverse  electron  transfer  in  the  cytochrome  region  when  oxygen  is  present  than 
when  oxygen  is  not  present. 

Klingenberg  :  This  was  at  the  initiation  of  state  4. 


Function  of  Flavoenzymes  in  Electron  Transport 
and  Oxidative  Phosphorylation*! 

Lars  Erxster 

Tlie  Wcuner-Cjven  Institute  for  Expen'inenta/  Biology, 
L  tiirersitv  of  Stocklio/iii,  Szvedeu 

Questions  concerned  with  reaction  pathways  and  mechanisms  bv  which 
flavoenzymes  hnk  substrate  oxidation  to  the  terminal  respiratory  chain 
currently  occupy  an  important  position  in  the  research  field  of  mito- 
chondrial electron  transport  and  oxidative  phosphorylation.  In  the  present 
paper  two  topics  relevant  to  these  problems  are  discussed. 

In  the  first  section,  data  relating  to  the  problem  of  involvement  of 
quinone  reductases,  and  in  particular  of  vitamin  K  reductase,  in  the 
mitochondrial  oxidation  of  reduced  pyridine  nucleotides  will  be  presented. 
Cogent  evidence  will  be  put  forward  that  the  dicoumarol-sensitive  flavo- 
protein,  described  by  Martius  and  collaborators  [14]  under  the  name 
phylloquinone  reductase  or  vitamin  K  reductase,  does  not  participate  in 
the  main  pathway  of  the  mitochondrial  oxidation  of  DPXH.  It  does 
constitute  the  major  pathway  of  direct  oxidation  of  TPNH  in  the  mito- 
chondria, and  presumably  in  the  cell.  Dr.  Conover  in  this  Svmposium  will 
present  data  bearing  on  this  latter  point  [5]. 

Recently  the  concept  was  developed  in  our  laboratory  [6,  7]  that  the 
aerobic  oxidation  of  succinate  in  mitochondria  involves  an  investment  of 
high-energy  phosphate,  i.e.  a  type  of  "  activation  reaction  ".  The  basic  lines 
of  evidence  underlying  this  concept  will  be  summarized  by  Dr.  Azzone 
later  this  afternoon  [8].  Some  recent  data  bearing  on  the  relation  of  this 
activation  mechanism  to  the  phenomenon  of  the  succinate-linked  reduction 
of  mitochondrial  pyridine  nucleotides,  described  some  time  ago  by  Chance 
and  HoUunger  [9,  10]  and  by  Klingenberg  and  co-workers  [11,  12],  are  the 
subject  of  the  second  section  of  this  paper. 

*  This  work  has  been  supported  by  grants  from  the  Swedish  Cancer  Society 
and  the  Swedish  Aledical  Research  Council. 

t  The  following  abbreviations  are  used:  AcAc,  acetoacetate ;  AMP,  ADP, 
ATP,  adenosine-5'-mono,  di-,  and  tri-phosphate ;  DCPIP,  2,6-dichlorophenolin- 
dophenol;  DPX.^DPXH,  TPN,  TPNH,  oxidized  and  reduced  di-  and  triphos- 
phopyridine  nucleotide;  EDTA,  ethylenediaminetetraacetate ;  P,,  inorganic 
orthophosphate. 


140  LARS   ERNSTER 

DT  Diaphorase :  properties  and  functional  aspects 

PROPERTIES,    AND    COMPARISON    WITH    VITAMIN    K    REDUCTASE 

In  1958,  the  occurrence  of  an  abundant  diaphorase  activity  in  the 
sohible  cytoplasm  of  rat  hver  was  detected  in  our  laboratory  [13,  14].  The 
enzyme  catalyzed  the  reduction  of  2,6-dichlorophenolindophenol  by 
DPNH  and  TPNH  at  equal  rates,  and  therefore  we  decided  to  call  it 
"  DT  diaphorase  ".  Early  this  year  [15],  we  reported  the  partial  purification 
of  the  enzyme  and  some  of  its  properties.  These  can  be  summarized  as 
follows:  (i)  The  enzyme  is  a  flavoprotein  with  a  very  high  turnover 
number,  of  the  order  of  ten  millions.  (2)  It  reacts  at  equal  maximal  rates 
with  DPNH  and  TPNH,  but  its  affinity  for  TPNH  is  slightly  higher  than 
for  DPNH.  (3)  Besides  various  dyestuffs  and  ferricyanide,  a  number  of 
naphtho-  and  benzoquinones  can  serve  as  electron  acceptors,  but  not 
vitamin  K^,  or  any  long-chain  substituted  quinones.  (4)  The  enzyme  is 
strongly  inhibited  by  dicoumarol,  and  the  inhibition  is  not  competitive 
with  regard  to  the  electron  acceptor.  (5)  It  is  inhibited  by  sulphydryl 
reagents ;  and  (6)  by  thyroxine  and  related  compounds.  (7)  It  is  activated 
by  bovine  serum  albumin,  which  increases  both  the  maximum  velocity  of 
the  enzyme  and  its  affinity  for  its  substrates.  (8)  Although  the  enzyme  is 
present  in  both  mitochondria  and  microsomes,  it  is  most  abundant  in 
the  soluble  cytoplasmic  fraction. 

Several  of  these  properties  resembled  those  of  various  bacterial  and 
plant  quinone  reductases  described  by  Wosilait  and  associates  [16-18],*  as 
well  as  those  of  the  vitamin  K  or  phylloquinone  reductase  of  Martins  [14]. 
However,  DT  diaphorase  clearly  differed  from  Martins 's  enzyme  in  that  it 
did  not  react  at  any  appreciable  rate  with  vitamin  K^,  whereas  this  com- 
pound is  the  only  electron  acceptor  specified  in  papers  published  between 
1954  and  1959  by  Martins  and  collaborators. 

This  situation  markedly  changed  a  few  weeks  ago.  In  a  paper  which  has 
just  appeared,  Miirki  and  Martins  [21]  now  report  properties  of  vitamin  K 
reductase  which  differ  in  several  important  respects  from  those  they 
previously  reported.  Moreover  the  newly  reported  properties  of  the  enzyme 
strongly  resemble  those  of  DT  diaphorase.  A  brief  summary  of  this  develop- 
ment (which  escaped  recognition  in  the  Miirki  and  Martins  paper)  is  shown 
in  Table  I.  In  fact,  except  for  its  insensitivity  to  — SH  reagents  and  to 
thyroxine,  vitamin  K  reductase  now  reveals  almost  identical  properties 
with  those  of  DT  diaphorase,  and  therefore,  we  are  strongly  inclined  to 
conclude  that  the  two  enzymes  are  identical.  During  the  last  two  years,  a 
considerable  amount  of  information  has  accumulated  in  our  laboratory 

*  A  similar  quinone  reductase  from  dog  liver  has  recently  been  described  by 
Wosilait  [19].  The  isolation  of  a  DT  diaphorase-like  flavoenzyme  from  brain  tissue 
has  been  briefly  reported  by  Giuditta  and  Strecker  [20]. 


FU^XTION   OF   FLAVOENZYMES    IX   ELECTRON   TRANSPORT 


141 


concerning  the  cellular  function  of  DT  diaphorase.  This  may  enable  us 
now  to  examine  critically  the  role  of  vitamin  K  reductase,  which,  as  is  well 
known,  has  been  postulated  by  Martins  [3,  4]  to  constitute  the  exclusive 
pathway  of  reduced  pvridine  nucleotide  oxidation,  and  of  oxidative 
phosphorylation,  in  normal,  intact  mitochondria.  The  experimental  data 
to  be  presented  have  been  obtained  in  collaboration  with  Dr.  T.  E.  Conover 
and  Air.  L.  Danielson. 

TABLE  I 
Comparison  of  Vitamin   K  Reductase  and  DT  Diaphorase 


Vitamin  K  Reductase 
(Martius  et  ol.,  1954-S9 

[1-4]) 


DT  Diaphorase 

(P^rnster  et  al.,  i960 

[15]) 


\"itamin  K  Reductase 
(Miirki  and  Martius,  i960 

[21]) 


Flavoenzyme 
(turnover  number, 
I  -2  X  10"). 

Reacts  with  DPXH  and 
TPNH  at  equal  rates. 


Reacts  specifically  with 
vitamin  Kj 


Strongly  inhibited  by 
dicoumarol. 

Inhibited  bv  thvroxine. 


Not  inhibited  by  p- 
chloromercuribenzoate. 


Present  in  mito- 
chondria. 


Flavoenzyme 

(turnover  number, 
-  lo'). 

Reacts  with  DPXH  and 
TPXH  at  ecjual  rates,  but 
affinitv  slightlv  higher  for 
TPXH. 

Reacts  with  dyestuffs, 
ferricyanide,  various 
naphtho-  and  benzo- 
quinones,  but  not  with 
vitamin  Ki  and  other 
long-chain  substituted 
quinones. 

Strongly  inhibited  by 
dicoumarol. 

Inhibited  by  thyroxine 
(and  related  compounds). 

Inhibited  by  /)-chloro- 
mercuribenzoate. 

Activated  by  bovine 
serum  albumin. 

Present  in  mitochondria 
and  microsomes,  but  bulk 
m  soluble  cytoplasm. 


Flavoenz\Tne 
(turnover  number, 

7  X  lO^). 

Reacts  with  DPXH  and 
TPX'H  at  equal  rates,  but 
affinitv  slightlv  higher  for 
TPXH. 

Reacts  with  dyestuffs, 
ferricyanide,  various 
naphtho-  and  benzo- 
quinones,  but  not  with 
vitamin  Kj  and  other 
long-chain  substituted 
quinones. 

Strongly  inhibited  by 
dicoumarol. 

Xot  inhibited  by 
thyroxine. 

Xot  inhibited  by  /)- 
chloromercuribenzoate. 

.Activated  by  bovine 
serum  albumin. 

Present  in  mitochondria, 
but  bulk  in  soluble 
cytoplasm. 


REL.\TION    TO    MITOCHONDRIAL    RESPIRATORY    CHAIN 

When  intact  rat  liver  mitochondria  were  incubated  with  glutamate,  and 
under  conditions  allowing  optimal  rates  of  respiration  and  phosphorylation, 
addition  of  5  x  10  "^  m  vitamin  K3  or  lO"''  m  dicoumarol  had  no  effect  on 
the  rate  of  oxygen  consumption  (Table  II).  However,  when  respiration 


142  LARS    ERNSTER 

was  blocked  by  the  addition  of  i  mM  amytal,  it  could  be  completely 
restored  by  vitamin  K3  and  was  then  strongly  inhibited  by  dicoumarol. 
The  vitamin  K3-induced  respiration  was,  in  accordance  with  the  observa- 
tions of  Colpa-Boonstra  and  Slater  [23],  sensitive  to  antimycin  A.  The 
antimycin  A  inhibition  could  be  overcome  to  some  extent  by  adding 
cytochrome  c  (not  shown). 

From  these  findings  it  was  concluded  that  in  intact  liver  mitochondria 
there  exist  two  pathways  of  antimycin  A-sensitive  DPNH  oxidase,  of 
which  only  one,  characterized  by  a  sensitivity  to  amytal,  functions  under 

TABLE  II 

Effect  of  Vitamin  K.,  and  Various  Inhibitors  on  the  Respiration  of 
Mitochondria  in  the  Presence  of  Amytal 

(from  Conover  and  Ernster  [22]) 

Additions  /xatoms  oxygen 

None  5  •  49 

Vitamin  K3  5   57 

Dicoumarol  5   32 

Amytal  0-22 

Amytal  +  vitamin  K3  5 '25 

Amytal  +  vitamin  K3  +  antimycin  A  1-57 

Amytal  +  vitamin  K3  +  KCN  118 

Amytal  +  vitamin  K3  +  dicoumarol  i  ■  1 3 

The  complete  system  contained  per  Warburg  vessel:  10  /unioles  glutamate, 
20  fimoles  tris  buffer  (pH  7 '4),  20  //.moles  orthophosphate  (pH  7  "4),  4  /xmoles 
MgClo,  2  /xmoles  adenosine  triphosphate,  24  /xmoles  glucose,  an  excess  of  yeast 
hexokinase,  50  /itmoles  sucrose,  and  mitochondria  from  200  mg.  rat  liver.  The 
amounts  of  the  additions  were  as  follows:  5  x  iq-^  /xmole  vitamin  K3,  lO"*  /itmole 
dicoumarol,  10  /tmole  amytal,  i  [xg.  antimycin  A,  and  i-o  /xmole  KCN.  Final 
volume,  I  o  ml.  Temperature  3o\  Reading  begun  after  6  min.  thermoequilibra- 
tion.  Time  measured,  20  min. 

normal  conditions.  Another  pathway,  characterized  by  a  sensitivity  to 
dicoumarol  and  proceeding  probably  via  DT  diaphorase,  is  not  functioning 
normally  in  the  terminal  oxidation  of  DPNH,  because  it  lacks  a  link  to  the 
cytochrome  system,  but  it  can  be  brought  into  reaction  by  adding  an 
artificial  link  such  as  vitamin  K.5. 

The  phosphorylation  arising  from  the  vitamin  Kg-induced  respiration 
in  the  amytal-blocked  system  yielded  a  P/0  ratio  that  was  about  one  unit 
lower  than  that  of  the  normal  system  (Table  III).  However,  the  P/0  ratio 
obtained  with  succinate  as  substrate  also  was  lowered  under  these  condi- 
tions. In  other  words,  it  cannot  be  decided  with  the  evidence  presently 
available,  whether  or  not  the  vitamin  Kg-induced  by-pass  of  the  amytal- 
sensitive  site  involves  the  loss  of  a  phosphorylation. 


FUNCTION    OF    FLAVOENZYMES    IN    ELECTRON    TRANSPORT  I43 

TABLE  III 

ESTERIFICATION    OF    PHOSPHATE    DURING    THE    OxiDATION 

OF  Substrate  in  the  Presence  of  Amytal  and  \"itamix  Kg 

(Conover  and  Ernster   unpublished) 

Experimental   conditions   as   in   Table   II.    Substrates  were   added   in   a   final 
concentration  of  o-oi  M. 


P  0 

ratio 

Substrate 

None 

\' 

itamin  K., 

Amytal 

Vitamin  K3 

+ 
amytal 

glutamate 

iS-OH-butyrate 

succinate 

2-63 

2-14 

1-78 

2-46 

1-40 
1-65 

1-53 

1-45 
1-04 
I-I9 

The  antimycin  A-sensitivity  of  the  Yitamin  Ky-induced  respiration  in 
the  amytal-blocked  system  indicated  that  the  electrons  mediated  by 
vitamin  K3  may  enter  the  respiratory  chain  at  the  level  of  cytochrome  b. 
This  point  could   be  tested   with  the   double-beam   spectrophotometer, 


400- 


300- 


200 


434- 490  m// 
cyt.  b 
reduction 


Jf=00lcm' 


Fig.  I.  Effect  of  amytal  and  vitamin  Kj  on  the  oxygen  consumption  and  on  the 
reduction  of  cytochrome  c  during  the  oxidation  of  glutamate  (Conover  and 
Ernster,  unpublished).  The  medium  contained  001  m  triethanolamine  buffer, 
pH  7-4,  o-oi  M  Pj,  pH  74,  0004  .M  MgCL,  0065  M  KCl,  and  o-i  M  sucrose. 
Mitochondria  from  200  mg.  rat  liver  were  used.  The  amounts  of  additions  were  as 
follows:  2-0  /amoles  L-glutamate,  o-8  /^mole  ADP,  2-0  /imoles  amytal,  and  0-04 
^mole  vitamin  K3.  Final  volume,  i  -2  ml.  Temperature,  25  ^ 


144  LARS   ERNSTER 

kindly  placed  at  our  disposal  by  Dr.  M.  Klingenberg  in  Marburg.  As  shown 
in  Fig.  I,  the  restoration  ot  respiration  by  vitamin  K3  in  the  amytal- 
blocked  system  was  accompanied  by  an  abrupt  increase  of  the  light 
absorption  difference  at  434  490  m/x,  indicative  of  a  reduction  of  cyto- 
chrome h. 

SEPARATION    OF    DT    DIAPHORASE    AND    DPNH    OXIDASE 

In  order  further  to  fortify  the  concept  that  DT  diaphorase  does  not 
participate  in  the  main  DPNH  oxidase  pathway,  an  attempt  was  made  to 
separate  the  two  systems.  This  proved  possible  by  exposing  liver  mito- 
chondria to  disruption  by  a  rapidly  rotating  Super-Thurrax  blendor, 
followed  by  differential  centrifugation,  essentially  according  to  the 
procedure  employed  by  Kielley  and  Kielley  [24]  in  their  studies  of  mito- 
chondrial ATPase.  The  procedure  results  in  three  submitochondrial 
fractions:  a  soluble  fraction,  a  light  pellet,  and  a  heavy  pellet. 


D   DPNH 
□    TPNH 


.£    2 


.9  Light      Heavy  >- 

-D  Sup.          pel.          pel.  % 

I      ^ V '  o 

o  Submitochondrial  fractions  <^ 


Fig.  2.  Diaphorase  activities  of  submitochondrial  fractions  prepared  according 
to  Kielley  and  Kielley  (1953)  (from  Danielson,  Ernster,  and  Ljunggren  [25]). 


In  examining  the  DPNH  and  TPNH  diaphorase  activities  of  these 
fractions  it  was  found  (Fig.  2)  that  the  soluble  fraction  contained  virtually 
the  entire  TPNH  diaphorase  activity  of  the  original  mitochondria,  accom- 
panied by  an  equal  DPNH  diaphorase  activity,  whereas  the  two  pellets 
exhibited  only  DPNH  diaphorase  activities.  Moreover,  the  activities  found 
in  the  soluble  fraction  were  markedly  activated  by  Tween  and  strongly 
inhibited  by  dicoumarol  (both  of  these  properties  are  characteristic  of 
DT  diaphorase),  whereas  the  activities  of  the  pellets  were  not  influenced  by 
these  agents  (Table  IV).  The  selectivity  of  the  inhibition  by  dicoumarol 
between  soluble  and  pellet  DPNH  diaphorase  activities  is  illustrated  in 
Fig.  3.  This  treatment  of  mitochondria  thus  resulted  in  a  selective  solubil- 
ization of  DT  diaphorase. 


FUNCTION    OF    FLAVOENZYMES    IN    ELECTRON    TRANSPORT 

TABLE   IV 

Properties  of  Diaphorase  Activities  of  Submitochondrial 
Fractions  Prepared  According  to  Kielley  and  Kielley  (1953) 

F"or  experimental  details,  see  Danielson,  F>nster  and  Ljunggren  [25]. 


145 


Submitochondrial 
fraction 


Additions 


Diaphorase  activity 

/Ltmoles  DCPIP  reduced/ 

min./g.  liver 


DPNH 


TPNH 


Supernatant  none  028  030 

8  mg.  Tween  0-70  071 

8  mg.  Tween  +  lo^**  M  dicoumarol  005  0-04 

Pellet  (light)  none  050  o-oo8 

8  mg.  Tween  050  0-038 

8  mg.  Tween  +  lo""  M  dicoumarol  0-46  o-oo6 


The  light  sediment  exhibited  a  DPXH  oxidase  activity,  as  shown  in 
Table  V.  This  was  sensitive  to  amvtal  and  antimvcin  A.  When  cytochrome 
c  was  added,  the  respiration  was  increased,  and  the  stimulated  respiration 
was  only  partially  inhibited  by  these  agents.  Thus  this  pellet  seems  to 


7        6        5        4 
-log  M  dicoumarol 
Fig.    3.    Effect   of  dicoumarol   on   diaphorase   activities   of  submitochondrial 
fractions  prepared  according  to  Kielley  and  Kielley  (1953).  Activities  measured  bv 
using  DPNH  as  substrate  and  DCPIP  as  hydrogen  acceptor.   For  experimental 
details,  see  Danielson,  Ernster,  and  Ljunggren  [25]. 

contain  both  the  "internal"  type  of  DPXH  oxidase  of  mitochondria,  and 
the  "external"  type  of  DPXH  cytochrome  c  reductase  (cf.  [26,  27]).  As 
Professor  Lindberg  reported  earlier  in  this  session  [28],  the  two  systems 
may  also  be  distinguished  by  their  different  degrees  of  sensitixitv  to  desa- 
minothyroxine. 

Using  the  "internal"  DPXH  oxidase  system,  that  is,  the  light  pellet 
without  supplementation  with  cytochrome  r,  it  was  found  that  the  amytal- 
block  now  could  not  be  by-passed  by  added  \  itamin  K3  (Fig.  4).  However, 

VOL.  II. — L 


146  LARS    ERNSTER 

TABLE  V 

Properties  of  DPNH  Oxidase  Activity  of  Liver  Mitochondrial 
Fragments  Prepared  According  to  Kielley  and  Kielley  (1953) 

(Ernster,  Danielson  and  Conover,  unpublished) 

The  test  system  contained  submitochondrial  particles  ("light  pellet")  from 
200  mg.  liver,  o-i  mM  DPNH,  0-02  m  phosphate  buffer,  pH  7-5,  and  where 
indicated,  i  mivi  amytal,  o-8  jug./ml-  antimycin  A,  0-33  mivi  KCN,  o-oi  mM 
cytochrome  c,  in  a  final  volume  of  3  ml.  The  oxidation  of  DPNH  was  followed  at 
340  m/Li  in  a  recording  Beckman  DK2  spectrophotometer. 


/xmoles  DPNH  oxidized/min./g.  liver 

Addition                      Without                           With 

cytochrome  c               cytochrome  c 

None                                    o-i6                               046 
Amytal                                 0-03                               0-23 
Antimycin  A                       003                                022 
KCN                                  O-OI                              0-02 

-A 

X 

Q 

+  " 
E. 

13 

\ 

j:    400 

0) 
Q. 

\ 

£     300 

en 

'e 

^ 

, 

J3 

13 

1 

°     200 

> 

-Vit.  K 

T    J. L 

5.     \ 

E 

E 
< 

■D 

\ 

"^     100 

_ 

Q 

\ 

-► 

Imin 

-^- 

1r- 

' 

- 

0 

- 

h 

Fig.  4.  Requirement  of  DT  diaphorase  for  the  vitamin  Kj-mediated  oxidation 
of  DPNH  by  mitochondrial  fragments  in  presence  of  amytal  (Conover  and 
Ernster,  unpublished).  The  medium  contained  19  /nmoles  orthophosphate  (pH  7-5) 
and  2  mg.  serum  albumin  in  i  -o  ml.  Submitochondrial  fragment  preparation  from 
I  g.  of  rat  liver  was  used.  The  amounts  of  the  additions  were  as  follows:  i-o 
/xmole  DPNH,  2-0  /^moles  amytal,  0005  jumole  vitamin  K3,  purified  DT  diaphor- 
ase capable  of  reducing  i  /imole  DCPIP  per  min.,  and  003  /imole  dicoumarol. 
Final  volume,  i  -3  ml.  Temperature,  20  . 

the    by-pass    could    be    achieved    by   adding  a  purified  sample   of  DT 
diaphorase. 

QUINONE    SPECIFICITY 

These  early  studies  were  carried  out  using  vitamin  K3  as  the  only 
quinone.  Then  Dr.  Conover  made  the  somewhat  surprising  observation 
that,  although  DT  diaphorase  can  react  with  a  number  of  both  naphtho- 


FUNCTION    OF    FLAVOENZYMES    IN    ELECTRON    TRANSPORT  1 47 

and  benzoquinones,  only  vitamin  K3,  out  of  a  great  number  of  quinones 
tested,  was  able  to  carry  out  the  above  described  by-pass  of  the  mito- 
chondrial amytal-sensitive  site.  This  phenomenon  is  illustrated  in  Table 
VI.  It  can  be  seen  that  there  was  a  clear  requirement  for  both  the  naphtho- 
quinone skeleton  and  the  2-AIe  substituent  when  the  mitochondrial 
respiratory  chain  served  as  terminal  electron  acceptor.  This  requirement 

TABLE  VI 

Requirement  for  2-Methyl-i,4-naphthoquinone  Structure  in  Mediation  of 

Electron  Transport  between  DT  Diaphorase  and  Mitochondrial 

Respiratory  Chain 

(from  Conover  and  Ernster  [22],  and  unpublished  data) 


Relative 

activity 

Quinone* 

As  terminal 
electron 
acceptor 

As 

electron  mediator 

to 

Cyto- 
chrome 

c 

"Cytochrome  b"l 
System  I     System  II 

Vitamin  Kg  (2-Me-i  ,4-naphtho- 

quinone) 
1 ,4-naphthoquinone 
1 ,2-naphthoquinone 

/i-benzoquinone 
2-Me-benzoqviinone 
2,6-diMe-benzoquinone 
CoQo  (2-Me-5,6-dimethoxy- 
benzoquinone) 

Vitamin  Ki 
Vitamin  K., 
CoQio 

I  oof 
93 

72 

174 
159 
168 

155 

0 
0 
0 

100 
140 

^7 

0 

6 

20 

22 

0 
0 
0 

100 

5 
0 

0 
0 
0 

0 

2 

I 
0 

100 
15 
19 

3 
3 
2 

3 

2 

2 

2 

*  The  quinones  were  used  in  final  concentrations  of  33  or  67  fiM  as  terminal 
acceptors,  and  8-33  or  10  /xM  as  mediators. 

t   This  activity  was  about  i    5  times  that  obtained  with  DCPIP  as  acceptor. 
+   System  I  :  Intact  mitochondria,  glutamate,  amytal. 

System  II :  Submitochondrial   DPNH   oxidase   (Kielley   and   Kiellev,    light 
pellet),  TPNH,  purified  DT  diaphorase,  KCN. 

was  valid  both  for  intact  mitochondria  and  for  the  reconstructed  submito- 
chondrial system,  thus  eliminating  effects  due  to  permeability  barriers. 
With  purified  cytochrome  c,  coupling  occurred  with  both  2-Me  substituted 
and  non-substituted  naphthoquinone,  and  to  some  extent  also  with  2-Me 
benzoquinones.  The  general  rule  that  we  presently  envisage,  after  having 
tested  a  large  collection  of  quinones,  is  schematically  illustrated  in  Fig.  5. 


148  LARS   ERNSTER 

It  is  obvious  that  any  type  of  quinone  specificity  involved  in  the  function 
of  DT  diaphorase  is  a  specificity  on  the  acceptor  and  not  on  the  donor 
side.  In  other  words,  vitamin  K  may  be  the  specific  coupler  of  DT  dia- 
phorase to  the  cytochrome  system  in  the  living  cell ;  however,  7iot  because 
DT  diaphorase  requires  vitamin  K  specifically  but  because  cytochrome  b 
does.  Whatever  the  reason  for  this  requirement  may  be,  it  is  clear  that 
the  name  "vitamin  K  reductase"  for  the  flavoenzyme  as  such  is  hardly 
adequate. 


Fig.  5.  Quinone-catalyzed  coupling  between  DT  diaphorase  and  cytochromes. 
NQ  =  naphthoquinone;  BQ  =  benzoquinone;  Me  =  methyl. 


CONCLUSIONS    AND    COMMENTS 

Figure  6  summarizes  in  a  schematic  form  our  conclusions  as  to  the 
relation  of  DT  diaphorase  to  the  main  pathway  of  mitochondrial  DPNH 
oxidation.  It  seems  to  be  clear  from  the  data  presented  above  that  the 
amytal-sensitive,  DPNH  specific  pathway  represents  the  main,  if  not 
exclusive,  route  of  DPNH  oxidation  in  normal,  intact  rat-liver  mitochondria. 
The  dicoumarol-sensitive  DT  diaphorase,  which  very  probably  is  identical 
with  Martins  and  collaborators'  vitamin  K  reductase,  is  present  in  these 
mitochondria  without  any  apparent  functional  link  to  the  terminal  electron 
transport  system.  In  order  to  establish  such  a  link,  the  external  supply  of  a 
catalytic  amount  of  vitamin  K3  (or  any  other  2-methylnaphthoquinone 
without  a  long  carbon-chain  substituent  in  the  3-position)  is  needed  for 
electrons  from  DT  diaphorase  to  enter  the  respiratory  chain  at  or  before 
the  level  of  cytochrome  b.  Alternatively,  DT  diaphorase  can  be  coupled  to 
the  respiratory  chain  by  way  of  naphthoquinones  without  a  2-methyl 
substituent,  and  to  some  extent  also  by  way  of  2-methylbenzoquinones. 
In  these  cases,  external  cytochrome  c  is  required  in  addition  to  the  quinone, 
and  the  entrance  of  the  electrons  takes  place  beyond  the  antimycin  A 
sensitive  site  of  the  chain,  probably  at  the  level  of  cytochrome  a. 

There  are  only  two  further  brief  comments  I  would  like  to  add  to  these 
conclusions.  First,  it  should  be  pointed  out  that  although  Martius's 
vitamin  K  reductase  does  not  seem  to  participate  in  the  main  pathway  of 


FUNCTION  OF  FLAVOENZYMES  IN  ELECTRON  TRANSPORT 


149 


DPNH  oxidation  in  liver  mitochondria  this  does  not  imply  that  Alartius's 
original  idea  [29]  of  the  participation  of  vitamin  K  in  the  main  respiratory 
chain  need  to  be  abandoned.  Indications  of  a  specific  role  of  vitamin  K  in 
DPN-linked  respiration  and  phosphorylation  have  been  reported,  both  in 
fractionated  bacterial  systems  [30]  and  in  U.V. -irradiated  liver-mito- 
chondrial  preparations  [31,  32].  Even  if  this  evidence  is  only  circum- 
stantial, its  validity  is  not  influenced  by  the  present  conclusions.  It  may 
well  be  that  bound  vitamin  K  does  participate  as  an  electron  carrier  in  the 
amvtal-sensitive,    main    pathway    of    mitochondrial    DPXH    oxidation. 


DPNH 


TPNH 


Amytal 


Antimycin  A 


Dicoumarol 


Fig.  6.  Rtlation  of  DT  diaphorase  to  the  main  pathway  of  DPXH  oxidation 
of  intact  liver  mitochondria.  Fp]j  =  DPXH  diaphorase;  Fpi,T  —  DT  diaphorase. 
Dotted  arrows  indicate  pathways  involving  externally  added  carriers. 


located,  tentativelv,  between  the  DPXH  diaphorase  and  cytochrome  b. 
Such  a  possibilitv  mav  actually  find  some  indirect  support  in  our  data, 
which  indicate  that  cytochrome  /;  might  require  a  2-methylnaphtho- 
quinone  as  a  specific  electron  donor.  Pertinent  to  this  possibility  may  also 
be  the  preliminary  findings,  illustrated  in  Table  VII,  that  the  DPXH 
diaphorase  reaction  of  the  Kielley  and  Kielley  preparation  reveals  a  marked 
sensitivity  to  amytal  when  measured  with  2-methyl-substituted  quinones 
as  electron  acceptors,  whereas  it  exhibits  only  a  slight  amytal  sensitivity 
with  non-substituted  quinones.  Thus,  the  role  of  vitamin  K  in  respiration 
and  phosphorylation  deserves  further  consideration. 

A  second  point  of  interest  is  that  of  the  well-known  uncoupling  effect 


150  LARS   ERNSTER 

of  dicoumarol  on  mitochondrial  oxidative  phosphorylation  [29].  The 
rational  standpoint  in  view  of  the  present  conclusions  would  seem  to  be 
that  this  effect  is  independent  of  the  inhibitory  effect  of  dicoumarol  on  DT 
diaphorase.  Alternatively,  one  could  think  that  DT  diaphorase,  although 
not  taking  part  in  the  main  pathway  of  terminal  electron  transport,  might 

TABLE  VII 
Capacity  of  Various  Quinones  to  Act  as  Terminal  Electron  Acceptors  in 

SUBMITOCHONDRIAL    DPNH    OXIDASE,    AND    THE    AmYTAL    SENSITIVITY    OF    THESE 

Reactions 

(lirnster,  Danielson  and  Conover,  unpublished) 

The  quinones  were  added  in  final  concentrations  of  0-04  mM.  Oxygen  uptake 
in  these  systems  was  blocked  with  03  mM  cyanide. 

P  .     .  "o  inhibition 

Terminal  electron  acceptor  by  2  mM 

activity  , 

amytal 


Oxygen 

I  -o 

81 

2-Me- 1 ,4-naphthoquinone 

1-3 

52 

2-Me-benzoquinone 

2-7 

64 

2-Me-5,6-dimethoxybenzoquinone  (CoQ„) 

2-8 

40 

1 ,4-naphthoquinone 

3-1 

12 

1 ,2-naphthoquinone 

99 

18 

/)-benzoquinone 

9-5 

5 

play  some  accessory  role,  such  as  a  regulation  of  the  redox-state  of  the  mito- 
chondrial pyridine  nucleotides,  during  coupling  of  respiration  to  phos- 
phorylation. However,  the  fact  that  certain  tissues,  e.g.  pigeon  liver  [21], 
seem  to  contain  very  little  or  none  of  the  dicoumarol-sensitive  flavoenzyme, 
and  still  exhibit  a  highly  active  phosphorylation,  would  seem  to  impose 
serious  obstacles  to  a  consideration  of  this  alternative. 


Activation  of  succinate  oxidation  and  succinate-linked 
reduction  of  DPN 

OBSERVATIONS    WITH    HIGH-ENERGY    PHOSPHATE-DEPLETED    MITOCHONDRIA 

I  wish  to  begin  the  second  section  of  my  report  by  quoting  a  finding 
that  Dr.  Low  and  I  described  in  1955  [33].  At  that  time  we  found  (Fig.  7) 
that  rat  liver  mitochondria  exposed  to  ageing  in  a  phosphate-containing 
medium  lost  some  of  their  succinoxidase  activity  in  the  course  of  the  ageing 
process.  The  decrease  was  of  a  transitional  character;  upon  prolonged 
ageing,  the  mitochondria  resumed  their  original  succinoxidase  activity. 
We  found,  moreover,  that  the  decreased  succinoxidase  activity  could  be 


FUNCTION  OF  FLAVOENZYMES  IN  ELECTRON  TRANSPORT 


K^l 


restored  to  its  original  level  by  the  addition  of  ATP.  From  these  and  other 
findings,  the  hypothesis  was  advanced  that  mitochondria,  above  a  certain 
level  of  structural  organization,  require  ATP  for  oxidizing  succinate,  and 
that,  at  this  transitional  stage  of  ageing,  the  mitochondria  had  already  lost 
their  endogenous  ATP  while  they  still  possess  part  of  their  organized 
feature. 


15-^A      o  o  o;^ 

^^  o 


£ 


10- 


o 


5- 


_0_ 


10 


1- 
20 


1^ 

30 


1^ 

60 


1^ 

70 


80 


=^1 

90 


120 


40  50 
min.  preincubation 
Fig.  7.  Effect  of  ageing  on  succinoxidase  activity  of  rat  liver  mitochondria  (from 
Ernster  and  Low  [33]).  The  continuous  lines  indicate  oxygen  (upper  line)  and 
phosphate  (lower  line)  uptake  in  absence  of  ATP  and  Mg++;  the  large  circles  indicate 
oxygen  uptake,  and  the  large  triangles  phosphate  uptake,  in  presence  of  ATP  and 
Mg++. 


These  findings  were  almost  forgotten  when  last  winter  Dr.  Azzone  in 
our  laboratory  made  the  interesting  obser\ation  [(y]  that  rat  liver  mito- 
chondria after  preincubation  in  the  presence  of  2  mM  arsenate  and  o  •  04  mM 
dicoumarol  (or  o- 1  mM  dinitrophenol)  for  a  period  of  3  to  4  min.  displayed 
a  strongly  inhibited  succinoxidase  activity,  which  could  be  greatly  en- 
hanced by  added  ATP.  In  extending  this  obser\  ation  (which  is  illustrated 
in  the  upper  part  of  Fig.  8)  it  was  found  [7]  that  the  decrease  of  the  succin- 
oxidase activity  was  correlated  with  an  exhaustion  of  the  endogenous  high- 
energy  phosphate  content  of  the  mitochondria;  and  conversely,  that  any 
means  of  pretreating  mitochondria  so  as  to  deplete  them  of  high-energy 


152  LARS   ERNSTER 

phosphate,  provided  that  it  caused  no  irreversible  damage  of  their  structural 
integrity,  led  to  a  decrease  of  the  succinoxidase  activity.  Furthermore,  what 
was  only  anticipated  in  1955  (and  even  questioned  later),  namely,  that 
the  observed  inhibition  was  not  due  to  an  accumulation  of  oxaloacetate, 
could  now  be  ascertained  by  rigorous  experimental  means  [7,  8]. 


(a) 


Jf=0020cm 


(b) 


Analytical 
data  © 

yumoles/g  prot. 

DPNH    078 
TPNH    3-60 


®     (D  © 


007  005 
0-47  0-38 


015 
083 


Fig.  8.  Effect  of  ATP  on  succinate  oxidation  and  pyridine  nucleotide  reduction 
in  rat  liver  mitochondria  preincubated  with  arsenate  and  dicoumarol  (from  Azzone, 
Ernster,  and  Klingenberg  [34]). 


From  work  along  these  lines  the  concept  was  developed  that  in  intact 
liver  mitochondria,  the  aerobic  oxidation  of  succinate  involves  an  activation 
reaction  by  means  of  high-energy  phosphate.  Since  this  mechanism  was 
visualized  as  involving  the  formation  of  a  high-energy  intermediate  in  the 
respiratory  chain  at  the  level  where  the  electrons  derived  from  succinate 
enter  the  terminal  respiratory  chain,  it  seemed  conceivable  that  this 
intermediate  might  also  be  involved  in  the  endergonic  reduction  of  the 


FUN'CTION    OF    FLAVOEXZYMES    IN    ELECTRON    TRANSPORT  1 53 

mitochondrial  DPN  by  succinate,  earlier  described  by  Chance  and 
Hollunger  [9,  10]  and  by  Klingenberg  et  al.  [11,  12].  It  was  therefore  of 
interest  to  investigate  whether  the  ATP-induced  activation  of  succinate 
oxidation  in  the  high-energy  phosphate  depleted  mitochondria  was 
reflected  in  an  increased  level  of  DPNH. 

In  this  part  of  our  investigation,  Dr.  Azzone  and  I  had  the  pri\ilege 
to  benefit  by  the  hospitality  and  collaboration  of  Dr.  Martin  Klingenberg 
at  Marburg.  A  typical  resulc  of  our  experiments,  a  detailed  account  of 
which  is  being  published  elsewhere  [34],  is  shown  in  the  lower  part  of 
Fig.  8.  As  can  be  seen,  the  ATP-induced  stimulation  of  the  aerobic 
oxidation  of  succinate  in  the  arsenate-dicoumarol  depleted  mitochondria 
was  paralleled  by  an  increase  of  the  340-380  absorption  difi^erence, 
indicative  of  the  reduction  of  pyridine  nucleotides.  Similar  results  were 
obtained  when  dicoumarol  was  replaced  by  io~^  M  dinitrophenol.  Dif- 
ferential analytical  data,  given  at  the  bottom  of  the  figure,  reveal  that  the 
increase  was  due  to  a  major  part  to  the  reduction  of  TPX,  and  to  a  minor 
part  to  the  reduction  of  DPX.  Admittedly,  the  observed  steady-state  levels 
of  the  reduced  pyridine  nucleotides  were  not  particularly  high,  about  10 
and  30",,  of  the  total  contents  of  DPX  and  TPX,  respectively.  On  the 
other  hand,  the  system  contained  a  fully  uncoupling  concentration  of 
dicoumarol  (or  dinitrophenol),  this  causing  a  maximal  flux  of  electrons 
towards  oxvgen ;  the  levels  of  DPXH  and  TPXH  found  may  thus  actually 
represent  the  maximal  values  obtainable  in  an  uncoupled  system. 

However,  the  main  importance  of  these  findings  in  our  opinion  is  the 
very  fact  that  an  ATP-dependent  reduction  of  pyridine  nucleotides  by 
succinate  could  occur  at  all  in  the  presence  of  a  fully  uncoupling  concen- 
tration of  dicoumarol  or  dinitrophenol ;  or  in  other  words,  that  a  high- 
energy  intermediate  at  the  level  of  the  respiratory  chain  could  be  formed 
at  the  expense  of  ATP  in  spite  of  the  uncoupled  state  of  the  oxidative 
phosphorvlation  svstem.  This  seems  to  imply  a  serious  challenge  to  those 
proposed  schemes  of  oxidative  phosphorylation  ([35],  [36],  [26];  cf.  [37] 
for  review)  which  invoke  the  participation  of  non-phosphorylated  high- 
energy  intermediates  at  the  level  of  the  electron  transport  chain,  and 
according  to  which  uncoupling  agents  act  by  disconnecting  the  interaction 
of  this  intermediate  with  inorganic  phosphate  and  ADP.  On  the  other 
hand,  the  present  finding  is  in  agreement  with,  and  even  lends  some 
support  to,  the  hypothesis  promulgated  by  our  group  [38-40]  that  phos- 
phorylated  reduced  electron  carriers  are  the  primary  high-energy  inter- 
mediates. According  to  this  hypothesis,  uncouplers  are  \'isualized  as  acting 
by  preventing  inorganic  phosphate  from  becoming  activated  in  connection 
with  the  energy-yielding  oxidative  step,  and  are  thus  not  expected  to 
interfere  with  the  reversal  of  this  reaction. 

On  the  basis  of  the  abo\  e  findings,  the  following  simple  reaction  scheme 


154  LARS   ERNSTER 

has  been  proposed  [7]  for  the  activation  of  succinate  oxidation  and  the 
succinate-Hnked  reduction  of  mitochondrial  DPN : 


succinate  +  A  +  ATP 
AH  -  P  +  DPN 


^  fumarate  +  AH  ~  P  +  ADP 
^A+DPNH  +  P: 


(I) 
(2) 


where  A  stands  for  the  electron  carrier  whose  reduction  by  electrons 
derived  from  succinate  requires  an  investment  of  high-energy  phosphate. 
Depending  on  the  nature  of  A,  Reaction  (i)  or  (2),  or  both  of  them,  may 
be  sum-reactions  involving  several  steps.  It  may  be,  thus,  that  A  is  succinic 
dehydrogenase,  in  which  case  Reaction  (i)  is  a  one-step  reaction,  and 
Reaction  (2)  a  several-step  one,  composed  of  a  transfer  of  P  from  AH  ~  P 
to  the  reduced  diaphorase-flavin,  and  a  subsequent  reversal  of  the  first 
respiratory  chain  phosphorylation.  It  may  also  be  that  A  is  the  diaphorase- 
flavin;  then,   Reaction  (i)  may  be  composed  of  a  reduction  of  succinic 


Succinate 


TPN 

\ 

DPN 


"Ps 


■ATP- 


-ADP- 


ATP- 


ADP- 


FPr 


(quinone) 


ATP- 


ADP- 


cytochromes 


-►O, 


Fig.  9.  Hypothetical  scheme  of  the  functional  link  of  succinic  dehydrogenase 
to  the  terminal  electron  transport  system  (from  Azzone,  Ernster,  and  Klingenberg 
[34]).  Fpf,  =  DPNH  diaphorase;  Fp^;  =  succinic  dehydrogenase. 

dehydrogenase  by  succinate,  followed  by  an  ATP-requiring,  phosphoryla- 
tive  reduction  of  A  by  the  reduced  succinic  dehydrogenase.  A  third 
alternative  may  be  that  A  is  a  quinone,  in  which  case  both  Reactions  (i) 
and  (2)  are  composed  of  two  steps;  Reaction  (i)  by  a  reduction  of  succinic 
dehydrogenase  by  succinate,  followed  by  an  ATP-dependent,  phos- 
phorylative  reduction  of  A  by  the  reduced  succinic  dehydrogenase;  and 
Reaction  (2)  by  a  transfer  of  P  from  AH  ~  P  to  the  reduced  diaphorase- 
flavin,  followed  by  a  reversal  of  the  first  respiratory  chain  phosphorylation. 
In  any  case,  AH  ~  P  must  be  of  such  a  nature  that  its  reoxidation  by  the 
subsequent  carrier  (B)  along  the  respiratory  chain  be  connected  with  a 
regeneration  of  ATP,  and  moreover,  that  the  further  oxidation  of  the 
resulting  BHo  by  molecular  oxygen  still  can  give  rise  to  two  net  phos- 
phorylations ;  otherwise,  the  aerobic  oxidation  of  succinate  in  mitochondria 
could  not  result  in  a  P/0  ratio  of  2.  These  reactions  may  then  be  written  as : 

AH~P  +  B  +  ADP ^A+BHo  +  ATP  (3) 

BH2  +  iO.3  4-  2ADP  +  2P, >  B  +  2ATP  +  H^O.  (4) 

A  schematic  illustration  of  these  concepts  is  found  in  Fig.  9. 


FUNCTION   OF   FLAVOENZYMES    IN   ELECTRON   TRANSPORT  1 55 

ENDERGONIC    REDUCTION    OF    ACETOACETATE    BY 
SUCCINATE    IN    LIVER    MITOCHONDRIA* 

Up  to  the  present,  the  succinate-hnked  reduction  of  mitochondrial 
DPN  has  exclusively  been  studied  by  measuring  the  increase  in  the  steady- 
state  level  of  endogenous  DPNH,  ensuing  upon  the  addition  of  succinate 
and  or  ATP.  One  obvious  limitation  of  this  system,  emerging  from  the 
above  reasoning,  may  be  that  any  increase  in  the  DPNH  level  that  one 
observes  is  a  resultant  of  two,  independent,  reaction  capacities;  on  one 
hand,  the  capacity  of  the  reaction(s)  feeding  in  electrons  from  succinate 
into  the  respiratory  chain  (Reaction  i  in  the  above  formulae),  and  on  the 
other,  the  capacity  of  the  chain  of  reactions  by  which  these  electrons  are 
transferred  from  their  point  of  entrance  to  oxygen  (Reactions  3  and  4). 
Clearly,  if  the  latter  capacity  is  equal  to  or  exceeds  the  former,  no  increase 
in  the  level  of  DPXH  may  be  expected  to  occur.  This  situation  may 
render  the  reproducibility  of  the  observations,  e.g.  from  one  tissue  or  one 
set  of  conditions  to  another,  dependent  on  irrelevant  circumstances,  and  in 
particular,  it  may  render  unrealistic  a  quantitative  evaluation  of  the 
number  of  high-energy  bond  equivalents  required  for  the  reduction  of  one 
molecule  DPX  bv  succinate.  Moreover,  using  the  above  test  system,  no 
conclusive  evidence  has  yet  been  presented  that  the  reducing  equivalents 
appearing  in  DPXH  actuallv  originate  from  succinate  and  not  from  some 
endogenous  substrate,  the  oxidation  of  which  has  been  facilitated,  in  a 
secondary  manner,  by  succinate  and /or  ATP. 

To  overcome  these  difficulties,  it  was  felt  desirable  to  devise  a  system 
in  which  the  DPX  reduced  by  succinate  was  continuously  reoxidized  by 
suitable  means,  e.g.  bv  wav  of  the  reversal  of  a  DPX'^-linked  dehydrogenase 
reaction.  The  reversal  of  the  /S-hydroxybutyric  dehydrogenase  reaction, 
consisting  of  a  reduction  of  acetoacetate  to  /3-hydroxybutyrate,  was 
considered  to  be  con^•enient  for  this  purpose,  since  this  is  the  only  known 
reaction  by  which  free  acetoacetate  can  be  metabolized  in  rat  liver  mito- 
chondria. It  was  in  fact  found  that  when  isolated  rat  liver  mitochondria 
were  incubated  in  the  presence  of  succinate  and  acetoacetate  under  aerobic 
conditions  and  in  the  absence  of  phosphate  acceptor,  there  occurred  a 
substantial  disappearance  of  acetoacetate  which  was  linear  with  time  and 
strictly  dependent  on  the  presence  of  succinate  (Fig.  10).  Furthermore, 
the  acetoacetate  reduction  was  completely  inhibited  by  2  niM  amytal, 
indicating  that  it  involved  an  electron  transfer  between  the  site  of  entrance 
of  electrons  from  succinate  into  the  respiratory  chain  and  DPX.  Replace- 
ment of  succinate  bv  malate,  with  or  without  amvtal,  resulted  onlv  in  an 

*  The  studies  reported  in  this  and  the  following  section  have  been  conducted 
in  collaboration  with  Drs.  G.  F.  Azzone  and  E.  C.  Weinbach. 


156  LARS   ERNSTER 

insignificant  reduction  of  acetoacetate,  probably  because  of  the  unfavour- 
able equilibrium  of  the  malate  +  acetoacetate  ^=i  oxaloacetate  + /S-hy- 
droxybutyrate  system.  This  finding,  together  with  the  amytal-sensitivity  of 
the  succinate-linked  reduction,  thus  clearly  eliminated  the  possibility  that 
malate  originating  from  succinate  rather  than  the  latter  itself  might 
constitute  the  reducing  agent  for  acetoacetate. 

As  could  be  expected  from  the  great  positive  diflFerence  in  redox 
potential  between  the  succinate /fumarate  and  DPNH/DPN  couples  (cf. 
[10]),  the  succinate-linked  reduction  of  acetoacetate  in  the  present  system 
was  strictly  dependent  on  an  active  oxidative  phosphorylation  coupled  to 


Malate 

-Succ.+  amytal 

No  substrate 

Malate 
+  amytal 


Succinate 


10  20 

Minutes 

Fig.  10.  Reduction  of  acetoacetate  by  succinate  in  rat  liver  mitochondria 
(Azzone,  Ernster,  and  Weinbach,  unpublished).  Each  flask  contained:  mito- 
chondria from  150  mg.  liver,  20  mM  glycylglycine  buffer,  pH  7*5,  8  mM  MgCla, 
62  mM  sucrose,  25  mM  KCl,  5  mM  P,,  5  mM  acetoacetate,  and,  when  indicated, 
10  mM  succinate,  10  mM  L-malate,  2  mM  amytal,  in  a  final  volume  of  i  ml.  Incuba- 
tion at  30'^  Acetoacetate  determined  according  to  Walker  [41]. 


the  terminal  oxidation  of  succinate.  Accordingly,  as  shown  in  Fig.  11,  the 
acetoacetate  reduction  was  abolished  by  dinitrophenol.  It  may  be  noticed 
that  half-inhibition  was  reached  at  a  concentration  of  about  5  x  io~^  M, 
which  is  considerably  below  that  required  for  a  corresponding  depression 
of  the  oxidative  phosphorylation.  This  finding  is  in  agreement  with  the  data 
of  Chance  and  Hollunger  [10],  from  their  spectrophotometric  studies  of 
the  succinate-linked  reduction  of  endogenous  DPN.  Since  dinitrophenol 
is  known  to  abolish  respiratory  control  at  a  concentration  lower  than  that 
needed  for  an  actual  depression  of  the  phosphorylating  capacity  [42,  43], 
these  data  indicated  that  the  reduction  of  acetoacetate  by  succinate  in  the 
present  system  was  dependent  not  only  on  an  active  oxidative  phos- 
phorylation but  also  on  a  state  of  respiratory  control,  the  latter  allowing 


FUN'CTIOX    OF    FLAVOEXZYMES    IN    ELECTRON    TRANSPORT 


O/ 


the  maintenance  of  adequate  levels  of  high-energy  intermediates  needed 
for  the  endergonic  reduction  of  DPN  by  succinate. 


5x10 

M  dmitrophenol 

Fig.  II.  Inhibition  of  succinate-linked  reduction  of  acetoacetate  by  2,4-dinitro- 
phenol  (Azzone,  Ernster,  and  Weinbach,  unpublished).  Each  flask  contained: 
mitochondria  from  400  mg.  liver,  10  mM  succinate,  5  mM  acetoacetate,  and 
dinitrophenol  as  indicated,  in  a  final  volume  of  2  ml.  Other  conditions  as  in  Fig.  10. 
Time  of  incubation,  20  min. 

TABLE  VIII 

Influence  of  Mg~~  .\nd  of  Terminal  Phosphate  Acceptor  on  the 
Succinate-linked  Reduction  of  Acetoacet.ate 

(Azzone,  Ernster  and  Weinbach,  unpublished) 

Each  Warburg  flask  contained:  mitochondria  from  250  mg.  liver,  20  niM  gly- 
cylglycine  bufi^er,  pH  7-5,  62  mM  sucrose,  50  mM  KCl,  10  mM  succinate,  5  mM 
acetoacetate,  15  mM  Pj,  and  where  indicated,  15  mM  AMP,  i  mM  .\TP,  15  mM 
glucose,  and  an  excess  of  yeast  hexokinase,  in  a  final  volume  of  2  ml.  Gas  phase, 
air;  in  centre  well,  02  ml.  2  M  KOH;  temperature,  30  .  Time  of  incubation, 
16  min. 


Additions 


8  niM  Mg 


Xo  Mg- 


—  JAcAc       Oxygen      „ ,  „      —  JAcAc       Oxygen      „,„ 

(/xmoles)      (/tatoms)        '  (/^tmoles)       (/xatoms) 


— 

2-6 

4-6 

AMP 

0 

II  -o 

1-63 

ATP,  hexokinase, 

glucose 

o- 1 

I  I  -2 

1-82 

8-7 
II-3 


1-79 


This  concept  was  further  emphasized  by  the  findings  recorded  in 
Table  \TII.  It  has  been  demonstrated  first  by  Baltscheffsky  [44]  that 
incubation  of  rat  liver  mitochondria  in  the  absence  of  ^Ig^~  abolishes  the 


158  LARS   ERNSTER 

requirement  for  phosphate  acceptor  in  maintaining  a  high  rate  of  respira- 
tion, without  parallel  loss  of  the  actual  phosphorylating  capacity.  Such  a 
"loose-coupling"  of  phosphorylation  from  respiration,  as  revealed  by  an 
increased  respiratory  rate  in  the  absence  of  phosphate  acceptor,  was  in  the 
present  case  accompanied  by  an  almost  complete  abolition  of  the  aceto- 
acetate  reduction.  The  phosphorylating  capacity  of  the  Mg++-deficient 
system  (measured  with  AMP  as  terminal  phosphate  acceptor)  was  the 
same  as  that  found  in  the  presence  of  Mg++.  As  anticipated,  no  aceto- 
acetate  reduction  took  place  in  the  presence  of  the  terminal  phosphate 

TABLE  IX 

Influence  of  Mg++  on  the  Succinate-linked  Reduction  of 
acetoacetate  under  various  conditions 

(Azzone,  Ernster  and  Weinbach,  unpublished) 

Experimental  conditions  as  in  Fig.  13,  except  that  mitochondria  from  300  (in 
Experiment  2,  200)  mg.  liver  were  added  per  flask. 


Experiment 

Additions 

No  Mg++ 

8  mM  Mg++ 

No. 

-  JAcAc 

(/xmoles) 

I 

— 

1-4 

4-0 

ATP  (i  mM) 

4-0 

3-8 

EDTA  (2  mM) 

I  -o 

4-4 

ATP  (i  mM),  EDTA  (2  mM) 

I  -o 

3-9 

2 

— 

06 

2-4 

ATP  (i  mM) 

2-7 

2-2 

Oligomycin  A  (i  /Ltg./ml.) 

0-7 

3-2 

ATP  (i  mM),  oligomycin  A  (i 

H-g-/rn\.) 

30 

3-2 

3 

NaF  (10  mM) 

0-4 
3-3 

3-9 

P;  omitted 

0-6 

4-2 

NaF  (10  mM),  Pj  omitted 

5-9 

acceptor  because  of  the  continuous  removal  of  the  high-energy  bonds 
generated  in  the  respiratory  chain.  These  findings  substantiate  the  above 
conclusion  that  "loosely-coupled"  respiration  cannot  give  rise  to  suc- 
cinate-linked DPN-reduction  even  though  the  phosphorylating  capacity 
of  the  mitochondria  remains  intact. 

Addition  of  ATP  to  the  Mg++-deficient  system  restored  the  aceto- 
acetate  reduction  (Table  IX),  but  this  effect  was  counteracted  by  EDTA, 
suggesting  that  it  was  dependent  on  endogenous  Mg++.  The  ATP  effect 
was  not  abolished  by  oligomycin  A,  and  thus  cannot  be  due  to  a 
supply  of  energy  to  the  respiratory  chain.  Furthermore,  10  mM  sodium 


FUNCTION    OF    FLAVOENZYMES    IN    ELECTRON    TRANSPORT 


159 


fluoride  gave  a  similar  efl^ect.  This  ATP-effect  may  be  analogous  to  the 
eff"ect  of  ATP  in  inducing  DPN-reduction  by  succinate,  recently  observed 
by  Chance  and  Hagihara  [45]  in  aged  pigeon-heart  mitochondria. 

It  was  brieflv  indicated  above  (Table  IX)  that  oligomycin  A  did  not 
inhibit  (in  fact  even  slightly  stimulated)  the  succinate-linked  reduction  of 
acetoacetate  in  the  present  system.  As  shown  in  Fig.  12,  this  compound 
was  also  able  to  restore  efficiently  acetocaetate  reduction  when  this  was 
suppressed  because  of  the  presence  of  a  terminal  phosphate  acceptor  (in 
this  case  ADP,  hexokinase  and  glucose).  Oligomycin  A  has  been  shown  by 


20 


16 


12  3  4  5 

/iq  Oligomycin  A 

Fig.  12.  Effect  of  oligomycin  A  on  succinate-linked  reduction  of  acetoacetate 
in  presence  of  terminal  phosphate  acceptor  (Azzone,  Ernster,  and  Weinbach, 
unpublished).  Experimental  conditions  as  in  .ATP-hexokinase-glucose  system  in 
Table  VIII. 


Lardy  et  al.  [46]  to  inhibit  mitochondrial  respiration  under  phosphorvlating 
conditions  but  not  if  the  phosphorylation  is  abolished  by  dinitrophenol ;  in 
extending  these  studies  we  found  that  oligomycin  A  onlv  inhibits  tightlv- 
coupled,  but  not  "loosely-coupled",  respiration.  Furthermore,  according 
to  Lardy  et  al.  [46],  oligomycin  A  also  strongly  inhibits  the  mitochondrial 
P,-ATP  exchange  and  dinitrophenol-induced  ATPase  reactions.  From 
these  observations,  oligomycin  A  appears  to  act  by  blocking  the  transfer  of 
phosphate  from  the  primary  high-energy  bonds  to  ADP.  This  mode  of 
action  fits  logically  with  the  present  findings  that  oligomycin  A  removed 
the  phosphate  acceptor  efi^ect  from  the  succinate-linked  reduction  of 
acetoacetate.  What  is  more  interesting,  however,  is  that  the  transfer  of 
energy  from  the  sites  of  the  succinate-linked  phosphorylations  to  the  site 


l6o  LARS   ERNSTER 

where  it  is  utilized  for  DPN-reduction  is  apparently  not  affected  by  the 
oligomycin  A-block.  Accepting  the  above  mode  of  action  of  oligomycin  A, 
this  would  mean,  either  that  this  transfer  can  take  place  directly,  without 
the  intermediary  of  ATP,  or  that  it  proceeds  via  a  fraction  of  intramito- 
chondrial  ATP  which  is  not  available  to  hexokinase  and  glucose  and  whose 
interaction  with  the  primary  high  energy  intermediates  is  not  blocked  by 
oligomycin  A. 

Based  on  the  conclusion,  reached  above,  that  "loosely-coupled" 
respiration  could  not  contribute  energetically  to  the  succinate-linked 
DPN-reduction,  it  was  considered  possible  to  estimate  the  stoicheiometry 
of  the  DPN-reducing  system  by  measuring  the  difference  in  rate  of 
succinate  oxidation,  observed  in  the  presence  and  absence  of  added 
acetoacetate.   It  may  be  assumed  that  the  respiration  observed  in  the 

TABLE  X 

Stimulation  of  Succinate  Oxidation  due  to  Reduction  of 
Acetoacetate  in  Mitochondria  in  Controlled  State 

(Azzone,  Ernster,  and  Weinbach,  unpublished) 

Experimental  conditions  as  in  Table  VIII,  except  that  mitochondria  from 
400  mg.  liver  and  25  mM  P,  were  added  per  flask.  All  flasks  contained  8  mM  MgCl2. 
Oligomycin  A,  when  present,  was  added  in  a  concentration  of  i  /xg./ml. 


Additions 


O2  consumption,  /xatoms  /xmoles  AcAc 


With         Without 
AcAc  AcAc 


JO,  Reduced 


None  9 '78  7-68  2-10  4-7 

Oligomycin  A  9-11  6-71  2-40  5-7 

ATP,  hexokinase,  glucose  i9"3  18-5  o-8  o 
Oligomycin  A,  ATP, 

hexokinase,  glucose  iO'i3  7'i7  2-96  5-4 

absence  of  both  phosphate  acceptor  and  acetoacetate,  being  "loosely- 
coupled",  cannot  contribute  energy  to  the  reduction  of  DPN;  and  conse- 
quently, that  addition  of  acetoacetate  to  the  phosphate  acceptor-free 
system  would  result  in  an  increase  of  the  respiratory  rate,  in  a  phosphate 
acceptor-like  manner,  to  the  extent  it  "trapped"  energy  from  the  respira- 
tory chain  by  reoxidizing  endergonically  reduced  DPN.  Data  presented  in 
Table  X  are  the  mean  values  of  duplicate  runs  and  are  reasonably  precise. 
It  is  seen  that  addition  of  acetoacetate  to  the  phosphate  acceptor-free 
system  resulted  in  an  increase  in  oxygen  consumption  by  2-10  /xatoms, 
and  this  was  accompanied  by  a  disappearance  4-7  /xmoles  of  acetoacetate. 
In   the   presence   of  oligomycin  A   (which  slightly  stimulated  both  the 


FUN'CTION    OF    FLAVOENZYMES    IN    ELECTRON    TRANSPORT  l6l 

respiratory  increase  and  the  acetoacetate  reduction)  the  corresponding 
values  were  2  •  40  /xatoms  and  5  •  7  /xmoles,  respectively.  Addition  of  ATP, 
hexokinase  and  glucose,  which  abolished  acetoacetate  reduction,  also 
significantly  diminished  the  respiratory  stimulation  due  to  acetoacetate, 
to  the  value  of  o  •  8  ^uatom ;  and,  finally,  addition  of  oligomycin  A  to  the 
hexokinase-glucose  system  restored  both  respiratory  stimulation  and 
acetoacetate  reduction  to  about  their  original  levels,  2-96  /xatoms  and  5-4 
/xmoles,  respectively.  Thus,  in  all  three  cases  where  acetoacetate  reduction 
occurred,  there  occurred  a  respiratory  stimulation  as  well,  the  rate  of 
which  was  approximately  o  •  5  /xatom  oxygen  per  /xmole  acetoacetate 
reduced.  Assuming  a  P/0  ratio  of  2  for  the  aerobic  oxidation  of  succinate 
to  fumarate,  this  implies  a  ratio  of  one  high  energy  bond  equivalent  per 

TABLE  XI 

Effect  of  Respiratory  Chain  Inhibitors  on  Succinate-linked 
Reduction  of  Acetoacetate  in  Rat  Liver  Mitochondria 

(Azzone,  Ernster,  and  Weinbach,  unpublished) 

In  each  flask:  5  niM  acetoacetate,  25  mM  succinate,  62  m.M  sucrose,  50  niM  KCl, 
20  mM  tris  buflfer,  pH  7  5,  8  mM  MgCL,  mitochondria  from  400  mg.  liver. 
When  indicated :  5  m.M  .ATP,  2  mM  amytal,  i  ■  25  /tg./ml.  antimycin  A,  1-25  /xg./ml. 
oligomycin  A,  0-5  mM  KCN.  P'inal  \olume,  2  ml.  Cjas  phase,  air.  Temperature, 
30".  Time  of  incubation,  20  min. 


.Additions 


J.-\cAc  (/xmoles) 


ATP  +ATP 


None  3-4  3-2 

Antimycin  A  00 

Cyanide  o  o 

No  as  gas  phase  o  o 

Oligomycin  .A  3-9  40 

molecule  of  acetoacetate  reduced.  This  value  is  in  agreement  with  that 
envisaged  by  the  reaction  mechanism  for  the  succinate-linked  reduction 
of  DPN,  discussed  above  (cf.  Reactions  i  and  2,  and  Fig.  9),  and  is 
considerably  lower  than  those  previously  arrived  at  by  Chance  and 
Hollunger  [10]  and  by  Chance  [47]. 

As  shown  in  Table  XI,  the  succinate-linked  reduction  of  acetoacetate 
in  the  present  system  was  completely  abolished  by  the  respiratory  inhibi- 
tors, antimycin  A  and  cyanide,  as  well  as  in  anaerobiosis.  Added  ATP, 
which  had  no  effect  on  the  aerobic  system,  did  not  remove  these  inhibi- 
tions. Hence,  in  contrast  to  the  succinate-linked  reduction  of  DPN  in  the 
high-energy  phosphate-depleted  mitochondria  (cf.  Fig.  10),  the  succinate- 
linked  reduction  of  acetoacetate  in  non-depleted  mitochondria  appears  to 

VOL.  II. M 


I 62  LARS    ERNSTER 

require  an  intramitochondrially  generated  supply  of  high-energy  com- 
pounds. As  will  be  indicated  below,  this  is  probably  due  to  compart- 
mentation  phenomena  in  the  intact  mitochondrion. 

It  was  of  special  interest  to  establish  whether  the  inhibitory  effect  of 
antimycin  A  was  merely  due,  like  those  of  cyanide  and  anaerobiosis,  to  a 
general  block  of  the  energy-generating  system,  or  whether  this  compound 
inhibited  the  succinate-linked  reduction  of  DPN  per  se.  The  latter  con- 
clusion has  recently  been  reached  by  Chance  and  Hollunger  [lo]  and  led 
them  to  postulate  that  the  succinate-linked  DPN-reduction  involves 
cytochrome  b.  The  reaction  scheme  proposed  by  us  (cf.  Fig.  9)  is  not 
compatible  with  such  a  conclusion. 

TABLE  XII 

Phosphorylation  Coupled  to  the  Antimycin  A  Insensitive  Oxidation 
OF  Succinate  by  Ferricyanide  in  Rat  Liver  Mitochondria 

(Azzone,  Ernster  and  Weinbach,  unpublished) 

In  each  flask:  mitochondria  from  300  mg.  liver,  50  mM  KCl,  20  mM  glycyl- 
glycine,  pH  7-5,  12-5  mM  P^,  8  mM  MgCL,  10  mM  succinate,  20  mM  ferricyanide, 
0-5  mM  KCN,  I  niM  ATP,  15  mM  glucose,  hexokinase,  and,  when  indicated, 
I  /xg./ml.  antimycin  A.  Final  volume,  2  ml.  Incubation  at  30    for  20  min. 


Without 
antimycin 

A 

With 
antimycin  A 

jLimoles  (Fe(CN)6)''+  reduced 
/xmoles  P,  esterified 
P/2^- 

i6-o 

5-5 
069 

i8-s 
4-2 
046 

Copenhaver  and  Lardy  [48]  reported  in  1952  that  oxidation  of  succinate 
in  rat  liver  mitochondria  with  ferricyanide  as  terminal  electron  acceptor  in 
the  presence  of  cyanide  gave  rise  to  a  phosphorylation  with  a  VJ2e  ratio  of 
0-6,  and  that  both  the  oxidation  and  the  coupled  phosphorylation  were 
insensitive  to  antimycin  A.  These  findings  were  now  confirmed  (Table 
XII)*  and,  using  this  system,  it  could  be  shown  that  the  succinate-linked 

*  Subsequent  to  the  studies  of  Copenhaver  and  Lardy  [48],  Pressman  [49] 
reported  that,  in  his  hands,  antimycin  A  did  inhibit  the  oxidation  of  succinate  by 
ferricyanide.  In  an  attempt  to  explore  the  reason  for  this  discrepancy,  we  found 
(cf.  also  Discussion,  p.  168)  that  the  antimycin  A-sensitivity  of  this  system  is 
dependent  on  the  Pj/ATP  ratio  prevailing  in  the  incubating  medium;  high  concen- 
trations of  Pj  favour  insensitivity,  and  high  concentrations  of  ATP  favour  sensi- 
tivity, to  antimycin  A.  Furthermore,  both  Pj  and  ATP  were  found  greatly  to 
stimulate  ferricyanide  reduction.  These  findings  are  strikingly  parallel  to  those 
reported  by  Hatefi  [50]  concerning  the  reduction  of  coenzyme  Q  in  beef  heart 
mitochondria  and  the  antimycin  A-sensitivity  of  this  reaction. 


FUNCTION   OF   FLAVOENZYMES    IN   ELECTRON   TRANSPORT  1 63 

TABLE  XIII 

Insensitivitv  of  Succinate-linked  Reduction  of  Acetoacetate  to 

axtimycin   a 

(Azzone,  Ernster,  and  Weinbach,  unpublished) 

In  each  flask:  mitochondria  from  300  mg.  liver,  50  m.M  KCl,  20  m.M  glycyl- 
glycine,  pH  7-5,  12-5  mM  Pj,  8  mM  MgCL,  10  mM  succinate,  3  mM  acetoacetate, 
and,  where  indicated,  20  mM  ferricyanide,  i  /xg./ml.  antimycin  A  and  0-5  niM 
KCN.  Final  volume,  2  ml.  Incubation  at  30"  for  20  min. 

Additions  /tmoles  AcAc  reduced 

None  2-6 

KCN  0-3 

KCN  +  (Fe(CN)6)'+  2-0 

KCN  +  (Fe(CN)6)'+  +  Antimycin  A  2-4 

reduction    of   acetoacetate    was    completely    insensitive    to    antimycin   A 
(Table  XIII). 


AMINATIVE    REDUCTION    OF    a-KETOGLUTARATE    BY    SUCCINATE 

Before  terminating  my  report,  let  me  briefly  show  you  some  pre- 
liminary data  obtained  with  another  dehydrogenase,  glutamic  dehydro- 
genase, as  a  trapping  system  for  the  DPNH  generated  by  succinate-linked 
DPN-reduction.  This  system  was  very  similar  to  that  used  by  Hunter  and 
Hixon  in  1949  [51]  for  the  demonstration  of  a-ketoglutarate-linked  sub- 
strate-level phosphorylation.  It  consisted  of  liver  mitochondria  incubated 
under  anaerobic  conditions  in  the  presence  of  x-ketoglutarate  and  am- 
monia. In  this  system,  as  Hunter  and  Hixon  ha\e  shown,  the  following 
reaction  takes  place : 

2a-ketoglutarate  -h  NH3  +  ADP  +  P, > 

glutamate  +  succinate  +  COo  +  ATP         (5) 

However,  unlike  Hunter  and  Hixon 's  system,  the  present  one  was  not 
supplemented  with  a  terminal  phosphate  acceptor.  It  was  thus  expected 
that  the  ATP  and  succinate  formed  in  the  a-ketoglutarate  oxidation  would 
give  rise  to  a  reduction  of  DPN,  after  which  the  DPNH  so  generated  would 
reduce  another  molecule  of  o^-ketoglutarate  ( +  ammonia)  to  glutamate. 
This  reaction  sequence  may  be  written  as  follows : 

succinate  +  ATP  +  DPN  ^=i  fumarate  +  ADP  +  P,  +  DPNH  (6) 

DPNH  +  a-ketoglutarate  +  NH3  t "  DPN  +  glutamate  (7) 

The  net  reaction  of  Reactions  57  is : 

3a-ketoglutarate  -I-  2NH3 >  2  glutamate  +  fumarate  +  CO2. 


164  LARS    ERNSTER 

Since  Reaction  6,  but  not  Reaction  5,  is  inhibited  by  amytal,  it  could  be 
expected  that,  if  Reactions  6  and  7  were  occurring,  the  disappearance  of 
a-ketoglutarate  would  be  diminished  in  the  presence  of  amytal.  The  data 
in  Table  14  show  that,  indeed,  there  occurred  an  amytal-sensitive  a-keto- 
glutarate utilization;  however,  unexpectedly,  this  was  dependent  on  the 
presence  of  externally  added  ATP.  As  anticipated,  on  the  other  hand, 
the  amytal-sensitive  part  of  the  a-ketoglutarate  utilization  could  be 
completely  abolished  by  hexokinase  and  glucose.  Further  data,  not  included 
in  Table  XIV,  show  that  the  CO.,  production,  in  contrast  to  the  a-keto- 
glutarate utilization,  was  not  influenced  by  amytal.  All  these  data  are  thus 
consistent  w^ith  the  above  reaction  sequence. 

TABLE  XIV 

ATP-DEPENDENT    AmINATIVE    REDUCTION    OF    a-KETOGLUTARATE   BY 

Succinate  in  Anaerobic  Mitochondria 
(Azzone,  Ernster,  and  Weinbach,  unpublished) 

In  each  flask:  0-05  M  KCl,  0-02  M  glycylglycine  buffer,  pH  7-5,  8  mM  MgClg, 
10  mM  Pj,  5  mM  NH4CI,  5  mM  a-ketoglutarate,  mitochondria  from  150  mg.  liver. 
When  indicated :  2  mM  amytal,  5  or  i  mM  ATP  (without  or  with  hexokinase- 
glucose).  Final  volume,  2  ml.  Temperature  30^.  Time  of  incubation,  20  min.  Gas 
phase.  No 


Additions 

^moles  a-ketoglutarate  consumed 

Without          With 
amytal          amytal 

None 
ATP 
ATP,  hexokinase,  glucose 

3-2 
6-3 

3-8 

3-4 
3-7 
3-9 

However,  regarding  the  requirement  for  external  ATP,  it  is  obvious 
that,  from  the  point  of  view  of  energy-transfer,  this  system  is  funda- 
mentally different  from  the  succinate-linked  reduction  of  acetoacetate.  The 
role  of  ATP  cannot  be  that  of  merely  "tightening"  the  mitochondrial 
structure  (as  in  the  case  of  the  Mg+ +-deficient  acetoacetate  system,  cf. 
Table  IX),  since,  in  the  present  case,  oligomycin  A  counteracted  the  ATP 
effect.  It  was  also  found  in  preliminary  experiments  that  ATP  could  be 
replaced  by  catalytic  amounts  of  AMP.  This  eliminates  the  possibility  that 
the  a-ketoglutarate-linked  phosphorylation  might  not  be  able  to  furnish 
energy  to  the  succinate-linked  reduction  of  DPN.  Whether  this  discrepancy 
in  ATP  requirement  between  the  acetoacetate  and  a-ketoglutarate  systems 
is  due  to  the  different  dehydrogenases,  /S-hydroxybutyric  and  glutamic,  or 
to  the  different  sites  of  phosphorylation,  respiratory  chain  and  substrate 
level,  involved  in  the  two  systems,  cannot  be  decided  at  this  time.  In  any 


FUNCTION  OF  FLAVOENZYMES  IN  ELECTRON  TRANSPORT         1 65 

case,  it  is  indicative  of  a  complex  pattern  of  compartmentation  of  energy- 
transfer  routes  within  the  mitochondria.  Similar  indications  have  recently 
been  obtained  in  our  laboratory  along  other  lines  of  approach  [52,  ^t,]. 


CONCLUSIONS 

The  main  conclusions  of  the  second  section  of  this  paper  may  be 
summarized  as  follows : 

1.  In  rat  liver  mitochondrial  preparations  depleted  of  high-energy 
phosphate  by  preincubation  with  arsenate  and  dicoumarol  or  dinitrophenol, 
the  oxidation  of  succinate  is  greatly  stimulated  by  ATP.  Parallel  to  the 
respiratory  stimulation,  the  mitochondrial  pyridine  nucleotides  become 
reduced  to  a  slight  but  significant  extent.  It  is  concluded  that  the  ATP- 
induced  activation  of  succinate  oxidation  and  the  ATP-induced  reduction 
of  DPN  by  succinate  involve  a  common  high-energy  intermediate,  which 
consists  of  a  phosphorylated,  reduced  electron  carrier,  and  whose  formation 
at  the  expense  of  ATP  and  succinate  is  not  inhibited  by  uncoupling  con- 
centrations of  dinitrophenol  and  dicoumarol.  The  reduction  of  DPN  by 
succinate  is  thought  to  involve  a  re\ersal  of  the  DPN-flavin-linked  oxida- 
tive phosphorylation,  and  its  extent  in  a  respiring  system  is  consequently 
a  resultant  of  the  rate  at  which  electrons  derived  from  succinate  enter  the 
respiratory  chain  and  the  rate  at  which  these  electrons  are  transferred  from 
their  site  of  entrance  towards  oxygen. 

2.  Intact  liver  mitochondria  incubated  under  aerobic  conditions  in  the 
absence  of  phosphate  acceptor  catalyze  a  reduction  of  acetoacetate  to  ^- 
hydroxybutyrate,  coupled  to  the  oxidation  of  succinate  to  fumarate.  The 
reaction,  which  provides  conclusive  evidence  for  a  substantial  transfer  of 
hydrogen  from  succinate  to  mitochondrial  DPN,  is  completely  inhibited 
by  amytal,  as  well  as  by  low  concentrations  of  dinitrophenol,  addition  of 
terminal  phosphate  acceptor,  or  omission  of  Mg^  *.  The  phosphate 
acceptor  effect  is  removed  by  oligomycin  A,  which  presumablv  acts  by 
blocking  the  transfer  of  phosphate  between  the  primary  high-energy 
intermediates  and  ADP.  In  the  Mg+ ^-deficient  system  acetoacetate 
reduction  is  restored  by  ATP  and  by  sodium  fluoride.  The  succinate- 
linked  acetoacetate  reduction  is  also  suppressed  by  antimycin  A,  cyanide, 
or  in  the  absence  of  oxygen ;  under  these  conditions,  the  reduction  is  not 
restored  by  added  ATP.  Ferricyanide,  in  the  presence  of  cyanide,  allows 
phosphorylation  coupled  to  succinate  oxidation,  and  also  restores  aceto- 
acetate reduction.  Under  appropriate  conditions,  both  the  coupled  phos- 
phorylation and  the  acetoacetate  reduction  of  the  ferricyanide  svstem  are 
insensitive  to  antimycin  A.  Respiration  with  succinate  as  substrate  in  the 
absence  of  phosphate  acceptor  is  stimulated  by  acetoacetate  and  the  stimu- 
lation corresponds  to  0-5  /^atom  oxygen  per  /xmole  acetoacetate  reduced. 


1 66  LARS   ERNSTER 

From  these  findings  it  is  concluded  that  the  reduction  of  acetoacetate  by 
succinate,  catalyzed  by  tightly-coupled  liver  mitochondria,  involves  a  reversal 
of  the  DPN-flavin-linked  oxidative  phosphorylation ;  that  the  energy 
required  for  this  process  is  equivalent  to  one  high-energy  bond  per 
molecule  of  acetoacetate  reduced;  that  this  energy  can  be  supplied  by  one  or 
both  of  the  two  terminal  respiratory  chain  phosphorylations  without  the 
intermediary  of  extramitochondrial  ATP;  and  that  the  succinate-linked 
reduction  of  mitochondrial  DPN  does  not  involve  the  antimycin  A- 
sensitive  site  of  the  respiratory  chain. 

3 .  Liver  mitochondria  under  anaerobic  conditions  catalyze  an  aminative 
reduction  of  a-ketoglutarate  to  glutamate,  coupled  to  the  oxidation  of 
succinate  to  fumarate,  which  proceeds  at  the  expense  of  high-energy 
phosphate  generated  in  the  a-ketoglutarate-linked  substrate-level  phos- 
phorylation. Some  preliminary  observations  are  presented  which  suggest 
the  existence  of  a  complex  pattern  of  compartmentation  of  mitochondrial 
energy-transfer  routes. 

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29,  392  (1958). 

39.  Grabe,  B.,  Biochim.  biophys.  Acta  30,  360  (1958). 

40.  Lindberg,  O.,  Grabe,  B.,  Low,  H.,  Siekevitz,  P.,  and  Ernster,  L.,  Acta  chem. 
scaud.  12,  598  (1958). 

41.  Walker,  P.  G.,  Biochem.  J.  58,  699  (1954). 

42.  Chance,  B.,  /;/  "  Ciba  Foundation  Symposium  on  Regulation  of  Cell  Meta- 
bolism"". J.  and  A.  Churchill  Ltd.    London,  91  (1959). 

43.  Azzone,  G.  F.,  Eeg-Olofsson,  O.,  Ernster,  L.,  Luft,  R.,  and  Szabolcsi,  G., 
Exp.  Cell  Res.  22,  415  (1961 ). 

44.  Baltscheffsky   H.,  Biochim.  biophys.  Acta  25,  382  (1957). 

45.  Chance,  B.,  and  Hagihara,  B.,  Biochem.  biophys.  Res.  Conini.  3,  6  (i960). 

46.  Lardy,  H.  A.,  Johnson,  D.,  and  McMurrav,  W.  C,  Arch.  Binchcm.  BiopJivs. 
78,  587  (1958). 

47.  Chance,  B.,  Biochem.  biopliys.  Res.  Co?}im.  3,  10  (i960). 

48.  Copenha^'er,  J.  H.,  and  Lardy,  H.  A.,jf.  biol.  Chem.  195,  225  (1952). 

49.  Pressman,  B.  C,  BiocJiim.  biophys.  Acta  17,  273  (1955). 

50.  Hatefi,  Y.,  Biochitu.  biopliys.  Acta  31,  502  (1959). 

51.  Hunter,  F.  E.,  and  Hixon,  W.  S.,  7-  biol.  Cheru.  l8l,  67  (1949). 

52.  Azzone,  G.  F.,  and  Ernster,  l^.,J.  biol.  Cheru.  236,  1501  (1961). 

53.  .Azzone,  G.  F.,  and  Ernster,  L.,_7.  biol.  Chem.  236,  1510  (1961). 

Discussion 

Williams:  I  should  like  to  ask  Dr.  Ernster  why  he  thinks  pyridine  nucleotide 
reduction  is  necessary  in  the  ATP-activating  succinate  respiration  ?  I  ask  this 
question  for  two  reasons,  first :  in  his  own  results  respiration  in  fact  seemed  to  be 
active  at  a  time  when  little  reduction  of  pyridine  nucleotide  had  taken  place; 
secondly,  because  in  collaborative  experiments  with  Dr.  Chance  this  summer  in 
Philadelphia,  we  were  also  able  to  reactivate  the  respiration  with  ATP  while  seeing 
essentially  no  change  in  the  steady  states  of  the  pyridine  nucleotides.  You  didn't 
speculate  on  the  reason  why  you  needed  pyridine  nucleotide  reduction. 

Ernster:  We  do  not  mean  that  we  need  pyridine  nucleotide  reduction.  Our 
point  is  that  the  two  mechanisms:  activation  of  succinate  oxidation  and  succinate- 
linked  DPX-reduction  may  be  correlateti  in  the  sense  that,  during  the  initiation  of 
succinate  oxidation,  a  reduced  high-energy  intermediate  is  generated  at  the  level 
of  the  respiratory  chain,  and  that  this  intermediate  is  identical  with,  or  in  close 
relationship   to,    the   high-energy   intermediate   involved   in   the   first   respiratory 


1 68 


LARS   ERNSTER 


chain  phosphorylation.  Then,  depending  on  how  large  the  electron  flux  is  toward 
oxygen,  you  may  or  may  not  get  a  DPN-reduction. 

Chance:  I  would  like  to  congratulate  Dr.  Ernster  on  the  excellent  results  he 
has  got  in  spite  of  the  very  difficult  experimental  problem  of  using  acetoacetate 
reduction  as  the  assay  for  the  intramitochondrial  reduction  of  pyridine  nucleotides. 
First,  we  agree  completely  on  the  amytal-sensitivity,  provided  ATP  is  used  as  the 
energy  source.  Secondly,  endogenous  substrate  is  a  real  factor  to  be  considered. 
Thirdly  it  wasn't  clear  to  me  in  the  experiments  of  ferricyanide  reduction  whether 
or  not  that  system  had  an  exogenous  ATP  requirement.  If  it  had  I  would  have 
expected  it  to  have  shown  antimycin-sensitivity. 

Ernster:  In  the  ferricyanide  experiment  there  was  no  ATP  added  and  no 
ATP  requirement.  These  conditions  were  apparently  suited  for  producing  suffi- 
cient energy  for  the  reduction  of  pyridine  nucleotide,  by  way  of  the  phosphoryla- 
tion occurring  between  succinic  dehydrogenase  and  ferricyanide.  That  phos- 
phorylation was  not  sensitive  to  antimycin  A. 

Chance:  It  is  possible  that  we  are  in  agreement  on  the  antimycin  A  because 
we  could  do  the  ferricyanide  experiment  in  the  way  that  requires  ATP. 


TABLE  I 

Effect  of  P,  and  ATP  on  Antimycin  A-Sensitivity  of 
Reduction  of  Ferricyanide  by  Succinate 

The  system  contained:  mitochondria  from  300  mg.  rat  liver,  100  jumoles  KCl, 
40  |umoles  glycylglycine  buffer  pH  7-5,  16  /j.moles  MgCl.,,  20  fxmoles  succinate, 
30  /Limoles  ferricyanide,  i  jumole  KCN,  and,  when  indicated,  i  ng.  antimycin  A, 
in  a  final  volume  of  2  ml.  Incubation  for  20  min.  at  30  . 


Additions  (/xmoles) 


Ferricyanide  reduced  (/u,moles) 


ATP 


■Antimycin  A 


+  Antimycin  A 


15 
60 

15 

60 


10 
10 

10 


10-3 
19-8 
21  -9 
16 -5 

22-3 
29-7^ 


IO-6 

20 -2 

24-1 

2-8 
22-3 

28-8^ 


*  Complete  reduction. 


Ernster:  Yes,  I  can  see  that.  Let  me  show  a  slide  here  which  illustrates  how 
complicated  this  system  is  (Table  I).  It  can  be  seen  that  the  ferricyanide  reduction 
by  succinate  may  or  may  not  be  antimycin  A  sensitive  depending  on  the  concentra- 
tions of  phosphate  and  ATP;  and  furthermore,  that  both  phosphate  and  ATP 
stimulate  ferricyanide  reduction,  in  an  additive  manner.  I  can't  explain  these  data 
but  they  do  illustrate,  I  think,  the  complexity  of  the  system. 


Coupling  of  Reduced  Pyridine  Nucleotide  Oxidation  to 
the  Terminal  Respiratory  Chain 

T.    E.    CONOVER* 

The   Wenner-Gren  Institute  for  Experimental  Biology, 
Lhiiversity  of  Stockholm,  Szceden 

As  was  reported  previously  [9],  a  soluble  enzyme  has  been  isolated 
which  can  couple  the  oxidation  of  both  TPNH  and  DPNHf  to  the 
reduction  of  a  number  of  dyes  and  other  electron  acceptors.  This  enzyme, 
which  has  been  called  "  DT  diaphorase  "  from  its  lack  of  specificity  towards 
the  pyridine  nucleotides,  has  been  found  in  all  cellular  fractions  examined, 
but  exists  predominately  in  the  cytoplasm.  The  properties  of  this  enzyme 
and  its  interaction  with  various  quinones  and  with  the  respiratory  chain  of 
mitochondria  have  been  studied  at  some  length.  In  this  paper  some  of 
these  studies  will  be  reported  with  the  hope  of  drawing  a  possible  inference 
as  to  the  role  of  this  abundant  enzyme  in  the  cell. 

The  importance  of  the  level  of  extramitochondrial  or  cytoplasmic 
reduced  pyridine  nucleotide  in  the  control  and  regulation  of  metabolism 
and  svnthesis  has  been  pointed  out  by  Krebs  [14],  Dickens  [6],  and  others. 
The  mechanism  of  the  regulation  of  the  levels  of  these  reduced  pyridine 
nucleotides,  however,  remains  incompletely  understood.  This  is  particularly 
true  in  the  case  of  TPNH,  a  substance  which  is  essential  for  cellular  syn- 
thetic reactions.  It  is  without  question  that  a  most  important  point  in  the 
regulation  of  these  levels  of  reduced  pyridine  nucleotide  is  the  control  of 
their  mitochondrial  oxidation.  Since  the  DT  diaphorase  reacts  with  both 
DPNH  and  TPXH  and  is  abundantly  present  in  the  cytoplasm,  the  ques- 
tion of  whether  this  enzyme  functions  in  the  oxidation  of  cytoplasmic 
reduced  pvridine  nucleotide  was  carefullv  considered. 

If  freshly  prepared  mitochondria  from  rat  liver  were  incubated  with 
soluble  cytoplasm  from  rat  liver  prepared  by  centrifugation  at  105  000  x  g 

*  Fellow  of  the  National  Foundation,  New  York,  N.Y.  Present  address : 
Division  of  Nutrition  and  Physiology,  The  Public  Health  Research  Institute  of  the 
City  of  New  York,  Inc.,  New  York,  N.Y. 

t  Abbreviations :  TPNH,  reduced  triphosphopyridine  nucleotide ;  TPN, 
triphosphnpyridine  nucleotide;  DPNH,  reduced  diphosphopyridine  nucleotide; 
DPN,  diphosphopyridine  nucleotide;  ATP,  adenosine  triphosphate;  DCPIP, 
2,6-dichlorophenolindophenol. 


170  T.    E.    CONOVER 

and  with  added  glucose-6-phosphate  and  TPN,  very  little  consumption 
of  oxygen  was  observed,  as  is  shown  in  Fig.  i.  This  is  due  to  the  fact 
that,  as  has  been  reported  previously  by  Pullman  and  Racker  [16],  TPNH, 
which  is  formed  from  the  oxidation  of  glucose-6-phosphate,  is  not  readily 
oxidized  by  mitochondria.  If  vitamin  K3  in  low  concentration  was  added, 
however,  there  was  initiated  a  rapid  oxygen  uptake,  indicating  a  rapid 


u 

,  1 

12 

/ 

10 

/ 

8 

/ 

6 

/ 

A 

-        / 

2 

"    /                        ^v^-^<^^  ^ 

zi^d2==^?— i> — ^?      ^ 

10        15 
minutes 


20 


Fig.  I.  Oxidation  of  glucose-6-phosphate  by  liver  mitochondria  in  the 
presence  of  soluble  cytoplasm  and  vitamin  K3.  The  complete  system  contained 
per  Warburg  vessel  i  -o  /^tmoles  TPN,  20  jumoles  glucose-6-phosphate,  50  jumoles 
tris  buffer  (pH  7-4),  30  /^moles  orthopbosphate  (pH  7  "4),  10  /imoles  MgCl,, 
5  /tmoles  adenosine  triphosphate,  60  ;u,moles  glucose,  an  excess  of  yeast  hexokinase, 
225  /xmoles  sucrose,  0-03  /tmole  vitamin  K^,  dialyzed  supernatant  fluid  centri- 
fuged  at  105  000  X  g  from  450  mg.  rat  liver,  and  mitochondria  from  500  mg.  rat 
liver.  Final  volume,  3-0  ml.  Temperature,  30  .  Reading  begun  after  5  min. 
thermoequilibration. 

1 .  Complete  system. 

2.  No  vitamin  Kg. 

3.  No  mitochondria. 


oxidation  by  the  mitochondria  of  the  TPNH  formed  in  the  incubation 
medium. 

This  respiration  was  insensitive  to  Amytal  but  was  inhibited  by  Anti- 
mycin  A  and  cyanide  (Table  I).  Most  importantly,  it  was  observed  to  be 
sensitive  to  dicoumarol  at  concentrations  of  io~^  M  or  less,  which  is  a  very 
characteristic  property  of  DT  diaphorase  [9]. 

The  implication  that  DT  diaphorase  was  involved  in  the  oxidation  of 
extramitochondrial  TPNH  in  this  system  was  supported  by  the  duplication 
of  this  system  with  isolated  enzymes.  Mitochondria  freshly  prepared  from 


COUPLING    OF   REDUCED    PYRIDINE   NUCLEOTIDE   OXIDATION 

TABLE  I 


171 


EiTECT  OF  Some  Inhibitors  on  Vitamin  Kj-Stimulated  Oxidation  of  Glucose- 
6-Phosphate  by  Mitochondria  and  Soluble  Cytoplasm 


Additions  (+  )  or  omissions  ( — ) 


Relative  oxygen  consumption 


Complete  system 

+   2   X    10  ^  m  Amytal 
+   I  ij.g.  Antimvcin  A 
+    10-3  ^i  J^C^T 

+   10  "^  m  dicoLimarol 
—  vitamin  K., 


100 
109 

39 
24 

43 
31 


Conditions   as  in   Fig.    i.   Respiration  of  complete  system  was  10-12   /^atoms 
oxygen/20  min. 


minutes 


Fig.  2.  Mitochondrial  oxidation  of  extramitochondrial  TPNH  mediated  by 
DT  diaphorase  and  vitamin  K;j.  The  complete  system  contained  per  Warburg 
vessel  0-5  jLimole  TPN,  20  /xmoles  glucose-6-phosphate,  i  unit  glucose-6-phosphate 
dehydrogenase  (Sigma),  20  |umoles  tris  buffer  (pH  7-4),  12  /imoles  orthophosphate 
(pH  7-4),  4  /umoles  MgCl.^,  2  /tmoles  adenosine  triphosphate,  24  /tmoles  glucose, 
an  excess  of  yeast  hexokinase,  50  /tmoles  sucrose,  mitochondria  from  200  mg. 
rat  liver,  o- 01  /nmole  vitamin  K^,,  and  an  amount  of  450-fold  purified  DT  diaphorase 
(together  with  i  mg.  serum  albumin)  capable  of  reducing  i  /^miole  vitamin  K3 
per  min.  Final  volume,  i  o  ml.  Temperature  2°-  Reading  begun  after  6  min. 
thermoequilibration.  [3] 

1.  Complete  system. 

2.  No  DT  diaphorase. 

3.  No  DT  diaphorase,  no  vitamin  Kg. 

4.  No  mitochondria. 


1 72  T.    E.    CONOVER 

rat  liver  were  incubated  in  an  isotonic  buffered  medium  containing  TPN, 
glucose-6-phosphate,  purified  Zwischenferment,  phosphate,  Mg  +  +,  ATP, 
hexokinase,  and  glucose.  As  is  shown  in  Fig.  2  the  extramitochondrially- 
generated  TPNH  was  not  oxidized  under  these  conditions  to  any  ap- 
preciable extent  by  the  mitochondria  in  agreement  with  the  previous 
experiment.  Addition  of  vitamin  K3  produced  a  two-  to  threefold  stimula- 
tion of  the  total  respiration.  Addition  of  purified  cytoplasmic  DT  dia- 
phorase  [9]  to  the  vitamin  Kg-stimulated  system  gave  a  further  two-  to 
threefold  stimulation  of  the  respiration.  This  rate  of  respiration  was  close 
to  that  obtained  maximally  with  succinate  or  glutamate  as  substrate  and 
it  may  represent  the  limit  of  the  cytochrome  system  to  react  with  oxygen 
rather  than  that  of  TPNH  to  react  with  the  cytochrome  chain. 

TABLE  II 
Effect  of   Some   Inhibitoks   on   the   Mitochondrial   Oxidation   of   Extra- 

MITOCHONDRIAL    TPNH    MEDIATED    BY    DT    DiAPHORASE    AND    VlTAMIN    K, 

Additions  (  +  )  or  omissions  ( — )  Relative  oxygen  consumption 

Complete  system  100 

+   2   X    10^  M  Amytal  118 

+   I  /xg.  Antimycin  A  20 

+   lO"^  M  cyanide  24 

+   lO""®  M  dicoumarol  24 

—  DT  diaphorase,  —  vitamin  K;,  20 

Conditions  as  in  Eig.  2.  Respiration  of  complete  system  was  5-56  /diatoms 
oxygen /20  min  [3]. 

The  pattern  of  inhibition  by  various  inhibitors  was  identical  in  this 
system  to  that  in  the  previous  one  (Table  II).  The  inhibition  of  the 
respiration  by  Antimycin  A  and  cyanide  is  in  agreement  with  the  report  of 
Colpa-Boonstra  and  Slater  [2]  on  the  oxidation  of  reduced  vitamin  Kg  by 
mitochondria.  This  inhibition  by  Antimycin  A  suggests  the  entrance  of 
electrons  into  the  respiratory  chain  at,  or  above,  the  site  of  Antimycin  A 
inhibition,  probably  at  the  level  of  cytochrome  h.  This  is  supported  by 
other  experiments  which  are  reported  elsewhere  [8]. 

As  DT  diaphorase  can  reduce  a  wide  variety  of  quinones  tested  [8,  9] 
it  was  assumed  that  this  stimulation  of  the  oxidation  of  TPNH  was  a 
general  property  of  such  quinones.  However,  as  is  shown  in  Table  HI,  it 
was  highly  specific  for  2-methyl-naphthoquinones,  and  in  particular,  for 
vitamin  K3.  Such  a  specificity  increased  the  anticipation  of  a  biological 
role  for  such  a  system.  While  it  was  recognized  that  vitamin  K;5  is  a  highly 
artificial  material  for  a  biological  system  it  was  felt  that  possibly  some 


COUPLING    OF   REDUCED    PYRIDINE    NUCLEOTIDE   OXIDATION 


173 


TABLE  III 

Effect   of   Various    Quinones   on   the    Mitochondrial   Oxidation   of 
extraahtochondrial   tpxh    in   the   presence   of   dt   diaphorase 


Additions 


Relative   oxygen   consumption 


vitamin  K3  (2-methyl-i,4-naphtho- 

quinone) 
1 ,2-naphthoquinone 
1 ,4-naphthoquinone 
2-hydroxy- 1 ,4-naphthoquinone 
2-methyl-3 -hydroxy- 1,4- 

naphthoquinone 

/)-benzoquinone 
2-methylbenzoquinone 
2,6-dimethylbenzoquinone 
coenz\TTie  Qo  (2-methyl-5,6- 
dimethoxybenzoquinone) 


100 
17 
14 
14 

24 

15 
10 

9 
14 
13 


Conditions  as  in  Fig.  2.  The  amount  of  quinone  was  10  -  /L^mole  added  in 
10  /d.  ethanol.  Respiration  of  the  system  with  vitamin  K3  was  5  "20  ^atoms  oxygen/ 
20  min.  [Cono\er  and  Ernster,  unpublished]. 


^  100 


2  3  A 

Amytal  (  M  xlO^  ) 

Fig.  3.  EflFect  of  Amytal  on  the  respiration  of  liver  slices.  Each  Warburg 
vessel  contained  50  /^moles  glucose,  200  mg.  rat  liver  slices  and  2-0  ml.  Krebs- 
ringer-phosphate  solution  (pH  7-4).  Gas  phase,  oxygen.  Temperature,  37  . 
Time  measured,  60  min.  [7]. 


174  T.   E.    CONOVER 

naturally-occurring  quinones  of  a  similar  type  might  serve  in  a  similar 
role  in  the  intact  cell. 

It  was  observed  by  Ernster  [7]  that  there  was  in  liver  slices  an  Amytal- 
resistant  respiration  which  amounted  to  about  30-40°,,  of  the  total  ob- 
served respiration  with  glucose  as  substrate  (Fig.  3).  This  is  in  contrast 
to  the  more  recent  report  of  Chance  and  Hess  [i]  with  ascites  tumour  cells 
where  the  respiration  was  completely  inhibited  by  Amytal.  Investigations 
were  initiated  by  Mr.  Kadenbach  in  this  laboratory  on  the  nature  of  this 
Amytal-resistant  respiration  with  particular  emphasis  on  whether  DT 
diaphorase  might  be  involved. 


10- 


6- 


D   no  add  itions 
Q    Vit,K3-b:sultit( 


i 

/  / 


12 


10 


D  no  addit  ions 
S   dicoumarol 


none       Amytal 


none       Amytal 


Fig.  4.  The  effect  of  vitamin  K3  and  dicoumarol  on  the  respiration  of  liver 
slices  in  the  presence  and  absence  of  Amytal.  Each  Warburg  vessel  contained  50 
/limoles  glucose,  100  mg.  wet  weight  rat  liver  slices,  and  o-8  ml.  Krebs-Ringer- 
phosphate  solution  containing  half  the  usual  concentration  of  CaCl,.  The  amounts 
of  the  additions  were  2  jumoles  Amytal,  10"'-  /itmole  vitamin  Kg-bisulphite,  and 
2  X  io~-  ^mole  dicoumarol.  The  final  volume  was  i  o  ml.  Gas  phase,  oxygen. 
Temperature,  37  "5  .  Time  measured,  60  min. 


Preliminary  investigation  quickly  showed  that  the  function  of  DT 
diaphorase  in  either  the  normal  or  the  Amvtal-resistant  respiration  of  rat 
liver  slices  with  glucose  as  substrate  would  be  difficult  to  demonstrate. 
Figure  4  shows  in  a  simple  manner  two  experiments  in  which  the  addition 
of  vitamin  K.j-bisulphite  had  no  stimulatory  effect  on  either  normal  or 
Amytal-resistant  respiration,  nor  did  addition  of  dicoumarol  show  any 
inhibitory  effect  on  respiration  in  these  two  conditions. 

The  water-soluble  vitamin  Kg-bisulphite  was  used  in  these  experi- 
ments in  order  to  avoid  addition  of  alcohol  to  the  system,  since  alcohol  is 


COUPLING    OF    REDUCED    PYRIDINE    NUCLEOTIDE    OXIDATION- 


ITS 


readily  used  as  substrate  by  the  slices.  It  was  shown  with  the  isolated 
enzyme  system  that  vitamin  K.j-bisulphite  may  also  be  used  in  stimtilating 
the  mitochondrial  oxidation  of  TPXH  presumably  because  the  bisulphite 
portion  is  readily  split  otf. 

In  yiew  of  the  yarious  possible  oxidative  pathways  open  to  the  DPXH 
which  would  be  generated  from  the  oxidation  of  glucose  it  was  felt  that 
the  DT  diaphorase  system  in  the  rat  liver  slice  might  better  be  demon- 
strated with  a  substrate  which  would  specifically  generate  TPXH. 

In  Fig.  5  is  shown  the  effect  of  dicoumarol  on  the  Amytal-resistant 
respiration  of  rat  liver  slices  in  the  presence  of  citrate  as  substrate.  It  can 
be  seen  that  while  there  was  some  stimulation  of  citrate  respiration  by 


-    10 


D  Without  dicoumarol 
Q  With  dicoumarol 


endcgep.  citrcte       citrate 

Vit   K,-bi5.  Vit.  K-,-bi 


Fig.  5.  The  dicoumarol  sensitivity  of  the  Amytal-resistant  respiration  of 
liver  slices  with  citrate  as  substrate.  Conditions  as  in  Fig.  4.  The  amounts  of  the 
additions  were  25  /tmoles  citrate,  5  x  10-  /^mole  vitamin  Ky-bisulphite,  i  /umole 
TPX,  and  5    x    lo"^  ^(mole  dicoumarol.  Time  measured,  60  min.  [13]. 


vitamin  K3,  there  was  very  little  dicoumarol  sensiti\ity  characteristic  of 
DT  diaphorase.  It  was  therefore  apparent  that  unless  there  was  an  im- 
permeability of  the  slice  to  dicoumarol  that  there  must  exist  alternative 
amytal-insensitive  pathways  of  TPNH  oxidation.  One  such  possibility 
considered  was  a  trans-hydrogenation  to  DPX  and  subsequent  oxidation 
of  the  DPXH  formed.  It  has  been  suggested  by  various  workers  [11,  15, 
17]  that  the  dehydrogenases  which  are  able  to  react  with  both  DPXH  and 
TPXH  may,  in  the  presence  of  their  substrate,  act  as  transhydrogenases. 
If  this  is  the  case  it  might  be  assumed  that  lactic  dehydrogenase  con- 
stitutes the  major  portion  of  the  transhydrogenase  activity  in  the  cytoplasm. 
On  this  basis  the  previous  experiment  was  repeated  on  rat  liver  slices 


176  T.    E.    CONOVER 

which  were  preincubated  with  iodoacetate  in  order  to  deplete  the  level  of 
lactate  and  pyruvate  and  therefore  lower  the  transhydrogenase  activity.  It 
can  be  seen  in  Fig.  6  that  this  preincubation  has  not  changed  greatly  the 
pattern  of  citrate  oxidation.  Vitamin  K3  still  stimulated  respiration  to 
some  extent.  Most  striking,  however,  is  that  the  citrate  respiration  was 
now  markedly  inhibited  by  dicoumarol. 

It  would  therefore  appear  that  the  DT  diaphorase  system  can  be  demon- 
strated in  the  intact  cell  with  liver  slices  in  the  presence  of  added  vitamin 
K3  under  conditions  where  the  transhydrogenase  activity  of  the  cytoplasm 
is  low.  It  is  difficult,  however,  to  ascertain  from  this  whether  DT  diaphorase 
is  able  to  function  in  the  respiration  and  oxidation  of  reduced  pyridine 
nucleotide  in  normal  conditions.  Under  usual  slice  conditions,  particularly 
in  the  presence  of  Amytal  where  lactate  levels  are  high,  the  activity  of 
DT  diaphorase  may  be  masked  by  the  transhydrogenase  activity. 


D  Without  dicoumarol 
Q  With    dicoumarol 


4- 


3- 


o     2 


I  - 


citrate       citrote  Citrate 

Vit   Kj-bis  Vit   Kj-bis      Vit  Kj-bis. 

TPN 


Fig.  6.  The  dicoumarol  sensitivity  of  the  Amytal-resistant  respiration  of 
iodoacetate-pretreated  liver  slices.  Conditions  as  in  Fig.  4  except  that  the  slices 
were  preincubated  for  20  min.  at  37  -5  in  the  presence  of  2  x  lo"'*  m  iodoacetate. 
Time  measured,  60  min.  [13]. 


Several  of  the  properties  of  DT  diaphorase  would  suggest  some 
limitations  in  its  function  in  the  cell.  From  a  Lineweaver-Burk  plot  for 
the  enzyme  it  was  seen  that  the  Michaelis  constants  for  the  enzyme  are 
high;  o-i8  mM  for  DPNH  and  0-13  mM  for  TPNH.  Albumin,  which 
activates  the  enzyme  in  the  purified  state,  gives  a  marked  lowering  of  the 
Kj^j  to  o-o8  mM  and  0-04  mM  respectively.  TPNH  in  both  cases  has  a 
somewhat  lower  K^^/  than  has  DPNH.  It  would  seem  that  though  the 


COUPLING    OF    REDUCED    PYRIDINE    NUCLEOTIDE    OXIDATION  1 77 

enzyme  has  a  very   high  turnover  number  [9]    it  requires    rather  high 
levels  of  substrate  in  order  to  function  efficiently- 

This  effect  may  be  illustrated  by  comparing  the  activity  of  two  types  of 
diaphorase  enzvmes,  the  purified  DT  diaphorase  and  the  D  diaphorase  of 
the  mitochondrial  respiratory  chain  prepared  by  extraction  of  mitochondria 
with  Lubrol  W.  The  diaphorase  activity  of  these  two  enzymes  was 
compared  in  the  oxidation  of  both  added  DPNH  and  of  DPNH  generated 
with  a  system  containing  alcohol  dehydrogenase  and  ethanol,  which  at  the 
pH  used  has  an  equilibrium  unfavourable  to  the  production  of  DPNH. 
As  may  be  seen  in  Table  IV  the  two  diaphorase  activities  were  chosen  so 
as  to  react  at  similar  rates  with  added  DPNH  as  substrate.  In  the  alcohol 
dehydrogenase  system  D  diaphorase  could  still  function  efficiently;  how- 
ever, the  reaction  rate  with  DT  diaphorase  was  greatly  reduced  as  com- 
pared with  the  activity  with  added  DPNH. 

TABLE  IV 

Comparison  of  the  Activities  of  DT  Diaphorase  axu  DPNH  Diaphorase  of 
Mitochondria  with  Added  DPNH  and  with  a  DPNH -Generating  System 

Diaphorase  activity 

Purified  DT  diaphorase     Mitochondrial  D  diaphorase 
(/Ltmoles  DCPIP  reduced/niin.) 

DPNH  0020  0017 

Ethanol,  DPN,  alcohol 

dehydrogenase  00024  00165 

The  assay  system  contained  004  m.M  DCPIP,  o-i  niM  DPNH  or  DPN,  33 
mM  ethanol,  excess  alcohol  dehydrogenase,  o  33  niM  KCN,  oi",,  albumin,  and 
0-05  mM  orthophosphate  (pH  75).  Reaction  followed  hy  JE^on-  [Ernster,  Daniel- 
son,  and  Ljunggren,  unpublished]. 

It  seems  then  that  DT  diaphorase  would  function  in  the  cell  only  when 
the  levels  of  reduced  pyridine  nucleotide  are  high.  As  Glock  and  AIcLean 
[10]  and  others  [12]  have  shown  this  is  generally  found  only  in  the  case  of 
TPNH.  It  is  assumed,  therefore,  that  the  function  of  DT  diaphorase  is 
primarily  with  regard  to  the  TPNH  of  the  cell. 

Although  the  bulk  of  this  enzyme  is  located  in  the  cytoplasm  it  can  also 
be  extracted  from  mitochondria  [5].  Here  the  function  of  the  enzyme  is 
perhaps  even  more  obscure  than  it  is  in  the  cytoplasm.  Numerous  trans- 
hydrogenases  have  been  reported  which  would  presumably  allow  the 
oxidation  of  TPNH  through  the  active  DPNH  oxidase  of  mitochondria. 

As  has  been  reported  pre\"iously  [4,  8]  DT  diaphorase  can  be  demon- 
strated in  mitochondria  by  b}  passing  the  site  of  Amytal  inhibition  in  the 

VOL.  II. — N 


178  T.    E.    CONOVER 

oxidation  of  pyridine  nucleotide-linked  substrates  by  the  addition  of 
vitamin  Kg.  However,  DT  diaphorase  seems  to  play  no  role  in  the  normal 
respiration  of  mitochondria.  Some  coupling  of  mitochondrial  DT  dia- 
phorase to  the  respiratory  chain  by  naturally  occurring  quinones  may 
occur  and  may  indeed  account  for  the  observed  TPNH  oxidase  and  cyto- 
chrome r-reductase  activity  in  mitochondria;  however,  the  necessity  for 
such  a  pathway  is  difficult  to  comprehend. 

In  conclusion,  it  must  be  said  that  though  a  role  for  DT  diaphorase  in 
cellular  respiration  has  been  diligently  searched  for,  the  evidence  of  its 
participation  in  this  role  is  rather  meagre.  Figure  7  is  recorded  with  some 
hesitation  as  it  probably  implies  more  than  may  actually  be  the  case.  It  is 
possible  that  when  TPNH  levels  are  high  that  DT  diaphorase  may  func- 
tion as  is  diagrammatically  shown  and  as  has  been  shown  to  occur  with 


Cytoplasm 


Mitochondria 


/3-OH-But.  dehyd,       Transhydrogenose 
Lactic  dehyd.  malic  dehyd.  glut,  dehyd 

etc  etc  etc 

TPNH  ^;=:^ DPNH  ^;^==^  DPNH  ^^=!=  TPNH 


Fig.   7.   Some   possible   pathways   of   reduced    pyridine    nucleotide   oxidation 
and  its  regulation. 


added  vitamin  Kg.  This  pathway  would  require  the  presence  of  some 
natural  mediator,  presumably  of  a  quinone  type,  to  function  between  DT 
diaphorase  and  the  respiratory  chain.  It  may  be  emphasized  that  in  liver 
at  least  it  is  the  absence  or  unavailability  of  sufficient  amounts  of  such  a 
low  molecular  weight  mediator  that  is  essential  for  the  maintenance  of  the 
high  levels  of  TPNH,  rather  than  the  absence  of  an  enzyme  which  can 
oxidize  TPNH.  Similar  conclusions  have  been  reached  by  Wenner  [18]  in 
regard  to  the  operation  of  the  glucose-6-phosphate  shunt  in  ascites  tumour 
cells. 

If  speculation  on  the  basis  of  the  quinone-specificity  of  this  pathway 
would  suggest  a  quinone  of  the  vitamin  K  type  in  this  role,  it  raises  the 
interesting  possibility  of  regulation  of  TPNH  levels  by  a  factor  of  nutritional 


COUPLING    OF    REDUCED    PYRIDINE    NUCLEOTIDE    OXIDATION  1 79 

importance.  On  the  other  hand,  if  a  relation  exists  between  the  function  of 
DT  diaphorase,  the  biological  activity  of  dicoumarol,  and  the  nutrition 
requirement  for  vitamin  K  in  the  animal  organism,  then  a  much  more 
specific  role  for  DT  diaphorase  must  be  found. 

References 

1.  Chance,  B.,  and  Hess,  B.,^.  biol.  Chcni.  234,  2404  (1959). 

2.  Colpa-Boonstra,  J.  P.,  and  Slater,  E.  C,  Biochitn.  biophys.  Acta  27,  122  (1958). 

3.  Conover,  T.  E.,  and  Ernster,  L.,  Biochem.  biophys.  Res.  Conim.  I,  26  (i960). 

4.  Conover,  T.  E.,  and  Ernster,  L.,  Acta  chem.  scand.  14,  1840  (i960). 

5.  Danielson,  L.,  Ernster,   L.,  and  Ljunggren,  ^L,  Acta  chetn.  scand.   14,  1837 
(i960). 

6.  Dickens,  F.,  Glock,  G.  E.,  and  McLean,  P.,  /;/  "  Ciba  Foundation  Symposium 
on  the  Regulation  of  Cell  Metabolism".  J.  and  A.  Churchill,   London,    150 

(1959)- 

7.  Ernster,  L.,  "Biochemical  Society  Symposia".  16,  54  (1959). 

8.  Ernster,  L.,  this  volume,  p.  139. 

9.  Ernster,  L.,  Ljunggren,  AL,  and  Danielson,  L.,  Biochem.  bi(jp/iys.  Res.  Comni. 
2,  88  (i960). 

10.  Glock,  G.  E.,  and  McLean,  P.,  E.\p.  Cell  Res.  II,  234  (1956). 

11.  Holzer,  H.,  and  Schneider,  S.,  Biochem.  Z.  330,  240  (1958). 

12.  Jacobson,  K.  B.,  and  Kaplan,  X.  0.,jf.  biol.  Chetn.  226,  603  (1957). 

13.  Kadenbach,  B.,  Conover,  T.  E.,  and  Ernster,  L.,  Acta  chem.  scand.  14,  1850 
(i960). 

14.  Krebs,   H.   A.,   and   Kornberg,   H.   L.,   "  P2nergy  Transformations   in   Living 
Matter".  Springer,  Berlin  (1957). 

15.  Xavazio,  F.,  Ernster,  B.  B.,  and  I^rnster,   L.,  Biochini.  biophys.  Acta  26,  416 

(1957)- 

16.  Pullman,  M.  E.,  and  Racker,  ¥..,  Science  123,  i  105  (1956). 

17.  Talalav,  P.,  and  Williams-.Ashman,  H.  G.,  Proc.  nat.  Acad.  Sci.,  Wash.  44,  15 
(195S)'. 

18.  Wenner,  C.  V^.,').  biol.  Chetn.  234,  2472  (1959). 

Discussion 

Peters  :  Were  these  citrate  experiments  done  \\  ith  mitochondria  ? 

Conover:  They  were  done  with  liver  slices. 

Peters:  I  wasn't  quite  sure  why  you  used  citrate  rather  than  isocitrate. 

Conover:  Well  the  primary  reason  was  that  the  isocitrate  we  had  was  in  the 
lactone  form  and  we  weren't  quite  sure  whether  this  would  be  readilv  oxidized. 

Low'ENSTEiN :  W^hat  concentration  do  you  add  ? 

Conover:  Usually  lo  ■'  ^L  Actually  the  experiments  shown  did  not  give  the 
effect  of  TPN  alone.  It  is  not  as  high  when  added  by  itself  as  when  added  in  the 
presence  of  vitamin  K:j. 

LowENSTEiN  :  The  other  question  I  wanted  to  ask  was :  are  the  TPN  diaphorase 
activities  found  in  the  cytoplasm  and  the  mitochondria  the  same  ? 

Conover:  They  are,  as  far  as  we  have  been  able  to  tell.  We  have  isolated  the 
mitochondrial  DT  diaphorase  and  it  exhibits  identical  properties  with  the  cvto- 
plasmic  DT  diaphorase. 


l8o  T.    E.    CONOVER 

LowENSTEiN :  What  I  was  going  to  ask  Dr.  Ernster  earlier  was  whether  the 
TPN  diaphorase  from  mitochondria  is  the  same  as  transhydrogenases  discussed 
by  Dr.  Kaplan  ? 

Ernster:  No. 

LowENSTEiN :  What  is  the  difference  ? 

Ernster:  That  it  doesn't  transfer  hydrogen  between  TPN  and  DPN.  Kaplan's 
transhydrogenase  is  reported  to  be  bound  to  the  mitochondria  (J.  biol.  Che?n.,  205, 
I  (1953))  whereas  about  95 °o  of  our  enzyme  is  in  the  soluble  cytoplasm  {Biochem. 
biophys.  Res.  Comni.,  2,  88  (i960)).  Furthermore,  DT  diaphorase  is  strongly 
inhibited  by  dicoumarol. 

LowENSTEiN :  Can  you  give  a  figure  for  the  comparative  rates  of  soluble  TPN 
diaphorase  and  mitochondrial  transhydrogenase  ? 

Ernster:  The  activity  of  DT  diaphorase  ranges  between  30  and  100  /umoles 
reduced  pyridine  nucleotide  oxidized  per  min.  and  per  g.  rat  liver  (wet  weight).  I 
don't  know  what  the  activity  of  transhydrogenase  is  if  you  measure  it  with  TPN 
and  DPN  and  not  with  the  analogues. 

Singer  :  I  am  wondering  if  you  could  refresh  my  memory  on  what  compelling 
reason  there  is  to  believe  that  the  transhydrogenase  activity,  as  measured  by  Kaplan 
and  others,  can  be  ascribed  to  the  action  of  the  respiratory  chain  DPNH-dehydro- 
genase  ?  I  might  add,  to  clarify  my  question,  that  while  considerable  transhydro- 
genase activity  follows  the  respiratory  chain  DPNH-dehydrogenase  throughout 
purification  when  the  transhydrogenation  of  DPNH  with  DPN  analogues  is  used 
as  an  assay,  no  DPNH-TPN  transhydrogenation  at  all  is  shown  by  the  purified 
enzyme.  Thus  the  enzyme  we  have  isolated  is  obviously  not  the  one  catalyzing 
transhydrogenations  observed  in  mitochondria  and  its  fragment  involving  TPN. 

Lowenstein:  It  has  been  purified  by  Kaufmann  and  Kaplan  and  has  been 
found  to  remain  intimately  associated  with  the  DPNH-electron  transport  system. 


Mitochondrial  Lipids  and  their 
Functions  in  the  Respiratory  Chain 

E.  R.  Redfearn 

Department  of  BiocJiemistry, 
The  University  of  Liverpool,  England 

Mitochondria  contain  relatively  large  amounts  of  lipids  and  there  is 
now  a  great  deal  of  evidence  which  suggests  that  they  play  both  structural 
and  functional  roles  in  mitochondrial  metabolism.  We  have  been  studying 
the  problem  of  lipid  function  in  the  respiratory  enzyme  system,  both  in 
intact  phosphorvlatmg  mitochondria  and  in  non-phosphorylating  mito- 
chondrial fragments. 

Nature  of  mitochondrial  lipids 

Non-phosphorvlating  preparations  of  pig-heart  muscle  [i]  were 
denatured  with  methanol  and  the  lipid  extracted  with  40-60  light 
petroleum.  The  extract  was  then  chromatographed  on  silicic  acid  (Mallin- 
krodt)  and  fractions  eluted  with  increasing  concentrations  of  diethyl 
ether  in  40-60  light  petroleum.  Stronglv  adsorbing  material  at  the  top  of 
the  column  was  eluted  with  methanol.  One  result  of  such  an  analysis  is 

TABLK  I 

Lipm  Composition  of  Pio  Heart-Miscle  Preparation 

....  Concentration  Percentage  of 

ivipid  ,  ,  .    ^  ,  1-    ■  1 

(mg./g.  protein)  total  lipid 


Total  lipid 

Phospholipid 
Sterol 

Neutral  lipid 
Ubiquinone 


420 

1000 

378 

90-1 

14-3 

3-4 

i6-3 

3-9 

4-1 

0-98 

shown  in  Table  I.  The  total  lipid  which  amounts  to  30",,  of  the  total  dry 
weight  of  the  preparation,  contains  90",,  phospholipid  while  smaller 
amounts  of  sterol,  neutral  lipid  and  ubiquinone  make  up  the  total. 

All  the  fractions  were  examined  spectrophotometricallv.  Apart  from 
ubiquinone  which  showed  intense  selective  absorption  at  275  m^tt,  a  number 


1 82  E.    R.    REDFEARN 

of  fractions  showed  weak  absorption  at  various  wavelengths  between  230 
and  300  m/i.  There  was,  however,  no  evidence  of  spectra  characteristic  of 
the  tocopherols  or  the  vitamins  K. 

Extraction  of  lipids  with  organic  solvents 

An  obvious  way  to  tackle  the  problem  of  lipid  function  in  mitochondrial 
particles  is  to  remove  the  lipid  by  a  suitable  extraction  procedure  and 
observe  the  effect  on  enzyme  activities.  Nason  and  Lehman  [2,  3]  did  this 

TABLE  II 

Effect  of  Number  of  E^xtractions  with  Light  Petroleum  on  the  Succinic 
Oxidase  and  Cytochrome  Oxidase  Activities  of  Pig  Heart- 
Muscle  Preparation 


Succinic  oxidase 

Cytochrome 

oxidase 

Number 

of 
ns 

(^1.0 

,/hr 

./mg. 

protein) 

(lA.O 

./hr./mg. 
Expt.  N 

protein) 

extractio 

Ex 

pt.  N 

0. 

0. 

I 

2 

3 

I 

2 

3 

0 

I 

259 
567 

320 

242 

810 
886 

1240 

628 

2 
3 
4 

373 

— 

• — 

842 

— 

— 

243 

— 

— 

902 

— 

— 

15 

— 

— 

492 

— 

— 

1056 

20 
30 



960 

540 

— 

1616 

828 

40 

— 

770 

— 

— 

1880 

— 

Expt.  No.  I  :   2  ml.   preparation   (age   3   days)   extracted  successively  with  4  ml. 

40-60    light  petroleum  for  i  min. 
Expt.  No.  2 :    I    ml.   preparation   (age  7   days)  extracted  successively  with    i   ml. 

40-60    light  petroleum  for  i  min. 
Expt.  No.  3 :    I    ml.   preparation   (age    i    day)   extracted   successively  with   5   ml. 

40-60    light  petroleum  for  i  -5  min. 
Enzyme  activities  determined  as  described  by  Redfearn  et  al.  [11]. 

simply  by  shaking  the  enzyme  preparation  with  an  organic  solvent  such 
as  isooctane.  After  such  a  treatment  it  was  found  that  the  succinic-  and 
DPNH-cytochrome  c  reductase  activities  had  fallen  considerably  but 
that  they  could  be  restored  to  their  original  levels  by  adding  a-tocopherol 
as  a  suspension  in  bovine  serum  albumin.  Although  it  was  later  shown  [4] 
that  other  substances  would  also  reactivate  solvent-extracted  preparations 
a  hypothesis  was  put  forward  implicating  y.-tocopherol  as  an  essential 
component  of  the  electron  transport  system  [5].  The  specificity  of 
the  reactivation  by  tocopherol  was  doubted  by  Deul  et  al.  [6].  Redfearn 
and  Pumphrey  [7]  then  showed  that  the  loss  of  enzymic  activity  after 


MITOCHONDRIAL   LIPIDS   AND   THEIR   FUNCTIONS    IN   THE   RESPIRATORY   CHAIN       1 83 

shaking  with  an  organic  solvent  was  due  principally  to  small  amounts 
of  the  solvent  retained  in  the  enzvme  suspension  acting  as  a  physical 
inhibitor.  Removal  of  this  residual  solvent  by  physical  means,  e.g.  dis- 
persion with  a  surface-active  agent,  gave  complete  restoration  of  enzymic 
activities.  These  findings,  which  have  since  been  confirmed  by  others  [8, 
9],  make  it  necessary  to  be  extremely  cautious  in  evaluating  the  results  of 
extraction-reactivation  experiments.  Thus  inactivation  of  an  enzvme  svstem 
due  to  an  inhibition  by  the  solvent  must  be  clearly  distinguished  from 
inactivation  brought  about  by  the  removal  of  lipid  essential  for  some 
structural  or  functional  role. 

In  a  study  of  the  effect  of  removal  of  lipid  on  enzyme  activities,  pig 
heart-muscle  preparations  were  extracted  with  organic  solvents  by  Nason's 

TABLE  III 

KXTRACTION    OF    A    PlG    HeART-MuSCLE    PREPARATION    WITH    DlETHVL    EtHER 


Number 

of 

extractions 


Succinic 

oxidase 

{%  original 

activity) 


Succinic- 
cytochrome  c 
reductase 
( "1,  original 
activity) 


L  hicjuinone 

extracted 

(",,  total 

extractable 

ubiquinone) 


Lipid 
extracted 
(",,  total 

lipid) 


100 

106 
75 
63 
50 


100 
73 
63 
61 

54 


50 
78 
79 

96 


o 
I 

7 
II 

16 


I  ml.  preparation  (35  mg.  proteinnil.)  extracted  successively  with  5  ml. 
peroxide-free  diethyl  ether  for  1-5  min.  Extracts  washed  with  water,  dried  and 
solvent  evaporated.  Lipid  residue  weighed.  Lipid  dissolved  in  40-60  light  petrol- 
eum and  ubiquinone  separated  and  determined  as  described  by  Pumphrey  and 
Redfearn  [i].  Enzyme  activity  determined  by  the  methods  described  by  Redfearn 
it  al.  [11]. 

technique  and  the  residual  soh ent  remo\ed  by  incubating  the  suspension 
in  a  Warburg  manometer  until  solvent  evolution  had  ceased.  In  this  way, 
the  effects  of  extraction  could  be  studied  without  the  additional  complica- 
tion of  the  inhibitory  effects  of  the  solvent  itself.  In  experiments  using  40- 
60  light  petroleum  as  the  solvent  it  was  found  that  one  or  two  extractions 
produced  marked  increases  in  the  succinic  oxidase  and  cytochrome  oxidase 
activities  (Table  II).  The  amount  of  total  lipid  removed  from  the  particles 
appeared  to  be  small  and  less  than  50",,  of  the  total  extractable  ubiquinone 
was  removed  even  after  forty  successive  extractions. 

With  diethyl  ether,  the  endogenous  ubiquinone  of  heart-muscle 
preparations  could  be  extracted  much  more  effectively.  The  results  of  an 
experiment  are  shown  in  Table  III.  96",,  of  the  total  extractable  ubiquinone 
was  removed  after  eight  extractions  with  ether;  the  succinic  oxidase  and 


184  E.    R.    REDFEARN 

succinic  cytochrome  c  reductase  activities  had  fallen  to  approximately  50% 
of  the  original  activities.  Polar  solvents,  such  as  ether  and  acetone,  differ 
from  non-polar  solvents  in  that  cytochrome  oxidase  is  much  more  readily 
inactivated.  Thus  after  one  or  two  extractions  with  ether,  cytochrome 
oxidase  becomes  the  rate-limiting  step  in  the  respiratory  chain. 

Although  much  remains  to  be  done  on  the  correlation  between  enzymic 
activites  and  lipid  content  of  respiratory  chain  particles,  certain  conclusions 
can  be  drawn  from  these  preliminary  experiments.  Treatment  of  heart- 
muscle  preparations  with  organic  solvents  appears  to  have  three  principal 
effects :  (i)  the  physical  action  of  the  solvent  producing  changes  in  particle 
size  and  morphology,  (ii)  the  removal  of  lipid  from  the  particle  by  solution 
in  the  solvent,  and  (iii)  the  retention  by  the  particles  of  small  amounts  of 
the  solvent  by  surface  adsorption  or  solution  in  the  lipid.  The  first  of  these 
is  undoubtedly  the  cause  of  the  increased  enzyme  activities  obtained  after 
shaking  the  preparation  with  the  solvent.  This  treatment  probably  results 
in  the  breaking  down  of  large  particles  or  aggregates  into  smaller  particles 
or  in  changes  in  particle  structure  which  allow  an  easier  access  of  the 
reactants  to  the  particles.  The  effecc  is  probably  analogous  to  the  action 
of  surface-active  agents  and  the  effect  of  freezing  and  thawing,  processes 
which  also  result  in  increased  enzyme  activities.  The  solvent  probably  also 
displaces  endogenous  cytochrome  c,  which  explains  why  solvent  treated 
particles  show  a  complete  requirement  for  added  cytochrome  c. 

With  regard  to  the  second  effect,  lipid  is  removed  only  with  great 
difficulty  by  non-polar  solvents  but  more  readily  by  polar  solvents,  e.g. 
certain  lipid  components,  such  as  ubiquinone,  can  be  almost  completely 
removed  by  extraction  with  ether.  Cytochrome  oxidase  activity  appears  to 
be  much  more  sensitive  to  polar  solvents  than  to  non-polar  solvents. 

The  third  effect,  inhibition  by  the  solvent  itself,  can  be  reversed  by  any 
one  of  a  number  of  methods  which  depend  on  the  removal  or  displacement 
of  the  solvent.  It  is  interesting  to  note  that  this  type  of  inhibition  depends 
upon  the  structure  of  the  particular  solvent.  Weber  and  Wiss  [10]  have 
show^n  with  the  //-alkanes,  those  with  6-7  carbon  atoms  are  the  most 
active.  Even  more  potent  inhibitors  are  the  vitamin  K.,  analogues  with 
short  side-chains.  Weber  and  Wiss  [10]  showed  that  like  the  organic 
solvents,  the  inhibition  due  to  these  substances  could  be  reversed  by 
vitamin  K,,  phvtol  and  ubiquinone.  Redfearn,  Pumphrey  and  Fynn  [11] 
suggested  that  the  action  of  naphthoquinone  inhibitors  described  by  Ball, 
Anfinsen,  and  Cooper  [12]  could  be  explained  in  terms  of  a  similar  non- 
specific physical  effect.  Thus  it  could  be  imagined  that  the  short  lipophilic 
side-chains  dissolve  in  the  lipid  phase  of  the  particle  with  the  projecting 
layer  of  large  naphthoquinone  nuclei  acting  as  a  barrier  to  the  reactants. 
Recently,  Herdlin  and  Cook  [13]  have  presented  evidence  which  appears 
to  support  this  idea. 


MITOCHONDRIAL   LIPIDS   AND   THEIR   FUNCTIONS   IN   THE   RESPIRATORY   CHAIN       1 85 

Ubiquinone  (Coenzyme  Q) 

There  is  now  a  large  amount  of  evidence  which  suggests  that  ubiquin- 
one is  a  functional  component  of  the  respiratory  chain.  It  is  widely  dis- 
tributed in  mitochondria,  it  undergoes  enzymic  oxidation-reduction  and 

TABLE   IV 

Concentration  of  Ubiquinone  in  Mitochondrial  Preparai  ions 


Preparation 


Ubiquinone 
(^moles/g.  protein) 


Pig  heart-muscle  preparation  4-0 

Horse  heart-muscle  preparation  40 

Guinea-pig  kidney  mitochondria  i  -6 

Rat  liver  mitochondria  i    4 

Pig  kidney  mitochondria  1-2 

Arum  viaciilatujii  spadix  mitochondria  i  -4 

it  is  able  to  restore  enzvme  acti\itv  to  soh ent-extracted  preparations  [14, 
15-  16]. 

A  survey  of  ubiquinone  concentrations  in  a  number  of  tissue  prepara- 
tions has  been  made  by  the  method  described  bv  Pumphrev  and  Redfearn 
[1]    and   some   results   are   shown   in   Table    W .    The   concentration    of 

TABLE  V 
Relative  Concentrations  of  the  Cytochromes  and  Ubiquinones 


Concentration 
Preparation  (/mioles  g.  protein) 


Ratic 


Ti         UQ  a  b  r  ci        UQ 


Pig  heart- 
muscle 
prepara- 
tion 0'74     o-b       00.S5   0-37     439      2-0        i-6        0-23      i -o      120 

Rat  liver 
mito- 
chondria     0-13        0-13        0-I2        0-14        I'4I        0-93        0^93        0-9  I-O        IQ-O 

The  concentrations  of  cytnchnimes  (/,  b,  and  c  were  determined  using  the 
wavelengths  and  molar  extinction  coefficients  given  by  Chance  and  Williams  [26] 
and  Ci  from  the  data  of  Green   et  al.   [27 J. 

ubiquinone  relative  to  the  cytochromes  has  also  been  determined  in  pig 
heart-muscle  preparations  and  rat  li\er  mitochondria  (Table  \).  The 
cytochrome  concentrations  were  determined  spectrophotometricallv  after 
solubilization  of  the  preparations  in  sodium  cholate.  It  will  be  seen  that  in 


1 86  E.    R.    REDFEARN 

both  cases,  on  a  molar  basis,  ubiquinone  is  present  in  a  considerable  excess 
over  the  cytochromes.  On  an  electron-carrying  basis  the  ubiquinone/cyto- 
chrome ratio  is,  of  course,  increased  further  by  a  factor  of  two  to  give 
ratios  of  20-24.  The  reason  for  this  large  excess  of  ubiquinone  is  not  clear 
at  the  moment  although  it  has  important  consequences  when  discussing  its 
possible  function  in  the  respiratory  chain.  It  is  interesting  to  note  the 
extraction  experiments  already  described  indicate  that  relatively  large 
amounts  of  ubiquinone  may  be  removed  from  mitochondrial  particles 
without  apparently  having  drastic  effects  on  enzymic  activities.  Thus  it  is 
possible  that  only  the  stoicheiometric  amount  is  necessary  for  efficient 
operation  of  the  respiratory  chain. 

The  results  of  a  study  of  the  kinetics  of  ubiquinone  reactions  in  heart- 
muscle  preparations,  the  action  of  inhibitors  on  these  reactions  and  a 
discussion  of  the  possible  function  of  ubiquinone  in  the  non-phosphorylat- 
ing  respiratory  chain  have  been  presented  recently  [17,  15,  16].  To 
summarize  briefly,  the  rate  of  reduction  of  ubiquinone  by  DPNH  or 
succinate  is  less  than  the  total  electron  flux  as  measured  by  the  substrate 
oxidase  rates ;  most  of  the  endogenous  ubiquinone  appeared  to  be  accessible 
to  both  substrates;  inhibitor  studies  indicate  that  its  site  of  action  is 
between  the  flavoproteins  and  the  antimycm-A-sensitive  region.  Three 
possible  schemes  for  the  position  of  ubiquinone  in  the  non-phosphorylating 
chain  can  be  put  forward  [16].  These  are:  (i)  that  ubiquinone  is  on  the 
main  respiratory  chain  mediating  the  reaction  between  the  flavoproteins 
and  the  cytochromes,  (ii)  that  it  reacts  only  with  the  flavoproteins  to  form 
a  blind-alley  pathway,  and  (iii)  that  it  is  on  a  branch  pathway  linking  the 
flavoproteins  with  cytochrome  (\  via  the  antimycin-A-sensitive  region. 

In  order  to  try  to  elucidate  the  mode  of  action  of  ubiquinone  in  the 
intact  p»hosphorylating  system  we  have  begun  experiments  with  rat-liver 
mitochondria.  The  mitochondria  were  prepared  by  a  modification  of  the 
method  of  Schneider  and  Hogeboom  [18]  and  ubiquinone  determined  by 
the  method  of  Pumphrey  and  Redfearn  [i].  Respiratory  control  and  P/0 
ratios  were  determined  with  the  oxygen  electrode  [19],  and  steady-state 
oxidation-reduction  levels  of  ubiquinone  were  measured  in  the  different 
metabolic  states  [20]  of  the  mitochondria.  Typical  spectra  are  shown  in 
Fig.  I.  It  can  be  seen  that  in  the  absence  of  added  substrate  or  ADP 
(State  i)  the  ubiquinone  is  45^0  reduced  while  on  adding  ADP  (State  2) 
it  becomes  38°(,  reduced.  In  the  presence  of  added  substrate  (succinate) 
but  no  ADP  (State  4)  the  ubiquinone  is  80",,  reduced  but  on  adding  ADP 
(State  3)  falls  to  72%  reduction.  The  results  of  experiments  on  four 
different  mitochondrial  preparations  are  shown  in  Table  VI.  When 
succinate  is  the  substrate  ubiquinone  is  largely  reduced  (80-89°,,)  in  State  4 
and  becomes  less  reduced  (70-86%)  in  State  3  while  the  corresponding 
figures  for  ^-hydroxybutyrate  are  53-72"^,  (State  4)  and  40  63",,  (State  3). 


MITOCHONDRIAL   LIPIDS   AND   THEIR   FUNCTIONS   IN   THE   RESPIRATORY   CHAIN       1 87 

Experiments  with  antimycin  A,  which  inhibits  oxidation  of  ubiquinol, 
have  shown  that  the  enzymically  reducible  ubiquinone  is  only  80-90*;' o  of 
the  total.  Thus  succinate  in  State  4  is  actually  giving  complete  reduction 


Oi2r 


260  280  300 

Wavelength  (m//) 

Fig.  I.  Steady-state  oxidation-reduction  levels  of  ubiquinone  in  rat-liver 
mitochondria. 

—  ^_^ —  total  ubiquinone  (oxidized); 

—  □ —  total  ubiquinone  reduced  with  XaBHj; 

—  X—  Pi-hair;  —  A—  Pj  +  ADP  +  air; 

— O —  Pi  + succinate -I- air;  — • —  Pj  +  succinate -I- ADP  +  air. 

of  the  enzvmicallv  active  material,  while  /S-hydroxybutyrate  in  State  4  is 
giving  about  80",,  reduction. 

These  changes  are  qualitivelv  similar  to  those  reported  by  Chance  [21] 
for  guinea-pig  kidnev  mitochondria  and  support  the  view  that  the  oxida- 
tion of  ubiquinol  is  blocked  in  the  absence  of  a  phosphate  acceptor  by  an 
inhibitorv  interaction  which  could  involve  energy  conservation  as  a  high- 
energv  intermediate  UQIL^I  bv  a  series  of  reactions  as  follows: 

UQ  +  I  -  UQ.I  (i) 

UQ .  I  +  FPH.,  =  UQH..  -  I  +  FP  (2) 

UQH.,^I^X  =UQH.,  +  I-X  (3) 

I~X  +  ADP  +  P,  -X+I  +  ATP  (4) 


i88 

E.    R 

.    REDFEARN 

TABLE  VI 

Steady- 

•State   Oxidation-Reduction    Levels 

OF   Ubiqu 

inone   in 

Various 

Metabolk 

::  States  of  : 

Rat-Liver 

Mitochondria 

Preparatic 

^n 

Ubiqui- 
none 
concentra- 
tion 
(/^moles/g. 
protein) 

Substrate 

Steady-state  percentage 
reduction  of  total  ubiquinone 

number 

ADP  +  O, 

+  Pi  +  0, 

+  ADP  + 
0, 

+  O2 

14 

1-3 

endogenous 

38 

45 

44 

— 

succinate 

72 

80 

80 

82 

15 

1-6 

endogenous 

45 

— 

— 

— 

8-hydroxy- 

44 

64 

68 

— 

butyrate 

succinate 

80 

85 

87 

88 

17 

1-3 

endogenous 
/3-hydroxy 

33 

— 

~ 

butyrate 

30 

53 

— 

— 

succinate 

69 

Si 

84 

— 

18 

2  ■  2 

endogenous 
/3-hydroxy- 

63 

— 

butyrate 

63 

72 

72 

— 

succinate 

S6 

89 

88 

— 

Steady-state  determinations  were  made  with  a  reaction  mixture  of  the  following 
final  composition:  Sucrose,  107  mM ;  MgCL,  15.5  mM ;  KCl,  25  mM ;  Na.,HP04- 
KHoPOj,  pH  7-4,  12-5  mM  (or  tris-HCl,  pH  7-4,  i8-8  mM);  ADP,  18  mM ; 
sodium  ^-hydroxybutyrate,  4-5  mM ;  sodium  succinate  3-0  mM;  mitochondrial 
protein,  approx.  6  mg./ml.  Total  volume  1  4  ml.  Mixture  aerated  for  30  sec. 
Temp.  17-20. 

Hatefi  [22]  has  also  described  results  of  experiments  on  beef-heart  mito- 
chondria which  support  such  a  role  for  ubiquinone.  He  found  also  that 
when  phosphate  in  the  medium  was  replaced  by  tris  the  ubiquinone  went 
into  the  completely  oxidized  state.  This  was  interpreted  as  being  due  to  the 
release  of  the  inhibitory  etfect  of  phosphate  on  the  oxidation  of  ubiquinol. 
In  the  present  work  this  phosphate  effect  could  not  be  demonstrated  in 
rat-liver  mitochondria ;  the  steady-state  levels  of  ubiquinone  were  almost 
the  same  in  the  absence  of  phosphate,  in  the  presence  of  phosphate,  and 
in  the  presence  of  ADP  without  added  phosphate  (Table  VI). 

Recently  a  number  of  workers  have  put  forward  hypotheses  implicating 
phosphorylated  derivatives  of  quinones  in  oxidative  phosphorylation  [23, 
24,  25].  Ubiquinol  monophosphate,  a  possible  intermediate  in  these 
postulated  reaction  mechanisms  has  been  synthesized  by  Dr.  K.  J.  M. 
Andrews  of  Roche  Products  Ltd.,  Welwyn.  In  a  preliminary  experiment 
this  substance  was  added  to  rat-liver  mitochondria  in  the  presence  of  ADP 
but  no  stimulating  effect  on  the  rate  of  oxygen  uptake  was  observed.  Also 


MITOCHONDRIAL    LIPIDS   AND   THEIR   FUNCTIONS    IN   THE   RESPIRATORY   CHAIN       1 89 

spectrophotometric  examination  of  light  petroleum  extracts  of  mito- 
chondria did  not  reveal  anything  with  the  spectral  characteristics  of 
uhiqiiinol  monophosphate. 

Summary 

1.  Mitochondrial  preparations  contain  relatixelv  large  amounts  of 
phospholipids  with  smaller  concentrations  of  neutral  lipid,  sterol  and 
ubiquinone. 

2.  The  extraction  of  mitochondrial  preparations  with  organic  solvents 
was  studied  and  three  principal  effects  were  distinguished. 

3.  The  concentrations  of  ubiquinone  in  a  number  of  mitochondrial 
preparations  were  measured.  Ubiquinone  concentrations  with  respect  to 
the  individual  cytochromes  were  shown  to  be  relatively  high. 

4.  The  steadv-state  oxidation-reduction  levels  of  ubiquinone  in  rat- 
liver  mitochondria  in  various  metabolic  states  have  been  measured.  The 
possible  role  of  ubicjuinone  in  oxidative  phosphorlvation  was  discussed. 

Acknowledgment 

The  author  is  indebted  to  Dr.  Alison  AI.  Pumphrey  and  Mr.  G.  H. 
Fynn  for  their  collaboration  in  this  work. 

References 

1.  Pumphrey,  A.  M.,  and  Rt-dfearn,  E.  R.,  B/ochetn.  jf.  76,  61  (i960). 

2.  Nason,  A.,  and  Lehman,  I.  R.,  Science  122,  19  (1955). 

3.  Nason,  A.,  and  Lehman,  L  R.,jf.  biol.  Cheni.  222,  511  (1956). 

4.  Donaldson,  K.  O.,  and  Nason,  A.,  Proc.  >iat.  Acad.  Sci.,  Wasli.  43,  364  (1957). 

5.  Donaldson,  K.  O.,  Nason,  A.,  and  Garrett,  R.  H.,^.  hial.  Client.  233,  572  (1958). 

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9.  Igo,  R.  P.,  Mackler,  B.,  and  Hanahan,  D.  ].,y.  biol.  C/ieni.  234,  1312  (1959). 

10.  Weber,  F.,  and  Wiss,  O.,  Hclv.  cliim.  acta  42,  1292  (1959). 

11.  Redfearn,  E.  R.,  Pumphrey,  A.  AL,  and   Fynn,  G.  IE,  Biocliini.  biopiJiys.   Acta 
44,  404  (i960). 

12.  Ball,  E.  G.,  Anfinsen,  C.  B.,  and  Cooper,  0.,y.  biol.  Chtni.  168,  257  (1947). 

13.  Hendlin,  D.,  and  Cook,  T.  M.,  Biochem.  biopJiys.  Res.  Conini.  2,  71  (i960). 

14.  Green,  D.  E.,  and  Lester,  R.  L.,  Fed.  Proc.  18,  987  (1959). 

15.  Redfearn,  E.  R.,  and  Pumphrey,  A.  AL,  Biochetn.  jf.  76,  64  (i960). 

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Transport",  (G.  E.  W.  Wolstenholme  and   C.  AL   O'Connor,   eds.)  p.   346, 

Churchill,  London  (1961). 

17.  Pumphrey,  A.  M.,  and  Redfearn,  E.  R.,  Biochem.  jf.  72,  3P  (1959). 

18.  Schneider,  W.  C,  and  Hogeboom,  G.  H.,jf.  biol.  Client.  183,  123  (1950). 

19.  Chance,  B.,  and  Williams,  G.  R.,  Xature,  Loud.  175,  1120  (1955). 

20.  Chance,  B.,  and  Williams,  G.  R.,  Advaitc.  Enzymol.  17,  65  (1956). 


190  E.    R.    REDFEARN 

21.  Chance,  B.,  "  Ciba  Foundation  Symposiuni  on  Quinones  in  Electron  Trans- 
port", (G.  E.  W.  Wolstenholme  and  C.  M.  O'Connor,  eds.)  p.  327,  Churchill, 
London,  1961. 

22.  Hatefi,  Y.,  Biochim.  biophys.  Acta  31,  502  (1959). 

23.  Harrison,  K.,  Nature,  Loud.  181,  1131  (1958). 

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(1959). 

Discussion 

ZiEGLER :  I  would  like  to  point  out  that  it  would  be  difficult  to  measure  the 
initial  rates  of  endogenous  Q  reduction  by  the  method  Dr.  Redfearn  used.  The 
particles  contain  a  large  excess  of  Q  relative  to  the  cytochromes  or  flavoprotein 
and  only  part  of  it  may  be  rapidly  reduced  in  a  blocked  system.  In  order  to  measure 
initial  rates  at  22'  the  reaction  would  have  to  be  stopped  in  less  than  a  second  and 
I  believe  your  reaction  times  were  of  the  order  of  several  seconds.  Later  in  this 
Symposium  we  will  present  data  which  show  that  the  rate  of  reduction  of  exogenous 
Q  is  fully  compatible  with  the  assumption  that  it  functions  as  an  electron  carrier 
between  the  flavoprotein  and  cytochrome  c.  The  turnover  of  the  flavoprotein  with 
Q  as  the  acceptor  is  more  rapid  than  it  is  in  the  intact  particle  with  oxygen  as  the 
acceptor. 

Redfe.'VRN  :  This  is  a  derivative  particle  that  you  are  using  ? 

Ziegler:  The  naturally  occurring  quinone,  Qjo,  is  reduced  as  rapidly  as  the 
synthetic  Q  homologues. 

Redfearn  :  We  have  measured  the  rate  of  reduction  of  exogenous  ubiquinone 
in  our  preparation,  and  found  the  rate  to  be  very  much  slower  than  that  of  the 
reduction  of  the  endogenous  material. 

Ziegler:  You  do  require  lipids.  Coenzyme  Qj,,  is  extremely  insoluble  in  water, 
and  by  adding  a  mixture  of  phospholipids  you  can  increase  its  effective  concentra- 
tion to  the  point  where  you  can  use  it  as  the  final  electron  acceptor. 

Redfearn  :  I  would  like  to  add  that  in  experiments  we  did  with  Dr.  Chance  we 
measured  the  rate  of  reduction  of  endogenous  ubiquinone  in  the  double  beam 
spectrophotometer  at  the  same  time  as  we  measured  the  rate  by  the  extraction 
procedure,  and  we  got  very  close  agreement. 

Chance  :  We  have  been  interested  in  the  maximal  rate  at  which  the  endogenous 
Q  could  be  reduced.  I  think  that  Dr.  Redfearn  and  I  had  already  observed  rates 
at  22'  of  about  5  micromoles  per  hour  per  milligram  protein  for  the  reduction  of 
endogenous  Q  on  adding  succinate  to  the  CN-inhibited  system.  By  using  a  rapid 
flow  apparatus  and  more  active  preparations  we  have  observed  values  of  Q-reduc- 
tase  activity  up  to  this  level,  which  is  a  rather  high  activity,  but  this  is  still  some- 
what less  than  the  rates  of  oxygen  reduction. 

Singer:  I  was  wondering,  since  you  did  not  commit  yourself,  which  of  the 
three  possible  hypotheses  of  the  mode  of  action  you  favour,  and  whether  the  sum 
total  of  the  data  presented  today  plus  those  you  published  in  the  Biochemical 
Journal  do  not  point  to  a  possible  function  of  Q  in  interchain  electron  transport  ? 


MITOCHONDRIAL   LIPIDS   AND   THEIR   FUNCTIONS   IN   THE   RESPIRATORY   CHAIN       191 

In  weighing  the  evidence  it  is  well  to  remember  that  what  matters  is  not  that  under 
certain  sets  of  conditions  the  rate  of  cycling  of  ubiquinone  approaches  that  of  the 
respiratory  chain,  but  rather  that  it  is  relatively  easy  to  produce  conditions  under 
which  the  turnover  of  ubiquinone  is  lower  than  the  rate  of  succinate  oxidation. 
The  latter  type  of  experiment  would  not  suggest  that  ubiquinone  is  an  obligatory 
component  of  the  electron  transport  chain,  but  it  would  by  no  means  exclude  its 
function  as  an  interchain  lipid.  This  function  would,  of  course,  also  lead  to  a  reduc- 
tion of  cytochromes  c  and  c-^  but  not  necessarily  in  the  same  chain. 

Redfearx  :  I,  of  course,  rather  favour  this  idea  and  I  discussed  it  at  some  length 
in  a  recent  paper,  but  I  didn't  want  to  commit  myself  here.  I  think  this  possibility 
fits  the  results  well  but  we  can't  exclude  the  other  possibilities. 

EsTABROOK  :  I  have  a  question  of  terminology  on  your  very  interesting  observa- 
tion. You  lose  only  20",,  of  the  succinate  oxidase  activity  on  removing  96  "o  of  what 
you  said  was  extractable  Qi,,.  Is  this  total  ubiquinone  or  that  extracted  bv  vour 
solvent  system  ?  Was  there  still  over  10",,  remaining  ? 

Redfearn  :  Yes. 

Williams:  I  wanted  to  ask  whether  it  would  be  a  logical  consequence  of  Dr. 
Singer's  hypothesis,  that  you  should  be  able  to  isolate  chains  which  do  not  contain 
ubiquinone  ? 

Redfearx:  Yes,  that  is  what  we  are  trying  to  do.  We  find  it  very  difficult  to 
remove  the  remaining  few  per  cent  of  ubiquinone.  As  you  continue  extracting  you 
remove  more  and  more  phospholipids  and  other  structural  lipids  and  then  you 
start  losing  activity.  It  is  very  difficult  to  remove  ioo"„  of  the  ubiquinone  without 
removing  other  lipids. 


The  Functional  Link  of  Succinic  Dehydrogenase  with  the 
Terminal  Respiratory  Chain 

Giovanni  Felice  Azzone* 

The  Wemier-Gren  Institute  for  Experimental  Biology, 
University  of  Stockholm,  Sweden 

It  is  our  purpose  to  examine  some  energetic  aspects  of  the  electron 
transport  system  catalyzing  the  aerobic  oxidation  of  succinate  in  intact 
liver  mitochondria.  It  has  been  generally  accepted  that  the  oxidation  of 
succinate  is  completely  independent  of  electron  transport  and  phosphoryla- 
tion in  the  DPN-flavin  region  of  the  respiratory  chain.  Support  for  this 
concept  came  from  the  findings  that  mitochondria  either  depleted  of  DPN, 
or  in  the  presence  of  amytal,  as  well  as  non-phosphorylating  submito- 
chondrial  preparations  are  fully  capable  of  catalyzing  the  aerobic  oxidation 
of  succinate.  Therefore  it  seemed  likely  that  cytochrome  b  was  the  site  of 
entrance  for  the  electrons  coming  from  succinic  dehydrogenase;  the  two 
phosphorylations  in  the  cytochrome  region  of  the  respiratory  chain  could 
then  account  for  the  net  phosphate  uptake  occurring  during  succinate 
oxidation. 

Renewed  interest  in  this  question  has  emerged  subsequent  to  the  recent 
work  of  Chance  and  Hollunger  [i,  2],  and  of  Klingenberg  et  al.  [3,  4],  who 
found  that  the  extent  of  reduction  of  mitochondrial  pyridine  nucleotide  is 
greatly  increased  by  the  addition  of  a  flavosubstrate,  succinate,  or  glycerol 
I -phosphate. 

A  different  approach  to  the  question  of  the  interaction  between  succinic 
dehydrogenase  and  the  DPX-flavin  region  of  the  respiratory  chain  recently 
has  been  possible  because  of  the  finding  [5,  6]  that  intact  liver  mito- 
chondria, when  depleted  of  high  energy  phosphate,  are  no  longer  capable 
of  oxidizing  succinate  at  any  appreciable  rate  unless  ATP  is  added,  or 
synthetized  in  the  system.  Furthermore,  the  beneficial  effect  of  ATP  is  not 
abolished  in  the  presence  of  uncoupling  agents. 

The  depletion  of  mitochondrial  high  energy  phosphate  and 
the  inhibition  of  succinate  oxidation 

An  experiment  illustrating  the  depression  of  the  capacity  for  succinate 
oxidation  in  rat  liver  mitochondria  is  shown  in  Fig.  i.  Addition  of  arsenate 

*  Fellow  of  the  Consiglio  Nazionale  delle  Ricerche.  Permanent  address : 
Istituto  di  Patologia  Generate,  Universita  di  Padova,  Italy. 

VOL.  II. — o 


194 


GIOVANNI    FELICE    AZZONE 


(Expt.  (a))  to  liver  mitochondria  in  the  presence  of  succinate  ehcited  a 
respiration  which  was  less  than  half  of  that  obtained  in  the  presence  of 
either  dicoumarol  (Expt.  (a)),  or  phosphate  and  a  phosphate  acceptor. 
The  increased  respiration  elicited  by  arsenate  was  due  to  a  partial  release 
of  respiratory  control.  Subsequent  addition  of  dicoumarol  released  the 
respiratory  control  completely.  When  the  mitochondria  were  preincubated 
(Expt,  (b) )  with  arsenate  for  3  min.  prior  to  the  addition  of  succinate  the 
respiration  was  about  half  maximal,  but  even  this  level  was  reached  only 
after  a  lag  phase.  If  on  the  other  hand  dicoumarol  was  added  during  the 
preincubation  (Expt.  (c))  together  with  arsenate,  the  rate  of  succinate 
oxidation  remained  low  upon  prolonged  incubation  (about  10%  of  the 
maximum). 


[02]= 0   - 


Fig.  I.  Inhibition  of  succinate  oxidation  in  rat  liver  mitochondria  incubated 
with  arsenate  and  dicoumarol  [6].  Concentrations  of  the  reagents  in  a  final 
volume  of  1-5  ml.  were  as  follows:  0-05  M  KCl,  0-033  ^  tris  buffer  pH  7-5, 
o-oo8  M  MgClo,  0-05  M  sucrose,  0-013  m  succinate  (Succ),  0-002  m  arsenate 
(Arsen.),  o- 00006  M  dicoumarol  (Die.)-  Mitochondria  from  400  mg.  rat  liver  wet 
weight  (about  8  mg.  protein).  The  substances  were  added  at  the  points  indicated. 
"1-3  Medium"  stands  for  KCl,  tris,  MgCl  and  sucrose  added  in  a  volume  of  i  -3 
ml.  Oxidation  rate  of  succinate  is  given  in  m/iatoms  oxygen  per  min. 


A  possible  interpretation  of  these  findings  was  suggested  by  experi- 
ments in  which  arsenate  was  found  to  deplete  the  mitochondria  of  their 
endogenous  phosphate.  When  mitochondria  labelled  with  ^'-P  were 
incubated  in  the  presence  of  arsenate  (Table  I)  an  almost  complete 
depletion  of  mitochondrial  phosphate  took  place  within  a  few  minutes. 
Addition  of  succinate  largely  prevented  this  effect  of  arsenate.  The  pre- 
vention was  ascribed  to  a  reincorporation  of  inorganic  phosphate  into 
ATP  by  way  of  aerobic  phosphorylation,  since  the  oxidation  of  succinate 
is  only  partly  uncoupled  by  arsenate.  Addition  of  respiratory  inhibitors 
such  as  antimycin  A  or  KCN  abolished  the  succinate  effect  on  the  arsenate- 
induced  depletion. 

Thus  the  initial  low  rate  of  succinate  oxidation  after  preincubation  with 
arsenate  was  considered  to  be  a  consequence  of  the  loss  of  high  energy 
phosphate  from  the  mitochondria,  and  the  gradual  increase  in  the  rate  of 


THE    FUNXTIONAL    LINK    OF    SUCCINIC    DEHYDROGENASE 


195 


[Oj]=0 


Fig.  2.  Stimulation  of  succinate  oxidation  by  ATP  in  rat  liver  mitochondria 
preincubated  with  arsenate  and  dicoumarol  [5].  Experimental  conditions  as  in 
Fig.  I.  0001  M  ATP  (ATP)  was  added  at  the  point  indicated. 

oxidation,  as  a  consequence  of  the  resynthesis  of  high  energy  intermediates 
taking  place  during  the  oxidation  of  succinate.  When  this  resynthesis  was 
aboUshed  by  the  presence  of  an  uncoupling  agent,  the  depression  of 
succinate  oxidation  became  permanent.  Under  these  conditions,  added 
ATP  was  required  for  stimulating  the  oxidation  of  succinate  (Fig.  2).  No 
stimulation  of  the  oxidation  rate  was  observed  when  ATP  was  replaced  by 
AMP  or  EDTA. 


TABLE  I 

Depletion    of    Mitochondrial    Endogenous    Phosphate    by    Arsenate    and 
Protection  by  Succinate 

"*-P-labelled  mitochondria"  from  500  mg.  rat  liver  (wet  weight)  were  incubated 
in  open  tubes  at  30.  After  7  min.,  i  ml.  of  the  incubation  mixture  was  filtered 
through  a  Celite  layer  as  reported  elsewhere  [12].  Each  tube  contained  in  a  final 
volume  of  5  ml.:  0-05  M  KCl,  0-03  M  tris  buffer  pH  7-5,  0-125  ^  sucrose,  and 
when  indicated  o-oi  M  succinate,  0-003  ^'  arsenate,  2  /(g.  antimycin  A,  o-ooi  M 
KCN.  As  in  Table  II  and  III,  the  number  of  counts  is  here  indicative  of  the 
amounts  of  endogenous  phosphate  which  remained  in  the  mitochondria  after 
preincubation. 


Additions 


Counts/min.  (  x  10   ^) 


None 
Arsenate 
Succinate 

Arsenate  +  succinate 
Arsenate  +  succinate  + 

Antimycin  A 
Arsenate  +  succinate  +  KCN 


700 

100  (14 •4°,,) 
521  (73-5"o) 
421  (6o-2<'o) 

223  (3i-9"o) 
128  (18-30,.) 


196  GIOVANNI    FELICE   AZZONE 

The  concept  that  intact  mitochondria  have  a  strict  requirement  for  high 
energy  phosphate  and  not  merely  of  inorganic  phosphate  in  order  to  main- 
tain a  high  rate  of  succinate  oxidation  is  supported  further  by  the  following 
findings. 


Fig.  3.  Correlation  of  the  inhibition  of  succinate  oxidation  with  the  depletion 
of  mitochondrial  phosphate.  Mitochondrial  endogenous  phosphate  measured  as 
described  in  Table  I. 


[O2]  =  0 


[O2]=0 


Fig.  4.  Prevention  of  the  inhibition  of  succinate  oxidation  by  amytal  [6]. 
Experimental  conditions  as  in  Fig.  i.  coca  m  amytal  was  added  at  the  point 
indicated. 


The  presence  of  a  five-fold  molar  excess  of  inorganic  phosphate  during 
the  preincubation  of  the  mitochondria  with  arsenate  completely  prevented 
the  inhibition  of  succinoxidase  activity.  On  the  other  hand,  if  the  same 
concentration  of  inorganic  phosphate  was  added  to  the  mitochondria  after 


THE    FUNCTIONAL    LINK    OF    SUCCINIC    DEHYDROGENASE  1 97 

they  had  been  depleted  of  high  energy  phosphate  by  the  arsenate-di- 
coumarol  pretreatment,  no  stimulation  of  the  oxidation  rate  was  observed, 
and  added  ATP  was  required  in  order  to  restore  the  succinoxidase  activity. 


Fig.  5.  Stimulation  of  succinate  oxidation  by  inorganic  phosphate  and  ATP 
in  pretreated  rat  liver  mitochondria  [6].  Experimental  conditions  as  in  Fig.  i.  The 
mitochondria  used  in  this  experiment  were  pretreated  for  5  min.  at  30"  with 
ooooi  M  DNP  plus  0001  M  AMP,  and  then  washed  with  025  M  sucrose. 

As  shown  in  Fig.  3,  the  time  of  preincubation  with  arsenate  necessary 
for  inhibiting  succinate  oxidation  corresponded  approximatelv  to  the  time 
required  for  depleting  the  mitochondria  of  high  energy  phosphate. 

Additional  support  for  the  above  conclusion  was  obtained  with  the  use 
of  amytal.  As  reported  elsewhere,  the  depleting  etfect  of  arsenate  on  the 


198  GIOVANNI    FELICE   AZZONE 

mitochondrial  high  energy  phosphate  was  almost  completely  abolished  in 
the  presence  of  2  mM  amytal.  In  agreement  with  this  experiment,  it  was 
found  (Fig.  4)  that  when  amytal  was  added  prior  to,  or  together  with, 
arsenate  a  high  respiration  ensued  upon  the  addition  of  succinate.  On  the 
other  hand,  if  amytal  was  added  afler  the  preincubation  with  arsenate,  the 
succinoxidase  activity  was  greatly  inhibited  and  again,  added  ATP  was 
required  to  increase  the  oxidation  rate. 

It  could  be  demonstrated  clearly  that  the  depression  of  the  capacity  for 
succinate  oxidation  was  caused  by  the  depletion  of  mitochondrial  high 
energy  phosphate  compounds,  and  not  by  the  presence  of  arsenate  itself. 
This  was  accomplished  by  the  use  of  2,4-dinitrophenol  (DNP)  plus  AMP 
to  pretreat  mitochondria  which  were  then  washed  free  of  the  depleting 
agents.  The  succinoxidase  activity  of  these  preparations  was  very  low  and 
could  be  stimulated  more  than  two-fold  by  the  addition  of  ATP  (Fig.  5  {a)). 
Inorganic  phosphate  also  could  increase  the  oxidation  rate  but  only  if 
added  before  the  uncoupling  agent  and  together  with  succinate  (Fig.  5  {b)). 
Under  these  conditions  high  energy  phosphate  compounds  could  be 
synthetized  by  the  mitochondria. 

The  question  of  oxaloacetate 

Oxaloacetate  is  known  to  be  a  competitive  inhibitor  of  succinic  de- 
hydrogenase, and  its  accumulation  has  been  considered  as  chiefly 
responsible  for  the  inhibitions  of  succinate  oxidation  observed  by  difi^erent 
workers.  Furthermore,  a  protective  effect  of  ATP  against  the  inhibition 
induced  by  oxaloacetate  has  been  reported  by  Pardee  and  Potter  [8]  and 
by  Tyler  [9].  Therefore  it  was  necessary  to  examine  in  greater  detail  the 
mechanism  by  which  energy  is  provided  for  the  activation  of  succinate 
oxidation,  and  also  the  possible  ways  by  which  oxaloacetate  may  interfere 
with  the  mitochondrial  oxidation  of  succinate. 

The  possibility  that  the  accumulation  of  oxaloacetate,  per  se,  could  be 
responsible  for  the  low  rate  of  succinate  oxidation  after  the  arsenate- 
dicoumarol  preincubation,  has  been  excluded  by  three  types  of  experiments : 

{a)  The  inhibition  was  not  relieved  by  the  addition  of  cysteine 
sulphinate,  in  the  presence  of  amytal.  Control  experiments  showed  that 
cysteine  sulphinate  did  remove  an  inhibition  of  succinate  oxidation  due  to 
added  oxaloacetate  in  agreement  with  the  finding  of  Singer  and  Kearney 
[10]  who  have  demonstrated  that  cysteine  sulphinate  transaminates 
oxaloacetate  to  aspartate. 

{b)  It  would  be  anticipated  that  ATP  could  be  replaced  by  GTP  (or 
ITP)  if  ATP  was  acting  by  removing  oxaloacetate  via  the  oxaloacetic 
carboxylase  reaction  since  this  reaction  specifically  utilizes  GTP  (or  ITP). 
Under  our  conditions  little  stimulation  of  succinate  oxidation  was  observed 


THE    FUNCTIONAL    LINK    OF    SUCCINIC    DEHYDROGENASE  1 99 

after  the  addition  of  GTP  or  ITP  as  compared  with  that  induced  by  ATP. 
Furthermore,  only  trace  amounts  (less  than  5  x  iq-^  m)  of  phosphoenol- 
pyruvate,  the  product  of  the  oxaloacetic  carboxylase  reaction,  could  be 
detected  after  the  addition  of  ATP. 

(c)  No  measurable  amounts  of  oxaloacetate  could  be  detected  after  the 
arsenate-dicoumarol  p  reincubation ;  the  assay  was  sensitive  to  concentra- 
tions of  oxaloacetate  in  the  incubation  mixture  as  low  as  5  x  10  ^^  m  [ii]. 

Although  these  findings  seem  to  preclude  a  possible  involvement  of 
oxaloacetic  acid  as  directly  responsible  for  the  observed  inhibition  of 
succinate  oxidation,  some  indication  was  obtained  that  the  presence  of 
oxaloacetate  was  necessary  during  the  preincubation  in  order  to  obtain 
the  inhibited  state.  Addition  of  cysteine  sulphinate  during  the  arsenate- 
dicoumarol  preincubation,  which  could  be  expected  to  remove  all  the 
oxaloacetate  formed  from  endogenous  substrates,  resulted  in  a  complete 
protection  of  succinate  oxidation. 

The  substrate  level  phosphorylation  compartment  and  the 
energy  source  for  the  activation  of  succinate  oxidation 

Evidence  has  been  presented  that  the  a-ketoglutarate-linked  substrate- 
level  phosphorylation  can  give  rise  to  an  ATP  which  is  not  directly  available 
to  the  DNP-induced  ATPase  [12].  From  this  finding  the  concept  was 
developed  that  the  ATP  originating  from  the  substrate  level  phosphory- 
lation is  compartmentalized  in  the  mitochondria  and  that  accessory  reac- 

TABLE    II 

Effect  of  AMP  and  of  Various  Substrates  on  the  DNP-Induced  Depletion 
OF  Mitochondrial  Endogenous  Phosphate  [12] 

Each  tube  contained  in  a  final  volume  of  3  ml.:  0001  M  AMP,  o-oooi  M 
DNP,  0-003  '^i  glutamatc,  0-003  ^1  /S-hydroxybutyrate,  0-003  -^i  oxaloacetate  and 
0-003  ^^  succinate;  other  experimental  conditions  as  in  Table  I;  o-ooi  m  MgCl., 
was  also  added  in  P^xpt.  2.  Time  of  incubation  7  min.  in  Expt.  i  and  5  min.  in 
Expt.  2. 

Additions  Counts /sec. 

Expt.  I  none  429 

DNP  277(65-0%) 

DNP  +  succinate  170(39-8%) 

DNP  +  AMP  114(26-7%) 

Expt.  2         none  326 

DNP  238(73-2%) 

DNP  +  Glutamate  2i2(65-2"o) 

DNP  +  /3-hydroxybutyrate  223  (68-6%) 

DNP  +  oxaloacetate  134  (41  - 1  %) 

DNP  +  succinate  1 30  (40  - 1  %) 


200  GIOVANNI    FELICE   AZZONE 

tions  are  required  for  transferring  phosphate  from  this  ATP  to  external 
ADP.  Indications  were  obtained  for  the  following  mechanisms  being 
operative  in  this  transfer  of  phosphate :  a  double  adenylate  kinase,  the 
oxaloacetic  carboxylase-pyruvic  kinase  and  the  activation  of  succinate 
oxidation  discussed  in  the  present  paper.  An  experiment  showing  the 
effectiveness  of  these  three  mechanisms  in  transferring  phosphate  in  order 
to  render  ATP  available  to  the  DNP-induced  ATPase  is  illustrated  in 
Table  II.  When  ^-P-labelled  mitochondria  were  incubated  in  the  presence 

TABLE  III 

Effect  of  Cysteine  Sulphinate  and  Oxaloacetate  on  the  Release  of  ^T 
FROM  Mitochondria  during  Incubation  with  Arsenate  [7] 

Each  tube  contained  in  a  final  volume  of  3  ml.:  0-05  M  KCl,  0-03  M  tris 
buffer  pH  7-5,  0-125  M  sucrose,  o-oi  M  MgCl.,  and,  when  indicated,  0-003  m 
arsenate,  0-005  M  cysteine  sulphinate,  0-002  M  amytal,  0-003  M  oxaloacetate. 
"^-P-labelled  mitochondria"  from  500  mg.  liver.  Time  of  incubation,  5  min. 
Temperature,  30^. 


Addition 

Counts 

/sec. 

Expt.  I 

Expt.  2 

None 

482 

525 

Arsenate 

76 

73 

Arsenate,  cysteine  sulphinate 

— 

211 

Amytal 

429 

— 

Amytal,  oxaloacetate 

203 

— 

Amytal,  oxaloacetate,  cysteine 

svilphinate 

404 

— 

Arsenate,  amytal 

339 

— 

Arsenate,  amytal,  oxaloacetate 

117 

— 

of  DNP  a  partial  release  (about  one-third)  of  the  ^-P  took  place.  Addition 
of  AMP,  succinate  or  oxaloacetate  enhanced  the  releasing  effect  of  DNP. 
No  such  effect  was  obtained  with  ^-hydroxybutyrate  or  glutamate,  indicat- 
ing that  the  release  induced  by  succinate  or  oxaloacetate  was  not  due  in 
an  unspecific  manner  to  the  presence  of  an  oxidizable  metabolite. 

The  capacity  of  oxaloacetate  in  removing  ^-P  from  the  mitochondria  is 
also  illustrated  in  Table  III.  When  the  depleting  effect  of  arsenate  was 
removed  by  the  presence  of  amytal  (see  Fig.  4),  addition  of  oxaloacetate 
induced  again  a  large  release  of  ^^P  from  the  mitochondria  (Expt.  i). 
Oxaloacetate  was  able,  alone  or  in  the  presence  of  amytal,  to  remove  ^^P 
from  the  mitochondria,  and  the  depleting  effect  of  oxaloacetate  was 
removed  by  the  addition  of  cysteine  sulphinate  (Expt.  i).  A  partial  but 
significant  protection  against  the  arsenate-induced  release  of  ^^P  from 
labelled  mitochondria  was  also  obtained  when  cysteine  sulphinate  was 
added  to  the  incubation  medium  (Expt.  2).  Thus  the  capacity  of  cysteine 


THE    FUNCTIONAL    LINK    OF   SUCCINIC    DEHYDROGENASE  201 

sulphinate  to  prevent  the  inhibition  of  succinate  oxidation  when  added  to 
the  mitochondria  during  the  incubation  with  arsenate  and  dicoumarol,  can 
be  explained  on  the  basis  of  the  abihty  of  cysteine  sulphinate  to  maintain, 
by  rendering  the  oxaloacetic  carboxylase  reaction  inactive,  high  energy 
compounds  in  the  substrate  level  phosphorylation  compartment  of  the 
mitochondria. 

On  the  basis  of  the  present  findings  it  appears  that  oxaloacetate  besides 
its  known  function  as  competitive  inhibitor  of  succinic  dehydrogenase, 
possesses  also  the  capacity  of  competing,  through  the  oxaloacetic  car- 
boxylase reaction,  in  the  utilization  of  the  high  energy  phosphate  com- 
pounds required  for  succinate  oxidation.  This  additional  property  of 
oxaloacetate  appears  to  be  of  particular  significance  in  conditions  where 
the  generation  of  high  energy  phosphate  compounds  from  respiratory 
chain  phosphorylations  is  blocked  by  the  addition  of  uncoupling  agents. 
In  these  conditions  the  oxidation  of  succinate,  which  otherwise  can 
dispose  of  the  high  energy  intermediates  formed  in  the  last  two  respiratory 
chain  phosphorylations,  will  be  dependent,  as  sole  source  of  energy,  on 
the  ATP  which  originates  from  the  a-ketoglutarate-linked  substrate-level 
phosphorylation. 

The  activation  of  succinate  oxidation 

The  experimental  evidence  reported  above  supports  the  concept  that  in 
intact  mitochondria  the  aerobic  oxidation  of  succinate  must  proceed 
through  a  thermodynamically  unfavourable  reaction  which  requires  the 
investment  of  energy.  This  energy-demanding  reaction  appears  to  be 
characteristic  only  of  phosphorylating  mitochondrial  preparations,  since  no 
such  requirement  has  been  demonstrated  for  non-phosphorylating 
succinoxidase  preparations. 

Although  no  conclusive  evidence  exists  at  present  regarding  the  nature 
of  the  high  energy  intermediate  formed,  it  seems  conceivable  that  this 
intermediate  must  possess  the  following  properties :  (a)  it  must  be  different 
from  the  two  high  energy  intermediates  which  provide  the  two  net  phos- 
phorylations occurring  during  the  aerobic  oxidation  of  succinate ;  (/;)  the 
energy  of  this  additional  intermediate  can  be  used  directly  or  indirectly  for 
the  reduction  of  the  mitochondrial  pyridine  nucleotide;  this  conclusion  is 
supported  by  the  findings  made  in  collaboration  with  KHngenberg  [ii] 
that  the  stimulation  of  succinate  oxidation  by  ATP  in  arsenate-dicoumarol 
pretreated  mitochondria  is  paralleled  by  a  reduction  of  mitochondrial 
pyridine  nucleotide ;  (c)  the  formation  of  this  high  energy  intermediate  is 
not  impaired  by  the  presence  of  uncoupling  agents.  This  finding  suggests 
that  the  energy  required  for  activation  of  succinate  oxidation  and  for 
reduction  of  pyridine  nucleotide  cannot  be  supplied  by  a  non-phos- 
phorylated  intermediate  of  the  X  ~  I  type,  because  the  latter  has  the 


202  GIOVANNI    FELICE   AZZONE 

property  of  being  hydrolyzed  by  DNP.  On  the  other  hand,  this  result  is 
consistent  with  those  hypotheses  in  which  a  reduced  phosphorylated 
electron  carrier  of  the  XH  ~  P  type  is  formed  at  the  expense  of  ATP  in 
the  presence  of  uncoupling  agents. 

Therefore,  it  appears  likely  that  once  the  reduced  phosphorylated 
intermediate  is  formed,  it  can  either  transfer  electrons  to  the  cytochrome 
system  by  which  ATP  is  regenerated,  or  to  DPN  in  which  case  reduced 
pyridine  nucleotide  is  formed  and  inorganic  phosphate  is  liberated. 
Further  work  is  in  progress  in  order  to  define  more  precisely  the  reactions 
by  which  the  oxidation  of  succinate  by  the  terminal  respiratory  chain 
requires  a  supply  of  high  energy  phosphate. 

References 

1.  Chance,  B.,  and  Hollunger,  G.,  Fed.  Proc.  l6,  163  (1957). 

2.  Chance,  B.,  and  Hollunger,  G.,  Nature,  Loud.  185,  666  (1960). 

3.  Klingenberg,  M.,  and  Slenczka,  W.,  Biochem.  Z.  331,  331  (1959). 

4.  Klingenberg,  M.,  Slenczka,  W.,  and  Ritt,  E.,  Biochem.  Z.  332,  47  (1959). 

5.  Azzone,  G.  F.,  and  Ernster,  L.,  Nature,  Loud.  187,  65  (i960) 

6.  Azzone,  G.  F.,  and  Ernster,  L.,_7.  biol.  Chem.  236,  1518  (1961) 

7.  Azzone,  G.  F.,  and  Ernster,  L.,^.  biol.  Chem.  236,  15 10  (1961) 

8.  Pardee,  A.  B.,  and  Potter,  V.  R.,y.  biol.  Chem.  176,  1085  (1948). 

9.  Tyler,  D.  B.,  J.  biol.  Chem.  216,  395  (1955). 

10.  Singer,  T.  P.,  and  Kearney,  E.  B.,  Arch.  Biochem.  Biophys.  61,  397  (1956). 

11.  Azzone,   G.    F.,   Ernster  L.,  and  Klingenberg,  M.,  Nature,  Lond.   188,  552 
(i960) 

12.  Azzone,  G.  F.,  and  Ernster,  \^.,y.  biol.  Chem.  236,  1501  (1961) 

Discussion 

Chance:  I  think  Dr.  Azzone's  paper  has  a  great  deal  of  information  in  it,  it 
takes  time  to  digest.  His  remark  about  the  non-phosphorylating  preparation 
oxidizing  succinate  directly  is  surely  one  that  we  must  not  forget  in  postulating 
mandatory  succinate  oxidation,  and  I  guess  that  it  is  one  thing  that  just  isn't 
explained  yet  by  your  mechanism.  We  have  the  feeling  that  the  ATP  requirement 
for  succinate  oxidation  and  ATP  requirement  for  reduction  of  pyridine  nucleotide 
may  not  be  identical  in  detail  although  they  appear  to  be  identical  in  the  kind  of 
experiments  that  you  have  been  doing.  I  think  that  the  amytal  sensitivity  that 
Dr.  Ernster  has  already  referred  to  gives  a  hint  that  this  process  does  involve 
carriers  of  the  respiratory  chain.  I  don't  quite  understand  the  basis  on  which 
you  conclude  the  uncoupling  agents  don't  impair  the  formation  of  DPNH,  or 
maybe  you  meant  they  didn't  impair  the  reactivation  of  succinate  oxidation, 
because  I  believe  it  is  clear  from  our  experiments  that  the  DPNH  formation  is 
highly  sensitive  to  uncoupling  agents. 

Azzone:  Well,  I  think  we  must  make  a  distinction  here.  If  you  mean  that  in 
State  3,  DPNH  gets  easily  oxidized,  then  I  agree  with  you  that  addition  of  dinitro- 
phenol,  which  induces  a  State  3  condition,  also  makes  it  more  difficult  to  observe 
DPN  reduction.  But  our  conclusion  that  addition  of  uncoupling  agents  does  not 


THE   FUNCTIONAL    LINK    OF    SUCCINIC    DEHYDROGENASE  2O3 

inhibit  DPN  reduction,  was  derived  from  the  finding  (reported  by  Dr.  Ernster) 
that  the  energy  of  ATP  could  be  used  for  reducing  DFX  in  the  presence  of 
dinitrophenol. 

According  to  the  phosphoryl-flavin  theory  [Low,  Siekevitz,  Ernster  and  Lind- 
berg,  Biochirn.  biophxs.  Acta  29,  392  (1958)]  ATP  can  react  with  the  diaphorase 
flavin  giving  rise  to  a  reduced  phosphorylated  electron  carrier : 

2H  -  +  ATP  +  2Fe  -  -  +  Fp >  ADP  +  zFe  "  '  -  +  FpH  -  P  (i ) 

It  has  been  suggested  that  Reaction  (i)  is  not  sensitive  to  the  uncoupling  agents, 
and  therefore  the  energy  of  ATP  can  be  used,  through  the  intermediate  FpH  -«-  P, 
for  reducing  DPN  (Reaction  (2)): 

FpH  ~  P  +  DPN >  Fp  +  DPXH  +  Pj  +  H  (2) 

In  the  presence  of  dinitrophenol  Reaction  (2)  will  take  place  from  right  to  left 
without  the  activation  of  inorganic  phosphate  (Reaction  (3)): 

DNP 

H+DPXH  +  Fp >DPX  +  FpH,  (3) 

Thus  the  sum  of  Reactions  (i),  (2)  and  (3)  will  account  for  an  ATP-ase  activity. 

Chance:  I  still  think  there  is  a  discrepancy  because  it  is  the  rate  of  reduction 
of  DPX  (the  second  equation)  that  we  observed  optically  to  be  inhibited. 

AzzoNE :  The  fact  that  the  rate  of  reduction  of  DPX  is  lower  in  the  presence 
than  in  the  absence  of  dinitrophenol  can  be  explained  on  kinetic  reasoning. 

Once  the  high  energy  intermediate  postulated  in  our  hypothesis  has  been  formed 
during  succinate  oxidation  [Azzone,  Ernster  and  Klingenberg,  Xattire,  Loud.  188, 
552  (i960)]  it  can  be  either  utilized  for  reducing  DPX,  or  reoxidized  by  the  cyto- 
chrome system.  The  higher  the  electron  flow  toward  the  oxygen  the  lower  will  be 
the  utilization  of  the  intermediate  in  the  backward  reaction  for  reducing  DPX. 

Ernster:  We  do  accept  the  fact  that  the  level  and  rate  of  DPX-reduction  may 
be  low  in  the  presence  of  an  uncoupling  agent  which  allows  full  respiration.  How- 
ever, the  point  we  wish  to  stress  is  this :  Is  it  at  all  possible  to  obtain  an  ATP- 
induced  DPX-reduction  (no  matter  how  little)  in  the  presence  of  a  fully  uncoupling 
concentration  of  dinitrophenol  or  dicoumarol  ?  I  think  our  data  clearly  show  that 
it  is. 

HoLTON :  Before  the  experiments  of  Chance  and  Hollunger  were  published, 
when  one  got  an  activation  of  oxidation  of  succinate  in  mitochondria  one  normally 
regarded  this  as  evidence  that  the  mitochondria  were  breaking  up.  One  knows  very 
well  that  in  intact  mitochondria  succinate  oxidation  is  rather  slow  while  in  mito- 
chondrial fragments  it  is  extremely  rapid,  so  it  is  clear  that  the  structural  state  of 
the  mitochondrion  can  have  some  influence  on  the  rate  at  which  it  oxidizes  suc- 
cinate cjuite  apart  from  mechanisms  of  the  type  postulated  by  Chance  and 
Hollunger.  I  wonder  whether  it  might  be  as  well  to  keep  in  mind  that  changes  in 
the  rate  of  oxidation  of  succinate  can  be  mediated  by  changes  in  the  structural 
state  of  the  mitochondrion,  that  changes  in  structural  state  can  be  brought  about 
by  changes  in  the  ATP: ADP  ratio  and  the  possibility  that  these  mechanisms  of 
succinate  oxidation  involving  energy  requirement  and  oxidation  via  DPXH  are 
not  the  only  way  of  explaining  an  acceleration  of  succinate  oxidation  under  any 
particular  experimental  conditions. 


204  GIOVANNI    FELICE   AZZONE 

Azzone:  We  have  not  yet  conclusive  evidence  about  the  chemical  reaction 
requiring  the  investment  of  energy.  However,  even  if  it  is  assumed  that  the  ATP 
stimulation  is  the  consequence  of  a  structural  effect  we  still  have  to  account  for 
the  formation  of  a  high  energy  intermediate,  controlling  the  oxidation  of  suc- 
cinate which  precedes  the  mechano-chemical  utilization  of  ATP. 

HoLTON :  We  must  consider  the  availability  of  the  succinate  to  the  succinic 
hydrogenase  besides  the  possibility  that  its  oxidation  requires  an  investment  of 
energy. 

AzzoNE :  According  to  the  mechanism  we  have  proposed,  in  non-phosphorylat- 
ing  preparations  it  is  possible  for  the  electron  derived  from  succinate  to  proceed 
through  a  sequence  of  electron  carriers  each  of  which  possesses  a  higher  redox 
potential  than  the  preceding  one ;  on  the  contrary,  in  intact  mitochondria  there  is  a 
thermodynamically  unfavourable  step  which  must  be  circumvented  by  the  invest- 
ment of  energy. 

Williams  :  I  should  like  to  say  that,  as  Dr.  Azzone  is  by  now  aware,  we  have 
found,  (a)  that  cysteine  sulphinic  acid  reactivates  rat  liver  mitochondria  under 
conditions  as  similar  as  we  could  get  to  the  ones  described  in  his  paper  in  Nature, 
(b)  that,  although  pyruvate  affords  some  degree  of  protection,  a-ketoglutarate  does 
not,  although  it  does  maintain  its  substrate  level  phosphorylation.  However,  I  think 
it  may  be  better  to  emphasize  the  measure  of  our  agreement  and  to  suggest  that 
you  are  now  coming  very  close  to  saying  that  oxaloacetate  is  necessary.  I  think  our 
disagreement  is  then  not  so  great  and  we  can  leave  open  the  details  of  how  oxalo- 
acetate exerts  this  inhibition  and  how  ATP  relieves  this  inhibition.  In  Tyler's 
work  ATP  relieved  the  inhibition  without  altering  the  oxaloacetate  concentration, 
so  looking  for  PEP  may  not  help  us,  and  here  I  had  wondered,  as  had  Dr.  Holton, 
whether  the  ATP  acts  by  segregating  the  oxaloacetate  from  the  succinic  dehydro- 
genase, although  there  are  no  major  optical  density  changes  during  this  process. 

Slater  :  I  should  like  to  support  what  Dr.  W^illiams  said  a  moment  ago,  that  a 
very  important  point  to  look  at  is  how  ATP  reverses  oxaloacetate  inhibition  and  I 
should  like  to  bring  over  a  suggestion  of  my  colleague  Dr.  Hiilsmann,  that  you 
look  into  the  possibility  that  ATP  is  activating  oxaloacetate  removal  by  reacting 
with  endogenous  substrate  and  forming  acetyl-coenzyme  A  which  promotes  the 
removal  of  oxaloacetate;  this  is  based  on  some  recent  experiments  and  explains 
quite  a  lot  of  phenomena  including  the  Amytal  experiments,  in  the  presence  of 
which  oxaloacetate  is  not  formed. 

AzzoNE :  We  have  not  been  able  to  demonstrate  the  presence  of  oxaloacetate 
in  the  arsenate-dicoumarol  preincubated  mitochondria  so  one  must  postulate  that 
oxaloacetate  is  compartmentalized  in  such  a  way  that  it  cannot  be  reached  by 
chemical  analysis. 

Mitchell  :  I  should  like  to  raise  a  point  that  may  bring  together  Dr.  Slater's 
views  and  those  of  Dr.  Holton.  The  suggestion  that  I  am  going  to  make  comes 
from  work  on  micro-organisms  which  Dr.  Moyle  and  I  have  been  doing  recently. 
It  so  happens  that  in  micrococci  you  can  show  that  the  oxidation  of  succinate  can 
be  blocked  by  DNP  when  the  membrane  is  intact.  It  can  also  be  blocked  by 
mercury  compounds  or  by  arsenite.  But,  it  can  be  demonstrated  that  the  blocking 
of  oxidation  by  these  inhibitors  is  not  a  direct  effect  on  the  oxidation  system  but  is 
due  to  blockage  of  the  system  by  which  "succinate"  passes  into  the  cell.  If  you 


THE    FUNCTIONAL    LINK    OF    SUCCINIC    DEHYDROGENASE  205 

give  a  short  period  to  allow  the  "succinate"  to  enter  the  cell  before  adding  the 
inhibitor,  the  succinate  that  disappeared  from  the  medium  before  the  inhibitor 
was  added  subsequently  becomes  oxidized  in  the  ceil  at  almost  the  normal  rate. 
If,  however,  you  break  the  membrane  before  adding  these  inhibitors  of  "suc- 
cinate" transport,  succinate  oxidation  is  not  inhibited.  There  is  not  time  to  go 
into  details,  but  we  have  good  reasons  for  believing  that  succinate  goes  through 
the  membrane  either  as  succinyl-CoA  or  as  succinyl-lipoate  or  some  closely 
related  substance.  Thus,  the  oxidation  of  succinate  requires  its  prior  "activation" 
to  allow  it  to  reach  the  oxidation  systems.  We  believe  that  the  "activation"  step 
is  catalyzed  by  an  enzyme  located  in  the  plasma  membrane. 

Hess  :  I  would  like  to  ask  a  short  question  to  one  of  the  last  three  speakers 
about  the  arsenate  treatment;  according  to  Dr.  Ernster  the  pyridine  nucleotides 
were  largely  reduced  after  the  arsenate  treatment.  How  do  you  explain  it  ?  And 
what  is  the  reductant  ? 

Packer:  I  would  like  to  comment  on  some  recent  experiments  which  Dr.  E.  E. 
Jacobs  and  I  have  been  doing,  as  they  may  have  some  bearing  on  this  problem. 
We  have  been  looking  at  certain  shunts  of  electron  transport  involving  the  oxidase 
end  of  the  chain.  For  example,  we  use  ascorbate  as  reducing  agent  and  catalysts 
such  as  tetramethylphenylenediamine ;  we  have  been  able  to  show  that  these  shunts 
can  be  tightly  coupled  to  phosphorylation  and  show  respiratory  control  with  ADP 
and  thus  can  calculate  P  to  O  ratios  by  measuring  O.,  utilization  (polarographically). 
If  the  mitochondria  are  carefully  washed  the  endogenous  substrate  can  be  removed 
so  that  P:0  ratios  come  out  to  be  about  10.  It  happens  that  this  shunt  is  capable 
of  reducing  pyridine  nucleotides,  and  in  the  steady  state  the  pyridine  nucleotides 
can  be  oxidized  and  reduced  by  initiating  a  brief  cycle  of  phosphorylation  by 
adding  a  small  amount  of  ADP.  The  oxidation  through  the  shunt  is  not  inhibited 
by  antimycin  but  the  reduction  of  pyridine  nucleotide  is  completely  blocked  in 
the  presence  of  this  substance.  I  would  commend  this  shunt  as  of  possible  interest 
in  connection  with  the  phenomenon  of  reversal  of  electron  transport  just  discussed. 

Estabrook:  I  should  just  like  to  state  that  in  particles  which  we  know  are 
devoid  of  endogenous  substrate,  DNP  preincubation  does  not  inhibit  succinate 
oxidation.  This  is  a  situation  with  particles  which  will  give  good  phosphorylation 
in  the  absence  of  DNP  and  also  a  situation  where  pyridine  nucleotide  is  reduced 
by  succinate.  This  is  one  more  additional  piece  of  evidence  for  the  complexity  of 
the  endogenous  substrate. 

Azzone:  I  don't  see  why  this  should  be. 

Estabrook:  You  didn't  produce  any  oxaloacetate. 

Chappell  :  The  same  is  true  in  kidney  mitochondria  which  has  very  little 
endogenous  substrate. 

Azzone:  We  think  that  the  ATP  requirement  for  succinate  oxidation  is  present 
only  above  a  certain  level  of  structural  integrity.  If  we  take  away,  by  destroying  or 
fragmenting  the  mitochondria,  the  structural  barrier  which  makes  necessary  the 
energy-requiring  reaction  then  we  find  neither  an  inhibition  nor  an  ATP  stimula- 
tion of  succinate  oxidation. 

Chappell:  This  is  not  true  in  kidney  mitochondria  which  responds  to  normal 
respiratory  control  and  P:0  ratios,  and  they  are  just  free  of  endogenous  substrate. 
The  addition  of  oxaloacetate  or  malate  will  induce  all  the  phenomena  which  you 


2o6  GIOVANNI    FELICE    AZZONE 

have  just  talked  about.  We  can  see  the  same  with  malate  in  the  digitonin-extracted 

particles. 

Williams  :  Can  I  just  make  one  more  point  because  I  think  this  is  going  to  be 
misunderstood  by  some  people,  the  question  is  not  whether  there  is  an  energy- 
Unked  reduction  of  pyridine  nucleotide  but  the  relation  of  this  reduction  to 
respiration,  and  to  this  Dr.  Packer's  data  are  totally  irrevelant  although  of  great 
intrinsic  interest. 


Pyridine  Nucleotides  in  Mitochondria 

E.  C.  Slater,  AI.  J.  Bailie  and  J.  Bouman 

Laboratory  of  Physiological  Chemistry, 
University  of  Amsterdam,  Xetherlands 

One  of  the  experimental  difficulties  in  studying  the  mechanism  of 
respiratory-chain  phosphorylation  is  that  the  concentration  of  the  high- 
energv  phosphate  compounds,  which  must  surely  be  intermediates  in  this 
reaction,  will  be  of  the  same  order  as  that  of  the  mitochondrial  enzyme- 
coenzvme  system  which  catalyzes  the  reaction.  This  situation  may  be  con- 
trasted with  that  appertaining  to  glycolytic  phosphorylation,  where  the 
high-energy  phosphate  intermediate  (diphosphoglyceric  acid)  can  accumu- 
late in  amounts  of  the  same  order  as  the  substrate  concentration. 

This  difficulty  can  be  decreased  by  about  one  order  of  magnitude  by 
studying  those  components  of  the  respiratory  chain  which  are  present  in 
the  greatest  concentration  in  the  mitochondria,  viz.  the  pyridine  nucleo- 
tides and  ubiquinone  (coenzyme  Q).  This  lecture  is  concerned  with  the 
pyridine  nucleotides. 

It  is  only  during  the  last  lo  years  or  so  that  the  pyridine  nucleotides 
have  been  considered  to  be  constituents  of  the  mitochondria.  In  the 
nineteen-thirties,  diphosphopyridine  nucleotide  at  least  was  thought  of  as 
a  dissociable  coenzyme  catalyzing  anaerobic  oxido-reductions  in  the 
"soluble"  fraction  of  the  cytoplasm.  The  first  indication  that  it  was  much 
more  firmly  bound  came  from  the  isolation  by  Cori  in  1948  of  crystalline 
phosphoglyceraldehyde  dehydrogenase  containing  firmly  bound  DPN  + 
[i].  Even  more  important  for  the  topic  of  this  lecture  was  the  observation 
of  Huennekens  and  Green  [2]  that  rabbit-liver  and  rabbit-kidney  "  cyclo- 
phorase"  preparations,  consisting  largely  of  mitochondria,  contained 
considerable  amounts  of  firmly  bound  pyridine  nucleotide,  sufficient  for 
maximal  respiration  in  the  absence  of  added  pyridine  nucleotide.  The 
amounts  of  the  pyridine  nucleotide  were  little  changed  after  prolonged 
incubation  in  the  presence  of  substrates.  The  importance  of  the  mito- 
chondrial pyridine  nucleotides  was  further  stressed  by  Lehninger's  [3] 
observation  that  extra-mitochondrial  DPNH  was  only  very  slowly  oxidized 


2o8  E.    C.    SLATER,    M.    J.    BAILIE   AND   J.    BOUMAN 

by  intact  liver  mitochondria,  and  by  Christie  and  Judah's  [4]  similar 
finding  with  respect  to  reduction  of  DPN.* 

The  intramitochondrial  pyridine  nucleotides  are  also  inaccessible  to 
DPN  +-destroying  enzymesf  which  are  present  in  the  microsome  fraction 
[5].  The  fact  that  the  pyridine  nucleotides  are  retained  within  the  mito- 
chondria, even  when  the  mitochondrial  suspension  is  diluted,  provides  the 
opportunity  of  studying  the  oxidation  and  reduction  of  the  mitochondrial 
pyridine  nucleotides  during  the  operation  of  oxidative  phosphorylation, 
an  opportunity  availed  of  with  great  success  by  Chance  and  Williams  [7], 
and  by  Klingenberg  and  his  co-workers  [8,  9]. 

For  some  time,  we,  like  others,  have  been  interested  in  the  possibility 
that  a  compound  of  DPN  with  some  substance,  variously  known  as  C,  I 
or  X,  might  be  an  intermediate  in  chemical  reactions  which  link  intra- 
cellular respiration  with  the  synthesis  of  ATP,  About  2  years  ago.  Dr. 
Purvis  announced  from  our  laboratory  that  he  had  evidence  that  rat-liver 
mitochondria  contained,  besides  DPN  +  and  DPNH,  a  third  form  of 
diphosphopyridine  nucleotide,  termed  "extra  DPN",  which  accumulated 
in  and  disappeared  from  the  mitochondria  in  a  manner  that  might  be 
expected  for  an  intermediate  of  oxidative  phosphorylation  [10,  11].  This 
conclusion  has  been  criticized  by  others,  whose  experiments  gave  no 
evidence  of  a  diphosphopyridine  nucleotide  compound  other  than  DPN  + 
and  DPNH. 

During  the  last  2  years.  Dr.  Purvis  has  been  continuing  this  investi- 
gation in  Dr.  Kaplan's  laboratory  in  Brandeis,  while  we  have  been  following 
up  other  aspects  in  Amsterdam.  Although  our  studies  are  not  yet  com- 
pleted, I  thought  it  only  fair  to  other  workers  in  the  field  that  we  bring  out 
an  interim  report  of  our  experiments. 

Pyridine  nucleotide  content  of  isolated  mitochondria 

Huennekens  and  Green's  [2]  measurements  of  the  pyridine  nucleotide 
contents  of  cyclophorase  preparations  were  followed  by  determinations  by 
Holton  [12]  of  the  DPN  +  content  of  isolated  rat-heart  sarcosomes,  and  by 
Glock  and  McLean  [13]  and  Jacobson  and  Kaplan  [14]  of  the  DPN +, 
DPNH,  TPN  +  and  TPNH  content  of  isolated  rat-liver  mitochondria.  The 

*  Birt  and  Bartley  [6]  have  recently  confirmed  by  direct  analysis  that  mito- 
chondria can  exclude  added  DPN  +  and  DPNH  from  participation  in  intramito- 
chondrial processes  of  oxidation  and  reduction.  TPN  +  and  TPNH,  on  the  other 
hand,  can  enter  the  mitochondria  readily. 

t  Abbreviations :  DPN  +,  DPNH,  oxidized  and  reduced  diphosphopyridine 
nucleotide ;  DPN,  all  forms  of  diphosphopyridine  nucleotide ;  TPN  +,  TPNH, 
TPN,  corresponding  symbols  for  compounds  of  triphosphopyridine  nucleotide ; 
ADP,  ATP,  adenosine  di-  and  triphosphate;  P,,  inorganic  phosphate;  EDTA, 
ethylenediaminetetraacetate. 


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I 


PYRIDINE   NUCLEOTIDES    IN    MITOCHONDRIA  211 

latter  two  groups  found  unexpectedly  high  concentrations  of  TPNH.  The 
mean  values  obtained  by  these  authors,  and  in  later  studies,  are  listed  in 
Table  I  (rat  liver)  and  Table  II  (heart). 

In  Table  I  are  shown  the  values  obtained  in  the  Amsterdam  laboratory 
over  the  last  4  years  by  five  different  workers.  DPN  +,  TPN  +  and  DPNH, 
TPNH  w'ere  determined  in  acid  and  alkali  extracts,  respectively,  of  the 
freshly  prepared  mitochondria.  Total  DPX  and  TPN  refer  to  the  amounts 
of  oxidized  nucleotides  found  when  the  mitochondria  were  treated  in  such 
a  way  as  to  convert  all  the  pyridine  nucleotides  into  the  oxidized  form  (see 
below).  The  total  DPN  and  TPN  contents  of  our  preparations  have 
remained  almost  constant  during  this  period.  Purvis  has  also  obtained  the 
same  values  for  mitochondria  prepared  by  our  procedure  in  Brandeis. 

The  Amsterdam  values  for  the  total  DPN  content  are  quite  similar  to 
those  reported  by  Birt  and  Bartley  [6]  for  DPN  ^+ DPNH,  but  are  con- 
siderably higher  than  the  others  in  Table  I.*  Our  total  TPN  values  are 
rather  higher  than  the  (TPN  "^  +  TPNH)  measured  by  other  workers.  It  is 
not  known  to  what  extent  these  differences  represent  differences  in  the 
nutritional  status  of  the  rats  used,  or  in  the  methods  used  to  isolate  the 
mitochondria.  All  workers  report  considerable  variation  from  preparation 
to  preparation. 

With  respect  to  the  DPNH/DPN+  and  TPNH/TPN+  ratios  our 
preparations  closely  resemble  those  of  Klingenberg  and  Slenczka  [8]. 
Birt  and  Bartley 's  preparations  contain  much  more  of  the  oxidized  pyridine 
nucleotide.  This  difference  is  probably  connected  with  the  method  of 
preparation  of  the  mitochondria. f 

Aliss  Bailie  has  found  essentiallv  the  same  values  by  our  enzymic 
fluorimetric  procedure,  which  is  similar  to  that  used  by  Jacobson  and 
Kaplan  [14]  and  Purvis  [11],  and  by  a  spectrophotometric  method,  which 
differs  somewhat  from  others  described  in  respect  to  the  determination  of 
the  reduced  pyridine  nucleotides.  The  alkali  extract  is  neutralized,  treated 
with  a-ketoglutarate,  NH4  and  glutamate  dehvdrogenase  to  oxidize  the 
DPNH  and  TPNH,  and  deproteinized  with  HCIO4.  DPN  +  and  TPN  + 
are  determined  spectrophotometrically  on  the  completely  deproteinized 
solution  by  successive  additions  of  ethanol  + alcohol  dehydrogenase  (at 
pH  10)  and  isocitrate  and  isocitrate  dehydrogenase  (at  pH  7-4).  The 
spectrophotometric  procedure  is  rather  more  reproducible  but  less 
sensitive  than  the  fiuorimetric. 

*  Klingenberg  et  al.  [9]  have  suggested  that  the  method  used  by  Holton  et  al. 
[15]  to  determine  the  DPN  +  content  of  rat-liver  mitochondria  might  also  estimate 
TPN  ^,  owing  to  traces  of  TPN  ^-specific  alcohol  dehydrogenase  in  our  prepara- 
tions of  this  enzyme.  This  was  not  the  case.  TPN  +  is  not  estimated  either  in  the  pro- 
cedure used  by  Holton  et  al.  [15]  or  in  our  recent  work  (see  Purvis  [11],  Table  I). 

t  A  more  recent  paper  {Biochem.  J.  76,  328  (i960))  reports  more  of  th^ 
reduced  nucleotides. 


212 


E.    C.    SLATER,    M.   J.    BAILIE   AND   J.    BOUMAN 


Oxidation  and  reduction  of  mitochondrial  pyridine  nucleotide 

The  first  study  of  the  oxidation  and  reduction  of  pyridine  nucleotides 
in  mitochondria  was  carried  out  by  Chance  and  WilHams  [7],  who  made 
the  important  discovery  that,  in  the  presence  of  substrate,  the  pyridine 
nucleotides  were  largely  reduced  when  the  respiration  of  the  mitochondria 
was  "  inhibited  "  owing  to  lack  of  ADP,  and  were  oxidized  by  the  additions 
of  ADP.  These  measurements  were  made  by  a  sensitive  spectrophoto- 
metric  technique,  which  enabled  the  determination  of  the  absorbancy 


o    4 


"T      I      I      I      I       I      I      I T 


/30H  15  mM 

STATE   3  /state  3^* 

\STATE 

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ADP  0  7mM 

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J — I I I I \ L 


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ADP  063mM 
PO4  4  OmM 


ADP  0  7mM     ^'*^^  ' 
PO4  4  OmM 

I     I     I     1     I 


0     12    3    4    5    6    7    8    0     1     2    3    4    5    6    7 

Time  (min) 

Fig.  I.  DPN  +  and  TPN  +  contents  of  rat-liver  mitochondria  in  different 
"States"  [7].  Rat-liver  mitochondria  (final  concn.  2-8  mg.  protein/ml.)  were 
suspended  in  o- 21  M  sucrose,  33  mM  nicotinamide,  2-5  mM  MgCU  final  volume 
2-7  ml.  The  following  additions  were  made:  at  zero  time,  07  mM  ADP  and 
4-0  mM  Pj;  at  2  min.,  15  mM  j8-hydroxybutyrate ;  at  4-5  min.,  0-63  mM  ADP 
and  4-0  mM  Pj.  DPN  +  and  TPN  +  were  determined  on  aliquots  of  the  same  sus- 
pension. Abbreviations:  PO4,  Pj;  j80H,  /3-hydroxybutyrate.  Temperature,  0°. 
Unpublished  experiment  of  Dr.  J.  L.  Purvis. 

changes  at  340  m/^i  in  a  mitochondrial  suspension.  Chance  and  Williams 
[16]  interpreted  these  absorbancy  changes  as  reflecting  changes  in  the 
oxido-reduction  state  of  DPN.  This  became  uncertain  when  Clock  and 
McLean  [13]  and  Jacobson  and  Kaplan  [14],  by  specific  enzymic  tests  on 
deproteinized  extracts,  showed  that  rat-liver  mitochondria  contained  much 
more  TPNH  than  DPNH.  However,  a  recent  study  by  Klingenberg  and 
Slenczka  [8],  who  used  both  Chance's  method  of  direct  spectrophoto- 
metric  observation  of  the  mitochondrial  suspension  and  the  less  sensitive 
but  more  specific  enzymic  assays  on  deproteinized  extracts,  have  confirmed 
the  most  important  findings  of  Chance  and  Williams.  In  particular,  they 
showed  that  the  rapid  absorbancy  changes  w^hich  follow   exhaustion  or 


PYRIDINE   NUCLEOTIDES    IX    MITOCHONDRIA 


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214  E.    C.    SLATER,    M.    J.    BAILIE   AND   J.    BOUMAN 

addition  of  ADP  are  indeed  due  to  changes  in  the  oxidation-reduction 
state  of  DPN,  changes  in  TPN  occurring  more  slowly.  The  finding  of 
TPNH  in  the  mitochondria  does,  it  is  true,  necessitate  rather  important 
quantitative  changes  in  the  interpretation  given  by  Chance  and  WiUiams, 
e.g.  whereas  the  latter  concluded  that  transition  from  State  3  (active  respira- 
tion) to  State  4  (respiration"  inhibited  "or  controlled  owing  to  lack  of  ADP) 
was  associated  with  a  change  in  DPN  from  50"  „  to  99" ,,  reduced,  Klingen- 
berg  and  Slenczka  found  that  the  corresponding  values  were  3"^  and  43% 
reduced.  Thus,  even  in  the  controlled  state  there  was  considerable  DPN 
in  the  oxidized  form. 

Purvis  (unpublished  observations)  has  obtained  similar  results,  which 
are  given  in  Fig.  i.  Addition  of  ADP  and  Pj  to  a  mitochondrial  suspension 
causes  oxidation  of  the  reduced  nucleotides  due  to  exhaustion  of  endo- 
genous substrate.  After  addition  of  substrate  (/S-hydroxybutyrate)  the 
ADP  is  soon  all  phosphorylated,  so  that  the  mitochondria  reach  State  4, 
in  which  about  5i"o  of  the  total  DPN  (measured  in  State  2)  and  6%  of 
the  total  TPN  are  in  the  oxidized  form.  Addition  of  more  ADP  brings  the 
mitochondria  into  State  3,  in  which  94'^',,  of  the  DPN  is  oxidized.  On  the 
other  hand,  there  was  little  formation  of  TPN  +.  These  results  confirm 
three  of  Klingenberg  and  Slenczka's  findings,  viz.  (a)  DPN  is  not  com- 
pletely reduced  in  State  4;  (h)  in  both  States  3  and  4  the  predominant 
reduced  pyridine  nucleotide  is  TPNH ;  (c)  DPNH  responds  to  addition  of 
ADP  much  more  rapidly  than  TPNH.  As  Klingenberg  et.  al.  [9]  have 
pointed  out,  the  presence  of  large  amounts  of  DPN^  in  the  controlled 
state  removes  one  of  the  main  arguments  of  Chance  and  Williams  [7] 
that  DPNH  is  present  in  an  inhibited  form  (DPNH~I). 

The  high  concentrations  of  reduced  pyridine  nucleotides  in  freshly 
prepared  liver  mitochondria  (cf.  Table  I)  are  presumably  caused  by  the 
presence  of  endogenous  substrate  and  an  "inhibited"  respiration,  owing  to 
the  absence  of  ADP.  The  DPNH  is  only  quite  slowly  oxidized  when  the 
preparation  is  diluted  in  isotonic  medium,  or  near  isotonic  (see  Table  HI — 
cf.  Kaufman  and  Kaplan  [5]),  but  it  is  rapidly  oxidized  in  hypotonic 
medium  without  further  addition,  or  in  isotonic  mediimi  by  addition  of 
dinitrophenol,  0-05  m  P|  or  lower  concentrations  of  P^  in  the  presence  of 
ADP  (Table  HI  and  Fig.  i—cf.  refs.  [7,  17,  8,  11,  5]). 

Purvis'  "extra  DPN" 

One  of  the  most  important  questions  is  whether  the  pyridine  nucleotide 
present  in  the  mitochondria  is  free  or  bound.  Huennekens  and  Green  [2] 
concluded  that  it  was  not  free  and  that  "the  principal  oxidases  of  the 
cyclophorase  complex  occur  as  conjugated  pyridinoprotein  enzymes". 
However,  the  concept  that  mitochondria  were  bound  by  a  semi-permeable 


PYRIDINE   NUCLEOTIDES    IN   MITOCHONDRIA  21 5 

membrane  was  not  then  accepted  by  these  workers.  Because  the  intra- 
mitochondrial  DPNH  could  not  be  oxidized  in  the  inhibited  State  4, 
Chance  and  WiUiams  [7]  and  Chance  and  H.  Bahscheffsky  [18]  concluded 
that  DPNH  was  in  an  inhibited  form  DPNH  ~  I.  Chance  and  Hollunger 
[19]  have  recently  stated,  "It  is  further  concluded  that  the  reduced  form 
is  bound  to  some  ligand,  for  example,  a  protein,  because  the  fluorescence 
maximum  is  at  443  m/x.  Thus  this  material  is  denoted  DPNH  ~  I.  .  .  .  "* 

It  is  doubtful  whether  the  sort  of  chemical  bonds  which  Huennekens 
and  Green  [2]  and  Chance  and  Hollunger  [19]  have  in  mind  could  sur- 
vive the  treatment  with  acid  or  alkali  used  to  prepare  deproteinized 
extracts  for  determinations  of  the  pyridine  nucleotides.  It  is  probable, 
therefore,  that  these  bound  forms  would  yield  free  DPN  +  and  DPNH  in 
the  extracts.  We  were  more  concerned  with  the  possibility  that  a  stable 
compound  of  DPN  of  low  molecular  weight,  which  could  survive  either 
the  acid  or  alkali  extraction,  might  be  present  in  mitochondria. 

Purvis  [10,  11]  found  that  the  total  amount  of  DPN  and  TPN,  deter- 
mined by  incubation  of  rat-liver  mitochondria  with  P;,  ADP  +  Pi,  or 
dinitrophenol,  appreciably  exceeded  the  amounts  of  (DPN  '+ DPNH) 
and  of  (TPN+  +  TPNH),  respectively,  determined  in  the  fresh  mito- 
chondria (see  Table  I).  The  amount  of  "extra  DPN"  found  in  this  way 
averaged  i  •  10  /imoles  g.  protein  for  the  Amsterdam  preparations  and 
1-37  yumoles/g.  protein  for  the  Brandeis  preparations.  The  corresponding 
values  for  "extra  TPN"  were  2-50  and  i  -42,  respectively.  Only  two  out 
of  sixty-three  preparations  examined  did  not  show  any  of  this  material. 

Klingenberg  and  Slenczka  [8],  who  did  not  find  any  evidence  for 
"extra  DPN",  in  somewhat  different  experiments,  concluded  that  Purvis' 
results  were  due  to  a  failure  of  his  fluorimetric  procedure. 

Table  I  shows  that  we  also,  on  the  average,  find  little  if  any  excess  of 

*  The  difference  spectrum  (anaerobic  minus  aerobic  steady  state)  shows  a 
pyridine  nucleotide  peak  at  320  m/:x  rather  than  at  340  m/i  [16  (see  Fig.  i),  15,  17]. 
This  does  not,  however,  prove  that  the  DPNH  found  in  the  mitochondria  is 
"bound  "  in  such  a  way  as  to  cause  a  displacement  of  the  absorption  peak,  as  un- 
fortunately appears  to  be  implied  by  Holton  et  al.  [15].  Biicher  and  Klingenberg 
[20]  have  with  justification  criticized  this  conclusion,  which  in  fact  we  had  not 
intended  should  be  made  from  our  results,  since  the  displacement  of  the  340-m;n 
peak  might  be  caused  by  the  contribution  of  the  S-bands  of  the  cytochromes  in 
this  region  of  the  spectrum,  as  already  discussed  by  Holton  [12].  Chance  and  M. 
Baltscheffsky  [17],  Chance  [21],  and  Chance  and  Hollunger  [19]  have  pointed  out 
that  the  DPNH  which  appears  when  ADP  is  exhausted  by  a  respiring  mito- 
chondrial preparation  has  an  absorption  peak  at  340  m/x.  The  changes  in  the  degree 
of  reduction  of  the  cytochromes  is  much  less  than  that  of  the  DPN  under  these 
conditions,  so  that  the  contribution  of  the  cytochrome  S-bands  to  the  spectrum 
in  the  near  ultraviolet  would  be  much  less.  It  should  be  pointed  out,  however,  that 
Chance  and  M.  Baltscheffsky  [17]  are  of  the  opinion  that  the  displacement  of  the 
DPNH  peak  in  the  anaerobic  spectrum  cannot  be  explained  by  the  S-bands  of  the 
cytochromes. 


2l6  E.    C.    SLATER,    M.   J.    BAILIE   AND   J,    BOUMAN 

"total  DPN"  over  and  above  (DPN  ++DPNH).  Since,  however,  we  have 
used  Purvis'  procedure  with  only  minor  modifications,  we  did  not  think 
it  so  likely  that  his  results  were  due  to  an  analytical  error.  A  few  prepara- 

TABLE  IV 

Percentage  of  Rat-Liver  Mitochondria  Preparations  Containing 

"Extra  DPN" 

Purvis  [ii]         Present  work 

No.  of  preparations  63  48 

Percentage  with  ''Extra  DPN" 

less  than  —  0-4  /xmole/g.  protein 

between  —  0-4  and  o  /.tmole/g.  protein 

o  and  0-4  /nmole/g.  protein 

0-4  and  0-8  /imole/g.  protein 

more  than  08  /imole/g.  protein 

99  99 


TABLE  V 

Effect  of  Added  Substrate  (90  sec.  at  0°)  on  Forms  of  Diphosphopyridine 
Nucleotide  in  Rat-Liver  Mitochondria 

Mitochondria  suspended  in  018  m  sucrose.  Unpublished  results  of  Dr.  J.  L. 
Purvis,  some  of  which  were  reported  by  Slater  and  Hiilsmann  [29].  Values  in 
jLtmoles/g.  protein. 


0 

10 

3 

46 

6 

23 

14 

12 

76 

8 

Expt. 

Substrate 

DPN  + 

DPNH 

"Extra 
DPN"* 

I 

None 

1-78 

1-56 

I  •  ID 

a-Ketoglutarate  (20  niM) 

0-70 

I   40 

2-34 

Glutamate  (40  mM) 

1-28 

1-56 

I   60 

2 

None 

I  -60 

1-94 

i-i8 

Succinate  (40  mivi) 

0-82 

1-77 

2-13 

3 

None 

1-73 

2-51 

0-70 

Succinate  (40  mM) 

0-74 

216 

2 -00 

4 

None 

I    55 

1-50 

1-42 

Fumarate  (20  mM) 

I  -99 

1-30 

I  -21 

5 

None 

1-74 

1-23 

0-97 

Malate  (20  mM) 

I  -64 

1-39 

0-91 

*  Total  DPN  minus  (DPN"  +  DPNH).  Total  DPN  determined  by  incubation 
in  absence  of  substrate  with  dinitrophenol  at  30  . 

tions  gave  results  resembling  those  of  Purvis.  Table  IV  shows  that  whereas 
Purvis  found  that  90%  of  his  preparations  contained  more  than  0-4 
/umole/g.  protein  "extra  DPN",  only  2o"o  of  our  preparations  contained 


PYRIDINE    NUCLEOTIDES    IN    MITOCHONDRIA  217 

this  amount  of  "  extra  DPN  ".  Although  we  are  under  the  impression  that 
some  of  our  preparations  of  Uver  mitochondria  have  the  properties 
ascribed  to  them  by  Purvis,  thev  turn  up  so  rarelv  that  we  could  not  be 
absolutely  certain  that  they  were  not  due  to  analytical  errors. 

Further  evidence  for  the  presence  in  liver  mitochondria  of  a  form  of 
diphosphopyridine  nucleotide  other  than  DPN  and  DPNH  was  obtained 
by  Pur\'is  by  studying  the  effect  of  adding  substrates.  Table  V  shows  that 
the  addition  of  a-ketoglutarate,  glutamate  or  succinate  for  90  sec.  at  o^ 
(without  added  substrate  there  is  no  change  in  the  amount  of  DPN  +  and 
DPNH  during  this  period)  caused  a  decline  in  the  amount  of  DPN  +, 
without  any  increase  in  the  amount  of  DPNH,  i.e.  some  pyridine  nucleo- 
tide disappeared.  Malate  and  fumarate,  on  the  other  hand,  caused  little 
change  in  the  DPN  +  or  DPNH  level. 

TABLE  VI 

Effect  of  Added  Succinate  (90  sec.  at  o  )  ox  Forms  of  Diphosphopyridine 
Nucleotide  in  Rat-Liver  Mitochondria 

Unpublished  experiments  of  J.  Bouman,  B.  Winter  and  M.  Bailie.  Mito- 
chondria suspended  in  o- 18-0-25  ^  sucrose.  Value  in  /(moles/g.  protein. 

Method  No.  ofexpts.  JDPN^  J  DPNH 

A.  Mean  results 

Fluorimetric  11  — o-6i  +0-66 

Spectrophotometric  4  —0-94  +o-6o 

B.  Single  experiment  (spectrophotometric  method) 

DPN  ^  DPNH  "  Extra  DPN  "* 


Fresh  mitochondria  2-34  i'54  0"57 

+   Succinate  (40  niM)  o-(Si  2-28  i'36 

*  Total  DPN  (determined  by  incubation  in  absence  of  substrate  with  0-05 
M   P,  for   10  min.)   minus  (DPN^    +    DPNH). 

The  results  with  succinate  appeared  to  be  in  conflict  with  those  of 
Chance  and  Hollunger  [22],  who  reported  extensive  reduction  of  DPN  + 
by  this  substrate. 

When  we  repeated  these  experiments  with  our  preparations  of  liver 
mitochondria,  which  contained  little  "extra  DPN",  the  DPN  +  which 
disappeared  nearly  always  appeared  as  DPNH  (see  Table  VL4).  These 
results  support  Chance  and  Hollunger  [22],  whose  findings  have  in  the 
meantime  been  confirmed  by  Klingenberg  et  al.  [9].  As  a  whole,  they  give 
no  support  to  the  existence  of  another  form  of  DPN.  When,  however, 
succinate  was  added  to  one  of  our  rare  preparations  which  appeared  to 
contain  "extra  DPN",  a  result  was  obtained  intermediate  between  that 


2l8 


E.    C.    SLATER,    M.    J.    BAILIE   AND   J.    BOUMAN 


reported  by  Purvis  and  the  bulk  of  our  results,  in  that  the  DPNH  content 
increased,  but  not  to  the  same  extent  as  the  decrease  of  DPN  +  (Table 
VIB).  This  result  suggests  the  possibility  that,  whenever  we  have  a  prepara- 
tion of  fresh  rat-liver  mitochondria  containing  some  "extra  DPN",  we 
can  increase  the  amount  by  adding  succinate.  However,  it  must  be  empha- 
sized that  Table  YIB  describes  a  single  result,  which  has  not  yet  been 
reproduced. 

At  this  stage,  we  must  conclude  that  the  tightly-coupled  rat-liver 
mitochondria  which  we  normally  prepare  in  Amsterdam  rarely  contain 
"extra  DPN",  and  that  this  substance  cannot  usually  be  induced  by  a 
short-term  incubation  with  succinate.  Further  progress  clearly  required  a 
reproducible  method  of  inducing  the  "extra  DPN". 

TABLE  VII 

Effect  of  Incubation  with  Glutamate  or  Succinate  in  Pi-Deficient  Med- 
ium,  AND  of  the   Subsequent  Addition   of   Dinitrophenol  on  DPN+  and 
DPNH  Contents  of  Rat-Liver  Mitochondria 

Unpublished  experiments  of  B.  Winter  and  M.  Bailie.  Mean  values  (/xmoles/g. 
protein.) 


Substrate : 


Glutamate 


Glutamate 


Succinate 


Analytical  Method: 


Fluorimetric 


Spectro- 
photometric 


Fluorimetric 


No.  of  expts. 


Changes  on  incubation  zvith  substrate 

Z1DPN+  -009 

JDPNH  -063 

J(DPN+  +  DPNH)  -072 

Changes  on  subsequent  addition  of  dinitrophenol 
JDPN+  1-88 

J  DPNH  -079 

J(DPN+  +  DPNH)  I   09 


-0-68 

-0-47 

+  0-23 

+  o-o6 

-0-45 

—  0-41 

I  -91* 

1-57 

-1-85 

—  I  -20 

o-o6* 

0-37 

*  Means  2   08  and  023,  respecti\ely,  if  one  doubtful  value  is  omitted. 

If  the  "  extra  DPN  "  is  really  an  intermediate  in  oxidative  phosphoryla- 
tion, as  we  hope,  we  should  expect  that  it  would  accumulate  in  the  absence 
of  inorganic  phosphate  or  ADP.  For  this  reason,  we  tried  incubation  with 
glutamate  in  the  presence  of  ADP,  hexokinase,  glucose,  and  nicotinamide. 
Under  these  conditions,  any  traces  of  inorganic  phosphate  are  rapidly 
esterified  and  accumulate  as  hexose  monophosphate.  The  rate  of  respira- 
tion is  only  about  one-seventh  that  obtained  on  the  addition  of  inorganic 
phosphate  [23,  24]. 

Figure  2  (A)  and  the  second  column  of  Table  VII  show  the  results  of  a 


PYRIDINE    NUCLEOTIDES    IN    MITOCHONDRIA  219 

series  of  eight  such  experiments  with  rat-Hver  mitochondria.  On  the 
average,  there  was  httle  change  in  the  DPX  ^  content  while  the  DPNH 
declined.  As  a  consequence,  the  (DPN++DPNH)  content  declined  in 
every  experiment,  by  an  average  of  0-73  jumoleg.  protein.  The  amount 
of  total  diphosphopyridine  nucleotide  which  disappeared  varied  con- 
siderably from  experiment  to  experiment — between  o  •  2  and  i  •  5  /xmoles/g. 
protein.  It  exceeded  0-4  ^nmole  g.  protein  in  six  of  the  eight  experiments. 


Time  (min)  |  Time  {mini 

Clu  +  ADP(-P,)  +DNP  Clu  +  ADP(-P,)  +  DNP 

Fig.  2.  DPN  +  and  DPNH  contents  of  rat-liver  mitochondria  after  incubation 
with  glutamate  in  the  absence  of  P;  and  after  adding  dinitrophenol. 

A.  A  set  of  eight  experiments,  in  which  the  fluorimetric  enzymic  assay  was 
used.  The  first  series  of  vertical  lines  show  the  range  of  values  for  the  DPN  +, 
the  DPNH  and  the  DPN  -  +  DPNH  contents  of  the  fresh  mitochondria.  The 
points  show  the  individual  values.  At  zero  time  the  mitochondrial  suspension  was 
added  to  a  mixture  containing  13-3  mM  KCl,  i  -8  mM  EDTA,  13  5  mM  glucose, 
4-5  mM  MgCl.2,  9  mM  L-glutamate,  o-8  mM  ADP,  38-7  mM  nicotinamide,  0-15  M 
sucrose  (derived  from  the  mitochondrial  suspension)  and  hexokinase.  The  final 
concentration  of  mitochondrial  protein  was  about  12  mg./ml.  After  15  min.  in  a 
manometer  flask  the  suspensions  were  analyzed  for  DPN  ~  and  DPNH.  The  values 
are  shown  on  the  second  set  of  vertical  lines.  Duplicate  flasks  were  treated  in  the 
same  way,  until  at  15  min.  o-  i-i  mM  dinitrophenol  was  added  from  the  side-arm. 
After  a  further  5  min.  the  DPN  +  and  DPNH  contents  of  these  suspensions  were 
determined.  The  values  are  shown  in  the  third  set  of  vertical  lines.  The  full  lines 
connecting  these  vertical  lines  show  the  changes  in  the  mean  values  of  DPN  +, 
DPNH  and  DPN  ^  +  DPNH.  The  discontinuous  lines  show  the  changes  in 
(DPN  *  +   DPNH)  in  the  individual  experiments. 

B.  A  set  of  four  experiments,  in  which  the  spectrophotometric  enzymic  method 
was  used.  Conditions  and  reaction  mixture  as  in  A,  except  that  a  concentration  of 
about  18  mg.  mitochondrial  protein /ml.  was  used.  The  full  lines  connect  the  mean 
values  of  DPN  +  and  DPNH.  The  discontinuous  lines  show  the  change  of  DPN  + 
+  DPNH  in  each  experiment.  One  of  the  DPN  +  values  after  dinitrophenol  is 
probably  a  mistake.  The  dotted  line  is  drawn  to  the  mean  obtained  by  ignoring 
this  value. 


220  E.    C.    SLATER,    M.    J.    BAILIE   AND   J.    BOUMAN 

It  should  be  emphasized  that  all  of  these  experiments  were  carried  out  in 
the  presence  of  nicotinamide,  which  prevents  the  destruction  of  DPN  +. 
In  six  of  the  eight  experiments,  we  added  dinitrophenol  which,  in  agree- 
ment with  Chance's  observations  with  other  uncoupling  agents,  brought 
about  oxidation  of  much  of  the  DPNH.  There  was  an  increase  in  the 
(DPN  +  +  DPNH)  content  of  between  0-3  and  2-4  (average  i  •  i)  /xmoles/g. 
protein. 

Thus,  during  the  incubation  with  substrate  a  part  of  the  total  DPN 
disappears  into  a  compound  which  does  not  react  in  our  enzymic  method. 
It  reappears  as  DPN  +  on  addition  of  dinitrophenol.  This  compound  is  not 
TPN  +.  In  three  experiments,  in  which  an  average  of  i  •  i  /^mole  (DPN  +  + 
DPNH)/g.  protein  disappeared  during  incubation,  0-25  /xmole  (TPN+  + 
TPNH)/g.  protein  also  disappeared.  In  four  experiments,  0-3  ju,mole 
(TPN  +  +  TPNH)/g.  protein  appeared  after  addition  of  the  dinitrophenol. 

Recently,  this  experiment  has  been  repeated  with  four  preparations 
using  a  spectrophotometric  method.  There  is  much  less  variation,  which 
may  be  partly  due  to  the  greater  accuracy  of  the  spectrophotometric  assay, 
but  is  also  probably  due  to  the  fact  that  the  experiments  were  carried  out 
in  close  succession  with  mitochondrial  preparations  which  were  probably 
very  similar  to  one  another.  Qualitatively,  the  same  picture  is  shown  (see 
Fig.  2  (B)),  but  the  amount  of  total  pyridine  nucleotide  disappearing  was 
rather  less — between  0-2  and  0-7  jumole/g.  protein. 

Three  experiments  (fluorimetric)  were  also  carried  out  with  succinate 
in  place  of  glutamate.  There  was  some  loss  of  DPN  +,  while  the  DPNH 
content  did  not  increase  (see  column  4,  Table  VII). 

From  these  experiments,  we  can  conclude  that  incubation  with  sub- 
strate in  the  absence  of  inorganic  phosphate  causes  some  of  the  diphos- 
phopyridine  nucleotide  to  disappear,  and  that  what  disappears  can  be 
largely  recovered  again  by  the  addition  of  dinitrophenol.  The  amount  of 
DPN  disappearing  in  this  way  was  sometimes  quite  large,  but  was  often 
only  small  in  experiments  which  appeared  to  be  carried  out  in  the  same 
way  with  identical  material.  We  do  not  understand  the  reasons  for  these 
differences  and,  at  present,  our  preparations  of  rat-liver  mitochondria  are 
discouraging  material  for  the  study  of  the  Purvis  compounds.  For  this 
reason,  we  have  recently  turned  to  other  mitochondria. 

However,  before  leaving  the  experiments  summarized  in  Fig.  2  and 
Table  VII,  it  is  worth  while  drawing  attention  to  the  state  of  oxidation  of 
the  pyridine  nucleotides  in  the  controlled  and  active  states.  In  these 
experiments,  respiration  was  controlled  or  inhibited  by  lack  of  inorganic 
phosphate.  In  this  controlled  state,  a  substantial  proportion  of  the  diphos- 
phopyridine  nucleotide  is  in  the  oxidized  form,  nearly  half  in  the  first 
series  of  experiments  (Fig.  2  (A)).  This  is  similar  to  Klingenberg  and 
Slenczka's  finding  when  respiration  was  inhibited  by  lack  of  ADP.  In  fact. 


PYRIDINE    NUCLEOTIDES    IN    MITOCHONDRIA  221 

we  found  \'ery  similar  results  with  glutamate  as  substrate  whether  lack  of 
Pj  or  ADP  was  responsible  for  the  inhibition  of  respiration  (see  Table  VIII). 
In  the  controlled  state,  the  degree  of  oxidation  of  the  pyridine  nucleotides 
is  probably  largely  controlled  by  equilibria  catalyzed  by  the  DPN-specific 
dehydrogenases,  such  as 

glutamate  +  DPN  +  =  a-ketoglutarate  +  NH+  +  DPNH 

Adding  dinitrophenol  to  a  mitochondrial  suspension  in  the  presence  of 
glutamate  and  absence  of  P,  causes  about  a  3-fold  increase  in  the  rate  of 
Oo  uptake  [23].  This  is  presumably  due  to  activation  of  DPNH  oxidation, 
with  a  consequent  increase  in  the  rate  of  oxidation  of  glutamate  to  a-keto- 
glutarate.  Further  oxidation  of  the  a-ketoglutarate  cannot  occur  at  an 

TABLE  VIII 

diphosphopyridlne  nucleotide  compounds  of  r.at-l1ver  mitochondria  in 

Controlled  State 

Glutamate  substrate.  The  P-deficient  medium  was  the  same  as  that  in  Fig. 
2.  The  ADP-deficient  medium  contained  15  mM  KCl,  5  mM  MgCl,,  30  mM 
nicotinamide,  40  mM  tris(hydroxymethyl)aminomethane — HCl  buffer,  pH  7-4, 
40  mM  potassium  phosphate,  pH  7  -4.  2  mM  EDTA,  o  •  i  mM  ADP,  0-12  M  sucrose, 
30  mM  glutamate.  Single  experiment  (fluorimetric).  Values  in  fimoles/g.  protein. 
Unpublished  experiment  of  M.  Bailie. 

State  of  mitochondria  DPN  +  DPNH  "  Extra  DPN  "* 

Fresh  1-51  2-30  0-54 

In  Pj-deficient  medium  1-72  i-8q  0'74 

In  ADP-deficient  medium  i-8.S  1-83  0-64 

*  Determined  as  in  Table  \\. 

appreciable  rate,  because  P,  is  necessary  for  the  substrate-linked  phos- 
phorylation step  of  a-ketoglutarate  oxidation  even  in  the  presence  of 
dinitrophenol  [2:;,  26].  The  marked  decrease  in  the  DPNH  concentration 
shown  in  Fig.  2  is  to  be  expected. 

In  other  experiments,  not  shown  in  Fig.  2,  respiration  was  fully 
activated  (sevenfold)  by  adding  P,  instead  of  dinitrophenol.  This  not  only 
activates  DPNH  oxidation,  but  also  DPN  ^  reduction  by  a-ketoglutarate 
and  by  malate  (the  oxidation  of  malate  to  oxaloacetate  is  involved  in  the 
oxidation  of  glutamate  bv  mitochondrial  preparations  [27].  In  fact,  the 
sevenfold  stimulation  of  the  respiratory  rate  was  accompanied  by  very 
little  change  in  the  degree  of  oxidation  of  the  diphosphopyridine  nucleotide 
(means  of  four  experiments  :  J  DPN  *,  +0-35  /u.mole/g.  protein  ;  J  DPNH, 
—  0-21  |Limole/g.  protein).  Thus,  under  the  conditions  of  our  experiments, 
the  diphosphopyridine  nucleotide  of  rat-liver  mitochondria  oxidizing 
glutamate  in  the  presence  of  Pj  and  ADP  is  about  50"  o  reduced.  This  is 


222  E.    C,    SLATER,    M.   J.    BAILIE   AND   J.    BOUMAN 

much  greater  than  the  degree  of  reduction  reported  by  KHngenberg  et  al. 
[9],  viz.  1-10*^0  reduced.  No  doubt,  a  difference  in  experimental  conditions 
is  responsible  for  this  discrepancy. 


Sarcosomes 

Muscle  mitochondria  (sarcosomes)  have  been  found  to  be  a  much 
more  reproducible  source  of  "extra  DPN  ".  Out  of  eleven  preparations  of 

TABLE  IX 
"Extra  DPN"  in  Sarcosomes 
Unpublished  experiments  of  M.  Bailie.  Values  in  /^imoles/g.  protein. 


"Extra   DPN"*    after 

incubation 

with  substratef 

"  Extra 
DPN"* 

ji 

Expt. 

DPN  + 

DPNH 

in  ADP- 

in  Pi- 

deficient 

deficient 

mediumj 

medium  J 

Rabbit  heart 

(fluorimetric 

method) 

E042 

6-2 

0-4 

1-8 

i-s 

— 

E044 

6-4 

0-6 

—  0-2 

1-3 

— 

E047 

4-7 

2-1 

1-9 

2-1 

— 

E053 

5-5 

I  *I 

1-3 

2-6 

— 

E055 

4-2 

1-8 

2-3 

3-1 

— 

E057 

44 

1-7 

i-S 

— 

— 

E065 

3-5 

2-1 

0-8 

— 

— 

E066 

4-5 

i-s 

1-3 

— 

— 

E067 

5-3 

i-o 

3-8 

— 

— 

E068 

3-9 

I  'O 

1-7 

— 

— 

E071 

2-8 

1-6 

1-4 

2-3 

3-2 

Means 

4-6 

1-4 

1-6 

2-2 

(3-2) 

Pigeon  breast 

(spectrophotometric  method) 

E063 

5-2 

I  -o 

06 

— 

— 

E070 

4-5 

1-5 

3-1 

2-5 

4-0 

E071 

3-7 

3-0 

I  -2 

— 

— 

E072 

5-5 

0-7 

0-4 

— 

— 

Means 

4-7 

1-5 

T7 

— 

■  — 

*  Determined  as  in  Table  VI. 

t   Succinate  (40  mivi),  or  succinate  (40  mM)  +  glutamate  (30  mM)  as  substrate. 

X   Composition  as  in  Table  VIII. 

rabbit-heart  sarcosomes,  and  four  of  pigeon-breast,  only  one  preparation 
was  found  not  to  contain  any  "extra  DPN"  (see  Table  IX).  The  amount 
v^as  increased  by  incubation  with  succinate  in  ADP-deficient  medium 


PYRIDINE   NUCLEOTIDES    IN   MITOCHONDRIA  223 

(State  4)  and  especially  in  a  P, -deficient  medium.  The  one  preparation 
(E044)  without  "extra  DPN "  contained  it  after  incubation  with  suc- 
cinate. It  is  noteworthy  that,  in  disagreement  with  our  finding  with  rat- 
liver  mitochondria  (Table  VIII),  more  "extra  DPN"  was  found  on 
incubation  of  heart  sarcosomes  in  P,-deficient  medium  than  in  ADP- 

TABLE  X 

Reduction  of  DPN  +  brought  about  by  adding  Succinate  to  ADP-Deficient 
Rabbit-Heart   Sarcosomes   in   Presence  of   Glutamate 

Unpublished  experiments  of  M.  Bailie.  Values  in  /Limoles/g.  protein. 


Expt. 

E042 

E044 

E047 

DPN+, 

before  succinate 

6-57 

4-07 

7-35 

after  succinate 

3-31 

1-82 

2-74 

JDPX  ^ 

-3-26 

-2-25 

—  4-61 

DPNH, 

before  succinate 

0-30 

I  -22 

I-I9 

after  succinate 

3  "62 

3-21 

3-82 

JDPNH 

3-32 

I  -99 

2-63 

deficient  medium.  This  is  probably  related  to  the  fact  that  the  degree  of 
respiratory  control  is  much  greater  with  sarcosomes  oxidizing  succinate  in 
a  Pj-deficient  medium  than  in  an  ADP-deficient  medium. 

The  "extra  DPN  "  is  not  derived  from  TPN.  In  Expt.  066,  the  freshly 
prepared  sarcosomes  contained  TPN +,  0-58;  TPNH,  0-73;  "extra 
TPN",  0-26  /xmole/g.  protein. 

TABLE  XI 

Reduction  of  DPN  +  by  Addition  of  Succinate  (90  sec.  at  o")  to  Rabbit- 
Heart  Sarcosomes 

Unpublished  experiment  of  M.  Bailie  (H071).  Sucrose,  o-i8  M;  succinate, 
40  m.M.  Values  in  /xmoles/g.  protein. 


DPN+, 

before  succinate 

2-8i 

after  succinate 

1-34 

JDPN  + 

-1-47 

DPNH, 

before  succinate 

1-57 

after  succinate 

4-24 

J  DPNH 

2-67 

In  the  course  of  these  experiments,  we  have  repeated  Chance  and 
Hollunger's  [19]  observation  on  the  effect  of  adding  succinate  to  sarco- 
somes in  State  4  (i.e.  deficient  in  ADP)  in  a  medium  already  containing 
glutamate.  Our  results  agree  with  those  of  these  authors  in  that  succinate 
brought  about  a  considerable  reduction  of  the  DPN  (Table  X).  Addition 
of  succinate  to  the  sarcosomal  suspension  for  only  90  sec.  at  o  (cf.  Tables  V 
and  VI)  also  caused  the  appearance  of  much  DPNH  (Table  XI). 


224  E-    ^'    SLATER,    M.    J.    BAILIE    AND    J.    BOUMAN 

Conclusion 

Our  results  agree  with  those  of  Klingenberg  and  Slenczka  [8]  in  that 
they  fail  to  show  the  presence  in  most  preparations  of  fresh  rat-liver 
mitochondria  of  detectable  amounts  of  the  "extra  DPN  "  of  Purvis.  We 
do,  however,  often  find  appreciable  amounts  of  a  compound  with  the 
properties  of  "extra  DPN"  after  incubation  of  the  mitochondria  with 
substrate,  in  the  absence  of  Pj.  Rabbit-heart  and  pigeon-breast  sarcosomes 
are  a  much  more  consistent  source  of  "extra  DPN". 

Further  work  is  required  to  establish  the  significance  of  the  "extra 
DPN"  and  it  would  be  premature  to  speculate  on  its  nature.  Concerning 
whether  it  is  more  likely  to  be  a  DPN  +  or  a  DPNH  compound,  the  reader 
is  referred  to  a  discussion  between  one  of  the  writers  and  Dr.  B.  Chance, 
published  in  the  Proceedings  of  the  International  Symposium  on  Haem 
Compounds  (Canberra,  1959). 

Acknowledgments 

These  studies  have  been  supported  in  part  by  a  grant  from  the  Life 
Insurance  Medical  Research  Fund.  One  of  us  (M.  J.  B.)  is  a  recipient  of 
personal  grants  from  the  Netherlands  Ministry  of  Education  and  the  Anti- 
Cancer  Council  of  Victoria,  Australia.  We  wish  to  thank  Dr.  J.  P.  Colpa- 
Boonstra  and  Mr.  A.  Perk  for  their  collaboration  in  some  of  these  experi- 
ments. Dr.  J.  L.  Purvis  for  making  available  Fig.  i,  and  Mr.  B.  Winter, 
Miss  M.  A.  Searle,  and  Mr.  J.  G.  Huisman  for  their  technical  assistance. 


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17.  Chance,  B.,  and  Baltscheffsky,  M.,  Biocheni.J.  68,  283  (1958). 

18.  Chance,  B.,  and  Baltscheffsky,  H..^.  biol.  Chem.  233,  736  (1958). 

19.  Chance,  B.,  and  Hollunger,  G.,  Nature,  Loud.  185,  666  (i960). 

20.  Bucher,  Th.,  and  Klingenberg,  M.,  Augetv.  Chem.  70,  352  (1958). 

21.  Chance,  B.,  in  "  Ciba  Foundation  S>Tnposium  on  Cell  Metabolism",  ed. 
G.  E.  W.  Wolstenholme  and  C.  M.  O'Connor.  J.  and  A.  Churchill  Ltd., 
London,  85  (1959). 

22.  Chance,  B.,  and  Hollunger,  G.,  Fed.  Proc.  16,  163  (1957). 

23.  Borst,  P.,  and  Slater,  E.  C,  Nature,  Lond.  184,  1396  (1959). 

24.  Borst,  P.,  and  Slater,  E.  C,  Nature,  Lond.  185,  537  (i960). 

25.  Hunter,  F.  E.,  Jr.,  Phosphorus  Metabolism  I,  297  (1951). 

26.  Judah,  J.  D.,  Biochern.jf.  49,  271  (1951). 

27.  Borst,  P.,  and  Slater,  E.  C,  Biochim.  biophys.  Acta  41,  170  (i960). 

28.  Chance,  B.,  and  Williams,  G.  R.,  Advanc.  Ensyniol.  17,  65  (1956). 

29.  Slater,  E.  C,  and  Hiilsmann,  W.  C,  in  "Ciba  Foundation  Symposium  on 
Cell  Metabolism",  ed.  G.  E.  W.  Wolstenholme,  and  C.  M.  O'Connor. 
J.  and  A.  Churchill  Ltd.,  London,  58  (1959)- 

30.  Lester,  R.  L.,  and  Hatefi,  Y.,  Biochim.  biophys.  Acta  29,  103  (1958). 


Discussion 

Chance  :  I  should  be  glad  to  clarify  an  ambiguity  which  Prof.  Slater  pointed  to ; 
in  the  experiments  published  in  the  work  with  Hollunger,  we  had  pretreated  the 
guinea-pig  kidney  mitochondria  with  glutamate.  Therefore  some  ATP  was  pro- 
duced by  the  a-ketoglutarate  step.  The  addition  of  succinate  therefore  produced  a 
rapid  and  complete  reduction  of  DPN.  For  the  last  8  to  10  months  we  have  been 
working  with  pigeon-heart  preparations  which,  when  freshly  prepared  or  after  a 
day's  ageing,  do  have  a  ATP  requirement  for  DPN  reduction  in  the  presence  of 
succinate.  They  will  respire  slowly  but  the  DPN  will  not  be  reduced  unless  you 
add  ATP.  If  they  are  suspended  in  a  medium  containing  phosphate,  the  respir- 
ation will  increase  and  the  DPN  will  slowly  be  reduced  in  several  minutes.  Thus 
this  preparation  is  admirably  suited  to  separate  the  substrate  and  energy  require- 
ments for  the  DPN  reduction. 

Slater  :  I  think  there  must  still  be  some  difference  between  our  rabbit-heart 
sarcosome  preparation  and  your  pigeon-heart  preparation  because  90  sec.  at  o'  is 
probably  a  short  time. 

Chance:  Yes;  I  think  it  is  a  question  of  endogenous  substrate  concentration. 


VOL.  II. — Q 


Nucleotides   and   Mitochondrial   Function:   Influence   of 
Adenosinetriphosphate  on  the  Respiratory  Chain 

Martin  Klixgenberg 

PhysiologiscJi-CJiemiscJies  Institut  der  Universitdt, 
Marburg,  Germa?iy 

It  has  been  known  since  the  discovery  of  oxidative  phosphorylation 
that  electron  transport  of  the  respiratory  chain  effects  the  phosphorylation 
of  the  adenine  nucleotide  system.  The  reverse  control  of  electron  transport 
bv  the  phosphorylation  of  the  adenine  nucleotides  has  been  demonstrated 
with  the  influence  of  ADP  on  respiration  and  on  the  redox  state  of  nucleo- 
tides and  cytochromes  of  the  respiratory  chain  [cf.  i,  2,  3].  It  could  be 
shown  only  recently  that  the  redox  state  of  the  respiratory  chain  can  also 
be   influenced  by  ATP  in   a   reversal  of  the  oxidative  phosphorylation 

[4,  5,  6]. 

The  relation  between  the  ATP  level  and  electron  transport,  as  followed 
by  the  respiration  and  the  redox  state  of  the  respiratory  chain,  is  the  subject 
of  studies  presented  in  this  article.  This  aspect  will  be  pursued  both  with 
respect  to  the  intramitochondrial  ATP  as  well  as  to  the  eflect  of  external 
ATP.  In  this  context  we  are  concerned  with  the  reversal  of  the  oxidative 
phosphorvlation,  which  was  postulated  to  take  place  in  the  succinate  and 
glycerolphosphate  induced  DPX  reduction  in  mitochondria  [7,  S,  9,  10]. 
Some  related  data  on  the  major  intramitochondrial  nucleotide  systems  will 
be  presented  first. 

Intramitochondrial  nucleotide  systems 

The  anion  exchange  chromatograms  of  Fig.  i  give  an  example  of  the 
pattern  and  behaviour  of  intramitochondrial  nucleotides  in  two  difl^erent 
functional  states  [11].  Only  the  pyridine  and  adenine  nucleotide  systems 
can  be  extracted  in  appreciable  amounts  from  these  muscle  mitochondria. 
The  concentration  of  reduced  pyridine  nucleotide  in  the  perchloric  acid 
extract  of  mitochondria  is  quantitatively  determined  bv  the  concentration 
of  the  acid  decay  products  of  DPXH  (ADP-ribose)  and  of  TPNH  (ADP- 
ribosephosphate)  [12].  In  confirmation  of  the  results  obtained  by  other 
methods,  in  the  presence  of  succinate  DPN  is  mostly  reduced,  whereas  in 
the  presence  of  dinitrophenol,  DPN  is  oxidized.  Similar  but  smaller 
changes  are  observed  with  the  TPN  system.  As  expected,  in  the  presence 


228 


MARTIN    KLINGENBERG 


of  succinate  a  higher  ATP-level  is  formed  than  with  dinitrophenol.  How- 
ever, it  is  remarkable  that  also  in  the  presence  of  succinate  appreciable 
amounts  of  ADP  and  AMP  are  found  together  with  inorganic  phosphate. 


100        125 
Fractions 

Fig.  I.  Anion  exchange  chromatograms  (Dowex  2)  of  perchloric  acid  extracts 
of  pigeon-breast  muscle  mitochondria.  Before  extraction  the  mitochondria  were 
incubated  for  60  sec.  in  about  15  ml.  of  the  reaction  medium,  containing  0-3  M 
sucrose,  10  mivi  triethanolamine-HCl-buffer,  pH  72,  25".  This  medium  was 
oxygen  (i  atm.)  saturated. 

Chromatogram  a.  Mitochondria  (47  mg.  protein)  incubated  under  addition 
of  4  mM  succinate. 

Chromatogram  b.  Mitochondria  (47  mg.  protein)  incubated  under  addition 
of  o  •  I  mM  dinitrophenol. 


In  a  first  attempt  to  correlate  functional  states  of  the  three  major 
mitochondrial  nucleotide  systems,  the  phosphorylation  state  of  the  adenine 


NUCLEOTIDES    AND    MITOCHONDRIAL    FUNCTION 


229 


nucleotide  system  is  compared  with  the  redox  states  of  the  pyridine  and 
flavin  nucleotide  systems  under  various  conditions,  as  shown  in  Table  I. 
These  data  have  been  obtained  by  enzymatic  analysis.  The  mitochondria 
are  saturated  with  substrate  in  the  presence  of  oxygen  and  thus  are  in  a 
condition  facilitating  the  phosphorylation  of  the  endogenous  adenine 
nucleotides.  The  data  refer  to  a  steady  state.  The  substrates  can  be 
divided  into  two  groups :  the  DPX-specific  substrates,  pyruvate  plus  malate, 


TABLE  I 

Nucleotide  Systems  in  Mitochondria 

Comparison  between  the  phosphorylation  state  of  the  adenosine  phosphates 
and  the  redox  state  of  DPN  and  fiavoprotein. 


Additions 

AlP 
i:AP* 

DPNH 
i:DPNt 

(/L.Mol/g.Prot.) 

Heart  muscle 

Succinate 

0-56 

0-75 

030 

Succinate,  PO4 

0-54 

0-70 

0-24 

Succinate,  antimycin  A 

0-07 

0-26 

0-14 

Pyruvate  +  malate 

0-33 

0-28 

002 

Pyruvate  +  malate,  PO4 

0-62 

Fh 

0-37 
ght  tniiscle 

0-03 

Glycerol- x-P 

0-45 

0-55 

051 

Glycerol- 1 -P,  PO4 

0-75 

049 

030 

Glycerol- 1 -P,  antimycin  A 

0-17 

o-o6 

0-13 

Pyruvate  +  malate 

059 

o-i8 

0-15 

Pyruvate  +  malate,  PO4 

0-73 

0-04 

o-o8 

*  Z-AP^ATP  +  ADP  +  AMP. 
t  rDPN  =  DPN  +  DPNH. 

J  Calculated  from  extinction  changes  at  468-500  m/t,  using  Jen-,;^,  =  8-5  cm~^. 

and  the  non-DPN-specific  substrates,  succinate  and  glycerol  phosphate. 
The  postulated  energy  supply  for  the  succinate  or  glycerol-phosphate 
linked  DPN  reduction  might  be  reflected  in  the  intramitochondrial  ATP- 
level.  However,  with  both  these  groups  of  substrates  about  50-70%  of  the 
adenine  nucleotides  are  present  as  ATP.  Addition  of  phosphate  on  top  of 
the  endogenous  phosphate  increases  the  phosphorylation,  as  is  to  be 
expected.  In  contrast,  there  are  large  difl"erences  between  the  two  groups 
of  substrates  with  respect  to  the  degree  of  reduction  of  the  DPN.  With 
succinate  or  glycerol  phosphate,  DPN  is  reduced  to  a  greater  extent  than 
with  pyruvate  plus  malate.  These  results  show  that  the  redox  state  of  the 
mitochondrial  DPN  appears  not  to  be  only  a  function  of  the  endogenous 
energy  supply,  as  expressed  in  the  phosphorylation  state  of  the  adenine 


230  MARTIN   KLINGENBERG 

nucleotide  system,  but  also  to  be  related  to  the  reducing  qualities  of  suc- 
cinate or  glycerol  phosphate.  This  is  supported  by  measurements  of  the 
absorption  of  mitochondrial  suspensions  at  the  flavin  nucleotide  wave- 
length, from  which  the  amount  of  flavin  nucleotide  reduced  has  been 
estimated.  With  succinate  or  glycerolphosphate  several  times  more  flavin 
nucleotide  is  reduced  than  with  pyruvate  plus  malate.  Thus  under  these 
conditions  of  optimum  intramitochondrial  energy  supply,  a  relation  of  the 
redox  state  between  the  pyridine  nucleotide  and  the  flavin  nucleotide 
systems  appears  to  exist. 

When  electron  transport,  and  thus  energy  supply  for  oxidative  phos- 
phorylation, are  inhibited  by  antimycin  A,  the  adenine  nucleotides  are 
phosphorylated  to  a  low  degree  only.  It  is  to  be  noted  that  under  these 
conditions  the  reduction  of  DPN  and  flavoprotein  in  the  presence  of 
succinate  or  glycerolphosphate  is  also  diminished.  This  indicates  that  not 
only  the  reduction  of  DPN,  but  also  of  a  part  of  flavoprotein  is  dependent 
on  functioning  oxidative  phosphorylation.  Again,  the  redox  states  of  the 
DPN  and  flavin  nucleotides  behave  in  a  parallel  manner. 

An  energy-dependent  reduction  of  flavoprotein  had  been  observed  and 
reported  previously  in  flight  muscle  mitochondria  [13,  14].  It  v^as  con- 
cluded that  this  flavoprotein  cannot  be  on  the  main  pathway  of  the  oxida- 
tion of  glycerolphosphate,  since  it  was  not  reducible  when  electron 
transport  was  inhibited  by  anaerobiosis  or  antimycin  A.  Thus  this  flavo- 
protein may  be  reduced  by  the  substrates  through  an  energy-dependent 
hydrogen  transfer  in  a  way  similar  to  the  mitochondrial  DPN. 

Influence  of  exogenous  ATP 

EFFECT    OF    ATP    ON    PYRIDINE    AND    FLAVIN    NUCLEOTIDES 

In  contrast  to  heart  muscle  and  flight  muscle  mitochondria,  the  pyridine 
and  flavin  nucleotides  in  isolated  skeletal  muscle  mitochondria  remain 
oxidized  if  succinate  or  glycerolphosphate  is  added,  although  both  sub- 
strates are  active  hydrogen  donors  for  these  mitochondria  [4,  15].  Only  the 
subsequent  addition  of  ATP  results  in  a  large  reduction  of  the  DPN,  as 
shown  in  Fig.  2.  On  addition  of  phosphate,  DPN  becomes  largely  re- 
oxidized.  A  similar  ATP  efl^ect  can  be  obtained  in  heart  muscle  mito- 
chondria with  another  type  of  flavin  specific  substrate,  in  the  presence  of 
capronate.  The  extent  of  the  DPN  reduction  is  quantitatively  measured  by 
enzymic  pyridine  nucleotide  analysis  (Table  II).  DPN  is  further  reduced 
to  45%  by  the  addition  of  ATP  in  the  presence  of  either  glycerolphosphate 
or  succinate.  With  the  DPN  specific  substrates,  pyruvate  plus  malate,  a 
dift'erent  picture  emerges.  In  this  case,  DPN  is  reduced  to  about  32*^0  by 
the  substrates  alone.  ATP  addition  does  not  increase  further  the  reduction 
of  DPN. 


NUCLEOTIDES    AND    MITOCHONDRIAL    FUNCTION  23 1 

These  results  show  that  added  ATP  can  influence  the  redox  state  of 
intramitochondrial  components  of  hydrogen  transfer  such  as  DPX  and 
flavoprotein.  It  is  to  be  assumed  that  ATP  exerts  its  influence  through  back 
reactions  of  oxidative  phosphorylation.  The  energy  requirement  for  the 
DPX  reduction  in  the  presence  of  succinate  or  glycerolphosphate — so  far 
only  postulated  on  the  basis  of  thermodynamic  reasoning — has  been 
directly  demonstrated  bv  these  experiments.  This  phenomenon  appears  to 


340- 
380  myu 


DPN 
reduction 


capronat 

Fig.  2.  The  reducing  effect  of  ATP  on  mitochondrial  pyridine  nucleotides 
in  the  presence  of  non-DPN-linked  substrates.  Double  beam  spectrophotometer 
recordings  of  suspensions  of  mitochondria,  a.  Skeletal  muscle  mitochondria,  2  •  8 
mg.  protein  ml.  b.  Heart  muscle  mitochondria,  2-0  mg.  protein/nil.  Incubated  in 
0-3  M  sucrose,  i  mM  EDTA,  10  m.M  triethanolamine-HCl-buffer,  pH  72,  25  , 
air-saturated. 

apply  to  all  substrates  that  can  transfer  hydrogen  to  the  respiratory  chain 
without  a  participation  of  DPX,  since  also  with  fatty  acids  DPX  can  be 
reduced  at  the  expense  of  energy  supply  from  .A.TP.  In  agreement  with 
this  picture  no  ATP  is  required  for  the  reduction  of  DPX  with  DPX- 
specific  substrates  such  as  pyruvate  plus  malate. 

Further  work  may  be  mentioned  which  was  aimed  at  exluding  other 
possible  explanations  of  this  ATP  effect.  Thus  the  specificity  for  ATP  and 
the  studies  on  the  conditions  for  the  ATP-effect  furnished  additional 
evidence  that  ATP  acts  in  a  reversal  of  oxidative  phosphorylation  [15]. 

In  particular,  the  question  arises  why  ATP  is  required  for  DPX  reduc- 
tion in  isolated  skeletal-muscle  mitochondria  in  contrast  to  mitochondria 


232 


MARTIN   KLINGENBERG 

TABLE  II 

Redox  State  of  Pyridine  Nucleotides  in  Skeletal  Muscle  Mitochondria 


Additions 


DPNH 

/xMol/g.Prot. 


TPNH 
/xMol/g.Prot. 


DPNH 
^DPN* 


Glycerol- 1 -P 
Glycerol- 1 -P  +  ATP 
Succinate 
Succinate  +  ATP 
Pyruvate  +  malate 


0-72 
2-05 
030 

1-85 
I -So 


0-47 
0-58 
0-39 
o-6o 
o-SS 


o- 17 
0-47 
o-o8 
0-44 
032 


Mitochondria  after  washing  with  serum  albumin 

Glycerol- 1 -P  2-02  044  042 

Succinate  3*  01  0-46  063 

Pyruvate  +  malate  2-28  0'44  0-48 

Incubated  in  03  M  sucrose,  10  mM  triethanolamine-HCl-bufFer,  i  mM  EDTA, 
pH  72,  25°.  Concentration  of  substrates:  4  mM;  ATP:  i  mM. 

*i;DPN  =  DPN  +  DPNH. 

isolated  from  some  other  organs.  The  second  part  of  Table  II  shows 
that,  after  washing  the  skeletal-muscle  mitochondria  with  serum  albu- 
min, no  ATP  is  required  for  DPN  reduction.  When  added  after  the  sub- 
strates, albumin  is  also  effective  in  facilitating  the  DPN  reduction,  as 
shown  in  Fig.  3.   It  is  assumed  that  albumin  reverses  an  endogenous 


DPN 
reduction 


Fig.  3.  The  effect  of  serum  albumin,  antimycin  A  and  ATP  on  the  redox  state 
of  the  mitochondrial  DPN  in  the  presence  of  glycerol- 1 -phosphate.  The  absorption 
trace  (dashed  curve)  is  corrected  for  the  absorption  due  to  albumin  and  by  a  shift 
of  the  pen  position,  for  the  absorption  due  to  antimycin  A  (cf.  legend  Fig.  2.). 


NUCLEOTIDES    AND    MITOCHONDRIAL    FUNCTION  233 

uncoupling  of  the  mitochondria.  In  consequence,  now  the  endogenous 
oxidative  phosphorylation  is  more  efficient  in  supplying  energy  for  DPN 
reduction.  No  external  ATP  is  required  any  more,  since  addition  of  ATP 
in  this  case  would  not  increase  the  DPN  reduction.  As  shown  further  in 
the  experiment  of  Fig.  3,  DPNH  becomes  oxidized  when  respiration,  and 
thus  oxidative  phosphorylation,  are  inhibited  by  antimycin  A.  The  energy 
for  DPN  reduction  now  has  to  be  supplied  by  external  ATP.  The  experi- 
ment shows,  firstly,  that  for  DPN  reduction  the  oxidative  phosphorylation 
system  has  not  only  to  be  intact,  but  also  operative.  Otherwise,  ATP 
addition  is  required.  Secondly,  albumin  acts  only  by  protecting  oxidative 
phosphorylation. 

A  similar  reaction  sequence  can  be  observed  with  flavoprotein  (Fig.  4). 
The  partial  oxidation  of  flavoprotein  on  addition  of  antimycin  A,  which 


468-500m;< 


00025 


Flavoprotei 
reduction 


"I 


^ 

^ 

\    alt 

5mg 

umin 

^  60  sec^ 

4mM'V 
gly-1-P^ 

\ 

1-^y 

1 
ImM 

' 

I         ant.  A 

A 

1  r 

1 

V 

/'^ 

V. 

Fig.  4.  The  effect  of  serum  albumin,  antimycin  A  and  ATP  on  flavoprotein 
in  the  presence  of  glycerol- 1 -phosphate  (cf.  legend  Fig.  2.). 

was  previously  mentioned  (cf.  Table  I),  demonstrates  that  the  reduction 
of  some  flavoprotein  depends  on  energy  supply  from  oxidative  phosphory- 
lation. When  inhibited,  oxidative  phosphorylation  can  be  replaced  by 
added  ATP,  which  then  effects  a  reduction  of  flavoprotein  in  the  same  way 
as  with  DPN. 


EFFECT    OF    ATP    ON    CYTOCHROMES    AND    RESPIRATION 

So  far  we  have  dealt  with  the  influence  of  the  ATP-level  on  the  redox- 
state  of  DPN  and  flavoprotein.  By  these  experiments  an  action  of  ATP  on 
the  DPN-flavin  region  only  is  conclusively  demonstrated.  That  means 
that  these  experiments  show  primarily  the  reversal  of  the  first  phosphory- 
lation step  of  the  respiratory  chain.  It  has  now  been  possible  to  demonstrate 
also  an  influence  of  ATP  on  the  redox  state  of  the  cytochrome  region  of 


MARTIN    KLINGENBERG 


the  respiratory  chain  and  on  the  overall  electron  transport,  i.e.  the  oxygen  up- 
take, in  the  presence  of  succinate  or  glycerolphosphate  (15).  Both  cytochrome 
h  and  flavoprotein  (Fig.  5),  in  the  presence  of  succinate  or  glycerolphos- 


468-490m/i 

^E  = 
0005 
cm-' 

Flavoprotein 
reduction 


434-490m/i 


0010 
cm  ' 


Cyt.b         I 
reduction  I 


Fig.   5.  The  reducing  effect  of  ATP  on  flavoprotein  and  cytochrome  h  in  the 
presence  of  succinate  or  glycerol- 1 -phosphate  (cf.  legend  Fig.  2). 

phate  are  further  reduced  on  the  addition  of  ATP.  The  degree  of  reduction 
of  cytochrome  b  increases  from  about  io"o  in  the  presence  of  substrate 
alone  to  40%  after  the  addition  of  ATP.  Cytochrome  c,  as  shown  both  with 


550-S4lm/x 


00025 


Cyt.  c 
reduction 


Fig.  6.  The  oxidizing  effect  of  ATP  on  cytochrome  c  in  the  presence  of 
glycerol- 1 -phosphate  and  succinate  (cf.  legend  Fig.  2). 


glycerolphosphate  and  succinate  (Fig.  6)  is  however  rapidly  oxidized  by 
addition  of  ATP.  The  degree  of  reduction  of  cytochrome  c  decreases  from 
i2°o  to  about  5  to  8%.  Also  cytochrome  a  is  oxidized  by  addition  of  ATP 
(Fig.  7).  In  this  experiment  a  low  concentration  of  azide  has  been  added 
in  order  to  increase  the  reduction  of  cytochrome  a  and  afterwards  the 


NUCLEOTIDES    AND    MITOCHONDRIAL    FUNCTION  235 

oxidizing  effect  of  ATP,  which  otherwise  is  very  small.  Azide  at  this 
concentration  does  not  yet  inhibit  noticeably  the  respiration  (cf.  also  [i6]). 
The  reductive  effect  of  ATP  on  cytochrome  h  and  the  oxidative  effect  of 
ATP  on  cytochromes  c  and  a  are  reversed  by  addition  of  phosphate,  ADP 
or  dinitrophenol,  as  was  found  with  the  ATP-dependent  reduction  of 
DPX  and  flavoprotein. 


Fig.  7.  The  simultaneous  inhibition  of  respiration  and  oxidation  of  cyto- 
chrome a  in  the  presence  of  succinate  on  addition  of  ATP.  Amperometric  record- 
ing of  oxygen  consumption  and  simultaneous  spectrophotometric  recording  at  the 
a-band  of  cytochrome  a. 

The  interaction  of  ATP  with  the  respiratory  chain  causes  a  "crossover 
point"  of  the  redox  changes  of  the  respiratory  components  between  cyto- 
chrome h  and  cytochrome  c.  The  "crossover  point"  can  be  interpreted  to 
indicate  the  reaction  step  at  which  the  electron  transfer  is  inhibited  by 
ATP.  Thus,  it  is  to  be  expected  that  in  the  overall  electron  transport  also, 
i.e.  in  the  oxygen  uptake,  an  inhibitory  effect  of  ATP  can  be  observed.  As 
shown  in  the  upper  recordings  of  Figs.  7  and  8,  after  addition  of  ATP,  the 
respiration  of  skeletal-muscle  mitochondria,  both  with  glvcerolphosphate 
and  succinate,  is  inhibited  to  about  50^' o-  After  further  addition  of  phos- 
phate or  dinitrophenol,  respiration  is  again  accelerated  synchronously  with 


236  MARTIN    KLINGENBERG 

the  increase  of  reduction  of  cytochrome  a.  It  appears  that  in  these  mito- 
chondria, which  are  partly  uncoupled,  the  respiratory  control  is  increased 
by  ATP.  The  "crossover  point"  between  cytochrome  h  and  cytochrome  c 
shows  further  that  the  respiration  is  inhibited  by  the  action  of  ATP  at  this 
point.  We  conclude  that  by  the  "crossover  point"  of  ATP  action  the 
reversal  of  oxidative  phosphorylation  also  at  this  step  of  the  respiratory 
chain  is  demonstrated.  It  should  be  noted  that  in  studies  of  ADP  action  on 
the  respiratory  chain  in  liver  mitochondria,  by  Chance  and  Williams,  a 
"crossover  point"  between  cytochrome  h  and  c  in  the  opposite  sense  had 


400  r 


//,  atom  O 


liter 


300  - 


200  L 


605- 
630  m/i 


i4E  = 
0-0025 


Cyt.  a 
reduction 


Fig.  8.  The  inhibition  of  respiration  and  oxidation  of  cytochrome  a  in  the 
presence  of  glycerol- 1 -phosphate  on  addition  of  ATP  (cf.  legend  of  Fig.  2  and  7). 


been  observed  [17],  which  led  to  assume  a  phosphorylation  step  at  this 
point. 

An  inhibition  of  respiration  with  glycerolphosphate  or  succinate  can 
also  be  obtained  by  albumin  addition  (Fig.  9).  This  inhibition  is  not 
abolished  by  phosphate  but  only  by  further  addition  of  ADP.  Thus 
albumin  can  also  increase  the  respiratory  control,  in  agreement  with  its 
assigned  role  of  binding  the  endogenous  uncoupling  agents  of  mitochondria. 

The  kinetics  of  the  redox  changes  initiated  by  ATP  or  albumin  can  also 
be  explained  on  the  basis  of  the  proposed  mechanism.  The  oxidation  of 
cytochrome  c  or  cytochrome  a  on  addition  of  ATP  is  very  rapid,  followed 


NUCLEOTIDES   AND   MITOCHONDRIAL    FUNCTION 


237 


by  a  slow  reduction ;  whereas  the  ATP-induced  reduction  of  DPN  or 
flavoprotein  is  comparatively  slow.  Also  the  respiration  is  inhibited 
immediately  after  the  addition  of  ATP.  Thus  the  rapid  inhibition  of  the 
overall  electron  transport  by  ATP  appears  to  be  reflected  in  the  cyto- 
chromes. This  was  to  be  expected  since  cytochromes  c  and  a  are  thought 
to  be  in  the  direct  electron  transport  of  succinate  or  glycerolphosphate 
oxidation.  On  the  other  hand,  the  assumed  reversal  of  electron  transport 


400 


//-atom  O 


liter 


300 


200- 


605- 
630  m/x 


/1E  = 
00025 


Cyt  a 
reduction 


Fig.   9.  The  effect  of  albumin  on  respiration  and  on  the  redox  state  of  cyto- 
chrome a  in  the  presence  of  glycerol- 1 -phosphate. 

can  account  for  the  lower  velocity  of  the  ATP-induced  reduction  of  DPN 
and  of  flavoprotein.  These  components  receive  reducing  equivalents  in  a 
reverse  reaction  from  succinate  of  glycerolphosphate  at  a  speed  which  may 
depend  on  the  velocity  of  the  energy  supply.  The  parallel  behaviour 
between  cytochrome  oxidation  and  the  inhibition  of  respiration  extends 
also  to  the  effect  of  albumin  (Fig.  9).  Both  the  inhibition  of  respiration  and 
initiation  of  cytochrome  oxidation  increase  slowly  after  the  addition  of 
albumin.  This  kinetic  behaviour  is  understandable  on  the  grounds  of  the 
proposed  mechanism  of  albumin  action. 


238  MARTIN   KLINGENBERG 

Conclusions 

The  experimental  results  of  an  influence  of  ATP  on  the  respiratory 
chain  are  summarized  as  follows: 

1.  ATP  can  affect  the  reduction  of  DPN  in  the  presence  of  flavin- 
specific  substrates,  such  as  succinate,  etc.  Thus  the  energy  requirement  of 
the    DPN    reduction    in   the    presence   of  these   substrates    is    directly 
demonstrated. 

2.  ATP  can  affect  also  the  reduction  of  a  flavoprotein,  the  reduction  of 
which  had  been  shown  to  depend  on  operative  oxidative  phosphorylation. 

3.  ATP  can  influence  the  overall  electron  transport  by  inducing 
respiratory  control. 

4.  ATP  addition  causes  a  "crossover  point"  of  redox  changes  at  the 
respiratory  chain  between  cytochrome  b  and  c.  cf.  (15). 

These  results  are  interpreted  as  demonstrating  the  reversibility  of 
oxidative  phosphorylative  reactions.  At  least  two  phosphorylation  steps 
of  the  respiratory  chain  are  shown  to  be  reversible. 


DPN<^-^Fp<^-(b)^-±-c a O2 


In  the  DPN-flavin  region  a  complete  reversal  of  the  oxidative  phosphory- 
lation, including  also  a  reversal  of  electron  transfer,  can  be  effected.  In  the 
cytochrome  region  the  reversal  of  the  reactions  between  ATP  and  the 
respiratory  chain  is  seen.  The  interaction  of  ATP  at  the  cytochrome 
level  also  controls  the  overall  electron  transport  of  succinate  or  glycerol- 
phosphate  oxidation. 

There  are  tw^o  aspects  which  should  be  briefly  mentioned  on  the  basis 
of  these  results.  Firstly,  the  elucidation  of  the  mechanism  of  oxidative 
phosphorylation  depends  greatly  on  the  knowledge  about  the  inter- 
mediates of  the  phosphate  transfer  reactions.  The  reversibility  of  oxidative 
phosphorylation  presents  in  principle  the  possibility  to  estimate  the  energy 
content  of  the  intermediates.  Second  is  the  physiological  meaning  of 
energy-dependent  hydrogen  transfer  from  flavin  to  pyridine  nucleotide  in 
mitochondria,  as  originally  proposed  by  Krebs  [18].  In  this  case,  hydrogen 
from  succinate  or  fatty  acid  oxidation  w^ould  not  be  transferred  to  oxygen, 
generating  ATP  in  oxidative  phosphorylation,  but,  with  expenditure  of 
energy,  to  the  DPN  or  TPN  systems  of  the  mitochondria.  It  appears 
possible  to  imitate  such  a  system  in  experiments  with  liver  mitochondria 
where  hydrogen  in  the  presence  of  ATP  can  be  transferred  from  succinate 
to  oc-ketoglutarate  with  the  formation  of  glutamate  [19]. 


NUCLEOTIDES    AND    MITOCHONDRIAL   FUNCTION  239 

References 

1.  Lardy,  H.  A.,  and  Wellman,  H.,y.  biol.  Chem.  195,  25  (1952). 

2.  Chance,  B.,  and  Williams,  G.  B^.,y.  biol.  Chem.  217,  383  (1955). 

3.  Chance,  B.,  and  Williams,  G.  R.,  J.  biol.  Chem.  217,  409  (1955). 

4.  Klingenberg,  M.,  "  nth  Mosbach  Colloquium",  Springer  Verlag  (i960). 

5.  Azzone,  G.  F.,  Ernster,  L.,  and  Klingenberg  M.,  Nature,  Loud,  (in  press). 

6.  Chance,  B.,  and  Hagihara,  B.,  Biochem.  biophys.  Res.  Cottmi.  3,  i  (i960). 

7.  Chance,  B.,  and  Hollunger,  G.,  Fed.  Proc.  16,  163  (1957). 

8.  Biicher,  Th.,  and  Klingenberg,  M.,  Angezc.  Chem.  70,  552  (1958). 

9.  Chance,  B.,  and  Hollunger,  G.,  Nature,  Loud.  185,  666  (i960). 

10.  Klingenberg,  M.,  vSlenczka,  W\,  and  Ritt,  E.  Biochem.  Z.  332,  47  (1959). 

11.  Held,  H.  W.,  Schollmeyer,  P.,  and  Klingenberg,  M.  (unpublished). 

12.  Papenberg,  K.,  Klingenberg,  ]\I.,  and  Held,  M.  W.,  Biochem.  Z.  (in  press). 

13.  Klingenberg,  M.,  and  Biicher,  Th.,  Biochem.  Z.  331,  312  (1959). 

14.  Klingenberg,  M.  BiocJietn.  Z.  (in  press). 

15.  Klingenberg,  M.,  and  Schollmeyer,  P.,  Biochem.  Z.  333,  335  (i960). 

16.  Chance,  B.,  and  Williams,  G.  R.,J.  biol.  Chem.  221,  477  (1956). 

17.  Chance,  B.,  and  Williams,  G.  R.,  Advanc.  Enzymol.  17,  65  (1956). 

18.  Krebs,  H.  A.,  and  Kornberg,  H.  L.,  Ergebn.  Physiol.  49,  271  (1957). 

19.  Klingenberg,  M.,  and  Schollmeyer,  P.  (in  preparation). 

Discussion 

Slater  :  Alay  I  take  up  the  point  we  were  discussing  with  Dr.  Chance  a  moment 
ago.  You  require  ATP  for  the  reduction  of  succinate  in  your  freshly  prepared  rat- 
skeletal-muscle  sarcosomes,  but  when  they  are  treated  with  albumin  ATP  is  no 
longer  required.  You  suggest  that  the  albumin  removes  an  uncoupler.  Now  Dr. 
Chance's  pigeon-heart  sarcosomes  require  ATP  and  our  rabbit-heart  sarcosomes 
do  not  require  ATP.  The  difference  between  Dr.  Chance's  results  and  ours  could 
then  be  explained  by  Dr.  Klingenberg  by  the  presence  or  absence  of  an  uncoupler 
whereas  Dr.  Chance  would  prefer  to  explain  it  by  the  absence  or  presence  of  endo- 
genous substrate.  Is  it  possible,  then,  that  albumin  is  removing  an  endogenous 
substrate  which  is  a  fatty  acid  rather  than  an  uncoupler  ? 

Klingenberg  :  I  would  say  that  albumin  removes  the  uncoupler  or  may  pre- 
serve endogenous  substrate  because  ATP  is  no  longer  required. 

Chance:  I  would  be  inclined  to  agree  with  Dr.  Klingenberg's  view  that  we 
either  add  or  preserve  the  endogenous  substrate  so  that  energy  would  be  available, 
however,  for  the  reduction  of  the  pyridine  nucleotide  on  addition  of  Dc-glycero- 
phosphate  or  succinate. 

Slater  :  I  thought  you  said  that  albumin  was  removing  an  uncoupler. 

Klingenberg  :  The  general  opinion  is  that  it  removes  an  uncoupler. 

Chance  :  You  can  remove  an  uncoupler  by  ATP. 

Klingenberg:  I  presume  that  skeletal  muscle  mitochondria  are  slightly 
uncoupled  and  also  have  in  the  presence  of  endogenous  substrate  a  rather  low  level 
of  ATP  or  high  energy  substances. 

Slater:  Have  you  in  mind  as  an  uncoupler  the  unsaturated  fatty  acids  ? 

Klingenberg  :  Possibly. 

Chance  :  What  is  the  effect  of  ATP  when  you  have  added  glycerophosphate  to 
the  skeletal  muscle  but  not  phosphate  or  ADP  ? 


240  MARTIN    KLINGENBERG 

Klingenberg:  ADP  cannot  induce  DPN  reduction.  The  respiration  is  not 
stimulated. 

Chance  :  We  have  been  studying  for  some  time  the  ADP-inhibition  of  succinate 
oxidation.  When  you  add  ATP  to  mitochondria  which  are  not  too  tightly  coupled, 
you  may  produce  ADP  and  phosphate.  Under  these  conditions  I  expect  the  same 
as  you  have  observed,  cytochrome  c  goes  oxidized,  and  respiration  may  be  inhibited. 
I  think  it  is  something  which  should  be  controlled  in  these  preparations  because 
they  are  sensitive  to  small  amounts  of  ADP  which  will  inhibit  respiration  strange 
as  it  may  seem. 

While  I  certainly  agree  that  ATP  can  reverse  electron  transfer,  I  do  not  know 
whether  you  can  do  it  in  the  presence  of  oxygen  and  thermodynamics  speaks 
against  it.  I  do  not  question  the  phenomenon  but  I  am  not  sure  whether  you  have 
demonstrated  it  to  me  at  the  cytochrome  level. 

Two  comments  occur  to  me :  first,  when  the  mitochondria  became  anaerobic, 
you  report  flavin  slowly  became  oxidized.  This  may  be  hard  to  measure  accurately. 
The  second  point  is  that  we  get  much  more  DPN  reduction  on  adding  ATP  in  the 
presence  of  succinate,  in  fact  there  isn't  just  any  more  DPN  to  be  reduced  when 
mitochondria  go  anaerobic  with  added  ATP,  but  you  only  get  40°,,.  Is  this  a  differ- 
ence in  preparations  ? 

Klingenberg  :  This  may  be ;  we  sometimes  get  60%  but  never  more ;  I  do  not 
think  this  is  very  significant.  In  the  presence  of  ADP  or  phosphate,  ATP  does  not 
inhibit  respiration  and  does  not  oxidize  cytochrome  c.  This  would  be  an  argument 
against  the  hypothesis  that  ADP  is  generated  by  the  addition  of  ATP  and  thus  has 
the  effect  on  cytochrome  c.  Also  in  the  presence  of  DNP  no  oxidation  of  cytochrome 
c  or  inhibition  of  respiration  occurs  on  addition  of  ATP.  I  do  not  think  that  we 
have  observed  an  electron  transport  reversal  in  the  cytochrome  region.  I  think  we 
have  observed  a  reversed  interaction  of  ATP  with  the  cytochromes  which  results 
in  an  increase  of  the  respiratory  control. 

Chance  :  In  the  experiments  I  reported  this  morning,  special  precautions  were 
taken  to  exclude  the  possibility  that  the  electron  acceptor  for  the  observed  oxidation 
of  cytochrome  c  in  the  presence  of  ATP  was  oxygen.  If  it  were  oxygen,  then 
obviously  no  "reversed  interaction  of  ATP  with  the  cytochromes"  could  have 
been  proved.  In  Dr.  Klingenberg's  excellent  experiments,  which  have  just  been 
reported,  oxygen  was  present  and  no  inhibitor  of  the  oxidase  was  added ;  the  system 
being  in  a  steady  state.  Thus,  an  ATP  induced  inhibition  of  electron  transfer 
would  be  sufficient  to  explain  the  observed  results;  there  being  no  demonstration 
that  the  acceptor  of  electrons  from  cytochrome  c  was  a  substance  at  a  lower  and 
not  at  a  higher  redox  potential  the  former  would  be  required  in  reverse  electron 
transfer. 

Klingenberg  :  I  had  stated  in  my  report  that  we  interpret  the  oxidation  of  the 
cytochromes  c  and  a  to  demonstrate  an  interaction  of  the  ATP  with  the  cyto- 
chromes by  a  reversal  of  oxidative  phosphorylation.  Although  oxygen  had  not  been 
excluded,  these  experiments  should  show  the  reverse  interaction  of  ATP  by  way 
of  oxidative  phosphorylation  reactions  as  clear  as,  in  the  opposite  manner,  an 
influence  of  ADP  and  phosphate  on  the  redox  state  of  the  cytochromes  shows  an 
interaction  by  way  of  oxidative  phosphorylation.  This  is  further  supported  by  the 
increase  of  the  respiratory  control  after  addition  of  ATP. 


The  Role  of  ATPase  in  Oxidative  Phosphorylation* 

Maynard  E.  Pullman,  Harvey  S.  Pexefsky  and  E.  Racker 

Division  of  Xutrition  and  Physiology, 
The  Public  Health  Research  Institute  of  the  City  of  Xew  York,  Inc., 

X.Y.,  U.S.A. 

In  previous  communications  we  reported  the  resolution  of  mechanically 
fragmented  beef  heart  mitochondria  into  a  particulate  and  a  soluble 
protein  component,  both  of  which  were  required  for  oxidative  phosphory- 
lation [i,  2].  The  particulate  fraction  catalyzed  the  oxidation  of  a  number 
of  substrates  with  little  or  no  concomitant  phosphorvlation.  The  addition 
of  the  soluble  component  to  the  particulate  fraction  recoupled  the  respira- 
tion to  phosphorylation.  A  summary  of  the  properties  of  the  reconstituted 
system  and  of  the  soluble  factor,  as  well  as  some  of  the  more  recent 
developments  with  this  system  will  form  the  subject  of  this  discussion. 
Since  a  detailed  description  of  the  experimental  procedures  was  presented 
elsewhere  [3,  4]  only  the  salient  features  will  be  considered  here. 

Beef  heart  mitochondria,  prepared  according  to  the  method  of  Green 
et  al.  [5],  were  disrupted  in  vacuo  in  the  presence  of  glass  beads  bv  means 
of  a  high-speed  reciprocal  Xossal  shaker  [6].  The  suspension  was  centri- 
fuged  for  20  min.  at  26  000  x  g  yielding  a  brown,  gelatinous  residue  which 
was  discarded  and  a  yellow,  turbid  supernatant  fluid.  The  supernatant 
solution  was  recentrifuged  at  105  000  x  g  for  30  min.  A  red-brown  gela- 
tinous residue  (residue  i)  and  a  faintly  turbid,  yellow  supernatant  fluid 
were  obtained.  The  supernatant  fluid  was  decanted  and  clarified  by 
centrifugation  for  an  additional  30  min.  at  105  000  x  g,  vielding  a  clear 
yellow  solution.  Residue  i  was  washed  by  homogenizing  in  0-2^  m 
sucrose  containing  0-002  M  EDTA  and  centrifuged  at  lo^oooxg  for 
30  min.  The  washing  procedure  was  repeated  with  0-2^  m  sucrose  and 
the  final  residue  i  was  suspended  in  0-25  M  sucrose. 

As  shown  in  Table  I  residue  i  catalyzed  the  oxidation  of  succinate  with 
little  or  no  accompanying  phosphorylation.  Addition  of  increasing  amounts 
of  the  supernatant  solution  (105  000  xg)  resulted  in  almost  a  tenfold 
increase  in  phosphate  uptake.  The  coupling  factor  had  no  significant  effect 

*  This  work  was  supported  by  Grants  Xos.  A- 12 19  and  C-3463  from  the 
National  Institutes  of  Health,  United  States  Public  Health  Service,  Bethesda, 
Maryland. 

VOL.  II. — R 


242  MAYNARD   E.    PULLMAN,    HARVEY    S.    PENEFSKY   AND    E.    RACKER 

on  respiration  and  appears,  therefore,  to  be  primarily  concerned  in  the 
phosphorylation  mechanism.  Phosphorylation  in  the  reconstituted  system 
is  uncoupled  by  2,4-dinitrophenol  as  well  as  by  a  number  of  other  recog- 
nized uncouplers  including  dicoumarol,  chlorpromazine  and  triiodo-L- 
thyronine.  The  maximal  P:0  ratios  obtained  in  this  particular  experiment 
are  somewhat  less  than  those  generally  observed.  The  average  maximal 
P :  O  ratio  was  o  •  6,  with  a  range  of  o  •  4  to  o  •  8.  During  the  early  phases  of 
this  work,  different  preparations  of  the  particulate  fraction  exhibited 
residual  and  variable  phosphorylation  activity.  Nevertheless,  the  addition 
of  the  supernatant  fraction  never  failed  to  result  in  a  marked  increase  in 

TABLE  I 

Effect  of  the  Supernatant  Solution  on  Phosphorylation  Accompanying 

Succinate  Oxidation 

Each  Warburg  vessel  contained  0-05  m  succinate,  pH  7-4,  0-004  M  MgCl2, 
0'002  M  ATP,  0-0I2  M  potassium  phosphate  buffer,  pH  7-4,  0-032  M  glucose, 
0-005  M  tris,  pH  7-4,  o-ooi  M  EDTA,  0-06  mg.  yeast  hexokinase  (25  to  40  units/ 
mg.),  I  -6  mg.  of  the  particulate  fraction  and  the  indicated  amounts  of  the  super- 
natant solution  (105  000  X  g)  in  a  final  volume  of  0-5  ml.  Where  added,  2,4- 
dinitrophenol  (DNP)  was  0-0005  J^i-  Incubations  were  carried  out  for  36  min. 
at  30°. 


Supernatant  solution 

O2  uptake 

Pj  uptake 

P:0 

/^g- 

protein 

/xatoms 

/j-atoms 

None 

5-6 

0-3 

0-05 

75 

5-0 

0-8 

o- 16 

150 

4-7 

1-8 

0-38 

300 

4-9 

2-2 

0-45 

2400 

5-9 

2-7 

0-46 

2400 

+  DNP 

6-1 

0-4 

0-04 

the  P:0  ratio.  In  subsequent  work,  disruption  of  the  mitochondria  was 
carried  out  in  the  presence  of  EDTA,  which  in  confirmation  of  the  results 
reported  by  Linnane  [7],  consistently  yielded  particles  in  which  phosphory- 
lation was  either  low  or  absent.  For  reconstitution,  these  particles  were 
preincubated  with  the  soluble  protein  and  Mg  +  +  and  an  aliquot  of  the 
mixture  was  then  added  to  a  Warburg  vessel  containing  the  otherwise 
complete  reaction  mixture  (cf.  Table  I). 

Table  II  illustrates  the  dependency  on  the  coupling  factor  for  phos- 
phorylation associated  with  the  oxidation  of  various  substrates.  It  may  be 
seen  that  isocitrate  was  oxidized  by  the  submitochondrial  particles  at  about 
one-half  the  rate  of  succinate  and  that  no  esterification  of  Pj  occurred  in 
the  absence  of  the  purified  coupling  factor.  The  oxidation  of  ^-hydroxy- 
butyrate  required  the  addition  of  DPN  while  glutamate  oxidation  occurred 


ATPase  in  oxidative  phosphorylation  243 

only  if  the  system  was  supplemented  with  both  DPX  and  glutamic  de- 
hydrogenase. Again,  both  /S-hydroxybutyrate  and  glutamate  were  oxidized 
without  uptake  of  P;  unless  the  coupling  factor  was  present.  The  maximal 
P:0  ratio  obtained  was  independent  of  the  nature  of  the  substrate  under- 
going oxidation,  suggesting  that  only  phosphorylation  sites  in  the  respira- 
tory chain  between  succinate  and  oxygen  contribute  to  the  P:0  ratio.  A 
more  precise  localization  of  the  phosphorylation  site(s)  is  currently  under 
investigation. 

Purification  and  characterization  of  the  coupling  factor  revealed  the 
presence  of  a  Mg^  ^-dependent,  dinitrophenol-stimulated  ATPase.  The 
generallv  accepted  concept  that  the  mitochondrial  ATPase  is  functionally 

TABLE  II 

Effect  of  the  Purified  Coupling  Factor  on  Phosphorylation  Associ.\ted 
WITH  THE  Oxidation  of  Various  Substrates 

The  experimental  conditions  have  been  previously  described  (cf.  Table  II  [4]). 

o    1  Coupling  ^  ,  r,  ^  r.    ^ 

Substrate  .  O.,  uptake         R  uptake  P  :0 

factor  ■     ^  '     ^ 


fiA  /min .  /mg.   /u A  /min .  mg. 


Succinate 
DL-isocitrate 
DL-^-hydroxybutyrate 
L-glutamate 


— 

0-25 

0  -00 

0-00 

+ 

0-29 

0  ■  I  2 

0-41 

— 

0-14 

0-00 

0-00 

+ 

0-I4 

0-052 

0-38 

— 

0-054 

0  ■  003 

0  -05 

+ 

0-041 

0  -020 

0-49 

— 

0-040 

0  -003 

0-07 

4- 

0  ■  050 

0-023 

0-46 

related  to  the  enzymic  mechanism  of  oxidati\e  phosphorylation  prompted 
us  to  examine  the  relationship  between  the  ATPase  and  coupling  activities. 
Since  the  most  highly  purified  preparations  which  hvdrolvzed  80  to  100 
/^moles  of  ATP  min.  mg.  protein  induced  phosphorylation  coupled  to 
oxidation  in  the  presence  of  the  particles,  the  question  arose  whether  these 
activities  were  located  in  the  same  protein. 

ATPase  activity,  which  was  measured  in  a  system  using  phosphoenol- 
pyruvate  and  pyruvate  kinase  as  a  regenerating  system  for  ATP,  was 
markedly  higher  in  the  presence  than  in  the  absence  of  the  regenerating 
system.  This  is  due,  at  least  in  part,  to  the  fact  that  the  pyruvate  kinase 
system  removes  the  ADP  which  is  inhibitory  to  the  enzyme.  ATPase 
activity  was  increased  50  to  75",,  by  the  addition  of  5  x  10 -*  m  2,4- 
dinitrophenol. 

Studies  on  the  purified  ATPase  revealed  properties  consistent  with  its 
participation  in  coupled  phosphorylation  and  similar  to  those  described 


244 


MAYNARD    E.    PULLMAN,    HARVEY    S.    PENEFSKY    AND    E.    RACKER 


for  the  particulate  enzyme  of  mitochondria  and  phosphorylating  mito- 
chondrial fragments.  Some  of  these  properties  are  summarized  in  Table 
III.  While  a  number  of  divalent  cations  including  Co  +  +,  Mn  +  +,  Fe  +  + 


TABLE  III 
Properties  of  ATPase 


Divalent  cation  required  for  activity 
Stimulated  by  2,4-dinitrophenol 
Hydrolyzes  ATP,  ITP,  GTP,  and  UTP 
Inhibited  by  ADP  but  not  IDP 
Stoicheiometry :  ATP  +  HoO^ADP  +  Pj 
Exhibits  "latent"  activity  phenomenon 


and  Ca  +  +  substituted  for  Mg  +  +  in  activating  the  enzyme,  only  Mg  +  + 
and  to  a  lesser  extent  Co  +  +  gave  rise  to  a  dinitrophenol  stimulation.  The 
enzyme  hydrolyzed  ITP,  GTP,  and  UTP  in  addition  to  ATP.  However, 
it  seems  significant  that  only  ATP  hydrolysis  was  stimulated  by  dinitro- 
phenol. Neither  the  nucleoside  mono-  nor  diphosphates  were  hydrolyzed. 


O 


0-5 


10        1-5        2-0 

Preincubation  time   (hr) 


200 


Fig.  I.  Effect  of  preincubation  temperature  on  ATPase  and  coupling  activity. 
The  purified  enzyme  was  preincubated  either  at  o^  or  30.  At  the  indicated  time, 
aliquots  were  removed  and  the  appropriate  activity  measured  at  30  as  described 
elsewhere  [4]. 


The  specificity  of  the  ADP  inhibition  is  of  interest  in  view  of  the  specificity 
of  this  nucleotide  in  oxidative  phosphorylation.  The  "latent"  activity 
phenomenon  referred  to  in  the  table  may  actually  be  related  to  the  well- 
known    "latent"    properties    of   mitochondrial    ATPase    [8-10].    It   was 


ATPase  in  oxidative  phosphorylation  245 

observed  that  incubation  of  the  enzyme  at  30'  or  in  the  presence  of  ATP 
at  considerably  higher  temperatures  resuhed  in  an  activation  of  the  enzyme. 
It  was  necessary,  however,  to  accumulate  more  direct  evidence  that 
the  ATPase  and  coupling  activity  were  in  fact  catalytic  expressions  of  a 
single  protein.  Compelling  evidence  in  favour  of  this  view  was  obtained 
by  a  number  of  procedures  designed  to  selectively  destroy  one  of  the 
activities.  Invariablv  these  procedures  resulted  in  a  parallel  destruction  of 
both  activities.  The  most  striking  of  these  parallelisms  was  noted  during 
the  later  stages  of  the  purification  procedure  when  the  ATPase  activity 
became  extremelv  unstable.  Further  investigations  revealed  that  the 
purified  preparation  was  markedly  cold-labile.  That  is,  the  activity  of  the 
ATPase  declined  rapidly  at  ice  bath  temperature  while  at  room  tempera- 
ture the  activitv  generallv  increased.  This  rather  unusual  lability  was  also 
displayed  by  the  coupling  activity.  These  results  are  shown  in  Fig.  i.  In 

TABLE  IV 

Protection  by  ATP  Against  Heat  Inactivation  of  ATPase  and 
Phosphorylation  Activity 

A  solution  of  the  purified  enzyme  containing  i  -6  mg.  protein/ml.  was  divided 
into  approptiate  aliquots  and  heated  for  4  min.  under  the  indicated  conditions. 
The  ATP,  when  present,  was  4  x  10^  M.  Assays  for  coupling  activity  (P:0) 
were  carried  out  as  describeci  previously  [4]  with  0-038  mg.  of  the  coupling  enzyme 
and  0-495  mg.  of  the  particulate  fraction.  In  the  absence  of  the  coupling  enzyme, 
the  P:0  ratio  was  0-05.  The  ATPase  assay  was  carried  out  in  the  presence  of 
the  ATP  regenerating  system  [3]. 

Pretreatment  P  :  O  ATPase 


None 

60^  +  ATP 


/mioles  Pi/mg./io' 

0-46 

342-0 

o-  16 

59 'O 

0  ■  5  5 

342-0 

this  experiment,  aliquots  of  the  purified  protein  fraction  were  preincubated 
either  at  o'  or  30  for  the  indicated  intervals  and  then  added  to  the  appro- 
priate assay  system.  Both  assays  were  carried  out  at  30  .  As  may  be  seen, 
the  rapid  rates  of  inactivation  of  these  two  activities  at  o  \\  ere  strikingly 
parallel  while  at  30  both  activities  were  retained  for  over  20  hr.  The 
enzyme  may  be  kept,  however,  at  4"  as  a  suspension  in  50",,  ammonium 
sulphate  for  3  weeks  without  appreciable  loss  in  either  ATPase  or  coupling 
activity. 

Exposure  of  the  purified  enzyme  to  elevated  temperatures  also  failed 
to  achie\e  a  separation  of  the  two  activities.  As  shown  in  Table  IV,  both 
activities  were  inactivated  at  60  to  a  similar  extent  and  were  completely 
protected  by  ATP. 


246  MAYNARD    E.    PULLMAN,    HARVEY    S.    PENEFSKY    AND    E.    RACKER 

Similarly,  a  2-hr.  dialysis  at  room  temperature  resulted  in  parallel 
losses  of  both  activities.  These  results  are  presented  in  Table  V.  The 
addition  of  ATP  to  the  dialyzing  solution  at  a  final  concentration  of 
0-005  ^^  again  resulted  in  considerable  protection  of  both  activities. 
Various  salts,  for  example,  ammonium  sulphate,  ammonium  chloride  or 
potassium  sulphate,  added  to  the  dialyzing  medium  at  concentrations  then 
ten  times  higher  than  that  of  the  ATP  also  prevented  to  a  large  extent  the  loss 
of  both  activities.  Attempts  to  reactivate  the  dialyzed  enzyme  by  the 
addition  of  boiled  enzyme,  cold  inactivated  enzyme  or  several  known 
cofactors  have  been  unsuccessful. 

TABLE  V 

Protection  by  ATP  Against  Dialysis  Inactivation  of  ATPase  And  Coupling 

Activity 

2-8  mg.  of  the  coupling  enzyme  were  dissolved  in  1-5  ml.  sucrose-tris-EDTA 
and  divided  into  three  o  •  5  ml.  aliquots.  Dialysis  was  carried  out  at  room  temperature 
for  2  hr.  vs.  0-25  M  sucrose-o-oi  M  tris,  pH  7-4.  ATP  was  0-005  M  when  added. 
Assays  for  coupling  activity  (P  :0)  were  carried  out  as  described  previously  [4] 
with  0-038  mg.  of  the  coupling  factor  and  0-620  mg.  of  the  particulate  fraction  per 
vessel.  In  the  absence  of  coupling  factor  the  P  :0  was  003.  ATPase  activity  was 
measured  with  the  ATP  regenerating  system  [3]. 

Dialyzing  solution  P:0  ATPase 


/Ltmoles  Pi/mg./io' 

None  (undialyzed  control) 

0-48 

360-0 

Sucrose-tris 

0-09 

26-0 

Sucrose-tris-ATP 

0-33 

172-0 

Additional  evidence  for  the  single  enzyme  concept  was  obtained  from 
an  examination  of  the  effect  of  uncouplers  on  the  two  activities.  Some  of 
these  results  are  summarized  in  Table  VI.  In  general  it  was  found  that  all 
compounds  which  affect  the  ATPase  (i.e.  either  inhibit  or  stimulate)  also 
uncoupled  oxidative  phosphorylation.  /)-Chloromercuribenzoate,  azide, 
Dicoumarol,  dihydrovitamin  K^  diphosphate  (a  water-soluble  derivative 
of  vitamin  Kj)  and  triiodothyronine  inhibited  both  the  phosphorylation 
activity  and  the  ATPase  activity  of  the  purified  factor  at  concentrations 
between  5  x  10  ^  and  5  x  io~^  m.  Dinitrophenol  and  pentachlorophenol, 
two  potent  uncouplers  of  oxidative  phosphorylation,  inhibited  the  phos- 
phorylation activity  and  stimulated  the  ATPase  activity.  Azide  inhibited 
the  ATPase  activity  to  a  greater  extent  in  the  absence  than  in  the  presence 
of  dinitrophenol  with  the  result  that  the  stimulation  by  dinitrophenol  was 
in  effect  increased  from  50  to  300  or  400",,.  The  opposite  effect  was 
observed  with  /)-chloromercuribenzoate  which  completely  eliminated  the 
dinitrophenol  stimulation. 


ATPase  in  oxidative  phosphorylation 


247 


Finally,  in  agreement  with  current  concepts  of  the  mechanism  of 
oxidative  phosphorylation  which  predict  that  the  ATPase  and  ^-P— ATP 
exchange  reactions  are  involved  in  a  common  reaction  sequence  in  oxida- 
tive phosphorylation,  it  was  found  that  the  purified  ATPase  was  required 
to  reconstitute  a  dinitrophenol  sensitive  ^-Pj-ATP  exchange.  Representa- 
tive data  are  presented  in  Table  VII.  Aliquots  of  a  given  particulate 
preparation  were  used  to  measure  either  the  esterification  of  P;  in  the 
presence  of  succinate  or  the  incorporation  of  ^'^Pj  into  ATP  in  the  absence 
of  added  substrate.  As  may  be  seen,  neither  the  particles  nor  the  purified 
ATPase  when  tested  alone  catalyzed  an  appreciable  ^'^P— ATP  exchange  or 
phosphate  esterification.  However,  the  addition  of  increasing  amounts  of 

TABLE  VI 

Effect  of  Various  Compounds  on  ATPase  and  Phosphorylation  Activity 


Compound 


P:0 


ATPase 


2,4-dinitrophenol  Inhibition 

Pentachlorophenol  ,, 

/)-Chloronnercuribenzoate  ,, 

Azide  „ 

Dicoumarol  „ 

Dihydrovitamin  K^  diphosphate  „ 

Triiodo-L-thyronine  ,, 

Amytal  — 

Potassium  cyanide  — • 

Potassium  fluoride  None 

Warfarin  None 


Stimulation 
Inhibition 


None 
None 
None 

None 


the  purified  ATPase  to  the  particles  resulted  in  parallel  increases  in  both 
of  these  activities.  2,4-dinitrophenol  abolished  both  reactions. 

Based  on  the  evidence  presented,  as  well  as  on  other  supporting  data, 
we  have  concluded  that  the  catalytic  site  or  sites  responsible  for  the 
hydrolysis  of  ATP  and  for  the  coupling  activity  reside  on  the  same  protein. 
It  became  necessary,  however,  to  explain  the  observation  that  during  the 
course  of  purification  the  ATPase  was  purified  to  a  greater  extent  than 
the  coupling  activity.  The  apparent  greater  purification  of  the  ATPase  was 
actually  not  based  on  the  removal  of  other  protein  impurities,  but  depended 
to  a  large  extent  on  an  absolute  increase  in  total  units.  These  results  indi- 
cate an  activation  of  a  hydrolytic  site  or  removal  of  an  inhibitor  rather  than 
a  physical  separation  of  the  two  components. 

We  look  upon  the  hydrolysis  of  ATP  by  the  coupling  factor  as  an 
aberrant  activity  which  the  enzyme  has  acquired  following  the  disruption 
of  the  mitochondria.  Bound  to  the  structure  of  the  undamaged  mito- 
chondria, the  hydrolytic  potentialities  of  this  protein  are  largely  masked, 


248  MAYNARD    E.    PULLMAN,    HARVEY    S.    PENEFSKY    AND    E.    RACKER 

and  during  oxidative  phosphorylation  the  enzyme  functions  primarily  as  a 
transfer  agent.  A  similar  suggestion  was  made  many  years  ago  to  explain 
the  latent  ATPase  of  intact  mitochondria  [8-10]. 

We  feel  that  the  most  logical  site  for  the  action  of  this  enzyme  in 
oxidative  phosphorylation  would  be  the  terminal  transphosphorylation 
step.  The  possibility,  therefore,  of  an  ADP-ATP  exchange  catalyzed  by 
this  enzyme  was  explored.  Numerous  attempts  under  various  experi- 
mental   conditions    have    thus    far    been    unsuccessful.    However,    these 

TABLE  VII 

Effect  of  the  Coupling   Enzyme  on  Oxidative  Phosphorylation  and  the 
^-Pj-ATP  Exchange  Reaction 

The  particulate  fraction  was  preincubated  with  the  coupling  enzyme  and  Mg  +  + 
as  described  previously  [4].  Aliquots  of  the  preincubation  mixture  containing 
0-580  mg.  of  the  particulate  fraction  and  the  indicated  amount  of  the  coupling 
enzyme  were  added  to  the  Warburg  vessel  for  the  assay  of  oxidative  phosphoryla- 
tion or  to  test  tubes  for  the  measurement  of  the  ^-Pj-ATP  exchange  reaction. 
Oxidative  phosphorylation  was  measured  at  30°  for  30  min.  with  succinate  as 
substrate  [4].  The  ^-Pj-ATP  exchange  reaction  was  measured  as  described  else- 
where [4].  Each  tube  contained  o-oi6  !m  ATP,  o-oi6  m  MgCU,  o-oi  m  tris,  pH 
7-4,  0-04  M  ^-Pj  (i-2  X  10^  c.p.m.//Lxmole),  o-ooi  M  EDTA,  pH  7-4  and  the 
preincubated  enzyme  mixture  in  a  final  volume  of  o  •  5  ml.  When  added,  dinitro- 
phenol  was  5    x    10^  m. 


Coupling 
enzvme 


O2 
uptake 


Pi 
uptake 


P:0 


3-P,-ATP 


/^g. 

/xatoms 

jumoles 

c.p 

.m./;umole 
ATP 

0 

6-3 

o- 1 

0-02 

90 

5 

6-2 

o- 1 

0-02 

190 

ID 

5-5 

I  -o 

o-i8 

440 

20 

5-5 

1-5 

0-27 

740 

40 

5 '5 

2-0 

0-36 

1030 

20  +  DXP 

5  '5 

o- 1 

0-02 

50 

failures  are  not  considered  decisive  in  view  of  the  predominance  of  the 
hydrolytic  activity  exhibited  by  the  purified  enzyme.  Since  azide  was 
found  to  inhibit  the  ATPase  activity  at  concentrations  which  do  not  un- 
couple phosphorvlation,  attempts  were  made  to  demonstrate  the  ^^C- 
ADP-ATP  exchange  in  the  presence  of  this  compound.  It  was  anticipated 
that  appropriate  concentrations  of  azide  might  inhibit  the  hydrolytic 
activity  without  aff^ecting  the  transfer  activity.  These  experiments  were 
also  unsuccessful.  We  have  recently  isolated  from  the  submitochondrial 
particles  a  substance  which  is  a  potent  inhibitor  of  the  ATPase  and  appears 
to  have  no  effect  on  oxidative  phosphorylation  in  the  reconstituted  system. 


ATPaSE    IX    OXIDATIVE    PHOSPHORYLATION 


249 


This  material  is  heat-stable,  precipitable  by  trichloroacetic  acid  and 
nondialvzable.  The  possibility  that  this  substance  may  restore  the  ADP- 
ATP  exchange  by  masking  the  hydrolytic  site  of  the  ATPase  and  thus 
convert  it  to  a  transfer  enzyme  is  being  explored. 

Finally,  we  would  like  to  present  the  results  of  some  recent  attempts 
to  resolve  further  the  submitochondrial  particles.  If  the  particulate  fraction 
which  is  recoupled  by  the  ATPase  is  further  disintegrated  by  sonic  oscilla- 
tion, a  new  particle  is  obtained  (residue  2)  which  requires  both  the  super- 
natant fluid  from  which  these  particles  were  separated  as  well  as  the 
purified  ATPase  in  order  to  restore  phosphorvlation.  These  results  are 

TABLE  VIII 

The  Requirement  of  Factor  2  for  Oxidative  Phosphorylation  Catalyzed 

BY  Residue  2 

Experimental  conditions  as  described  in  the  text  and  [4].  Factor  2  was  added 
directlv  to  the  vessels. 


Residue 


Coupling 
Factor  i 


Factor  2 


JO 


AP: 


P:0 


Mg- 

l^g- 

Mg- 

/xatoms 

/xmoles 

I  (310) 

Xone 

Xone 

6-2 

0-4 

o-o6 

I  (310) 

148 

Xone 

5-9 

3-2 

0-55 

2  (310) 

Xone 

Xone 

5-3 

0-4 

o-o8 

,, 

148 

Xone 

6-1 

0-4 

0-07 

,, 

296 

Xone 

6-4 

04 

o-o6 

" 

148 

212 

5-8 

3-7 

0-64 

presented  in  Table  VIII.  The  top  half  of  the  table  merelv  shows  the 
response  of  the  original  particles  to  the  purified  ATPase  or  as  it  is  referred 
to  here,  coupling  factor  i.  The  lower  portion  of  the  table  shows  that  both 
the  ATPase  and  the  supernatant  solution  obtained  after  sonic  disintegra- 
tion (factor  2)  is  required  to  restore  oxidative  phosphorvlation.  Factor  2  is 
heat-labile,  non-dialyzable,  is  precipitated  at  pH  5-4  and  exhibits  no 
ATPase  activity.  It  does  not  catalyze  either  the  ''-P,  ATP  or  the  ADP- 
ATP  exchange  even  when  supplemented  with  the  ATPase,  but  is  required 
together  with  the  ATPase  for  the  restoration  of  a  dinitrophenol  sensitive 
^'■^P,-ATP  exchange  to  the  particles. 

Little  is  known  concerning  the  mechanism  bv  which  the  ATPase  is 
linked  to  the  electron  transport  chain,  nor  is  anvthing  known  of  the 
enzymic  function  of  factor  2.  Nevertheless,  the  resolution  of  the  enzvmes 
of  oxidative  phosphorylation  represents  a  necessarv  first  step  toward  the 
ultimate  goal  of  demonstrating  the  mechanisms  of  this  complex  process. 


250  MAYNARD   E.    PULLMAN,    HARVEY   S.    PENEFSKY   AND   E.    RACKER 

References 

1.  Pullman,  M.  E.,  Penefsky,  H.  S.,  and  Racker,  E.,  Arch.  Biochem.  Biophys.  76, 
227  (1958). 

2.  Penefsky,  H.  S.,  Datta,  Anima,  and  Pullman,  M.  E.,  Fed.  Proc.  18,  300  (1959). 

3.  Pullman,   M.  E.,  Penefsky,   H.   S.,   Datta,  Anima,   and  Racker,   E.,  J.  biol. 
Chem.  235,  3322  (i960). 

4.  Penefsky,  H.  S.,  Pullman,  M.  E.,  Datta,  Anima,  and  Racker,  E.,^.  biol.  Chem. 
235»  3330  (i960). 

5.  Green,  D.  E.,  Lester,  R.  L.,  and  Ziegler,  D.  M.,  Biochim.  biophys.  Acta  23, 
516  (1957). 

6.  Nossal,  P.  M.,  Aiist.J.  exp.  Biol.  med.  Sci.  31,  583  (1953). 

7.  Linnane,  A.  W.,  Biochim.  biophys.  Acta  30,  221  (1958). 

8.  Lardy,  H.  A.,  and  Elvehjem,  C.  A.,  Annu.  Rev.  Biochem.  14,  i  (1945). 

9.  Hunter,  F.  E.,  Jr.,  in  "Phosphorus  Metabolism",  ed:  W.  D.  McEIroy  and  B. 
Glass,  Vol.  I.  Johns  Hopkins  Press,  Baltimore,  297  (195 1). 

10.   Lardy,  H.  A.,  and  Wellman,  W.^J.  biol.  Che?u.  201,  357  (1953). 

Discussion 

CoNOVER :  As  the  cold  lability  of  the  ATP-ase  might  suggest  a  lipoprotein  of 
some  sort,  I  was  wondering  if  you  have  checked  the  lipid  contents  of  the  protein  ? 

Pullman:  No  we  haven't;  until  recently  we  haven't  been  able  to  obtain  the 
amount  of  purified  enzyme  required  for  many  of  the  physical  and  chemical  deter- 
minations which  we  would  like  to  carry  out.  We  think  we  have  now  solved  this 
problem  and  plan  to  examine  this  aspect  of  the  problem  in  the  near  future. 

Conover:  Also  I  might  add  that  I  have  tried  to  demonstrate  ADP-ATP 
exchange  in  magnesium-stimulated  ATP-ase  as  prepared  by  Kielley  and  Kielley, 
and  we  have  run  into  similar  difficulties  in  trying  to  find  an  exchange  in  this 
enzyme. 

Lardy:  It  is  very  interesting  that />-mercuribenzoate  inhibits  the  DNP  portion 
of  the  ATP-ase.  This  is  a  property  which  is  shown  in  partly  aged  mitochondria. 
We  have  found  several  years  ago  that  we  could  inhibit  endogenous  ATP-ase  activity 
and  in  some  experiments  with  these  mitochondria  it  actually  increased;  it  did, 
however,  eliminate  the  DNP-stimulated  portion. 

Pullman  :  In  a  few  experiments  we  too  have  observed  that  PCMB  stimulated 
the  magnesium  activated  portion  of  the  activity.  However,  by  far  the  most  consis- 
tent effect  of  PCMB  was  to  eliminate  the  DNP  stimulation  without  affecting  the 
Mg++  activation. 

Lehninger  :  It  seems  to  me  that  the  factors  which  Dr.  Pullman  and  I  have  been 
studying  in  our  laboratories  are  coming  closer  and  closer  together.  I  think  we  also 
share  the  view  that  this  coupling  mechanism  has  its  critical  point  where  the  water 
site  is  created,  possibly  by  an  inhibitor  such  as  U  factor.  I  would  like  to  suggest, 
however,  the  possibility  that  his  coupling  factor  or  ATP-ase  is  a  complex  enzyme 
in  the  same  sense  as  actomyosin  is,  and  is  composed  of  two  or  more  pieces.  I  have 
been  thinking  myself  that  our  C  factor  is  similar  to  your  ATP-ase  and  that  it  in  turn 
consists  of  two  components  one  which  may  be  M,  and  of  course  it  will  take  further 
work  to  clarify  all  these  things.  One  point  I  wanted  to  ask  you  was  when  you  have 


ATPase  in  oxidative  phosphorylation  251 

this  latency  phenomenon  and  you  develop  yourATP-ase  activity,  is  this  preparation 
capable  of  restoring  phosphorylation  even  after  the  latent  ATP-ase  has  appeared  ? 

Pullman  :  Yes,  it  always  maintains  its  coupling  activity.  The  only  discrepancy 
with  regard  to  the  two  activities  is  that  during  purification  the  ATP-ase  is  "acti- 
vated" while  the  coupling  activity  is  not. 

Packer  :  It  is  interesting  that  the  nucleotide  specificity  for  ATP-ase  is  also  very 
similar  to  that  for  actomyosin,  namely  that  the  all  four  nucleotides  catalyze  the 
reaction,  but  only  the  ATP-ase  is  DNP-stimulated.  These  are  similar  to  the  pro- 
perties of  actomyosin  B  as  reported  by  Blum  and  Alorales. 

Pullman  :  Yes,  there  is  a  remarkable  parallelism  between  the  mitochondrial 
ATP-ase  and  myosin  ATP-ase.  However,  I  think  that  in  the  case  of  myosin,  the 
hydrolysis  of  some  of  the  other  triphosphonucleosides  is  also  stimulated  by  DNP 
or  is  that  what  you  said  ? 

Packer:  I  only  wish  to  comment  that  there  are  remarkable  similarities  between 
this  ATP-ase  and  actomyosin  and  it  would  appear  that  your  enzyme  may  be  a  very 
prorpising  choice  as  the  mechano-protein  which  has  received  so  much  discussion 
this  afternoon. 


The  Mechanism  of  Coenzyme  Q  Reduction  in  Heart 

Mitochondria 

Daniel  M.  Ziegler* 

Institute  for  Enzyme  Research, 
University  of  Wisconsin,  Wis.,  U.S.A. 

Our  laboratory  and  the  Liverpool  group  have  previously  presented 
evidence  [i,  2,  3]  that  Coenzyme  Q  (CoQ)  is  positioned  between  the  flavo- 
protein  and  cytochrome  c^  in  the  mitochondrial  succinoxidase  system. 
Furthermore,  it  is  generallv  accepted  that  cytochrome  b,  at  least  in  non- 
phosphorylating  particles,  is  not  an  obligatory  electron  carrier  either  in  the 
reduction  of  CoQ  or  in  its  reoxidation  by  cytochrome  c^  [i].  However, 
CoQ  does  not  react  directly  with  the  flavoprotein  since  the  primary 
succinic  flavoprotein  isolated  by  the  method  of  Singer  et  a/.  [4]  does  not 
catalyze  the  reduction  of  CoQ.  We  have  isolated  a  soluble  form  of  the 
succinic  flavoprotein  that  can  catalyze  this  reaction  [5],  and  in  this  report 
we  will  present  evidence  that  the  non-haem  iron  associated  with  the 
succinic  dehvdrogenase  functions  as  an  electron  carrier  between  the  flavo- 
protein and  the  quinone. 

tablp:  I 

SucciN!c-CoQ  Reductase  Activities* 


Preparation 


/imoles  CoQ  reduced 
(min.,  mg.  protein) 


Beef  heart  mitochondria 
Succinic-CoQ  reductase  [5] 
Primary  succinic  fla\'()protein  [4] 


I  •  I 

560 

o-o 


*  The    succinic-CoQ    reductase    activities    were    measured    b) 
described  in  ref.  [2]. 


the    method 


Table  I  lists  the  succinic-CoQ  reductase  activities  of  heart  mito- 
chondria, the  soluble  succinic-Co(}  reductase  and  the  primary  succinic 
flavoprotein.  The  succinic-CoQ  reductase  is  about  fifty  times  more  active 
than  the  starting  heart  mitochondria  while  the  primary  succinic  flavo- 
protein does  not  catalyze  this  reaction.  It  is  apparent  that  the  site  necessary 
to  link  CoQ  to  the  flavoprotein  is  still  present  in  the  Q  reductase  but  is 
either  lost  or  non-functional  in  the  primary  flavoprotein. 

*  The  author  is  indebted  to  Dr.  D.  K.  Green  for  his  advice  during  the  course 
of  this  work. 


254 


DANIEL   M.    ZIEGLER 


The  turnover  of  the  flavoprotein  in  CoQ  reductase  with  CoQ  as  the 
electron  acceptor  is  shghtly  faster  than  that  of  the  same  enzyme  in  the 
electron  transport  particle  (ETP)  with  any  of  the  electron  acceptors  listed 
in  Table  II.  The  calculated  turnover  of  the  succinic  flavoprotein  in  ETP 
is  based  on  the  assumption  that  all  the  flavin  in  the  particle  released  by 
acid  only  after  tryptic  digestion  is  part  of  the  succinic  dehydrogenase  [4]. 
Either  this  assumption  is  not  valid  or  some  activation  of  the  enzyme  occurs 
during  its  isolation. 

The  soluble  succinic-CoQ  reductase  contains  4-2  to  4-6  m/^moles 
flavin,  4-4  to  4-8  m/xmoles  haem,  34  to  38  m^amoles  non-haem  iron,  and 
o- 18  to  0-20  mg.  lipid  per  mg.  protein.  The  ratio  of  flavin  to  protein  in  the 

TABLE  II 

Turnover  Rates*  of  the  Succinic  Flavoprotein  in  Soluble  and  Particulate 

Preparations 


Preparation 


Flavoprotein 

concn. 

(m/nmoles/mg. 

prot.) 


Electron  acceptor 


CoQ 


Phenazine 
methosulphate 


O, 


Primary 
succinic 
flavoprotein 

[4] 

Succinic  CoQ 
reductase  [5] 

Electron 
transport 
particle  [6] 


4-3 


4-2 


o-  ig 


12  600 


4100 


II  300 


9  700 


*  The  turnover  rates  are  expressed  as  moles  of  succinate  oxidized  per  min. 
per  mole  of  succinic  flavin. 

reductase  is  almost  identical  with  that  of  the  primary  succinic  flavoprotein 
and  both  forms  of  the  dehydrogenase  contain  non-haem  iron.  Singer  and 
his  associates  have  reported  that  the  ratio  of  iron  to  flavin  in  the  primary 
flavoprotein  is  4 :  i ;  whereas  in  the  reductase  the  ratio  is  8:1.  About  one- 
half  of  the  non-haem  iron  can  be  removed  from  the  reductase  by  prolonged 
aerobic  dialysis  against  10  ~^  M  ethylenediamine  tetraacetate,  but  the  CoQ 
reductase  activity  of  the  enzyme  is  destroyed  by  this  procedure. 

The  iron  that  is  removed  by  aerobic  dialysis  is  probably  not  adventi- 
tious iron  since  the  enzyme  can  be  dialyzed  anaerobically  for  the  same 
length  of  time  without  the  loss  of  either  iron  or  activity.  Addition  of  ferric 
or  ferrous  ions  to  the  enzyme  after  aerobic  dialysis  does  not  restore  activity. 
It  is  possible  that  some  functional  group  (i.e.  thiol)  required  for  iron 
binding  is  oxidized  during  prolonged  aerobic  dialysis. 


THE  MECHANISM  OF  COENZYME  Q  REDUCTION  IN  HEART  MITOCHONDRIA      255 

In  contrast  to  the  primary  flavoprotein  the  CoQ  reductase  contains 
haem  and  Hpid.  All  the  haem  can  be  extracted  with  acid-acetone  and  its 
reduced  pyridine  haemochromogen  is  identical  with  that  of  protohaem. 
The  spectrum  of  the  dithionite  reduced  enzyme  (Fig.  i),  indicates  that 
the  haem  prosthetic  group  is  similar  to  the  mitochondrial  cytochrome  b; 
however,  not  all  the  cytochrome  b  in  mitochondria  can  be  associated  with 
the  succinic  flavoprotein  since  mitochondria  contain  about  three  times  as 
much  protohaem  as  succinic  flavoprotein. 

The  cytochrome  present  in  the  CoQ  reductase  is  not  reduced  by 
succinate  (Fig.  i)  so  it  cannot  function  as  an  electron  carrier  between  the 


0  6- 


0-4- 


Q     0-2 

O 


-0- 


4: 

562 
.lO.D.  =  0  li"> 

1 

'        529     \\ 

; 

^-'''     '   i 

B 

500        550 

\        +  Succinate 

1 

\        +  Dithionite 

\                                         '' 

A    ; 

\:^ 

400 


450 


500        550 


(m// , 


Fig.  I.  Difference  spectra  (jf  succinic-CoQ  reductase  (oxidized  vs.  reduced). 
The  enz\Tne  was  dissolved  in  o  •  i  M  phosphate  buffer  pH  7  -4  at  a  final  concentra- 
tion of  I -16  mg.  protein  per  ml.  The  enzyme  was  first  reduced  with  succinate 
(100  /tmoles /ml.)  and  then  with  dithionite. 


flavoprotein  and  CoQ.  If,  however,  the  enzyme  is  first  reduced  with 
limiting  amounts  of  dithionite  the  cytochrome  is  reoxidized  by  fumarate 
(Fig.  2) — an  observation  which  suggests  that  a  functional  link  still  exists 
between  the  flavin  and  haem  groups.  We  have  not  been  able  to  define  the 
function  of  the  cytochrome  associated  with  the  flavoprotein.  One  possi- 
bility suggested  by  the  work  of  Conover  and  Ernster  [14]  is  that  cvtochrome 
b  is  an  intermediate  electron  carrier  between  extramitochondrial  oxidative 
enzymes  and  the  electron  transport  system. 

Beinert  and  Sands  have  examined  the  succinic-CoQ  reductase  by 
electron  paramagnetic  resonance  spectroscopy  and  they  have  reported  that 
the  enzyme  contains  a  paramagnetic  species  that  can  be  reduced  by 
succinate  and  reoxidized  by  CoQ  [7].  Since  iron  is  the  only  transition  metal 


256  DANIEL   M.    ZIEGLER 

present  in  significant  amounts  in  the  isolated  enzyme,  we  have  determined, 
by  a  chemical  method,  the  oxidation-reduction  state  of  the  enzyme  bound 
non-haem  iron  in  the  isolated  flavoproteins  and  a  number  of  submito- 
chondrial  particles  (Table  III). 

In  agreement  with  the  earlier  work  of  Massey  [8],  we  have  found  that 
the  non-haem  iron  associated  with  the  primary  succinic  fiavoprotein  is  not 
reduced  by  succinate,  but  approximately  30"',,  of  the  non-haem  iron  in  the 
isolated  Q  reductase  is  reduced  by  substrate.  Succinate,  but  not  DPNH, 
reduces  significant  amounts  of  the  iron  in  the  succinic-cytochrome  c 
reductase  particle  prepared  by  the  method  of  Green  and  Burkhard  [9]. 
This  particle   does  not  contain  a  functional  DPNH   chain  and  cannot 


0-4 

1 T'                 1                1                          1 

•      •            +  Fumarate 

0-2 

:       :             +  Na2S204 

0 

\                ^^^•■•"" 

••1                1              11        1 

400 


450  500 

Wavelength  (m/i) 


600 


Fig.  2.  Difference  spectra  (oxidized  vs.  reduced)  of  the  succinic-CoQ  reduc- 
tase. The  enzyme  was  first  reduced  with  Umiting  amounts  of  dithionite  and  then 
fumarate  (10  /xmoles/ml.)  was  added. 


catalyze  the  reduction  of  CoQ  by  DPNH ;  whereas,  in  the  DPNH  cyto- 
chrome c  reductase  particle  [10]  which  is  essentially  free  from  the  succinic 
fiavoprotein,  only  DPNH  reduces  significant  amounts  of  the  non-haem 
iron.  Either  substrate  can  reduce  approximately  the  same  amount  of  the 
non-haem  iron  in  ETP  where  both  the  DPNH  and  succinate  electron 
transport  chains  are  intact.  All  the  iron  potentially  reducible  in  these 
preparations  is  reduced  in  a  few  seconds  at  5".  Our  studies  on  the  rates  of 
iron  reduction,  indicate  that  at  5'  the  non-haem  iron  is  reduced  as  rapidly 
as  CoQ. 

Under  the  conditions  given  in  Table  III,  the  reduction  of  enzyme- 
bound   iron   is   strictly   substrate-dependent.    The   possible   non-specific 


THE  MECHANISM  OF  COENZYME  Q  REDUCTION  IN  HEART  MITOCHONDRIA       257 

reduction  of  iron  by  known  redox  components  of  the  electron  transport 
chain  after  the  enzyme  is  denatured  by  ethanol  can  be  excluded  in  a 
number  of  ways.  Since  in  ETP  the  same  amount  of  iron  is  reduced  either 
in  the  presence  of  antimycin  or  anaerobically,  it  follows  that  cytochromes 
c^,  c  and  a  cannot  participate  in  this  reduction.  The  succinic-CoQ  reduct- 
ase does  not  contain  CoQ,  and  the  cytochrome  b  present  is  not  reduced; 
therefore,  neither  of  these  components  reduces  the  non-haem  iron  during 
its  extraction  from  the  denatured  protein.  The  possible  interference  of  the 

TABLE  III 

Reduction  of  Xon-Haem  Iron  in  Mitochondrial  .Subfractions* 

™       ,  Percentage  of  total  non- 

„  ,  haem  Fe  reduced 

rreparation  non-haem 

Fe 


by  DPXH      by  succinate 


Primary  succinic  flavoprotein  [4]  17  o  o 

Succinic-CoQ  reductase  [5]  34  o  23 

Succinic-cytochrome  c  reductase  [9]  22  <  i  23 

DPNH-cytochrome  f  reductase  [10]  15  30  <2 

ETP„  [11]  9  39  30 

*  The  procedure  for  measuring  the  redox  state  of  enzyme-bound  non-haem 
iron  will  be  described  in  detail  elsewhere  [12].  A  brief  summary  of  the  method  is 
as  follows :  (i)  The  preparation  is  treated  with  KCN  or  antimycin  A  to  block 
oxidation ;  (ii)  substrate  is  added  at  zero  time ;  (iii)  the  reaction  is  stopped  by  adding 
CdCh  ;  (iv)  the  reduced  non-haem  iron  is  extracted  with  a  mixture  of  ethanol  (70*^0), 
o-chloromercuriphenol  (5  mg./ml.),  sodium  acetate  (100  /tmolesml.,  pH  4  "6), 
and  bathophenanthroline  (o-i  mg./ml.);  (v)  the  ferro-bathophenanthroline  colour 
is  measured  at  535  m/i  against  a  control  to  which  substrate  was  added  after  the 
CdClo. 

The  cadmium  ions  [13]  and  the  organic  mercurical  are  necessary  to  prevent 
the  non-enzymic  reduction  of  iron  by  thiols. 

fiavoproteins  cannot  be  entirely  eliminated,  but  in  most  preparations  the 
amount  of  iron  reduced  is  considerably  in  excess  of  the  fiavoproteins  and 
the  non-haem  iron  is  reduced  in  the  succinic-CoQ  reductase  but  not  in 
the  primary  succinic  flavoprotein.  The  ratio  of  flavin  to  protein  in  the 
latter  two  preparations  is  identical,  which  also  indicates  that  non-enzymic 
reduction  of  the  iron  by  reduced  flavoprotein  does  not  occur. 

Amytal,  a  specific  inhibitor  of  DPXH  oxidase,  blocks  the  reduction  of 
non-haem  iron  by  DPXH  (Table  IV),  but  malonate  increases  the  total 
amount  of  iron  reduced  by  this  substrate.  The  reverse  is  true  with  suc- 
cinate as  substrate.  Malonate  blocks  reduction  of  iron  by  succinate  in 
ETP,  but  amytal  increases  the  amount  of  iron  reduced  by  succinate.  In 
the  presence  of  both  succinate  and  DPXH  the  total  amount  of  iron 

VOL.  II. S 


2s8 


DANIEL   M.    ZIEGLER 


reduced  in  ETP  is  the  sum  of  the  amounts  obtained  with  either  substrate 
alone.  We  do  not  have  an  explanation  for  these  phenomena  but  the  results 
demonstrate  that  the  non-haem  iron  compounds  are  closely  associated 
with  the  flavoproteins  in  the  electron  transport  system,  since  all  of  the 
components  after  iron  in  the  electron  transport  secjuence  can  be  completely 
reduced  by  either  substrate. 

TABLE  IV 

The  Effect  of   Inhibitors  on  the  Reduction  of  Non-Haem  Iron  in  ETP 


Inhibitor 


Concentration 


Percentage  of 

total  iron  reduced 

bv  DPNH 


Percentage  of 

total  iron  reduced 

by  succinate 


None 

Amytal 

Malonate 


1  X     lO" 

2  X     lO 


39 
o 

6S 


3 

59 

o 


The  compound  2-thenovltrifluoroacetone  which  chelates  with  iron  is 
a  highly  efficient  inhibitor  of  succinic-CoQ  reductase  activity  (Table  V). 
The  level  of  the  inhibitor  required  to  block  the  reduction  of  CoQ  by  the 
reductase  has  only  a  small  effect  on  the  reduction  of  phenazine  metho- 
sulphate  and  does  not  affect,  at  all,  the  phenazine  reductase  activity  of  the 

TABLE  V 

Inhibition  of  Succinic-CoQ  Reductase  Activity  by  2-Thenoyl- 
trifluoroacetone* 


Electron  acceptor       (percentage  inhibition) 


Preparation 


CoQ 


Phenazine 
niethosulphate 


Primary  succinic  flavoprotein  [4] 
Succinic-CoQ  reductase  [s] 
ETP„[ii] 


97 


o-o 
17  -o 
i8-o 


*  The  final  concentration  of  the  inhibitor — i    x    10  ^  m. 


primary  succinic  flavoprotein.  These  data  demonstrate  that  this  metal 
chelating  compound  blocks  a  site  required  to  link  CoQ  to  the  flavoprotein. 
The  reagent  does  not  combine  with  the  flavin  since  it  does  not  aflPect  the 
phenazine  methosulphate  reductase  activity  of  the  primary  succinic  flavo- 
protein. Thenoyltrifluoroacetone  does  not  remove  non-haem  iron  but 
appears  to  form  a  strong  complex  with  the  enzyme-bound  iron,  which  can 
no  longer  undergo  oxidation  and  reduction.  These  data  are  consistent  with 


THE  MECHANISM   OF  COENZYME  Q  REDUCTION   IN   HEART  MITOCHONDRIA       259 

the  assumption  that  the  non-haem  iron  is  an  intermediary  in  electron  flow- 
between  flavoprotein  and  CoQ  but  not  between  flavoprotein  and  phenazine 
methosulphate. 

Spectra  of  the  purified  CoQ  reductase  suggest  that  the  preparation 
contains,  in  addition  to  the  flavin  and  haem  prosthetic  groups,  some  other 
component  that  can  undergo  oxidation  and  reduction.  In  addition  to  the 
fl.avin  band  at  450  m/x,  succinate  also  reduces  a  component  at  approxi- 
mately 415  m/jL  (cf.  the  difference  spectra  shown  in  Fig.  i).  The  change  in 
the  spectrum  upon  reduction  of  the  enzyme  by  succinate  cannot  be 
entirely  due  to  the  flavin  even  in  the  450  460  m/a  region  of  the  spectrum. 


450  500 

Wovelength  (m//) 


600 


Fig.   3.  These  spectra  were  obtained  under  the  same  conditions  as  those  of 
Fig.  2  except  that  CoQ.,  (0-05  ;umole)  was  added  instead  of  fumarate. 


Even  if  all  of  the  flavin  is  reduced,  which  is  very  unlikely,  the  decrease  in 
optical  density  at  450  460  m/n  is  considerably  greater  than  could  be 
attributed  to  the  flavin  alone. 

The  band  at  415  m^a  is  not  contributed  by  flavin,  since  it  is  not  re- 
oxidized  by  fumarate  (Fig.  2).  All  the  haem  is  reoxidized  and  essentially  all 
the  flavin  should  be  reoxidized  by  fumarate.  It  is  unlikely  that  the  bands 
remaining  after  the  addition  of  fumarate  can  be  attributed  to  the  flavin 
prosthetic  group.  In  addition  to  the  main  band  at  415  m/^i  a  broad  band 
persists  at  450  m/^,  which  again  suggests  that  not  all  the  reduction  observed 
in  this  region  is  due  to  the  flavin. 

The  components  of  the  enzyme  that  remain  reduced  after  the  re- 
oxidation  of  the  flavin  and  haem  by  fumarate  are,  however,  reoxidized  by 


26o  DANIEL   M.    ZIEGLER 

CoQ  (Fig,  3).  These  data  again  demonstrate  that  the  succinic-CoQ 
reductase  contains  a  component  other  than  the  haem  group  that  can 
function  as  an  electron  carrier  between  the  flavoprotein  and  CoQ.  Since 
non-haem  iron  is  the  only  other  known  compound  present  in  the  enzyme 
that  undergoes  oxidation  and  reduction  it  is  probable  that  the  iron  is 
responsible  for  the  415  m/x  band  observed  in  the  reduced  enzyme. 

References 

1.  Green,  D.  E.,  Ziegler,  D.  M.,  and  Doeg,  K.  A.,  Arch.  Biochem.  Biophys.  85, 

280  (1959). 

2.  Doeg,  K.  A.,  Krueger,  S.,  and  Ziegler,  D.  M.,  Biochim.  biophys.  Acta  41,  492 
(i960). 

3.  Pumphrey,  A.  M.,  and  Redfearn,  E.  R.,  Biochem.  jf.  72,  2P  (1959). 

4.  Singer,  T.  P.,  Kearney,  E.  B.,  and  Bernath,  P.,  J.  biol.  Chem.  223,  599  (1956). 

5.  Ziegler,  D.  M.,  and  Doeg,  K.  A.,  Biochem.  biophys.  Res.  Comm.  I,  344  (1959). 

6.  Crane,  F.  L.,  Glenn,  J.,  and  Green,  D.  E.,  Biochim.  biophys.  Acta  22,  475 
(1956). 

7.  Beinert,  H.,  and  Sands,  R.  H.,  Biochem.  biophys.  Res.  Comm.  3,  41  (i960). 

8.  Massey,  V.,  Biochim.  biophys.  Acta  30,  508  (1958). 

9.  Green,  D.  E.,  and  Burkhard,  R.  K.,  Arch.  Biochem.  Biophys.  (in  press). 

10.  Hatefi,  Y.,  Haavik,  A.  G.,  and  Jurtshuk,  P.,  Biochem.  biop/iys.  Res.  Comm.  3, 

281  (i960). 

11.  Linnane,  A.,  and  Ziegler,  D.  M.,  Biochim.  biophys.  Acta  29,  630  (1958). 

12.  Ziegler,  D.  M.,  and  Doeg,  K.  A.  (manuscript  in  preparation). 

13.  Fluharty,  A.  L.,  and  Sanadi,  D.  R.,  Fed.  Proc.  19,  608  (i960). 

14.  Conover,  T.  E.,  and  Ernster,  L.,  Acta  chem.  scand.  (in  press). 


Discussion 

Redfearn  :  This  hypothesis  raises  the  problem  of  the  transfer  of  the  electrons 
from  flavoprotein  which  carries  two  electrons  through  a  one  electron  carrying 
system  (the  iron)  then  again  to  a  two-electron  carrier.  It  was  nice  to  think  that  the 
quinone  could  function  as  a  semi-quinone  and  thus  mediate  the  reaction  between 
two-electron  carriers  and  the  one  electron  carrying  cytochromes.  Secondly,  I 
noticed  that  you  used  Q.,  in  your  assay  system.  According  to  Crane's  results  on 
acetone-extracted  preparations  Q.,  prodviced  an  antimycin-insensitive  pathway, 
which  suggests  that  the  site  of  action  of  Qo  was  not  the  same  as  that  of  the  naturally 
occurring  Qio-  Would  you  care  to  comment  on  this  ? 

Ziegler  :  Is  it  necessary  to  assume  that  the  flavoprotein  is  fully  reduced  during 
active  electron  transport  ?  I  would  be  more  inclined  to  believe  that  in  the  intact 
succinoxidase  particle  the  flavoprotein  may  be  reduced  only  to  the  semiquinoid 
form,  and  if  this  is  the  case  you  would  have  one  electron  transfer  during  the 
oxidation  of  succinate. 

With  reference  to  the  last  point  you  raised,  the  reoxidation  of  reduced  Q2  is 
partly  antimycin-insensitive  and  as  you  increase  the  length  of  the  side  chain  you 
induce  full  antimycin  sensitivity.  However,  in  the  reduction  of  Q  the  succinic-Q- 
reductase  will  react  rapidly  with  either  Qio  or  homologues  of  Q  and  we  have  used 


THE  MECHANISM  OF  COENZYME  Q  REDUCTION  IN  HEART  MITOCHONDRIA      26 1 

Q2  in  some  of  this  work  because  it  is  far  more  soluble  than  Qm,  and  in  this  way 
we  can  eliminate  the  necessity  of  adding  the  extra  phospholipids  required  to 
solubilize  Qio,  but  we  can  always  replace  the  Q.,  with  Qio  plus  phospholipids,  and 
we  have  not  detected  a  difference  in  specificity  in  the  reduction  of  Qo  or  Qk,  by 
succinate. 

Chance:  I  was  struck  by  the  small  degree  of  reduction  of  the  iron  in  the 
various  preparations  in  spite  of  the  fact  that  they  are  inhibited  by  cyanide  or 
antimycin  A.  Under  these  conditions  I  believe  all  the  flavin  would  be  reduced,  and, 
if  they  contain  Q,  that  would  be  similarly  reduced.  So  do  you  have  an  explanation 
of  values  of  only  30  to  60*',,  reduction  of  the  iron  ? 

Ziegler:  Some  data  which  I  did  not  show  indicate  that  if  you  add  both  sub- 
strates to  an  ETP,  which  contains  the  two  intact  chains,  the  total  amount  of  iron- 
reduced  summates.  DPNH  reduces  between  25  and  45^0  of  the  iron  and  succinate 
reduces  about  30*^0;  if  you  add  both  substrates  60  to  7o"o  of  the  iron  is  reduced. 
Chance:  This  is  however  considerably  smaller  than  the  percentage  reduction 
of  Q,  and  thus  suggests  that  not  all  the  non-haem  iron  is  active  in  the  pathway 
you  are  considering. 

Ziegler:  Yes,  I  would  agree  that  not  all  of  the  iron  functions  in  this  capacity. 
Chance  :  How  many  atoms  of  iron  per  molecule  of  Q  are  there  ? 
Ziegler:  In  ETP  there  is  twice  as  much  non-haem  iron  as  Q.  In  other  words, 
per  electron  equivalent  they  are  almost  equal. 

Chance:  I  am  still  not  clear  how  many  irons  per  Q  are  in  the  electron  transfer 
pathway. 

Ziegler  :  This  would  be  a  very  difficult  question  to  answer  at  this  time  since 
we  do  not  know  how  much  of  the  endogenous  Q  is  involved  in  the  oxidation  of 
either  succinate  or  DPNH.  A  more  pertinent  question  would  be,  how  many  irons 
per  flavoprotein  are  involved  in  the  pathway  to  Q,  and  in  all  of  our  preparations 
capable  of  reducing  Q  at  least  2  moles  of  iron  per  flavin  undergo  oxidation  and 
reduction.  In  ETP  the  ratio  of  reducible  iron  to  flavin  is  much  greater  than  2  and 
with  both  succinate  and  DPXH  present  the  ratio  can  be  as  high  as  15. 

EsTABROOK  :  On  the  same  point,  I  was  wondering  whether  you  have  an  explana- 
tion for  your  table  (p.  258)  of  inhibitors  where  you  show  that  when  you  used 
succinate  as  a  substrate  in  the  presence  of  amytal,  which  is  an  inhibitor  of  DPNH 
oxidation,  you  get  the  sunimation  of  iron  reduced.  In  the  same  way  as  in  the 
presence  of  malonate  with  DPNH  as  a  substrate,  you  also  find  this  summation. 
Ziegler:  I  have  no  adequate  explanation  for  these  phenomena. 
Singer  :  Your  slide  shows  eight  atoms  of  iron  per  mole  of  flavin  but,  if  I  am 
not  mistaken,  your  publication  on  the  highly  purified  enzyme  showed  the  same 
ratio  as  in  the  flavoprotein  itself,  that  is  four  to  one,  and  I  am  wondering  what  has 
happened  in  between  to  change  these  analytical  data  and  whether  this  might 
throw  some  light  on  Dr.  Chance's  question. 

We  have  been  studying  for  some  years  the  transformations  that  occur  during 
the  extraction  of  succinic  dehydrogenase  from  the  respiratory  chain  preparations. 
Since  our  results  are  relevant  to  the  function  of  the  metal  in  this  enzyme,  I  might 
sum  up  the  salient  points.  There  are  three  main  differences  in  the  behaviour  of 
succinic  dehydrogenase  between  particulate  (respiratory  chain-bound)  prepara- 
tions and  soluble  ones.   One  is  that  the  dehydrogenase  is  cyanide-sensitive  in 


262  DANIEL    M.    ZIEGLER 

respiratory  chain  preparations  but  not  in  the  extracted,  soluble  form ;  second,  that 
it  has  two  reaction  sites  for  phenazine  methosulphate  in  particulate  preparations 
but  only  one  in  soluble  ones  and  thus  in  the  particulate  form  the  enzyme  has  twice 
the  Qo,  in  the  phenazine  assay  that  it  has  in  soluble  preparations;  and,  third,  that 
in  particulate  preparations,  but  not  in  purified,  soluble  ones,  it  reacts  with  methy- 
lene blue,  brilliant  cresyl  blue,  and  related  dyes.  In  regard  to  all  three  criteria  the 
CoQ-reductase  of  Ziegler  and  colleagues  behaves  like  a  respiratory  chain  prepara- 
tion, as  expected  from  the  fact  that  the  enzyme  is  still  linked  to  cytochrome  b  in 
this  particle.  Work  in  our  laboratory  suggests  that  those  properties  of  succinic 
dehydrogenase  which  are  lost  on  solubilization  are  not  fundamental  characteristics 
of  the  flavoprotein  itself  but  may  be  the  consequence  of  the  binding  of  some  of 
the  non-haem  iron  of  the  flavoprotein  to  the  respiratory  chain.  We  proposed  some 
years  ago  that  at  least  two  of  the  four  iron  atoms  of  the  isolated  enzyme  may  act  as 
ligands  of  the  flavoprotein  to  the  respiratory  chain  in  particulate  preparations.  It 
is  established  that  at  least  two  of  the  four  irons  in  the  isolated  flavoprotein  do  not 
function  in  oxido-reduction  in  purified  preparations,  although  they  might  do  so 
when  bound  in  a  particle.  If  so,  they  might  also  be  involved  in  the  catalytic  cycling 
of  CoQ  in  such  particulate  preparations  as  the  CoQ  reductase. 

The  anomalous  absorption  changes  in  the  flavin  region  which  Dr.  Ziegler  has 
observed,  are,  probably  not  fortuitously,  very  similar  to  those  which  occur  in 
a-glycerophosphate  dehydrogenase,  a  flavoprotein  rich  in  iron,  in  the  succinic 
dehydrogenase  of  Micrococcus  lactilyticiis,  where  iron  has  been  shown  to  undergo 
oxido-reduction  by  the  substrate,  and  in  a  rat  liver  enzyme  which  oxidizes  inositol 
and  which  doesn't  even  have  flavin  but  is  an  iron  enzyme.  These  considerations 
would  again  suggest  that  part  of  the  iron  complement  of  the  flavoprotein  might 
undergo  oxido-reduction  in  respiratory  chain  preparations. 

Ziegler:  The  properties  of  the  soluble  succinic  CoQ-reductase  appear  to  be 
identical  with  those  of  the  particle-bound  dehydrogenase;  the  phenazine  metho- 
sulphate reductase  activity  of  the  isolated  enzyme  is  partly  sensitive  to  cyanide. 
Cyanide  also  blocks  the  reduction  of  Q.  We  have  tested  a  number  of  compounds 
that  have  been  used  to  inhibit  the  particle-bound  dehydrogenase,  and  disulphide 
compounds  such  as  lipoic  acid  are  very  effective  inhibitors  of  Q  reduction. 

The  discrepancy  between  the  concentration  of  iron  in  the  enzyme  reported 
here  and  in  our  earlier  publication  is  due  to  a  change  in  the  method  of  estimating 
enzyme-bound  iron.  The  ratio  of  4  irons  per  flavin  was  obtained  on  a  preparation 
that  had  been  thoroughly  dialyzed  against  a  versene  solution.  However,  the  activity 
of  the  enzyme  is  destroyed  by  prolonged  dialysis.  Currently  we  remove  extraneous 
iron  by  passing  the  enzyme  through  a  column  of  Dowex  A-i  chelating  resin.  This 
procedure  does  not  destroy  the  Q-reductase  activity  of  the  enzyme  and  the  ratio 
of  non-haem  iron  to  flavin  is  consistently  8:1. 

Ernster  :  I  would  like  to  hear  how  you  visualize  the  relationship  of  this 
mechanism  to  that  prevailing  in  phosphorylating  preparations,  especially  with 
respect  to  the  participation  of  cytochrome  b. 

Ziegler:  I  have  discussed  this  previously  with  Dr.  Ernster  and  I  think  we  are 
in  full  agreement.  Could  he  put  the  mechanism  we  discussed  on  the  board  ? 

Ernster  :  Well,  all  I  meant  to  ask  is  this  :  is  this  form  of  succinic  dehydrogenase, 
which  is  now  a  Q-reductase,  a  cytochrome  b  reductase  as  well  ? 


THE  MECHANISM  OF  COENZYME  Q  REDUCTION   IX   HEART  MITOCHONDRIA       263 

ZiEGLER :  No,  the  cytochrome  bound  to  the  enzyme  is,  of  course,  not  reduced 
and  the  enzyme  will  not  catalyze  the  reduction  of  a  number  purified  cytochrome 
h's  we  have  tested. 

HoLTON :  Could  I  ask  you  whether  your  conclusion  that  the  succinate-Q 
reductase  does  not  reduce  cytochrome  b  is  based  on  a  difference  spectrum  of  the 
reductase  in  the  presence  and  absence  of  succinate  ? 

Ziegler:  This  is  one  piece  of  evidence,  yes. 

HoLTOX:  Is  it  not  just  possible  that  your  isolated  reductase  has  cytochrome  b 
present  in  the  reduced  form  already  without  there  being  succinate  present,  and 
that  is  why  the  only  change  you  show  on  addition  of  succinate  is  the  reduction  of  Q. 
It  seems  to  me  to  be  very  odd  that  succinate  does  not  reduce  cytochrome  b  in  the 
presence  of  an  enzyme  which  catalyzes  the  oxidation  of  cytochrome  b  by  added 
fumarate. 

ZlEGLER :  No.  This  is  not  a  possibility.  Most  of  the  spectra  we  have  of  this 
enzyme  are  direct  spectra  and  in  no  instance  have  we  been  able  to  keep  the  haem 
in  the  reduced  form.  Cytochrome  h  is  quite  auto-oxidizable. 

Ch.\nce  :  This  cytochrome  b  which  comes  along  with  the  succinate-Q  reductase 
has  an  absorption  band  at  an  appreciably  different  wavelength  from  the  cyto- 
chrome b  in  the  particle.  I  think  it  is  very  close  to  what  Dr.  Holton  and  I  call 
"inactive"  cytochrome  6,  because  it  is  reduced  only  by  dithionite,  so  I  think  it  is 
a  little  premature  to  say  that  the  properties  of  this  kind  of  cytochrome  h  identify 
it  with  a  particular  pathway  of  electron  transfer  or  phosphorylation. 

HoLTOX :  Its  reoxidation  by  fumarate  indicates  that  this  cytochrome  b  is  in 
direct  connection  with  the  succinate-fumarate  system. 

Chance:  But  it  may  be  by  a  different  pathway. 

Ernster  :  The  mechanism  we  have  been  thinking  about  in  connection  with 
this  activation  of  succinic  oxidase  in  phosphorylating  systems  is  this : 

Succ.  '^ ^  Fps-^ 

^^  -\  Noii-p/iosp/iory/dting  system 

phusplwrylating  system  ^ ATP        "~-- 

DPNH  . Fpi,  ^ cyt.  b  >  CoQ 

(Fps  =  succinic  dehydrogenase;  Fpi,  =  DPNH  dehydrogenase.) 

Ziegler:  I  agree  with  Dr.  Ernster  that  this  is  one  possibility  we  have  to 
consider.  However,  as  Dr.  Chance  pointed  out,  the  haem  attached  to  the  enzyme 
may  have  been  modified  during  isolation  of  the  enzyme  since  all  of  the  bands  have 
been  shifted  to  slightly  lower  wavelengths. 

Slater:  Is  this  cytochrome  b  reduced  by  succinate  in  the  presence  of 
antimycin  ? 

Ziegler:  No,  it  is  not. 

Singer:  Much  is  made  in  discussions  of  this  type  of  the  reduction  of  flavin  as 
measured  at  450  or  460  m/t  with  or  without  a  reference  label.  Perhaps  I  am  merely 
voicing  Prof.  Keilin's  recent  caution  in  stating  that  the  reduction  of  flavin  in 
succinic  dehydrogenase,  etc.,  shuttles  between  the  oxidized  and  reduced  forms  in 
its  normal  catalytic  action.  Since  the  isolated  dehydrogenase  does  not  undergo 
anything  like  a  full  bleaching  even  after  activation  by  succinate  and  since  its  rate 


264  DANIEL   M.    ZIEGLER 

of  bleaching  is  not  commensurate  with  the  flavin  undergoing  such  a  complete 
cycle,  although  per  mole  of  flavin  the  turnover  number  of  the  enzyme  is  exactly 
the  same  as  in  intact  phosphorylating  mitochondria,  I  think  we  must  entertain  the 
possibility  that  the  enzyme  shuttles  between  oxidized  form  and  semiquinone  and 
not  between  the  oxidized  and  the  reduced  form  which  make  all  such  measurements 
highly  dubious.  I  think  we  should  bear  in  mind  that  measurements  at  450  m/x  do 
not  indicate  participation  of  flavoprotein  in  the  respiratory  chain. 

ZlEGLER :  I  agree  with  Dr.  Singer  that  a  considerable  amount  of  reduction  we 
observe  in  the  450  m/x  region  is  not  due  to  the  flavin. 

EsTABROOK :  A  few  years  ago  you  reported  a  very  powerful  inhibition  by 
propionyl-CoA  indicating  that  it  acts  in  the  flavin  region  of  the  respiratory  chain. 
Is  the  iron  reduced  or  not  reduced,  is  the  Q  reduced  or  not  reduced  by  propionyl- 
CoA? 

ZlEGLER :  We  have  not  studied  the  effect  of  propionyl-CoA  on  non-haem  iron 
reduction. 


Reactions  Involved  in  Oxidative  Phosphorylation 
as  Disclosed  by  Studies  with  Antibiotics 

Henry  Lardy 

Institute  for  Enzyme  Research, 
University  of  Wisconsin,  Madison,  TT'/V.,  U.S.A. 

Despite  the  great  progress  that  has  been  made  in  understanding  the 
process  of  oxidative  phosphorylation,  the  number  of  reactions  involved  and 
the  identity  of  all  but  a  few  of  the  reaction  components  remain  unknown. 
Most  of  the  information  extant  has  been  gained  from  studies  with  intact 
mitochondria.  Ultimately  the  process  must  be  examined  in  terms  of  the 
individual  enzymes  involved  and  the  reactions  they  catalyze.  But  while 
isolation  is  in  progress,  new  approaches  to  experiments  with  intact  mito- 
chondria may  tell  us  how  many  components  to  look  for. 

To  this  end  we  have  examined  nearly  one  hundred  highly  toxic  anti- 
biotics for  possible  effects  on  respiration  and  phosphate  transfer  by 
mammalian  mitochondria.  Approximately  lo",,  of  the  compounds  tested 
have  interesting  effects  on  these  processes  (cf.  [i]). 

Two  of  the  antibiotics — oligomycin  and  aurovertin — at  concentrations 
of  less  than  i  fig.  per  ml.  strongly  inhibit  mitochondrial  oxidation  of  all 
pyridine  nucleotide-linked  substrates.  The  inhibition  is  reversed  by  2,4- 
dinitrophenol  (DNP)  indicating  that  these  two  antibiotics  block  enzymes 
involved  in  the  energy-coupling  mechanism,  and  have  no  effect  on  the 
respiratory  enzymes. 

Oligomycin  was  found  [i]  to  inhibit  the  mitochondrial  ATPase  activity 
induced  by  either  DXP,  thyroid  hormones,  deoxycholate  or  Ca  +  +.  Since 
dinitrophenol  overcame  the  effect  of  oligomycin  on  respiration,  and 
oligomycin  nullified  the  effect  of  dinitrophenol  on  ATPase,  it  seemed 
possible  that  these  two  agents  acted  on  the  same  enzymic  site  as  com- 
petitors. However,  a  direct  test  of  this  hypothesis  demonstrated  that 
oligomycin  did  not  act  competitively  in  overcoming  the  effect  of 
dinitrophenol  [2]. 

Aurovertin  differs  from  oligomycin  in  its  effect  on  ATPase  (Fig.  i).  It 
depresses,  but  does  not  completely  inhibit,  the  ATPase  activitv  induced  by 
DXP,  Ca  ~  +,  or  TCAP.  It  has  no  effect  on  the  ATPase  induced  by 
Valinomycin,  Triac,  0-AIe-Triac,  DCA  or  ageing.  Oligomycin  overcomes 
all  these  (Fig.  i). 


266 


HENRY    LARDY 


Both  oligomycin  [i]  and  aurovertin  strongly  inhibit  the  exchange  of  '^^F- 
with  the  phosphate  of  ATP  and  the  exchange  of  ^^O  between  P^^04H  = 
and  water. 

For  purposes  of  discussion,  these  experimental  results  may  be  examined 
in  the  context  of  the  accepted,  but  hypothetical,  reactions  involved  in  fixing 
and  transferring  phosphate. 

X  -  I  +  pi«04H=  ^X  -  Pi«03=  +  P80H  (i) 

X  -  Pi«03  + ADP=  ^  XH  +  ATP=  =  (2) 

P«OH  +  H,0  ^  lOH  +  Hai^O  (3) 

X  -^  I  represents  a  product  whose  formation  required  the  energy 
available  from  an  oxidation-reduction  reaction.  Reactions   1+3  account 


<U        4 


ATP  ase  induced   by  uncoupling  agents 

5xlO"^M  2,4-dinitrophenol 
10     M  tncyano  amino  propene 
02  fig  valinomycin 
SxlO-'iM  O  ME  triac 
lO'^M   Mg   aged  M^ 


/ig  aurovertin 


0  I 

//g  oligomycin 


Fig.  I. 


for  the  exchange  of  ^^O  between  phosphate  and  water  [3] ;  reaction  2 
accounts  for  exchange  between  ADP  and  ATP  [4].  Reactions  i  +2  account 
for  ^'^Pj-ATP  exchange  [5].  Reaction  3  is  assumed  to  be  spontaneous. 

If  we  assume  that  reaction  i  is  blocked  by  oligomycin  and  aurovertin, 
we  learn  that  DNP  must  act  prior  to  the  stage  at  which  Pj  enters  the 
sequence.  If  DNP  prevents  formation  of  X  ~  I  or  catalyzes  the  hydrolysis 
of  X  ~  I  or  some  earlier  intermediate,  it  would  prevent  the  inhibition  of 
respiration  by  oligomycin  and  aurovertin.  Likewise  these  antibiotics  would 
block  the  effect  of  DNP  on  ATP  hydrolysis. 

But  this  scheme  does  not  adequately  explain  the  different  effects  of 


REACTIONS    INVOLVED    IN    OXIDATIVE    PHOSPHORYLATION  267 

these  two  antibiotics  on  i\TPase  induced  by  thyroid  hormones,  by  valino- 
mycin  or  by  ageing  (Fig.  i). 

One  manner  of  explaining  the  data  would  be  to  assume  that  two 
reactions  are  involved  in  the  ^'^O  exchange  reaction. 

X  -  I  +  Pi^OjH-  ^  XisOPi^Og- +  IH  (la) 

Xi80pi803=+YH  ;=^Xi80H  +  YPi803-  (ib) 

YPO3  +  ADP  ^  YH  +  ATP  (2a) 

If  oligomycin  blocked  reaction  (ib)  and  aurovertin  blocked  (la),  each 
would  block  the  effect  of  DNP  on  ATPase  since  DNP  acts  above  reaction 
(la).  We  are  then  led  to  the  conclusion  that  thyroid  hormones,  valinomycin 
and  ageing  bring  about  ATP  hydrolysis  by  catalyzing  the  hydrolysis  of 
XOPO3  .  Their  effect  would  thus  be  blocked  by  oligomycin  but  not  by 
aurovertin. 

There  are  some  data  which  detract  from  the  appeal  of  this  scheme. 
For  example,  valinomycin  reverses  the  inhibition  of  mitochondrial 
oxidation  by  aurovertin.  But  perhaps  some  uncoupling  agents  act  at  both 
the  DNP  site  and  on  XOPO.j'".  We  are  now  making  a  more  detailed 
comparison  between  aurovertin  and  oligomycin  to  determine  whether  they 
act  at  two  different  sites  or  whether  there  is  some  other  explanation  for  the 
differential  eifect  of  these  antibiotics  on  various  ATPase  activities  of 
mitochondria. 

References 

1.  Lardy,  H.  A.,  Johnson,  D.,  and  McMurray,  W.  C,  Arch.  Biochem.  Biopliys. 
78,  587  (1958). 

2.  Lardy,  H.  A.,  and  McMurray,  W.  C,  Fed.  Proc.  18,  269  (1959). 

3.  Cohn,  M.,  and  Drysdale,  G.  W.,'}.  biol.  Chem.  2l6,  831  (1955);  Boyer,  P.  D., 
Falcone,  A.  B.,  and  Harrison,  W.  H.,  Nature,  Land.  174,  401  (1954). 

4.  Wadkins,  C.  L.,  and  Lehninger,  A.  L.,  J',  biol.  Cheyn.  233,  1589  (1958). 

5.  Boyer,  P.  D.,  Luchsinger,  W.  W.,  and  Falcone,  A.  B.,^.  biol.  Chem.  223,  405 


(1956). 


Discussion 


Lehninger:  These  are  very  interesting  results.  In  the  ^**0  exchange  experi- 
ments on  dignitonin  preparations  we  recently  reported  [Chan,  Lehninger,  and 
Enns,  J.  biol.  Cheyn.  235,  1790  (i960)]  that  our  reaction  scheme  for  oxidative 
phosphorylation  could  not  explain  the  higher  incorporation  of  ^**0  from  H.,^**© 
into  ATP  than  we  were  getting  in  the  inorganic  phosphate.  The  2-stage  mechanism 
Dr.  Lardy  suggests  might  offer  some  possibiHty  of  explaining  this  ''^O  exchange, 
which  otherwise  can  be  explained  only  on  a  compartmentation  basis. 

EsTABROOK :  Is  the  arsenate  stimulation  of  oxidation  inhibited  by  oligomycin 
as  well  as  by  aurovertin  ? 


268  HENRY   LARDY 

Lardy  :  We  obtained  aurovertin  very  recently.  Of  a  large  number  of  ATP-ase 
stimulations  which  we  have  tested  including  arsenate  all  are  inhibited  by  oligo- 
mycin  but  we  haven't  tested  them  all  with  aurovertin.  In  addition  to  arsenate,  we 
haven't  tested  dicoumarol  on  aurovertin  yet. 

HoLLUNGER :  In  a  study  of  the  effect  of  guanidine  on  oxidative  phosphorylation 
[Acta  Pharmacol,  et  Toxicol.  ll,  Suppl.  i  (1Q55)]  I  came  to  the  conclusion  that 
guanidine  decreased  the  respiration  of  mitochondria  by  inhibiting  reactions  con- 
necting electron  transport  and  ATP-generation.  As  Dr.  Lardy  now  suggests  the 
same  point  of  attack  for  oligomycin  it  is  perhaps  of  some  interest  to  note  in  con- 
nection with  Dr.  Estabrook's  question  that  guanidine  inhibits  the  arsenate- 
stimulated  respiration  of  mitochondria. 

Lardy:  The  experiments  we  have  done  with  oligomycin  parallel  exactly 
those  of  Dr.  Hollunger,  and  the  compounds  behave  very  much  alike.  However, 
there  are  discrepancies,  e.g.  endogenous  ATP-ase  is  depressed  by  guanidine  but 
DNP-stimulated  ATP-ase  is  not  completely  depressed. 


STRUCTURE  AND  FUNCTION  OF 
CHLOROPLASTS  AND  CHROMATOPHORES 


Chairman's  Opening  Remarks 

T.  W.  GooDwix 

Department  of  Agricultural  Biochemistry,  University  of  Wales, 
Aberystwyth,  Wales 

A  glance  at  the  list  of  distinguished  speakers  in  today's  proceedings 
quickly  made  me  realize  that  it  would  be  an  act  of  supererogation  if  I 
attempted  to  discuss  chloroplasts  and  chromatophores  in  general  terms  as 
an  introduction  to  the  session.  I  feel  that  it  would  be  much  more  profitable 
if  I  made  some  general  observations  on  one  member  of  those  inseparable 
photosynthetic  twins — the  carotenoids  and  the  chlorophylls.  Dr.  Smith 
will  discuss  certain  aspects  of  chlorophyll  biochemistry,  so  I  shall  confine 
myself  to  the  carotenoids.  The  invariable  co-existence  of  carotenoids  and 
chlorophylls  in  all  photosynthetically  active  units  strongly  indicates  an 
important  function  of  carotenoids  in  photosynthesis.  It  has  been  known 
for  a  long  time  that  they  play  an  ancillary  role  in  photosynthesis ;  they 
absorb  light  in  the  region  of  the  spectrum  least  efl^ectively  used  bv  chloro- 
phyll and  pass  it  on,  with  \arying  degrees  of  efficiency  in  different 
organisms,  to  chlorophyll  for  use  in  the  primary  photosynthetic  act.  This, 
however,  does  not  make  the  carotenoids  essential  to  photosynthesis,  but 
only  allows  the  more  efficient  use  of  the  energy  of  the  visible  spectrum. 
However,  the  invariable  association  of  carotenoids  and  chlorophylls  in 
photosynthetic  organisms  suggests  a  more  fundamental  role  than  this.  As 
Stanier  [i]  has  put  it,  "In  the  long  run  natural  selection  ruthlessly 
eliminates  non-functional  gadgetry  from  living  organisms  and  as  biologists 
we  may  therefore  be  fully  confident  that  the  carotenoids  of  the  photo- 
synthetic apparatus  are  not  merely  the  organic  equivalent  of  tail  fins". 
The  work  of  Stanier  and  others  strongly  indicates  that  the  essential  func- 
tion of  carotenoids  in  the  photosynthetic  units  is  to  prevent  photo- 
oxidative  damage  by  chlorophyll.  I  do  not  intend  to  discuss  this  further 
now,  but  no  doubt  various  aspects  of  this  work  will  be  considered  during 
today's  session. 

I  wish  to  devote  the  remainder  of  my  time  to  considering  how  caro- 
tenoids are  synthesized.  Carotenoids  are  one  class  of  a  wide  group  of 
natural  products  known  as  terpenoids ;  these  have  a  common  characteristic 
in  that  they  are  built  up  from  isoprenoid  (branched  5-C  units).  It  is  clear 
from  the  work  of  Lynen,  Popjak,  and  Bloch  and  their  collaborators  that 
the  5-C  unit  from  which  steroids  and  other  triterpenes  are  formed  is 
isopentenyl  pyrophosphate  (see  Goodwin  [2,  3]  for  details).  The  mech- 
anisms  involved   in   the   formation   of  isopentenyl   pyrophosphate   from 


272  T.    W.    GOODWIN 


2CH3COOH >  2CH3COSC0A >  CH3COCH,COSCoA  +  CoASH 

Co/ 
( 

r 


Acetate  Acetyl-CoA  Acetoacetyl-CoA 

CH3COSC0A 


CoASH 


CH3C(OH)CH,CH20®<p-^CH3C(OH)CH,CH,OH  y^^  CH3C(OH)CH,COSCoA 

I  /       \  I  /       \  I 

CH2COOH  ADP    ATP         CH.COOH  TPN    TPNH,       CH.COOH 

Mevalonic  acid  Mevalonic  acid  /3-Hydroxy-^- 

5 -phosphate  methylglutaryl-CoA 


Mn'+ 


-ATP 
'A  DP 


ATP    A  DP 

CH3C(6h)CH,CH,0-®-0-®  s^<f  >  CH3C-CH,CH,0-®-0-® 


CH.COOH  ^0=     ^'  CH, 

Mevalonic  acid  Isopentenyl  pyrophosphate 

5  -pyrophosphate 

Fig.   I.  The  conversion  of  acetate  into  isopentenyl  pyrophosphate. 

acetate  are  outlined  in  Fig.  i.  Experiments  showing  that  isopentenyl 
pyrophosphate  is  also  involved  in  carotenoid  biosynthesis  have  not  yet 
been  reported ;  however,  there  seems  little  doubt  that  it  is  an  intermediate 
because  isotope  experiments  with  various  carotenogenic  preparations  have 
shown  that  acetate,  ^-hydroxy-/3-methylglutarate  and  mevalonate  are  all 
incorporated  in  the  expected  manner  (see  Goodwin  [2,  3].) 

CH3\ 

C— CH  .,CH..O— ®— O— ® > 

CH./ 

Isopentenyl  pyrophosphate 

CH3X  CH3\ 

^C=CHCH.,0®-0-®+  ;CCH,CH,0-e/-0— ®- 

CH3/  "  chX 

Dimethylallyl  pyrophosphate 


-®— ® 


-      '^CCH,,CH..Orpi— O— (P)+      '^)C=CHCH.,CHC=CHCH,0®— O— ® 
CH./  "       '  CH3/  1 

CH3 

,^->-® — ®  Geranyl  pyrophosphate 

CH3\ 

/C=CHCH..CHC=CHCH.,CHC=-CHCH,0— ®— O— ® 

CH3/  I  I 

CH3  CH3 

Farnesyl  pyrophosphate 
Fig.  2.  The  conversion  of  isopentenyl  pyrophosphate  into  farnesyl  pyrophosphate. 


STRUCTURE    AND    FUNCTION    OF    CHLOROPLASTS    AND    CHROMATOPHORES      273 


7,8,1  i,i2,i5,i5',i2',ii',8',7'-decahydrolycopene 

(lycopersene) 


l\ 


Phvtoene 


Phytofluene 

I 


^-Carotene 


Neurosporene 


Lycopene 
Fig.   3.  The  conversion  of  C-40  polyenes  into  lycopene. 

VOL.  II. — T 


274  '"'•    "^^'^    GOODWIN 

In  steroid  biosynthesis  isopentenyl  pyrophosphate  is  first  isomerized 
to  /3^-dimethylallyl  pyrophosphate  which  acts  as  a  starter  for  polymeriza- 
tion. This  compound  then  reacts  with  two  molecules  of  isopentenyl 
pyrophosphate  as  indicated  in  Fig.  2,  eventually  yielding  the  15-C  com- 
pound farnesyl  pyrophosphate  which  dimerizes  to  yield  the  hydrocarbon 
squalene,  the  acyclic  steroid  precursor.  By  analogy  the  corresponding 
compounds  concerned  with  carotenoid  biosynthesis  would  be  the 
20-C  compound  geranylgeranyl  pyrophosphate  and  lycopersene  respect- 
ively; neither  has  yet  been  unequivocally  identified.  If  we  accept  the 
assumption  that  this  lycopene  derivative  is  the  basic  40-C  carotenoid 
precursor  then  three  main  problems  arise  which  are  unique  to  the  bio- 
synthesis of  carotenoids:  (a)  what  is  the  pathway  of  dehydrogenation  of 


y  \  /\ 


"OH 


Lycopene  Rhodopin  [Dehydrorhodopin]* 


I 


'OMe    HO"^  \,  /     ^OMe     HO 

Spirilloxanthin 


[Dehydrorhodovibrin]  *     Rhodovibrin 

*  These  compounds  have  not  yet  been  identified. 

Fig.  4.  The  conversion  of  lycopene  into  spirilloxanthin  in  RJiodospirilliim 
rubrinn. 

the  40-C-precursor  ?  {b)  how  do  the  acyclic  (lycopene)  and  cyclic  (^-caro- 
tene) derivatives  arise  .''  and  (f)  how  do  the  oxygenated  derivatives  (xantho- 
phylls)  arise  ? 

With  regard  to  the  first  tw'o  queries  lycopene  almost  certainly  arises  by 
the  sequential  dehydrogenation  of  phytoene  via  the  route  indicated  in 
Fig.  3.  The  pathway  of  /S-carotene  synthesis  is  much  less  certain  because 
of  the  doubts  about  the  point  at  which  cyclization  takes  place.  My  own 
view,  which  has  been  given  in  detail  previously  [4],  is  that  cyclization  is  an 
early  event  in  the  conversion  of  the  basic  40-C  precursor  into  ^-carotene. 

Hydroxylated  xanthophylls  are  formed  in  the  photosynthetic  bacteria 
by  the  insertion  of  oxygen  following  the  completion  of  the  parent  hydro- 
carbon. For  example,  in  Rhodospirillum  riibrum  the  formation  of  spiril- 
loxanthin from  lycopene  has  been  proved,  and  the  pathway  is  probably 
that  indicated  in  Fig.  4  [i,  3].  There  are  no  reports  of  experiments  indicat- 


STRUCTURE    AND    FUNCTION    OF    CHLOROPLASTS    AND    CHROMATOPHORES      275 

ing  how  hydroxylated  xanthophylls  are  formed  in  higher  plants  and  algae, 
but  it  is  clear  that  /S-carotene  epoxides  are  formed  in  excised  leaves  by 
epoxidation  of  the  parent  hydrocarbon. 

Having  given  a  very  brief  summary  of  what  we  know  about  the 
mechanism  of  carotenoid  formation,  I  shall  conclude  by  mentioning  two 
specific  problems  which  should  be  of  direct  interest  to  this  symposium. 
Both  are  concerned  with  the  action  of  light  on  carotenoid  formation  in 
photosynthetic  organisms  and  both  are  only  in  the  embryonic  state  of 
development.  The  first  problem  is  the  synthesis  of  carotenoids  in  illu- 
minated etiolated  maize  seedlings.  Etiolated  seedlings  produce  only 
small  amounts  of  xanthophylls ;  on  illumination  they  immediately  begin  to 
synthesize  the  typical  plastid  carotenoids,  mainly  /3-carotene,  lutein,  and 
neoxanthin,  along  with  the  chlorophylls  as  the  functional  chloroplasts 
develop.  Isotope  experiments  show  that  mevalonate  and  acetate  are 
ineffectively  incorporated  into  /3-carotene  during  this  period,  but  that  CO^ 
is  specificallv  incorporated.  Etiolated  seedlings  synthesize  considerable 
amounts  of  sterols  and  mevalonate  and  acetate  are  incorporated  into  these 
compounds  both  in  the  dark  and  on  illumination  of  the  seedlings ;  CO2  on 
the  other  hand  is  less  effectively  incorporated  into  the  sterols  than  into  the 
carotenoids  [5].  We  are  now  trying  to  find  out  the  biochemical  reason  for 
the  sudden  switch  of  terpenoid  precursors  from  steroid  synthesis  to 
carotenoid  synthesis  and  for  the  effectiveness  of  CO2  as  a  carotenoid 
precursor.  A  possible  explanation  is  that  TPNHo  is  required  for  the  later 
stages  of  carotenoid  synthesis  (  }  dehydrogenation)  and  this  would,  of 
course,  become  available  in  increasing  amounts  in  the  developing  chloro- 
plasts. Furthermore,  it  has  recently  been  observed  (H.  Yokoyama,  personal 
communication)  that  in  an  enzyme  preparation  from  PJiycomyces  blakeslee- 
anus  which  incorporates  labelled  mevalonate  into  /3-carotene  and  ergosterol, 
the  addition  of  TPNH.,  to  the  suspending  medium  results  in  relatively 
more  label  appearing  in  the  /3-carotene  fraction. 

The  second  problem  deals  with  the  purple  photosynthetic  bacterium 
Rlwdospirillum  riihrum.  When  grown  photosynthetically  this  normally 
produces  in  its  unsaponifiable  fraction  a  carotenoid  spirilloxanthin  and  a 
terpenoid  recognized  at  the  moment  only  by  its  Rf  value  [6].  When  R. 
riibnim  is  grown  heterotrophically  in  the  dark  it  is  colourless  and  syn- 
thesizes only  the  terpenoid  compound;  on  illumination  spirilloxanthin  and 
bacteriochlorophyll  are  synthesized  together  as  functional  chromatophores 
develop.  This  situation  is  obviously  very  similar  to  that  encountered  in 
etiolated  seedlings.  However,  a  somewhat  different  situation  can  be 
demonstrated  under  suitable  conditions.  Dr.  June  Lascelles  m  Oxford  [7] 
showed  that  washed  colourless  Rhodopseudomonas  spheroides  resuspended 
in  a  medium  containing  small  amounts  of  glycine,  a-ketoglutarate, 
fumarate  and  salts,  including  Fe^ +,  and  with  a  gas  phase  containing  b^/^y 


276  T.    W.    GOODWIN 

oxygen  and  the  remainder  nitrogen,  rapidly  synthesized  bacteriochloro- 
phyll.  Mr.  Brian  Davies  in  my  laboratory  has  repeated  these  experiments 
using  Rhodospirillum  rubriim  and  found  the  same  situation  to  exist ;  further- 
more he  has  extended  the  experiments  and  has  found  what  one  would  have 
anticipated,  that  spirilloxanthin  is  synthesized  alongside  bacteriochloro- 
phyll.  Two  possible  explanations  exist:  {a)  either  a-ketoglutarate  and/or 
fumarate  and  the  carbon  residue  of  glycine  are  providing  carotenoid 
precursors,  or  {b)  the  conditions  are  such  as  to  stimulate  the  conversion  of 
an  existing  precursor  into  spirilloxanthin.  If  the  latter  is  the  true  explana- 
tion then  the  precursor  must  be  far  back  in  the  biosynthetic  pathway 
because  resuspension  of  colourless  R.  riibrum  cells  in  the  Lascelles  medium 
to  which  the  carotenoid  inhibitor  diphenylamine  has  been  added,  results 
in  the  accumulation  of  the  partly  saturated  polyenes,  phytofluene,  etc.  If 
the  first  possibility  is  correct  then  compounds  other  than  CO.,  are  actively 
concerned  in  carotenogenesis  and  thus  the  situation  is  to  some  extent 
dilTerent  from  that  in  etiolated  leaves.  These  differences  of  detail  aside, 
the  effect  of  light  on  both  organisms  is  essentially  the  same,  and  it  is  hoped 
that  soon  a  biochemical  explanation  of  this  important  action  of  light  in 
controlling  terpenoid  synthesis  in  photosynthetic  organisms  will  be  forth- 
coming. I  think  that  vou  will  agree  that  this  is  a  key  problem  because,  if 
this  re-routing  of  terpenoid  intermediates  on  illumination  did  not  occur 
then  functional  chloroplasts  and  chromatophores  would  not  result. 

References 

1.  Stanier,  R.  Y.,  "The  Harvey  Lectures"  (1958-9)  Academic  Press,  New  York, 
219  (i960). 

2.  Goodwin,  T.  W.,  "Recent  Advances  in  Biochemistry",  London,  Churchill 
(i960). 

3.  Goodwin,  T.  W.,  Auu.  Rev.  Plant  Physiol  (in  press)  (1961). 

4.  Goodwin,  T.  W.,  Advanc.  Enzy?noL  21,  295  (i959)- 

5.  Goodwin,  T.  W.,  Biochern.J.  70,  612  (195^)- 

6.  Davies,  B.  H.,  and  Goodwin,  T.  W.,  Biachcni.y.  73,  loP  (1959). 

7.  Lascelles,  J.,  Binchnn.'J.  72,  508  (1959). 


Haem  Protein  Content  and  Function  in  Relation  to 

Structure  and  Early  Photochemical  Processes  in 

Bacterial  Chromatophores* 

AIartix  D.  Kamen 

Brandeis  L  uiversity, 
Waltham,  Mass.,  U.S.A. 

The  essential  process  in  photosynthesis  is  the  absorption  and  conversion 
of  radiant  energy  into  chemical  free  energy  with  subsequent  storage  in  a 
form  which  can  be  used  for  biosynthesis.  Currently,  the  most  widely 
studied  example  of  this  process  in  cell-free  systems  is  "  photophosphoryla- 
tion" — the  chromatophore,  or  chloroplast,  catalysis  of  ATP  synthesis 
from  ADP  and  inorganic  phosphorus,  utilizing  energy  absorbed  by  the 
photoactive  pigments  contained  in  these  particles. 

The  time  during  which  the  energy  conversion  and  stabilization  phase 
of  photosynthesis  occurs  is  subsumed  between  ^  lo  "  sec.  and  ~  lo  ~-  sec. 
The  former  limit  is  set  by  the  time  required  for  initial  quantum  absorption 
and  subsequent  migration  of  the  energy  packet  to  the  active  centre  of  the 
system  by  processes  such  as  exciton  migration,  or  induced  resonance, 
which  depend  in  detail  on  the  specific  structural  features  of  the  photo- 
active particles.  It  is  possible  that  stabilization  of  the  excitation  energy  in 
the  so-called  "triplet"  state  of  the  photoactiye  pigment  can  extend  the 
upper  boundary  in  time  from  lo^^  sec.  to  lo^  sec.  [i].  The  latter  limit 
is  given  by  the  ayerage  turno\"er  number  of  enzymes  invoked  in  the 
biochemical  processes  which  lead  eventually  from  assimilation  of  CO.,  or 
organic  material  to  synthesis  of  cell  material. 

We  know  very  little  about  the  photochemical  processes  which  occur 
during  this  critical  interval  in  time.  A  basic  question  which  highlights  this 
ignorance  is :  What  molecular  composition  and  placement  is  both  necessary 
and  sufficient  to  bridge  the  gap  between  quantum  absorption  and 
biosyntheses  ? 

We  may  suppose  that  some  sub-unit  of  the  plant  granum  or  bacterial 

*  Communication  Xo.  i  i  i  in  the  series  "Publications  of  the  Graduate  Depart- 
ment of  Biochemistry,  Brandeis  University".  Researches  in  this  laboratory  on 
which  this  paper  is  based  have  been  supported  by  grants  from  the  National 
Science  Foundation  (Grant  Xo.  Cj-6441)  and  the  Xational  Institutes  of  Health 
(Grant  Xo.  C-3649). 


ayS  MARTIN    D.    KAMKN 

chromatophore  is  a  minimal  structure.  The  usual  experimental  approach 
is  to  fragment  cells  to  photoactive  particles  and  then  continue  fragmenta- 
tion until  some  photochemical  process,  such  as  the  Hill  reaction,  or  photo- 
phosphorvlation,  is  no  longer  supported  by  the  particle  preparation 
obtained.  Then,  re-activation  by  the  addition  of  external  factors  is  used  to 
define  the  biochemical  system.  Unfortunately,  such  an  approach  merely 
defines  a  system  that  can  work,  but  not  necessarilv  one  that  functions  in 
normal  photosynthesis.  As  an  example,  we  may  recall  the  remarkable 
activation  of  photophosphorylation  by  phenazine  methosulphate,  first  seen 
in  bacterial  chromatophore  preparations  [2,  3,  4]. 

A  refinement  on  this  methodology  is  to  examine  soluble  factors 
originally  present  in  the  intact  cell,  or  chromatophore,  and  which  were 
removed  during  fractionation.  A  number  of  investigators  have  found  that 
the  washings  from  chromatophores  and  chloroplasts  contain  activators  for 
photophosphorylation,  but  the  nature  of  these  factors  remains  obscure. 
The  soluble  system  obtained  by  washing  fragmented  chromatophores  is 
complex,  containing  numerous  enzvmes  associated  with  activities  such  as 
adenylate  kinase,  exchange  of  ATP  with  inorganic  P,  nucleotidase,  nucleic 
acid  depolvmerase,  catalase  and  peroxidase,  etc.  In  addition,  there  are  non- 
specific reductants,  as  well  as  fiavins,  quinones  and  haem  proteins  which 
have  been  dissociated  from  their  binding  sites  in  the  chromatophore. 
Dr.  Horio  and  I  have  found  recentlv  (unpublished)  that  thoroughly 
washed  chromatophores  from  RhoduspirilliDn  riibnim,  which  are  wholly 
inactive  in  photophosphorylation,  can  be  reactivated  by  addition  of  the 
purified  haem  protein,  a  pure  vellow  flavin  enzyme  which  is  a  pyridine 
nuclcotidc-linked  haem  reductase,  and  a  \olatile  reductant  obtained  by 
distillation  of  acetone  extracts  of  fresh  cells.  These  results  are  an  improve- 
ment on  those  previouslv  obtained  using  crude  extracts,  or  artificial 
electron  transport  mediators  like  phenazine  methosulphate,  because  they 
demonstrate  the  ability  of  single  factors  originally  present  in  the  chro- 
matophore to  participate  in  the  normal  metabolic  process.* 

Other  approaches  can  be  based  on  synthesis  rather  than  breakdown  of 
the  photochemical  apparatus.  Possible  methods  include  extraction  of  pre- 
cursor particles  from  colourless  mutants,  physical  treatments  of  normal 
cells  which  interfere  with  chloroplast  or  chromatophore  development  (e.g. 
heat  [5],  u.v.  irradiation,  variation  in  oxygen  tension  [6],  heterotrophic 
growth  conditions  [6],  etc.).  Immunochemical  approaches  have  been 
described  in  which  sera  specifically  directed  against  components  extracted 
from  light-grown  and  dark-grown  bacterial  svstems  have  been  prepared 
[7,  8]. 

*  H.  Baltscheffsky  {Biochim.  hiopliys.  Acta  41,  i  (i960))  has  published  recently 
results  of  studies  of  this  type  implicating  flavin  adenine  dinucleotide  as  an  inter- 
mediate in  electron  transport  coupled  to  photophosphorylation. 


HAEM    PROTEIN    CONTENT    AND    FUNCTION    IN    RELATION    TO    STRUCTURE        279 

However,  despite  the  many  researches,  particularly  on  chloroplast 
development,  emphasis  has  been  mainly  on  morphology.  Few  chemical 
analvses  at  the  molecular  level  ha\e  been  performed.  Xo  structural  studies 
have  been  conducted  simultaneouslv  with  exhaustive  molecular  analyses. 
The  development  of  the  bacterial  chromatophore  system,  the  study  of 
which  is  just  beginning  [9,  10],  may  pro\ide  a  well-defined  test  system  for 
future  research. 

There  is  one  study  available  on  molecular  composition  as  a  functi(Mi 
of  fragmentation.  While  the  data  are  incomplete  in  many  respects,  they  are 
sufficient,  taken  together  with  gleanings  from  various  other  researches  in 
the  literature,  to  base  a  \alid  discussion  of  possible  relations  between 
molecular  composition  and  the  primary  processes  in  photosynthesis. 

These  data  were  obtained  by  Xewton  and  Newton  in  our  laboratory 
three  years  ago  and  are  concerned  w  ith  the  composition  of  the  photoactive 
sub-cellular  particles  derived  by  various  fragmentation  procedures  from 
the  obligate  photo-anaerobe,  Chyomatiiun  [11].  The  gross  composition  and 
characterizations  of  some  components  of  chromatophores  and  chromato- 
phore fragments,  as  isolated  by  differential  centrifugation,  were  studied. 
Qualitative  kinetic  analyses  of  the  progressive  fragmentation  of  cells  into 
small  subcellular  aggregates  were  conducted,  together  with  molecular 
analyses  for  each  fraction. 

I  have  recast  these  data  so  as  to  summarize  briefly  the  essential  results 
in  a  single  table  (Table  I).  These  data  may  be  expanded  by  borrowing 
some  figures  from  other  researches.  'I'hus,  Lester  and  Crane  [12]  give  a 
figure  of  2-9  jxM  Coenzyme  "Q7"  (or  "ubiquinone"  [13])  per  g.  dry 
weight  of  cells.  This  approximates  to  ^0-5  /xM  per  g.  wet  weight  of 
chromatophores,  a  relatively  great  quantity  of  this  benzoquinone.  For 
R.  rubniin,  a  somewhat  higher  but  comparable  figure  is  given,  the  quinone 
found  being  "Q,, "  and  the  concentration  approximately  twice  that  of  the 
Chrojfuit/ 1(1/1  "();". 

Inasmuch  as  all  the  ph(jtoactive  structures  known  are  supposed  to  be 
self-duplicating  units,  it  can  be  expected  that  nucleic  acids  are  present. 
As  seen  in  Table  1,  acid-soluble  nucleotides  are  found  and  in  addition 
there  is  residual  phosphate  which  is  associated  with  protein  and  with 
insoluble  nucleic  acid.  A  reasonable  treatment  of  these  data  indicates  that 
out  of  the  total  P  present  (85  [.im)  probably  no  more  than  20  /tM  can  be 
ascribed  to  nucleic  acid.  This  can  be  contrasted  with  the  nucleic  acid  P 
content  of  chromatophores  originally  obtained  from  R.  ruhruni  by  Schach- 
man,  Pardee,  and  Stanier  [14],  who  found  for  the  same  protein  content  a 
value  of  ~  200  /tM. 

Of  course  it  is  not  surprising  that  large  variations  in  content  of  par- 
ticular fractions  will  occur  as  the  source  of  particles  is  varied.  This  is  true 
of  all  fractions  examined  to  date  such  as  the  chlorophylls  [15],  the  caro- 


28o  MARTIN   D.    KAMEN 

TABLE  I 

Molecular  Composition  of  Chromatium  Chromatophores 

(after  Newton  and  Newton  [ii]) 

Based  on  i  gm.  wet  weight  of  washed  chromatophores 


Protein  (nig.) 

Cytochrome  (/um) 

Carbohydrate  (mg.) 
Acid-soluble 
Insoluble 

Lipid  (mg.) 

Pigtnents  (i-im) 

Bacterio-chlorophyll 
Bacterio-carotenoid 

Nucleotides  (/^tivi) 
Pyridine 

Flavin  (/xm) 

Phosphorus  (/xm) 
Acid-soluble 

Insoluble 

Iron  (imM) 

Acid-soluble 


1 66  (modified  biuret  reaction) 

o- 18     (determined  as  pyridine  hemochromogen) 


62 

5 

57 

87 


3-4 
I  -6 

9-5 
02 

o- 17 

85 
9-4 

73 

12 
5 


(anthrone  reaction) 
(mostly  pentose) 
(galactose  polymer) 

(mostly    phospholipid ;    only   base   detected- 
ethanolaniine) 

(spectrophotometric  assay) 
(spectrophotometric  assay) 

(based  on  u.v.  absorption  as  adenine) 
(fluorimeter  assay  of  TCA  extract) 


(8-5    /uM    of    this    fraction    accounted    for    as 

inorganic  P) 
(51  /tM  of  this  fraction  accounted  for  as  lipid  P) 

(mostly  non-haem) 

(mostly  present  in  ferrous  form) 


tenoids  [16,  17],  the  haem  proteins  [18,  19]  and  the  quinones  [12].  The 
major  finding  appears  to  be  that  the  bacterial  chromatophores  are  relatively 
rich  in  RNA  [14]  and  depleted  in  DNA  [14],  which  suggests  a  basic 
composition  like  that  of  microsomes,  as  regards  gross  composition.  In 
fact,  the  overall  P  distribution  in  various  fractions  of  silver  beet  micro- 
somes, as  obtained  by  Martin  and  Morton  [20],  are  much  like  those  found 
by  Newton  and  Newton  [11]  for  Chrumatium  chromatophores.  Results 
given  by  Nakamura,  Chow,  and  Vennesland  [21]  for  spinach  chloroplast 
preparations  also  do  not  differ  significantly  from  those  reported  for  the 
chromatophores.  The  relation  of  nucleic  acids  to  development  of  photo- 
active structures  remains  to  be  elucidated.  A  beginning  has  been  made  by 
Brawerman  and  Chargoff  [5]  whose  interesting  work  I  can  only  mention 
in  passing  because  of  time  limitations.  It  is  noteworthy,  as  far  as  photo- 
chemical function  is  concerned,  that  Nakamura  et  ah  [21]  reported 
extensive  enzymic  depolymerization  of  nucleic  acid  in  chloroplast  frag- 
ments failed  to  impair  the  photophosphorylation  capacity.  This  indicates 


HAEM    PROTEIN    CONTENT    AND    FUNCTION    IN    RELATION    TO    STRUCTURE     28 1 

that  the  relation  between  nucleic  acid  and  the  photochemical  function  is 
indirect. 

The  carbohydrate  and  lipid  fractions  deserve  much  more  mention  than 
I  can  give  at  this  time.  Briefly,  the  Newtons  found  (Table  I)  that  in 
Chromatium  chromatophores,  the  major  fraction  of  the  carbohvdrate 
present  was  in  the  form  of  a  polysaccharide,  the  monomer  unit  of  which 
appeared  to  be  galactose.  The  presence  of  a  galactose  moietv  as  a  charac- 
teristic component  of  the  photosynthetic  carbohydrate  fraction  in  both 
chloroplasts  and  chromatophores  as  well  as  a  component  found  in  galacto- 
sidyl  lipids  has  been  well-documented  by  Benson  and  his  co-workers  [21, 
22].  Progressive  fragmentation  of  the  chromatophores  to  smaller  fragments 
resulted  in  a  loss  of  most  of  this  polysaccharide  with  a  corresponding 
relative  increase  in  lipid  [11]. 

The  lipids  present  in  most  photosvnthetic  tissues  appear  to  be  pre- 
dominantly of  neutral  or  cationic  type  [22].  Mono-  and  digalactosyl 
monoglycerides  predominate.  There  are  also  some  new  sulpho-lipids,  one 
of  which  has  been  identified  as  a  sulphonic  acid  analogue  of  the  major 
plant  glycosyl  monoglyceride  [23],  e.g.  the  structure  assigned  by  Benson 
et  al.  is  i-0-(i'-deoxy-i'-sulphoketopyranosyl)-3-0-oleoylglyceride.  The 
basic  phospholipid  present  in  Chromatium  appears  to  be  almost  wholly  a 
cephalin — namely,  ethanolamine  phosphatidyl  glycerol  [11].  The  nature 
of  the  fatty  acids  which  are  presumably  bound  as  esters  to  the  glycerol  is 
still  unknown.  This  phospholipid  is  held  to  account  for  practically  all  the 
fat  in  the  Chromatium  chromatophore  [11,  24]. 

It  seems  evident  that  the  photosynthetic  structures  elaborate  special 
lipids  and  carbohydrates  which  in  many  cases  appear  unique  to  the  photo- 
active particle  systems.  Very  probably  a  major  role  involves  stabilization 
of  chromatophore  and  chloroplast  structures  which  contain  both  polar  and 
non-polar  groupings.  It  may  be  mentioned  that  plastids  from  various  plant 
sources  appear  to  contain  hydrolytic  enzymes  (phosphatidases)  which 
attack  lecithin  and  other  lipids  [25]. 

Major  interest  resides  at  present  in  another  feature  of  data  such  as  are 
exemplified  in  Table  I.  It  will  be  noted  that  cvtochrome  (in  this  case,  a 
cytochrome  complex  made  up  of  a  modified  haem  protein  called  "  RHP" 
and  a  cytochrome  of  the  c-type  [26])  accounts  for  an  appreciable  fraction 
of  the  total  protein.  Thus,  out  of  166  mg.  total,  there  are  o- 18  /tivi  cyto- 
chrome. Most  of  this  cytochrome  is  the  "r"  component  which  has  mole- 
cular weight,  as  isolated  in  pure  form,  of  95  000  [24].  This  means  that 
approximately  17  mg.,  or  10" ,,  of  the  protein,  is  accounted  for  as  cyto- 
chrome. In  addition,  there  are  trace  amounts  of  haem  proteins  with  which 
are  associated  catalase  and  peroxidase  activities.  A  flavin  component  is 
associated  to  a  major  extent  with  a  yellow  enzvme  which  can  be  prepared 
from  both  R.  rubrum  [27]  and  Chromatium  (R.  G.  Bartsch,  unpublished) 


282  MARTIN   D.    KAMEN 

and  which,  as  mentioned  previously,  is  a  pyridine-nucleotide  linked  haem 
protein  reductase. 

These  results  relating  to  the  cytochrome  content  of  the  Chromatiiim 
chromatophores  are  applicable  generally  to  all  photoactive  particles, 
whether  of  bacterial  or  plant  origin.  Surveys  of  all  the  typical  species  of 
photosynthetic  bacteria  [28]  and  of  a  large  variety  of  plants  and  algae  [29, 
30]  reveal  that,  regardless  of  aerobic  or  anaerobic  habit,  these  systems  all 
contain  relatively  large  amounts  of  haem  proteins.  Further,  although  the 
major  component  invariably  is  a  cytochrome  of  the  "c"  type,  no  corre- 
sponding oxidase  of  the  "fl"  type  is  found  associated  with  chromatophores 
or  chloroplasts.  Significant  aspects  of  these  findings  have  been  discussed 
sufficiently  elsewhere  [19,  31].  Let  us  proceed  to  the  central  topic  of  this 
paper — a  possible  relation  between  haem  protein  content  and  the  early 
photochemistry  of  the  photosynthetic  process. 

The  ultimate  consequence  of  the  photochemical  act  may  be  thought  of 
as  the  establishment  of  a  voltage  gap  between  two  systems.  This  gap  is 
sufficiently  large  in  the  case  of  the  green  plants  and  algae  so  that  one 
system  can  operate  at  a  "mid-point"  potential  reducing  enough 
(negative  £"0)  to  drive  reductive  assimilation  of  CO.,  (and  perhaps  generate 
ATP  simultaneously)  while  the  other  can  provide  a  sufficiently  high 
oxidizing  "mid-point"  (positive  is^)  potential  eventually  to  liberate 
oxygen  from  water.  In  bacteria,  a  small  gap  may  be  all  that  is  necessary 
because  oxygen  is  not  liberated  during  COg  assimilation.  The  significance 
of  our  question  about  a  sufficient  and  necessary  molecular  composition 
and  placement,  posed  in  our  previous  discussion,  is  that  if  we  know  what 
molecules  are  present,  their  relative  concentrations,  and  their  disposition, 
we  may  begin  to  develop  and  examine  hypotheses  for  identifying  reactants 
in  the  primary  photochemistry.  In  Chromatiiim  chromatophores,  Newton 
and  Newton  [11]  have  shown  that  the  major  constituents  present  in  both 
chromatophores  and  chromatophore  fragments  include,  in  addition  to  the 
photoactive  pigments  and  the  major  gross  fractions  of  protein,  lipid,  and 
carbohydrate,  components  typical  of  a  mitochondrial  respiratory  chain, 
e.g.  pyridine  nucleotides,  flavins,  quinones,  and  cytochromes.  Associated 
with  these  compounds  are  a  variety  of  enzyme  activities  typical  of  an 
electron-transport  system,  as  noted  previously. 

In  Chromatiiim  chromatophores,  there  are,  for  every  20  bacterio- 
chlorophyll  molecules,  11  carotenoids,  1-5  haem  protein,  i  flavin,  and  i 
pyridine  nucleotide.  We  have  remarked  that  further  fragmentation  to 
small  particles  results  in  the  loss  of  a  major  part  of  the  polysaccharides, 
some  protein,  but  less  lipid,  so  that  the  fragmented  particles  became 
relatively  enriched  in  lipid.  However,  the  haem  protein  content  relative  to 
chlorophyll  remains  unchanged,  both  doubling  relative  to  total  protein 
content.  Thus,  in  the  chromatophore  fragment  (which  is  still  capable  of 


HAEM    PROTEIN    CONTENT    AND    FUNCTION    IN    RELATION    TO    STRUCTURE     283 

supporting  photophosphorylation  when  incubated  with  certain  external 
factors  [3]),  there  are  40  bacteriochlorophylls,  17  carotenoids,  2-8  haem 
proteins,  o  •  s  flavins,  and  i  pyridine  nucleotide.  Data  on  the  quinone 
contents  of  the  two  preparations  are  not  available.  We  may  generalize 
these  observations  to  the  statement  that  similar  molecules  are  present  as 
major  components  in  all  photoactive  structures. 

Now,  we  may  ask  what  mechanism  we  can  assume  for  energy  storage 
and  which  molecules  of  those  mentioned  as  major  constituents  are  likely 
reactants  for  production  of  molecular  species  sufficiently  stable  to  couple 
to  the  biochemical  phase  of  photosynthesis.  Of  course,  there  is  little  doubt 
that  one  reactant  will  be  excited  chlorophyll.  The  reactions  it  may  undergo 
upon  excitation  are  many  but  a  most  likely  type  of  reaction  is  one  involving 
electron  transfer.  It  is  not  possible  that  electron  ejection  (photo-ionization) 
will  occur  because  the  quantum  energy  in  actinic  light  is  insufficient  for 
such  a  process.  However,  electron  donation,  or  acceptance,  from  a  neigh- 
bouring molecule  is  possible.  Some  theories  [32]  are  built  on  the  notion 
that  chlorophyll  loses  an  electron  to  some  acceptor  and  so  becomes  a  strong 
oxidizing  agent.  An  alternative  notion  is  that  it  gains  an  electron  and 
becomes  a  strong  reducing  agent.  There  is  no  way  at  present  of  deciding 
between  these  two  alternatives. 

On  the  basis  of  some  arguments  based  on  comparative  biochemistry 
and  the  physical  chemistry  of  the  haem  proteins  (see  later  discussion  in 
this  paper)  and  results  obtained  by  Duysens,  Chance  and  others,  using  an 
approach  based  on  differential  spectrophotometry  of  fast  reactions  in 
suspensions  of  cells  and  extracts  [33,  34,  35,  36],  I  have  suggested  [37,  38] 
that  the  primary  electron  transfer  act  involves  reduction  of  chlorophyll  by 
the  iron  haem  protein  complex,  resulting  in  a  reduced  chlorophyll- 
chlorophyll  couple  on  the  one  hand  (£",;  ~  —  i-o  V.)  and  an  oxidized- 
reduced  haem  protein  couple  on  the  other  (Fig.  i).  The  potential  developed 
depends  on  whether  the  oxidation  of  the  central  iron  atom  proceeds  to  a 
formal  valence  state  of  three  positive,  or  whether  it  goes  to  a  higher 
effective  valence  (  +  4  or  +5,  as  in  catalytic  processes  catalyzed  by  haem 
protein).  In  the  former  case,  E^  will  vary  from  ~  o  to  +  o  •  3  V.  In  the  latter, 
it  may  rise  as  high  as  +  i  -o  V.  There  is  insufficient  energy  in  the  infrared 
quanta  (~  1-3  V.)  effective  in  bacterial  photosynthesis  to  provide  the  gap 
created  by  the  reduced  chlorophyll  and  oxidized  Fe^  ^  or  Fe'  +  systems, 
which  are  separated  by  ~  i  -7  to  2  -o  V.,  depending  on  what  potentials  are 
assumed  for  the  reduced  chlorophyll.  Hence,  it  is  not  expected  that  the 
haem  protein  in  the  purple  photosynthetic  bacteria  will  be  oxidized  to  a 
valence  state  higher  than  3  + ,  so  that  the  high  positive  potential  required 
to  liberate  oxygen  (  +  o-8  V.)  is  not  reached.  In  this  way,  we  may  account 
for  the  absence  of  oxygen  as  a  product  in  bacterial  photosynthesis  and  for 
the  requirement  of  an  added  H-donor,  other  than  water.  On  this  view 


284  MARTIN   D.    KAMEN 

there  is  a  cyclic  process  involving  first  photo-oxidation  of  haem,  then 
thermal  reduction  by  reducing  equivalents  supplied  from  the  H-donor 
through  a  chain  of  intermediates. 

The  bases  for  these  suggestions  may  be  reviewed  briefly.  We  know 
from  a  voluminous  literature  on  electron  transfer  processes  in  systems 
containing  organic  metal  conjugates  or  chelates,  that  the  presence  of  a 
macrocyclic  resonating  system  can  induce  rapid  electron  exchange  between 
ions  otherwise  shielded  by  solvent  [39,  40].  In  all  photoactive  systems  a 
situation  exists  in  which  an  efficient  resonating  macrocyclic  system — 
porphyrin  or  a  derivative  reduced  porphyrin  ring — is  chelated  to  mag- 
nesium or  iron  as  the  central  metal  ion.  If  we  suppose  that  the  magnesium 
chelate  (chlorin)  is  close  to  the  iron  chelate  (haem),  then  excitation  of  the 


C  /e  C,         ,Fe  JZ  ,Fe 

— C  N  OH'       ^-^        -C—      N  OH-  ^°'^   — C  N  OH" 

I         /  I        /  I        / 


C  Mq''"'  -C  Mg' 


-C  N 


/  I         / 


Fig.  I.  Electron  transfer  reaction  proposed  as  part  of  the  primary  photo- 
chemical process  in  photosynthesis. 

magnesium  chelate  by  a  photon  which  gives  rise  to  the  characteristic  red 
absorption  band  will  result  in  an  excited  chlorophyll  system  with  energy 
equivalent  ~  i-8  to  2-0  e.V.  above  the  ground  state.  De-excitation  can 
occur  immediately  by  electron  transfer  from  the  neighbouring  haem 
system.  If  the  iron  complex  is  one  which  is  originally  in  the  formal  valence 
state  of  Fe"  +,  it  will  be  oxidized  to  a  formal  valence  of  Fe"  +^.  Likewise, 
the  chlorophyll  acquires  an  excess  negative  charge  which  makes  it  equiva- 
lent to  a  "semichlorinogen  "  (see  Fig.  i). 

This  process,  which  most  probably  leaves  both  products  in  their  ground 
states,  results  in  two  systems  separated  in  energy  content  by  an  amount 
close  to  the  original  energy  of  excitation  of  chlorophyll,  the  "mid- 
point" potential  of  the  semichlorinogen  system  is  more  reducing  than 
that  of  the  oxidized  haem  system  by  ~  i  -8  e.V.  Stabilization  against  back 
reaction  may  require  0-2-0-3  e.V.,  so  that  we  may  assume  safely  a  maxi- 
mum of  ~  I  •  5  e.V.  available  for  the  spread  in  potential. 


HAEM    PROTEIN    CONTENT    AND    FUNCTION    IN    RELATION    TO    STRUCTURE     285 

We  can  infer  consequences  of  such  a  process  by  analogy  with  many 
observations  available  in  the  literature,  even  though  nothing  is  known 
directlv  about  solution  chemistry  of  higher  oxidation  states  of  iron. 
George  and  Irvine  [41]  have  shown  that  metmyoglobin  treated  by  a 
varietv  of  oxidizing  agents  (peroxide,  permanganate,  chloriridate)  gives  a 
product  spectroscopically  identical  with  the  intermediate  "complex  II" 
formed  when  metmvoglobin  acts  as  a  peroxidase.  They  have  established 
the  £"|j  as  ~  +0-9  V.  This  \alue  is  ~  o- 1  V.  more  oxidizing  than  that  for 
the  standard  oxvgen  electrode.  Hence,  the  oxidizing  equivalents  present 
in  this  complex  can  extract  an  electron  from  water.  If  it  is  assumed  that 
the  haem  chelate-protein  complex  acquires  a  similar  E'^,  then  the  E'q  for 
the  semichlorinogen  formed  would  be  ~  —  o-6  to  —0-7  e.V.,  assuming 
I  •  ^  e.V.  as  the  value  of  J/s^  between  the  reducing  and  oxidizing  com- 
ponents. Such  a  strong  reducing  potential  would  be  more  than  sufficient 
to  provide  an  electron  transfer  step  to  pyridine  nucleotide  (£",',=  —0-3) 
which  could  be  coupled  to  formation  of  ATP  from  ADP  and  inorganic 
phosphate  ("photophosphorylation ")  [42,  43].  On  this  basis,  the 
"  photoreductase  "  of  San  Pietro  and  Lang  [44]  would  have  assigned  as  its 
substrate  the  semichlorinogen  as  the  photoreductant  generated  by  the  light 
reaction. 

The  reactions  initiated  by  the  presence  of  the  Fe'^-haem  complex 
depend  on  the  environment  presented,  h  simple  combination  of  chloro- 
phvll  and  haem  protein  would  have  only  the  possibility  of  back  reaction, 
or  reversal  of  the  process  shown  in  Fig.  i .  However,  if  an  enzymic  pathway 
(such  as  through  the  photoreductase  to  pyridine  nucleotide)  is  available  to 
remove  the  electrons  from  the  semichlorinogen,  then  it  can  be  expected 
there  will  be  a  preferential  flow  of  electrons  to  the  enzyme  substrate.  If  a 
source  of  electrons  is  present  in  the  haem  complex,  either  in  the  protein  or 
as  a  simple  ligand  (water),  then  reduction  of  the  Fe^^-haem  to  its  original 
state  would  occur  with  the  production  of  a  free  radical. 

The  evidence  available  from  paramagnetic  spin  resonance  studies  of 
the  metmyoglobin  oxidation  complex,  while  somewhat  ambiguous,  appears 
to  be  consistent  with  this  postulated  sequence  of  events.  Gibson,  Ingram, 
and  Nichols  [45]  have  shown  that  the  complex,  studied  by  George  and 
Irvine  and  produced  by  peroxidation  of  metmyoglobin,  exhibits  an  ESR 
signal  with  a  »- value  close  to  that  for  the  free  electron.  The  precise  value 
for  g  is  somewhat  smaller  than  expected  for  a  7T-electron  localized  at  a 
methine  bridge  carbon.  It  is  more  consistent  with  the  presence  of  a 
delocalized  electron  in  an  orbital  spread  over  the  whole  macrocyclic 
structure,  or  of  a  substrate  free  radical,  such  as  OH.  At  the  same  time  the 
signal  at  ff  =  6  corresponding  to  the  unpaired  electrons  at  the  Fe  site  is 
quenched,  indicating  a  change  in  the  bonding  at  the  metal  ion  site.  George 
and  Irvine  [41]  have  presented  evidence  for  this  change  as  a  production 


286  MARTIN   D.    KAMEN 

of  "  ferryl  iron"  (FeO  +  +)  or,  alternatively,  as  in  the  formulation  of  Fig.  i. 
At  present  it  is  not  necessary  to  postulate  production  of  a  ferryl  complex 
which  requires  movement  of  two  protons  off  the  ligand  water.  This  is 
indicated  in  Fig.  i  by  leaving  the  iron  in  a  formal  valence  state  equivalent 
to  Fe^+,  without  alteration  of  the  chemical  nature  of  the  ligand. 

An  alternative  reaction  scheme,  which  has  been  discussed  by  Calvin 
[46],  begins  with  loss  of  an  electron  from  excited  chlorophyll,  concomitant 
with  generation  of  a  positive  hole  in  the  chlorophyll  complex.  This 
postulate  necessitates  a  delayed  oxidation  of  the  cytochrome,  or  at  least 
reduction  of  some  acceptor,  such  as  pyridine  nucleotide  before  oxidation 
of  haem  iron  occurs.  There  is  no  conclusive  evidence  at  present  to  refute 
this  notion,  although  the  low-temperature  measurements  of  Chance  and 
Nishimura  [35]  on  the  photo-induced  oxidation  of  the  Chromatium  cyto- 
chrome system,  together  with  the  quantum  yield  data  of  Olson  and 
Chance  [36],  seem  to  favour  prior  oxidation  of  haem  iron  as  a  primary 
reaction  following  quantum  absorption. 

A  variety  of  interesting  problems  comes  to  mind  when  predictions  are 
attempted  for  the  chemical  behaviour  of  a  higher  oxidation  state  such  as 
postulated  in  Fig.  i .  Fe^  +,  which  is  isoelectric  with  Mn^  +  would  contain 
four  unpaired  electrons  distributed  in  the  five  3d  orbitals  of  the  metal  ion. 
Upon  combinations  with  the  ligand  groups,  at  least  two  could  pair  leaving 
two  unpaired  electrons  and  the  two  free  3d  orbitals,  so  that  the  Fe'*  + 
orbitals  could  hybridize  as  usual  to  give  the  octahedral  complexes  found 
for  Fe^  +  and  Fe^  +.  There  is  evidence  from  the  studies  on  magnetic 
susceptibility  of  metmyoglobin-peroxide  complexes  that  this  occurs  [47, 
48].  If  all  the  electrons  paired,  then  seven  orbitals  rather  than  six  would 
be  available  with  the  Fe*  +  in  a  diamagnetic  state.  A  ligand  such  as  OH  ", 
but  not  HoO,  would  favour  such  an  arrangement,  if  analogy  with  the  lower 
valence  forms  holds. 

The  stabilizing  effect  of  both  the  porphyrin  ring,  and  possibly  the 
protein  moiety,  in  a  higher  valence  form  can  be  inferred  from  many  well- 
known  examples  such  as  the  metal  porphyrin  complexes  of  silver,  bismuth, 
cobalt,  etc.  Winfield  and  King  have  emphasized  this  possibility  [49]. 
Dwyer  [40]  has  discussed  similar  situations,  especially  the  case  of  the 
nitroprusside  ion,  and  it  is  from  his  discussion  that  the  suggestion  of  a 
possible  diamagnetic  complex  structure  is  drawn. 

One  point  which  should  be  made  is  that  until  direct  data  can  be 
obtained  on  the  chemistry  of  iron  haem  chelates  when  in  a  state  of  oxida- 
tion formally  higher  than  Fe^  +,  it  is  unsafe  to  assume  that  a  molecule  such 
as  CO  is  specific  for  the  Fe  +  +  state.  The  criterion  of  a  light  sensitive  CO- 
binding  has  been  used  universally  to  establish  the  presence  of  ferrous  iron, 
but  the  possibility  that  Fe^  +  could  bind  CO  in  a  similar  fashion  is  not 
excluded. 


HAEM    PROTEIN    CONTENT    AND    FUNCTION    IN    RELATION    TO    STRUCTURE     287 

In  terms  of  the  chromatophore  structure,  we  may  visualize  an  aggregate 
of  bacteriochlorophyll  molecules  [15]  together  with  the  accessory  pigments 
such  as  carotenoids,  which  for  the  most  part  are  not  attached  to  molecules 
with  which  thev  can  undergo  irreversible  electron  transfer  reactions  upon 
excitation.  Most  of  these  chlorophylls  upon  excitation  merely  transfer 
energy  by  some  obligatory  mechanism,  such  as  inductive  resonance. 
Migration  of  the  energy  quantum  proceeds  through  the  pigment  aggregate 
until  a  particular  chlorophyll  molecule  is  reached  which  can  be  de- 
excited  by  electron  transfer  in  such  a  way  as  to  produce  the  two  electro- 
chemical svstems  postulated  above.* 

Of  all  the  molecules  mentioned  as  analogues  of  the  respiratory  chain 
previously,  the  most  plausible  reactants  which  can  produce  both  highly 
positive  electrochemical  systems  while  affording  the  possibility  of  stabiliza- 
tion are  the  haem  proteins.  They  possess  the  necessary  electron  source — 
the  metal  atom — the  necessary  protein  component  for  close  coupling  to  the 
chlorophyll  and  the  porphyrin  ring  for  stabilization.  Hence,  we  may 
assume  it  is  the  haem  protein  that  reacts  with  the  excited  chlorophyll, 
rather  than  a  quinone,  a  flavin,  or  a  pyridine  nucleotide. 

The  rest  of  the  reaction  sequence  requires  that  back  reaction  between 
the  reduced  chlorophvU  and  the  oxidized  haem  be  slow  relative  to  the 
reduction  by  reduced  chlorophyll  of  pyridine  nucleotide  or  some  other 
H-acceptor.  This,  as  mentioned  above,  may  be  the  role  of  the  "photo- 
nucleotide  reductase"  discovered  by  San  Pietro  in  chloroplasts.  A  similar 
enzyme  may  exist  in  bacterial  chromatophores,  but  so  far  has  not  been 
found.  It  may  be  that  the  H-acceptor  in  the  bacteria  is  not  a  pyridine 
nucleotide,  but  rather  a  SH-compound.  The  presence  of  large  quantities 
of  the  yellow  flavin  enzyme  which  can  not  only  function  as  a  haem  protein 
reductase,  but  also  can  show  very  great  diaphorase  activity  [27]  suggests 
that  some  SH-compound  may  be  involved;  on  the  basis  of  the  recent 
demonstrations  by  Massey  [50]  and  by  Sanadi  and  Searls  [51]  regarding 
the  possible   coupling  of  SH-groups  to  flavin   in    diaphorase,   it   seems 

*  A  possibility  is  that  such  a  reactive  site  is  chlorophyll  dimer.  S.  S.  Brody  (see 
Science  128,  835  (1958),  also  Brody,  S.  S.  and  Brody,  M.,  Arch.  Biochem.  Biophys. 
82,  161  (1959))  have  shown  that  in  many  plant  systems  an  appreciable  fraction  of 
the  chlorophyll  is  in  the  form  of  a  non-fluorescent  dimer.  While  it  is  not  clear  how 
such  a  complex  could  react  to  give  two  systems  sufficiently  stable  and  separated 
by  a  sufficient  equivalent  voltage,  participation  of  such  dimers  in  photochemistry 
certainly  is  not  excluded. 

It  is  also  of  interest  that  on  the  basis  of  an  approach  based  wholly  on  analysis 
of  fluorescence  depolarization,  G.  Weber  (see  ref.  [37],  p.  408)  has  arrived  at  a 
scheme  for  the  energy  conversion  mechanism  in  photosynthesis  which  is  similar 
to  the  one  proposed  in  this  report  in  requiring  resonance  transfer  to  bring  an 
excited  electron  in  a  chlorophyll  singlet  in  contact  with  an  electron  donor.  In  later 
steps,  he  postulates  separation  and  transfer  of  an  electron  from  the  chlorophyll- 
donor  complex  to  an  electron  acceptor. 


288  MARTIN    D.    KAMEN 

quite    reasonable    to    suggest    participation     of    an     SH-compound     or 
grouping. 

At  any  rate,  back  reduction  of  haem  by  reduced  chlorophyll  would  be 
slowed  because  both  reactants  would  be  expected  to  have  reached  their 
ground  states  after  the  primary  deactivation  by  electron  transfer,  e.g. 

Chi,,,  +  Haem-^,„d ^^— >ChlH  +  Haem°-,„, 

Hence,  some  activation  energy  would  be  required  to  initiate  the  back 
reaction,  despite  the  great  energy  difference  of  some  2  e.V.  tending  to 
drive  it.  The  presence  of  a  specific  enzyme  which  would  give  the  reduced 
chlorophyll  the  alternative  of  a  reduction  process  requiring  little  or  no 
excitation  energy  compared  with  the  uncatalyzed  back  reduction  of  the 
oxidized  haem  could  represent  one  of  the  stereochemical  requirements  for 
stabilizing  the  reduced  product  in  the  presence  of  the  oxidizing  system 
created  by  the  electron  transfer. 

With  the  electron  now  located  in  some  molecule  at  the  reducing  end 
of  a  "  respiratory"  chain,  electron  migration  through  the  flavins,  quinones, 
and  various  haem  enzymes  to  the  terminal  oxidant,  created  by  the  initial 
photochemical  electron  transfer,  would  complete  the  cycle.  As  we  will  hear 
in  the  other  papers,  this  type  of  electron  transport  coupled  to  the  quantum 
excitation  process  is  generally  assumed  to  be  the  basic  mechanism  for 
photophosphorylation.  An  impressive,  if  not  conclusive,  accumulation  of 
data  is  at  hand  to  support  this  notion.  Some  of  these  data  undoubtedly  will 
be  presented  at  this  session. 

An  alternative  scheme  presented  by  Hill  and  Bendall  [52]  suggests  that 
the  phosphorylation  step  is  coupled  to  a  flow  of  electrons  against  the 
potential  gradient  between  "tie  points"  on  the  respiratory  chain  repre- 
sented by  the  haem  proteins,  in  this  case,  cytochrome  6g  and  cytochrome/. 
The  cytochromes  are  assumed  to  be  involved  in  back  reactions  which 
restore  the  system  to  its  original  state  before  photo-excitation.  Hill  and 
Bendall  consider  this  type  of  mechanism  necessary  because  of  the  fact 
demonstrated  by  Arnon  et  al.  [53]  that  photophosphorylation  increases, 
rather  than  decreases,  the  yield  of  molecular  oxygen  in  the  chloroplast 
reaction.  However,  there  are  alternative  explanations  for  this  phenomenom, 
which  do  not  require  the  concept  of  "reductive"  phosphorylation.  Thus, 
if  phosphorylation  occurs,  as  we  have  discussed,  below  the  nucleotide 
level,  rather  than  between  nucleotide  and  haem,  then  the  consumption  of 
oxygen  precursor  assumed  by  Hill  and  Bendall  as  obligatory  for  photo- 
phosphorylation, will  not  occur.  Rather  the  assumption  required  is  that 
coupling  of  phosphate  esterification  to  reduction  of  pyridine  nucleotide 
increases  the  amount  of  pyridine  nucleotide  reduced  and  hence  of  oxygen 
precursor  formed. 

There  are  a  few  points  I  think  need  brief  discussion  relating  to  the 
generalization  of  haem  proteins  as  H-donors  in  the  fundamental  photo- 


HAEM    PROTEIN    CONTENT    AND    FUNCTION    IN    RELATION    TO    STRUCTURE     289 

chemical  process.  There  are  abundant  data  showing  that  in  Chyomotium 
[36]  and  in  R.  rubriim  [:;4]  the  primary  oxidation  involves  the  cytochrome 
c-type  haem  protein  which  has  been  isolated,  purified,  and  characterized 
in  our  laboratorv  [26,  27].  The  evidence  includes  not  only  kinetic  studies 
in  the  presence  and  absence  of  a  variety  of  inhibitors,  but  also  the  demon- 
stration that  the  cvtochrome  oxidation  involves  several  components,  one 
of  which  is  oxidized  as  rapidly  at  —  180"  as  at  room  temperature  [35]. 
Data  for  comparable  changes  in  oxidation  state  of  chloroplast  haem  pro- 
teins remain  meagre  [^s\. 

Now,  it  mav  be  that  there  are  qualitative  difi'erences  between  plant  and 
bacterial  photosyntheses,  primarily  owing  to  the  ability  of  the  former  to 
produce  molecular  oxygen.  There  certainly  may  be  factors  not  considered 
in  the  previous  discussion,  which  are  of  crucial  importance  in  the  process 
of  oxygen  production.  One  possibility  is  the  metal,  manganese,  which  is 
present  in  very  large  amounts  in  chloroplasts,  and  which  appears  to  be 
required  for  green  plant  photosynthesis,  whereas  it  does  not  seem  essential 
(at  least  in  more  than  trace  amounts)  in  bacterial  photosynthesis  [56]. 
Kessler  has  presented  some  preliminary  evidence  [57]  correlating  man- 
ganese with  the  oxygen-producing  system.  Very  recently,  Treharne, 
Brown,  Eyster,  and  Tanner  [^8]  have  found  that  an  electron  spin  resonance 
arising  from  manganese  ion  in  Chlorella  kept  in  the  dark  disappears  upon 
illumination,  and  that  this  phenomenon  can  be  linked  with  a  photo- 
oxidation  of  A  In  +  -\* 

It  is  also  known  from  a  discovery  by  the  late  R.  Emerson  that  two 
quanta  can  co-operate  over  relatively  long  time  intervals  to  increase  the 
yield  of  molecular  oxvgen.  In  the  chromatophore  there  is  a  relatively  small 
ratio  of  chlorophvU  to  protein.  From  Table  I  we  can  see  the  ratio  of 
chlorophvll  to  cvtochrome  is  ~  15.  This  ratio  is  usually  greater  than 
several  hundred  in  most  chloroplasts  [15].  Similarly  the  ratio  of  chloro- 
phyll to  pyridine  nucleotide  is  20  in  the  Chrotnatium  chromatophore, 
whereas  it  can  be  no  less  than  2500  in  spinach  chloroplasts  [59].  This 
greatlv    increased  ratio  of  chlorophyll   to   other  components   in   oxygen 

*  We  may  recall,  if  only  in  a  footnote,  the  remarkable  reaction,  first  noted  by 
R.  H.  Kenten  and  P.  J.  G.  Mann  in  1949  and  studied  since  by  them  (see  Biocliem.  J. 
45»  255;  46,  67;  52,  125;  6l,  279)  in  which  manganous  ion  is  oxidized  photo- 
chemically  in  the  presence  of  plant  peroxidase,  hydroperoxide,  and  a  peroxidase 
substrate,  such  as  a  monohydric  phenol.  Pyrophosphate  is  added  to  trap  the 
manganic  ion  formed  as  the  insoluble  manganic  pyrophosphate.  These  authors 
have  found  that  chloroplasts  can  catalyze  this  reaction,  and  suggest  that  in  photo- 
synthesis a  cycle  occurs  involving  alternate  photo-oxidation  of  manganous  ion  to 
manganic  and  reduction  by  plant  material  of  manganic  to  manganous.  W.  F. 
Andreae  (see  Arch.  Biochem.  Biophys.  55,  584)  has  determined  that  this  reaction, 
which  depends  on  the  presence  of  catalase  or  peroxidase,  can  be  induced  by  cataly- 
tic amounts  of  a  hydrogen  donor  in  the  presence  of  a  variety  of  light  sensitizers. 
He  has  noted  further  the  nature  of  hydrogen  donors  most  effective  in  catalysis. 

VOL.  n. — u 


290  MARTIN    D.    KAMEN 

producing  systems  may  be  a  consequence  of  the  need  to  funnel  more  than 
one  exciton  to  a  given  reaction  site  to  produce  molecular  oxygen.  It  is 
reasonable  to  suppose  that  in  a  process  involving  multiple  electron  dona- 
tion, as  in  the  production  of  molecular  oxygen,  a  mechanism  for  delivering 
the  energy  of  more  than  one  quantum  to  an  active  site  may  be  required. 

Perhaps  the  puzzling  inability  of  the  green  sulphur  bacteria  to  produce 
molecular  oxygen,  despite  their  utilization  of  quanta  with  energies  as  high 
as  those  absorbed  effectively  by  green  plants,  is  owing  to  relatively  low 
chlorophyll  content. 

Returning  to  haem  protein  function  in  photosynthesis,  the  failure  to 
observe  shifts  in  spectra  in  the  chloroplast  upon  illumination  which  can 
be  interpreted  as  oxidation  of  haem,  can  be  rationalized  on  the  basis  of  the 
reaction  scheme  of  Fig.  i.  The  spectroscopic  methods  employed  at  present 
permit  only  observation  of  changes  associated  with  the  ferrous  to  ferric 
transition.  Transitions  from  ferric  haem  to  ferryl  or  pentavalent  iron  haem 
do  not  involve  changes  in  characteristic  maxima  in  difference  spectra 
which  are  sufficient  to  allow  detection  by  present  procedures.  If  the  cyto- 
chrome f  (chloroplast  cytochrome  c)  is  in  its  ferric  state  to  begin  with, 
then  the  photo-oxidation  may  proceed  to  the  higher  valence  state  of  iron, 
required  for  generation  of  the  system  which  oxidizes  water,  without  being 
accompanied  by  a  visible  shift  in  absorption. 

Leaving  sheer  speculation  for  the  more  solid  ground  of  physical 
chemistry,  it  should  be  emphasized  that  our  knowledge  of  the  chemical 
potentialities  of  haem  proteins  is  limited ;  it  is  derived  solely  from  studies 
of  specimens  obtained  from  a  restricted  set  of  unique  biochemical  struc- 
tures— the  mitochondrial  respiratory  systems.  As  discussed  elsewhere  [60] 
haem  proteins  derived  from  a  variety  of  bacterial  and  plant  sources,  where 
metabolism  is  in  no  way  associated  with  obligatory  reduction  of  oxygen, 
exhibit  a  great  diversity  of  physico-chemical  properties  quite  unexpected 
on  the  basis  of  the  classical  cytochrome  preparations.  There  is  a  great 
urgency  to  isolate  in  pure  form  in  sufficient  quantities  as  many  of  these 
haem  proteins  as  possible  to  enable  intensive  chemical  studies. 

As  an  example.  I  may  cite  the  unusual  haem  protein  we  know  as 
"RHP",  which  appears  to  be  present  only  in  the  purple  photosynthetic 
bacteria  [19].  R.  J.  P.  Williams  has  presented  some  elegant  studies  on 
haem  models  from  which  he  has  been  able  to  make  some  remarkable 
correlations  between  oxidizing  potential,  spectra,  magnetic  proper- 
ties, and  haem  binding  and  structure  in  the  haem  proteins  [61].  RHP 
represents  a  class  of  haem  protein,  hitherto  unknown,  which  can  be 
rationalized  in  the  Williams  scheme,  provided  one  of  the  ligands  in  the 
co-ordination  position  out  of  the  porphyrin  plane  is  a  group  with  a  rela- 
tively high  proton  affinity  (e.g.  carboxyl,  hydroxyl,  etc.).  RHP  is  a  myo- 
haematin  protein  with  a  typical  myoglobin-like  spectrum  and  electro- 


HAEM    PROTEIN    CONTENT   AND    FUNCTION    IN   RELATION    TO    STRUCTURE     29 1 

chemical  potential.  It  cannot  bind  oxygen  reversibly,  but  appears  to 
function  as  an  oxidase  or  as  an  electron  carrier  in  the  photorespiratory 
chain  [27].  It  contains  2  haems  per  molecule  (MW  =  28  000-35  °°°' 
depending  on  the  source).  One  or  both  of  these  haems  may  be  bound  by 
only  a  single  thio-ether  linkage  such  as  is  characteristic  of  cytochrome  c, 
which  has  two  such  links  to  a  single  haem. 

It  is  evident  that  this  protein  provides  a  good  test  object  for  the  present 
theories  about  haem  protein  structure.  For  instance,  Williams  has  pre- 
dicted RHP  would  be  a  "high-spin"  complex  [62].  His  prediction  seems 
to  be  correct  on  the  basis  of  work  by  A.  Ehrenberg  (unpublished)  using  a 
crystalline  sample  of  pure  RHP  provided  by  Dr.  Horio  and  myself. 

Further  work  on  the  amino  acid  sequence  of  haem  peptides  obtained 
from  RHP  as  well  as  from  other  bacterial  cytochromes,  should  provide 
important  data  for  rationalizing  the  structural  aspects  of  haem  protein 
chemistry,  and  is  now  proceeding  in  our  laboratory.  We  expect  that  work 
on  the  bacterial  and  plant  cytochromes  will  greatly  modify  and  extend 
present  concepts  of  the  chemistry  inherent  in  the  combination  of  iron 
tetrapyrrolic  chelates  and  proteins. 

However,  it  is  unlikelv  that  any  future  developments  will  support  a 
notion,  such  as  put  forward  by  Arnon  [63],  that  chloroplast,  or  any, 
cytochrome  in  its  ¥e^  ^  state,  will  possess  sufficient  positive  electro- 
chemical potential  to  extract  electrons  from  the  hydroxyl  ion  or  water. 
Hill  and  Bendall  [^2]  point  the  fallacy  of  this  notion  properly  in  reference 
to  the  cytochromes  known  at  present  to  exist  in  chloroplasts — namely,  the 
r-tvpe  haem  protein,  cytochrome  /,  and  the  6-type  haem  protein — cyto- 
chrome b^.  George  and  Irvine  have  found  [64]  that  mammalian  cyto- 
chrome r,  as  a  representative  of  the  haemochrome  type  haem  proteins, 
does  not  react  with  strong  oxidizing  agents  to  give  the  higher  valence 
(Fe'*  +)  form  of  haem,  as  appears  to  be  the  case  with  peroxidase  or  metmyo- 
globin.  It  would  seem,  then,  that  we  must  search  for  the  sort  of  haem 
protein  postulated  in  the  scheme  of  Fig.  i  among  the  haem  compounds  of 
plant  chloroplasts  which  are  contained  in  the  haem  fraction  which  is  not 
accounted  for  as  either  cytochrome  /  or  cytochrome  b^. 

So  far,  the  only  plausible  haem  compound  found  which  resembles 
myoglobin  and  other  myohaematin  proteins  is  the  RHP  of  the  bacterial 
chromatophores.  Its  presence  in  chloroplasts  remains  to  be  demonstrated. 
However,  even  if  a  myoglobin-type  compound  is  absent,  there  are  still 
both  peroxidases  and  catalases  present  in  appreciable  quantities  in  chloro- 
plast tissues ;  any  of  these  may  reveal  the  requisite  properties  upon  isolation 
and  purification.*  Even  the  attainment  of  a  Fe^-  state  in  cytochrome/  is 

*  It  is  possible  that  the  peroxidase  and  catalase  activities  found  in  chloroplasts 
are  functional,  at  least  in  part,  in  the  manganese  cycle  suggested  by  Kenten  and 
Mann  (see  previous  footnote). 


292  MARTIN    D.    KAMEN 

not  excluded.  In  the  reactions  studied  by  George  and  Irvine  movement  of 
two  protons  is  required  and,  most  likely,  a  "ferryl"  (FeO  + +)  state  is 
formed.  This  is  not  required  in  a  photo-induced  electron  transfer  reaction 
of  the  type  shown  in  Fig.  i.  It  may  be,  therefore,  that  the  only  way  to 
reach  the  Fe^  +  state  in  the  chloroplast  cytochrome,  or  other  haem  protein, 
is  by  a  photochemical  oxidation  which  proceeds  by  electron  transfer 
unaccompanied  by  proton  transfer. 

The  search  for  new  haem  proteins  in  photosynthetic  tissues  and 
intensive  study  of  their  structures  should  be  intensified.  At  the  same  time, 
experiments  designed  to  reveal  photochemical  capacities  of  haem  proteins 
should  be  pursued.  The  present  status  of  knowledge  about  haem  proteins 
seems  well  suited  to  application  of  Charles  Darwin's  admonition  that 
"without  speculation  there  is  no  good  and  original  observation". 

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Disc.  Faraday  Soc.  No.  27  (1959). 

2.  Geller,   D.   M.,   "  Photophosphorylation  by  Rhodospirilhim  nihnini  Prepara- 
tions", Ph.D.  Thesis,  Harvard  University,  Cambridge,  Massachusetts  (1957). 

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17.  Goodwin,  T.  W.,  Ibid.  24,  313  (1956). 

18.  Kamen,  M.  D.,  Bacterial.  Rev.  19,  250  (1955). 

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20.  Martin,  E.  M.,  and  Morton,  R.  K.,  Biochem.  J.  64,  221  (1956). 

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(1958). 

46.  Calvin,  M.,  in  McCollum-Pratt  Sympos.  "Light  and  Life",  ed.  B.  Glass  and 
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51.  Searls,  R.  L.,  and  Sanadi,  D.  R.,jf.  biol.  Chem.  235,  PC32  (i960). 

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294  MARTIN   D.    KAMEN 

62.  Williams,  R.  J.  P.,  in  "  Intl.  Sympos.  on  Haematin  Enzymes"  ed.  R.  Lemberg, 
R.  K.  Morton,  and  J.  Falk.  Canberra,  Australia;  Pergamon  Press,  London 
(in  press). 

63.  Arnon,  D.  I.,  Nature,  Loud.  184,  10  (iqSQ)- 

64.  George,  P.,  and  Irvine,  D.  H.,  in  "  Symposium  on  Co-ordination  Chemistry", 
Copenhagen,  C.  Christensen,  publ.,  135,  (i953)- 

Discussion 

Chance  :  This  is  a  very  exciting  mechanism  which  Dr.  Kamen  has  presented 
and  I  am  sure  that  it  is  just  as  he  says :  without  speculation  we  can't  get  anywhere. 
But  I  want  to  pull  him  back  a  little  bit  if  I  may  because  he  has  galloped  off  with 
the  wrong  haem  protein.  I  think  this  mechanism  is  intuitively  based  on  RHP  being 
the  initial  electron  donor  and  I  think  the  initial  electron  donor  is  cytochrome  c.  In 
studies  of  Chrotttatium  even  at  temperatures  of  liquid  nitrogen  we  observed  on 
illumination  of  the  bacteria  the  disappearance  of  a  band  which  has  a  peak  at 
420  m/x  which  suggests  that  the  primary  event  involves  a  c-type  cytochrome  and 
not  RHP. 

Kamen:  What  I  was  actually  talking  about  was  oxygen  evolution.  In  the 
bacterial  chromatophores,  we  do  not  have  oxygen  evolution  so  there  is  no  necessity 
to  go  to  the  higher  valency  states.  You  just  start  with  the  Fe-  +  and  go  to  the  Fe''  +. 
I  did  say  that  the  system  produced  in  bacteria  was  not  sufficiently  electropositive 
to  produce  the  Fe*  +  state.  One  could  suppose  that  if  in  green  plants  cytochrome 
begins  in  the  Fe^  +  state,  you  won't  see  the  change  because  it  involves  going  from 
Fe^  ^  to  Fe*,  whereas  in  the  bacteria  which  are  anaerobic,  the  steady  state  of  the 
cytochromes  is  predominantly  Fe-  +.  The  cytochromes  would  be  reduced  in  the 
dark,  and  when  you  add  light  you  would  go  from  the  Fe-  +  to  the  higher  valency 
state,  maybe  even  Fe*  +  for  all  I  know,  but  certainly  to  Fe^  +  and  you  would  see 
the  usual  difference  spectrum.  I  should  say  that  Dr.  Chance's  group  has  provided 
the  best  evidence  for  this  reaction  in  the  bacteria  by  showing  that  it  goes  at  liquid 
air  temperature.  As  regards  the  RHP  question  I  should  say  that  cytochrome  r-type 
proteins  cannot  be  peroxidized  to  Fe*  +.  Philip  George  tried  this  with  cytochrome 
c :  you  take  cytochrome  c  and  add  permanganate  to  it  and  all  you  get  is  destruction 
of  protein,  unlike  what  happens  with  myoglobin,  so  I  think  this  makes  it  improb- 
able that  cytochrome  c  is  involved  in  the  primary  reaction.  I  think  it  must  be  some- 
thing like  RHP,  or  even  something  quite  remote  from  a  haem  protein  like  man- 
ganese. I  don't  think  there  is  any  way  of  telling  at  this  time.  I  think  we  should  be 
looking  for  a  myohaematin-type  protein  in  green  plants.  There  are  catalases  and 
peroxidases  present  in  chloroplasts  and  chromatophores.  These  may  be  the  things 
which  are  reacting  in  trace  amounts,  and,  if  they  are  you  might  never  see  a  spectrum 
corresponding  to  the  reactions  of  these  components  but  rather  only  the  results 
arising  eventually  from  oxidation  of  cytochrome  c. 


Observations  on  the  Formation  of  the  Photosynthetic 

Apparatus  in  Khodospirillum  ruhriim  and  Some  Comments 

on  Light-Induced  Chromatophore  Reactions* 

Douglas  C.  Pratt,  Albert  W.  Frexkel,    and  Donald  D.  HICK^L\^" 

Department  of  Botany,  University  of  Minnesota, 
Minneapolis  14,  Minn.,  U.S.A. 

Formation  of  photochemically  active  chromatophores  in 

the  dark 

Cells  of  R/iodospiri/hini  rnhruni  when  cultured  aerobically  in  the  dark 
were  found  to  be  free  of  chromatophores  by  Schachman  et  al.  [i].  Vatter 
and  Wolfe  [2]  and  Hickman  and  Frenkel  [3]  confirmed  this  observation. 
I\Iore  recently,  Cohen-Bazire  et  al.  [4]  demonstrated  that  when  this 
organism  was  grown  at  very  low  oxygen  tensions  it  was  capable  of  produc- 
ing chlorophyll.  In  a  more  detailed  study  Cohen-Bazire  and  Kunisawa  [5] 
studied  chlorophyll  formation  in  dark-  and  light-grown  organisms  and 
also  measured  light-induced  phosphorylation  carried  out  by  chromato- 
phores isolated  from  these  organisms.  In  the  study  presented  here  we  have 
concentrated  on  the  efi^ects  of  oxygen  tension  on  growth,  chlorophyll 
formation,  and  on  structure  of  dark-grown  organisms.  We  present  some 
preliminary  observations  [6]  which  we  intend  to  expand  and  present  in 
greater  detail  at  a  later  time. 

EFFECT    OF    OXYGEN    TENSION    ON    GROWTH    AND    CHLOROPHYLL 
FOR\L\TION    BY    DARK-GROWN    CELLS 

In  a  previous  studv  [7]  it  was  observed  that  Rhodospirillum  grown  either 
in  the  dark  or  in  the  light  had  a  pronounced  CO2  requirement  which, 
except  for  large  additions  of  either  yeast  extract  or  casein  hydrolysate, 
could  not  be  replaced  by  many  common  metabolic  intermediates  which 
were  added  either  singly  or  in  various  combinations  to  CO2  free  culture 
media.  This  CO.,  requirement  was  found  to  saturate  at  about  i"„  COg, 
and  all  gas  mixtures  employed  subsequently  were  enriched  with  either  i 
or  5*^1,  COo.  In  our  first  experiments  dark-grown  cultures  were  gassed 

*  Work  supported  by  a  grant  from  the  Graduate  School  of  the  University  of 
Minnesota,  and  by  grants  from  the  National  Institute  of  Allergy  and  Infectious 
Diseases  (E-2218),  and  the  National  Science  Foundation  (G-c 


296       DOUGLAS    C.    PRATT,    ALBERT    W.    FRENKEL,    AND    DONALD    D.    HICKMAN 

with  air  enriched  with  CO.,,  and  the  rate  of  chlorophyll  formation  and 
growth  was  varied  by  adjusting  the  flow  of  air  through  the  spargers  of  the 
culture  tubes.  With  this  method,  however,  it  was  difficult  to  obtain 
reproducible  results,  except  for  the  conditions  when  the  cultures  were 
gassed  rapidly*  with  air  or  with  nitrogen.  To  obtain  more  reproducible 
results  we  secured  the  following  gas  mixtures:  0-5,  1-55,  and  7-3% 
oxygen  in  nitrogen  supplemented  with  1%  CO2  (the  values  for  oxygen 
indicated  here  were  obtained  by  mass  spectrometric  analysis).  All  cultures 
were  gassed  at  sufficiently  rapid  rates*  so  that  a  further  increase  in  gassing 


Fig.  I.  Effect  of  oxygen  tension  in  the  gassing  mixtures  on  growth  of  R. 
riibriwi  in  the  dark  (based  on  change  in  packed  cell  volume  per  ml.  of  culture 
suspension)  measured  four  days  after  inoculation.  Initial  concentration  o-ii  /nl. 
of  packed  cells  per  ml.  of  culture  suspension.  Rate  of  gassing  through  spargers  at 
35  ml.  of  gas  mixture  per  minute  per  40  ml.  of  culture  suspension  in  100  ml. 
culture  tubes.  Culture  tubes  incubated  at  30". 


rates   caused    little    if   any   effect    on    rates   of  growth    and    chlorophyll 
production. 

Figure  i  shows  the  effect  of  oxygen  tension  in  the  gassing  mixtures  on 
total  growth  per  culture  as  measured  after  four  days  of  incubation  in  the 
dark.  Growth  was  measured  by  measuring  changes  in  cell  mass  or  by 
measuring  changes  in  turbidity  at  680  m/x  calibrated  against  packed  cell 
volumes  of  aliquots  of  the  culture  suspensions  which  were  centrifuged  for 
35  minutes  at  2000  times  gravity  in  Hopkins  vaccine  tubes.  While  growth 
saturates  at  about  2",,  oxygen  (Fig.  1),  chlorophyll  concentration  on  a  cell 
volume    basis    decreases    with    increasing   oxygen    tensions    and    reaches 

*   35  40  ml.  of  gas  per  minute  per  40  cm^  of  liquid  volume. 


OBSERVATIONS    ON    THE    FORMATION    OF    THE    PHOTOSYNTHETIC    APPARATUS      297 

practically  zero  at  7-8"  o  oxygen  in  the  gassing  mixture  (Fig.  2).  A  com- 
bination of  the  curves  in  Figs,  i  and  2  results  in  the  curve  of  Fig.  3  which 
shows  that  chlorophyll  concentration  per  volume  of  culture  reaches  a 


10- 


0) 

u 

tt 

^ 

CD 

6 

•V 

-< 



4 

>^ 

cz 

Cl. 

0 

2 

1_ 

_o 

0 

Va 


u 


0      2 


v/- 


20 


%  Oxygen 


Fig.  2.  Effect  of  oxygen  tension  in  the  gassing  mixtures  on  the  chlorophyll 
content  (in  jug  bacteriochlorophyll  per  ^\.  of  packed  cells)  of  dark-grown  cells 
measured  four  days  after  inoculation.  Original  inoculum  contained  0-024  P-S- 
bacteriochlorophyll  per  jul.  of  packed  cells.  Culture  conditions  identical  with  those 
indicated  for  Fig.  i. 


Fig.  3.  Effect  of  oxygen  tension  in  the  gassing  mixtures  on  the  chlorophyll 
content  per  litre  of  bacterial  culture  suspension  incubated  in  the  dark,  measured 
four  days  after  inoculation.  This  graph  represents  a  combination  of  Figures  i  and  2. 


Fig.  4.  Sections  of  cells  cultured  anaerobically  in  the  dark  for  96  hours  at  30'', 
gassed  with  5%  CO2  in  N2  (residual  O2  :  0-036",,)  at  a  rate  of  35  to  40  ml.  of  gas 
per  hour  (cf.  Fig.  14,  ref.  [3]).  The  bacteriochlorophyll  content  at  the  end  of  the 
period  of  9-5  fig.  per  /xl.  of  packed  cells.  The  culture  had  been  derived  from  an 
inoculum  of  almost  colourless  aerobically  dark-grown  cells  which  contained  0-024 
/xg.  of  bacteriochlorophyll  per  /A.  of  packed  cells.  The  section  of  the  anaerobically 
cultured  cells  shows  abundant  and  distinct  chromatophores  (C).  Magnification  as 
indicated  for  Fig.  6. 

Figs.  5  and  6.  Sections  of  cells  cultured  for  66  hr.  aerobically  in  the  dark  at 
30°  (cf.  Fig.  9,  ref.  [31]).  The  cultures  were  gassed  with  5^0  CO2  in  air;  the  gassing 
rate  was  somewhat  slower  than  indicated  in  the  text  and  synthesis  of  a  small 
amount  of  chlorophyll  had  taken  place.  A  few  scattered  chromatophores  (C)  are 
apparent  in  some  of  the  cells.  A  lamellar  system  (L)  of  unknown  composition  and 
function  (cf.  Figs.  21-25,  ref.  [3])  also  can  be  observed. 


OBSERVATIONS    ON    THE    FORMATION    OF    THE    PHOTOSYNTHETIC    APPARATUS      299 

maximum  of  about  2"  o  oxygen  even  though  chlorophyll  concentration  per 
unit  volume  of  cells  is  highest  under  near  anaerobic  conditions  (0-036% 
Oo)  (Fig.  2).  The  observed  maximum  in  chlorophyll  concentration  per 
volume  of  culture  at  about  2%  is  due  to  the  rapid  growth  of  the  organism 
at  this  oxygen  tension  where  chlorophyll  synthesis  is  only  partly  inhibited. 
As  the  oxygen  tension  is  raised  beyond  this  level,  there  is  no  appreciable 
change  in  the  amount  of  growth  but  chlorophyll  synthesis  is  increasingly 
inhibited  and  comes  to  a  standstill  at  about  8%  oxygen. 

STRUCTURE    OF    DARK-GROWN    CELLS    AS    REVEALED    BY    ELECTRON 

MICROSCOPY 

Electron  micrographs  of  thin  sections  of  cells  grown  in  the  dark  at 
various  oxygen  tensions  reveal  the  following  picture.  Under  highly  aerobic 
conditions  the  nearly  complete  absence  of  chlorophvll  is  accompanied  by 
a  virtually  complete  lack  of  chromatophores  in  the  sectioned  cells  (Figs.  15, 
6).  Cells  derived  from  near  anaerobic  cultures  gassed  with  nitrogen  con- 
taining 0-036/0  oxygen  and  5",,  carbon  dioxide  contained  a  high  concen- 
tration of  bacteriochlorophyll  (Fig.  2)  and  when  sectioned  revealed  a 
great  abundance  of  chromatophores  (Fig.  4).  It  was  possible  to  isolate 
photochemically  active  chromatophores  from  such  cells  (Table  I)  which 
could  carry  out  both  light-induced  formation  of  ATP*  and  the  photo- 
reduction  of  DPN  in  the  presence  of  suitable  cofactors.  Observations  on 
cultures  grown  at  intermediate  oxygen  tensions  indicate  a  decrease  in 
chromatophore  concentration  with  increasing  oxvgen  tension.  It  appears 
that  both  chlorophyll  concentration  and  the  abundance  of  chromatophores 
can  be  controlled  w-ithin  certain  limits  by  controlling  the  oxygen  tension 
at  which  the  organisms  are  grown  in  the  dark. 

PHOTOCHEMICAL    ACTIVITIES    OF    DARK-GROW'N    CELLS    AND    OF    CELL- 
FREE    PREPARATIONS    DERIVED    FROM    THEM 

Intact  cells,  derived  from  initially  chlorophyll-free  cells,  which  had 
developed  chlorophyll  in  the  dark  at  reduced  oxygen  tensions,  show  an 
active  CO.,  fixation  (Table  I).  Cell-free  preparations  obtained  from  such 
cells  carry  out  active  light-induced  phosphorylation  and  the  photo- 
reduction  of  diphosphopyridine  nucleotide  [6,  8].  Cohen-Bazire  and 
Kunisawa  [5]  who  have  carried  out  similar  work  with  R.  riibriim  also  have 

*  Abbreviations  used:  ADP,  adenosine  diphosphate;  ATP,  adenosine  tri- 
phosphate; Pi,  inorganic  orthophosphate ;  DPN,  DPNH,  diphosphopyridine 
nucleotide  and  its  reduced  forms ;  TPN,  triphosphopyridine  nucleotide ;  FMN, 
FMNH2,  flavin  mononucleotide  and  its  reduced  form;  PPNR,  photosynthetic 
pyridine  nucleotide  reductase. 


300       DOUGLAS    C.    PRATT,    ALBERT    W.    FRENKEL,    AND    DONALD    D.    HICKMAN 

examined  rates  of  light-induced  phosphorylation  and  have  reported  rates 
for  preparations  from  both  dark-  and  light-grown  cultures  which  are  a 
good  deal  higher  than  the  ones  reported  here,  and  in  fact  appear  to  be  the 
highest  ones  reported  in  the  literature  for  bacterial  photo-phosphorylation. 
Vernon  and  Ash  [9]  have  reported  that  light-induced  reduction  of 
DPN  by  extracts  from  R.  riibrum  is  increased  in  the  presence  of  magnesium 
ions.  We  have  also  observed  a  small  stimulatory  effect  of  magnesium  (or 
manganese)  ions  on  the  photoreduction  of  DPN.  We  have  noticed,  how- 
ever, that  this  effect  is  much  more  pronounced  with  cell-free  preparations 

TABLE  I 

Photochemical  Activity  of  Intact  Cells  of  Rhoduspirillum  rubrum  and  of 
Chromatophores   Derived  from   Cells   Cultured   in  the   Dark  and  Light 

Initial  rates  (at  light  saturation) : 

/xM 

hr.   X   juM  bacteriochlorophyll 
Preparations  from  cultures  grown  in  the 

A 

Dark  Light 


215 


Intact  cells : 

CO  2  uptake 

30* 

Isolated  chromatophores : 

(a)  ATP  formation 

184 

(6)  DPN  reduction: 

+  Mg  +  +  (final  concn.: 

I  -3    X     lO"^  m) 

129 

-  Mg  +  + 

6-8 

21-6 

i8-8 


*  Corrected  for  CO 2  production. 

from  dark-grown  cells  (Table  I).  Repeated  washing  of  the  dark-grown 
preparations  almost  completely  eliminates  the  photoreducing  activity,  but 
such  activity  can  be  restored  (more  or  less  completely)  by  the  addition  of 
magnesium  (or  manganese)  salts.  Repeated  washing  of  preparations 
obtained  from  light-grown  cells  produces  a  much  more  gradual  loss  of 
activity  which  can  be  restored  by  the  addition  of  magnesium  ions.  We 
believe  that  this  behaviour  toward  magnesium  (or  manganese)  may  reflect 
some  subtle  differences  in  the  particles  derived  from  light-  and  dark-grown 
cells,  indicating  that  either  less  magnesium  (or  manganese)  is  bound  by  the 
chlorophyll  containing  particles  from  dark-grown  cells,  or  that  it  is  leached 
out  more  easily.  We  hope  to  obtain  more  information  about  this  ion  effect 
in  the  course  of  work  on  the  development  of  the  bacterial  chromatophore. 


OBSERVATIONS    ON    THE    FORMATION    OF    THE    PHOTOS YNTHETIC    APPARATUS      3OI 

Some  comments  on  light-induced  pyridine  nucleotide 
reduction  by  bacterial  chromatophores 

REDUCTION    OF    PYRIDINE    NUCLEOTIDES    BY    CHROMATOPHORES    AND 
BY    MITOCHONDRIA 

In  line  with  the  discussion  carried  out  by  Dr.  Chance  and  by  other 
participants  at  this  meeting  on  the  reduction  of  pyridine  nucleotides  by 
mitochondria  in  the  presence  of  ATP  (reaction  i)  (10-12), 

Succinate  +  DPX  -  +  nATP  ^^ 

fumarate  +  DPNH  +  H  ++  nADP  +  nP,         (i) 

one  may  compare  this  reaction  with  the  hght-induced  reduction  of  di- 
phosphopyridine  nucleotide  carried  out  by  R.  ruhrum  chromatophores 
which  carry  out  the  following  reactions  [8,  13-15]: 

light  .    . 

ADP^P ^ATP  (2) 

DPX  -  +  FMXH,  ^  DPXH  +  H    +  FAIN  (3) 

dark 
light 

DPX  ^  +  succinate  7 — ^  DPXH  +  H  *  +  fumarate  (4) 

dark 

Consequently,  it  might  be  postulated  that  the  photoreduction  of  DPX 
(reaction  4)  bv  R.  ruhrum  chromatophores  could  be  due  to  the  participation 
of  ATP  formed  according  to  reaction  2,  and  that  the  ATP  thus  formed 
would  be  utilized  for  the  dark  reduction  of  DPX  according  to  reaction  4. 
It  is,  therefore,  of  interest  to  examine  the  existing  evidence  for  and  against 
such  a  reaction  scheme  involving  a  combination  of  reactions  2  and  i  to 
account  for  the  light-induced  reduction  of  DPX  by  bacterial  chromato- 
phores. 

The  photoreduction  of  DPX  (reaction  4)  by  bacterial  chromatophores 
and  the  dark  reduction  of  DPX  (reaction  i)  by  mitochondria  have  the 
following  characteristics  in  common:  both  require  DPX,  succinate  and 
possibly  Alg*^  (or  Aln*^),  while  ADP  appears  to  be  inhibitory  to  DPX 
reduction  in  both  svstems,  although  there  is  not  complete  agreement  on 
the  behaviour  of  the  chromatophore  system,  as  will  be  described  later. 
The  two  reaction  svstems  appear  to  differ  in  the  following  respects: 
(rt)  the  mitochondrial  system  requires  ATP,  but  such  a  requirement  has 
not  been  established  for  the  chromatophore  system,  although  the  partici- 
pation of  endogenous  ATP  cannot  be  excluded  at  the  present  time ;  {b)  it 
appears  that  added  ATP  will  not  substitute  for  light  in  bringing  about  a 
dark  reduction  of  DPX  by  washed  R.  ruhrum  chromatophores  [15];  {c)  in 
mitochondria  there  is  a  direct  relationship  between  the  amount  of  DPX 


302       DOUGLAS   C.    PRATT,    ALBERT   W.    FRENKEL,    AND   DONALD   D.    HICKMAN 

reduced  and  the  amount  of  ATP  utilized  in  the  reaction.  Chromatophores 
in  the  hght  may  form  only  ATP  (reaction  2),  or  only  reduced  DPN 
(reaction  4),  or  both  ATP  formation  and  DPN  reduction  may  occur 
simultaneously  in  the  presence  of  the  required  cofactor  for  both  reactions. 
Vernon  and  Ash  [13]  have  studied  reactions  2  and  4  in  some  detail  and 
have  found  that  the  amount  of  inorganic  phosphate  esterified  in  the  light 
was  the  same  regardless  of  whether  their  preparations  carried  out  a 
simultaneous  reduction  of  DPN,  and  they  concluded  that  the  light- 
induced  phosphorylation  reaction  and  the  photoreduction  of  DPN  occur 
independently  of  each  other.  We  have  observed,  on  the  other  hand,  that 
the  rate  of  photoreduction  of  DPN  is  inhibited  under  conditions  where  the 
preparations  carry  out  the  light-induced  formation  of  ATP  at  the  same 
time  [15],  indicating  a  possible  relationship  between  these  two  processes, 
but  this  interaction  appears  to  be  different  from  the  one  exhibited  by  mito- 
chondria. Only  a  more  detailed  analysis  of  the  kinetics  of  the  chromato- 
phore  reactions  can  clarify  the  conflicting  reports  in  the  literature. 

As  mentioned  earlier  there  is  disagreement  about  the  effect  of  ADP  on 
the  photoreduction  of  DPN  by  R.  rubrimi  chromatophores.  Vernon  and 
Ash  [9]  initially  reported  that  ADP  inhibited  the  photoreduction  of  DPN ; 
in  a  later  paper  [13]  such  an  inhibition  was  not  observed.  We  have  noticed, 
however,  that  ADP  alone  does  not  bring  about  an  inhibition  of  DPN- 
photoreduction.  A  marked  inhibition  is  observed  only  when  inorganic 
phosphate  and  Mg  +  +  are  added  and  the  preparation  carries  out  active 
photophosphorylation  [15]. 

The  observations  available  thus  far  would  indicate  that  the  light- 
induced  reduction  of  DPN  by  R.  riihrinn  chromatophores  may  be  achieved 
without  the  utilization  of  ATP.  Except  for  the  requirement  of  an  exo- 
genous reducing  agent  [16]  and  the  absence  of  oxygen  production,  this 
reduction  appears  to  be  more  closely  akin  to  the  photoreduction  of  pyridine 
nucleotides  by  chloroplasts  than  to  the  dark  reduction  carried  out  by 
mitochondria. 


PHOTOREDUCTION    OF    PYRIDINE    NUCLEOTIDES    AND    THEIR   POSSIBLE 
ROLE    IN    METABOLIC    REGULATION 

On  several  occasions  Dr.  D.  I.  Arnon  has  raised  the  question  as  to  the 
curious  specificity  of  the  purified  photosynthetic  pyridine  nucleotide 
reductase  (PPNR)  of  San  Pietro  for  triphosphopyridine  nucleotide  [17]. 
The  specificity  of  R.  rubrum  chromatophores  for  diphosphopyridine 
nucleotide  is  equally  puzzHng  [15,  18].  It  may,  therefore,  be  of  interest  to 
consider  whether  these  observations  can  be  brought  in  line  with  recent 
views  on  the  role  of  these  two  pyridine  nucleotides  in  metabolic  regulation 
which  have  been  reviewed  most  recently  by  Klingenberg  and  Biicher  [19]. 


OBSERVATIONS    ON    THE    FORMATION    OF    THE    PHOTOSYNTHETIC    APPARATUS      303 

The  concept  has  developed  in  recent  years  that  TPNH  furnishes  reducing 
power  to  a  great  many  synthetic  reactions  in  metabohc  pathways  (ref.  [19], 
Table  IV),  while  DPNH  represents  the  prime  energy  source  for  oxidative 
phosphorylation  carried  out  by  mitochondria,  and  thus  only  indirectly 
supports  and  controls  a  great  variety  of  synthetic  metabolic  reactions 
through  the  production  of  ATP. 

In  oxvgen-producing  plants  there  is  an  obvious  relation  between  the 
TPN  specificity  of  the  PPXR  and  the  TPN  specific  triosephosphate 
dehydrogenase  present  in  leaves  [20  23],  as  has  been  pointed  out  by 
Arnon  [24].  Is  it  possible  that  the  primacy  of  TPN  reduction  over  DPN 
reduction  by  green  plant  photosynthesis  makes  it  feasible  to  channel 
photosynthetic  reducing  power  more  effectively  into  many  biosynthetic 
pathwavs  in  addition  to  those  of  carbohydrate  synthesis  ?  This  primacy  of 
TPN  photoreduction  over  that  of  DPX,  in  oxygen-producing  plants,  may 
represent  an  important  e\olutionary  advance  over  the  situation  that  exists 
in  Rhodospirilliim,  where,  at  least  in  vitro,  isolated  chromatophores 
specifically  photoreduce  DPX. 

It  remains  to  be  seen  whether  there  is  any  relevance  to  the  hypothesis 
proposed.  A  beginning  has  been  made  in  studies  on  the  effect  of  light  on 
oxidized  and  reduced  pyridine  nucleotides  in  green  plants  [25],  and  on  the 
metabolic  fate  of  hydrogen  in  illuminated  algae  [26],  but  comparisons  with 
photosynthetic  bacteria  are  not  yet  available.  One  thing  we  do  know  is  that 
there  appears  to  be  a  much  closer  relation  between  respiration  and  photo- 
synthesis in  the  non-sulphur  purple  bacteria  than  there  is  in  most  oxygen- 
producing  plants  [27  30].  While  the  reasons  for  this  can  be  manifold,  the 
pvridine  nucleotide  specificity  in  light-induced  reactions  may  represent  an 
important  aspect  in  considerations  of  over-all  metabolic  control  in  photo- 
synthetic organisms. 

References 

1.  Schachman,  H.  K.,  Pardee,  A.  B.,  and  Stanier,  R.  Y.,  Arch.  Biuchetn.  Biophys. 
38,  245  (1952). 

2.  Vatter,  A.  E.,  and  Wolfe,  R.  S.,J.  Bact.  75,  480  (1958). 

3.  Hickman,  D.  D.,  and  Frenkel,  A.  'W.,y.  biophys.  biochem.  Cytol.  6,  277  (i959)- 

4.  Cohen-Bazire,  G.,  Sistrom,  W.  R.,  and  Stanier,  R.  Y.,  J.  cell.  comp.  Physiol. 

49,  25  (1957)- 

5.  Cohen-Bazire,  G.,  and  Kunisawa,   R.  Proc.  not.  Acad.  Sci.,  Wash.  16,  1543 

(i960). 

6.  Pratt,  D.  C,  Hickman,  D.  D.,  and  Frenkel,  A.  W.,  Plant  Physiol.  35,  Suppl.  x 
(i960). 

7.  Pratt,  D.  C,  Ph.D.  thesis.  University  of  Minnesota  (Dec.  1959). 

8.  Frenkel,  A.  W.,  Ann.  Rev.  Plant  Physiol.  10,  53  (1959). 

9.  Vernon,  L.  P.,  and  Ash,  O.  K.,y.  biol.  Chem.  234,  1878  (1959). 

10.  Chance,  B.,  and  Hollunger,  G.,  Fed.  Proc.  16,  163  (1957). 

11.  Chance,  B.,  and  Hagihara,  B.,  Biochem.  biophys.  Res.  Connn.  3,  6  (i959)- 

12.  Chance,  B.,  Biochem.  biophys.  Res.  Comm.  3,  10  (1959). 


304      DOUGLAS    C.    PRATT,    ALBERT   W.    FRENKEL,    AND    DONALD    D.    HICKMAN 

13.  Vernon,  L.  P.,  and  Ash,  O.  K.,y.  biol.  Chem.  235,  2721  (i960). 

14.  Frenkel,  A.  W.,  J.  biol.  Chem.  222,  823  (1956). 

15.  Frenkel,  A.  W.,  Brookhaven  Symp.  Biol.  11,  276  (i959)- 

16.  Frenkel,  A.  W.,  Plant  Physiol.  33,  Suppl.  xvii  (1958). 

17.  San  Pietro,  A.,  and  Lang,  H.  M.,  J.  biol.  Chem.  231,  211  (1958)- 

18.  Frenkel,  A.  W.,7.  Amer.  chem.  Soc.  80,  3479  (1958). 

19.  Klingenberg,  M.,  and  Biicher,  T.,  Anmi.  Rev.  Biochem.  29,  669  (i960). 

20.  Gibbs,  M.,  Nature,  Loud.  170,  164  (1952). 

21.  Arnon,  D.  I.,  Science  116,  635  (1952). 

22.  Arnon,  D.  I.,  Rosenberg,  L.  L.,  and  Whatley,  F.  R.,  Nature,  Lond.  173,  1132 

(1954)- 

23.  Rosenberg,  L.  L.,  and  Arnon,  D.  \.,y.  biol.  Chem.  217,  361  (i955)- 

24.  Arnon,  D.  I.,  Brookhaven  Symp.  Biol.  ll,  181  (1959). 

25.  Oh-hama,  T.,  and  Miyachi,  S.,  Plant  Cell.  Physiol.  I,  155  (i960). 

26.  Moses,  v.,  and  Calvin,  M.,  Biochim.  biophys.  Acta  33,  297  (i959)- 

27.  van  Niel,  C.  B.,  Advanc.  Enzymol.  I,  263  (1941). 

28.  Johnston,  J.  A.,  and  Brown,  A.  H.,  Plant  Physiol.  29,  177  (i954)- 

29.  Brown,  A.  H.,  Amer.  J.  Bot.  40,  719  (i953)- 

30.  Brown,  A.  H.,  and  Weis,  D.,  Plant  Physiol.  34,  224  (i959)- 

31.  Frenkel,  A.  W.,  and  Hickman,  D.  D.,jf.  biophys.  bioclmn.  Cytol.  6,  285  (i959)- 


Discussion 

Bergeron  :  In  the  small  particle  preparations  from  the  dark-grown  cells  where 
you  get  pyridine  nucleotide  reduction  if  you  add  magnesium,  is  this  reduction 
inhibited  if  substrates  for  phosphorylation  are  present  as  it  is  with  the  regular 
Rhodospirillum  rubrum  chromatophores  ? 

Frenkel  :  We  have  not  worked  with  the  small  particle  preparations. 

Arnon  :  Dr.  Frenkel  said  that  Rhodospirillumrubrumwould  not  grow  on  acetate  in 
the  dark  but  we  have  grownC/;raw<7^m?«,  aphotosynthetic  sulphur  bacterium,  on  ace- 
tate without  added  CO^  or  under  conditions  when  COo,  which  might  be  formed  from 
acetate,  would  be  swept  out  by  continuously  bubbled  gas.  Under  these  conditions 
there  would  be  enough  COo  for  it  to  act  as  a  catalyst  but  not  as  a  substrate. 
Apparently,  there  are  differences  between  these  two  organisms.  My  second  point 
concerns  the  reduction  of  pyridine  nucleotides  in  the  dark,  i.e.  a  case  when  the 
photosynthetic  process  becomes  limited  to  ATP  formation  or  to  what  we  call 
cyclic  photophosphorylation.  As  we  shall  discuss  this  afternoon,  Chromatium  can 
use  hydrogen  gas  to  reduce  pyridine  nucleotides  in  the  dark.  In  Chromatium 
supplied  with  hydrogen  gas  light  is  required  only  for  ATP  formation.  If  exogenous 
ATP  is  substituted  for  light,  then  Chromatium,  which  unlike  R.  rubrum  is  normally 
a  strict  phototroph,  now  becomes  able  to  assimilate  carbon  dioxide  in  the  dark. 

Frenkel  :  I  was  very  surprised  to  hear  that  Chrcjmatium  can  get  along  without 
CO.j.  We  have  found  that  Rhodospirillum,  when  grown  on  standard  media  in  the 
dark  or  light,  has  a  definite  CO2  requirement  which  saturates  at  about  i  per  cent 
COo.  Thus  far  we  have  not  been  able  to  replace  this  COo  requirement  by  inter- 
mediates of  the  Krebs  tricarboxylic  acid  cycle,  or  by  a  number  of  other  well- 
defined  chemicals.  Only  high  concentrations  of  yeast  extract  or  casein  hydrolysate 
were  effective   in   relieving  this   CO.,  requirement.   With  regard  to  Dr.  Arnon's 


OBSERVATIONS    ON    THE    FORMATION    OF    THE    PHOTOSYNTHETIC    APPARATUS       305 

second  point  it  may  not  always  be  safe  to  generalize  about  the  mechanism  of 
photosynthesis  from  one  special  case.  The  observation  that  Clironiatiurn  can 
reduce  pyridine  nucleotides  in  the  dark  with  molecular  hydrogen,  does  not  appear 
to  preclude  the  possibility  that  Chroniatiwn  could  reduce  pyridine  nucleotides 
directly  or  indirectly  by  a  photochemically  generated  reductant. 

Arnon  :  The  point  is  that  hydrogen  gas  has  been  known  for  almost  30  years 
as  a  physiological  electron  donor  for  Chroynatium.  The  utilization  of  hydrogen  gas 
by  this  organism  is  not  to  be  regarded  as  an  experimental  artifact.  We  do  not  build 
but  merely  support  our  theory  with  the  facts  of  Chroma tiiini  photosynthesis.  We 
have  presented  other  lines  of  evidence  elsewhere. 

Frenkel:  We  have  tried  to  grow  Rhodnspirillnni  ruhrutu  with  hydrogen  but  it 
does  not  grow  very  well. 

Arnon:  Chromatium  grows  very  well  with  hydrogen  gas. 

Bergeron  :  Coming  back  to  the  question  of  acetate-grown  CJirumatium  I  should 
like  to  point  out  the  truth  in  both  points  of  view.  Recently  Dr.  Benedict  was  trying 
to  study  carotenoid  biosynthesis  in  Cluomatiinn  using  labelled  acetate,  and  he  was 
using  a  medium  containing  a  very  small  percentage  of  CO^.  They  were  growing 
very  nicely.  Some  objection  was  raised  to  the  carbonate  so  they  were  transferred 
to  a  medium  which  was  identical  except  for  the  fact  that  the  minimum  amount  of 
CO2  was  taken  away.  The  new  cultures  grew  slowly  but  finally  got  going  again  on 
the  pure  acetate. 

Arnon  :  Let  me  make  it  clear  again  that  when  I  say  that  Cluamatiuni  grows 
without  CO.j,  I  do  not  imply  that  CO._,  is  not  used  catalytically ;  I  firmly  believe  it 
is.  What  I  am  saying  is  that  we  have  grown  Chromatium  without  any  added  supply 
of  CO.,  and  under  conditions  where  any  large  concentrations  of  endogenous  CO2 
would  be  swept  out  by  bubbling  gas. 

Frenkel:  What  is  the  pH  of  the  medium  ? 

Arnon  :  They  grow  at  pH  between  7  and  7  •  8. 

Frenkel  :  Under  these  conditions  it  may  not  be  too  simple  to  remove  the  CO.^ 
which  is  produced  metabolically  at  a  rate  adequate  to  prevent  its  re-utilization. 

Arnon  :  As  I  said  earlier  I  firmly  believe  that  the  CO.,  is  used  catalytically. 

Frenkel:  In  studies  on  the  effect  of  COj  on  the  growth  of  micro-organisms, 
experimental  conditions  are  not  always  adequately  described.  At  low  gassing  rates, 
with  actively  metabolizing  cells  a  steady  state  concentration  of  CO^  may  be  built- 
up  permitting  continued  growth  of  the  micro-organisms. 

Kamen:  I  should  mention  that  in  practically  all  the  chloroplasts  structures 
which  are  known  there  is  a  very  high  concentration  of  chlorophyll  held  to  the 
protein.  In  the  bacteria,  as  you  may  have  noticed  in  Table  i  (p.  2S0),  the  ratio 
of  chlorophyll  to  protein  is  about  15,  whilst  in  the  case  of  chloroplasts,  it  is  some- 
thing of  the  order  of  1500.  In  the  case  of  the  nucleotides,  also,  the  concentration 
of  the  nucleotides  in  chromatophores  is  about  i  to  15  chlorophylls,  while  in 
spinach  chloroplasts  it  is  over  i  to  2500  as  Krogman  showed,  so  there  is  a  quantita- 
tive difference  between  structures  producing  oxygen  and  those  which  don't.  I 
believe  that  this  may  have  something  to  do  with  the  Emerson  effect  which  indicates 
that  at  least  two  quanta  are  funnelled  to  each  active  site  to  get  the  oxygen  off.  It  is 
very  difficult  for  an  organism  with  limited  amounts  of  chlorophyll  to  funnel  the 
quanta  to  where  it  wants  it. 

VOL.  n. — X 


306      DOUGLAS    C.    PRATT,    ALBERT   W.    FRENKEL,    AND    DONALD    D.    HICKMAN 

Vernon  :  I  can  give  you  some  information  about  the  relation  between  phos- 
phorylation and  photoreduction  reactions.  In  our  laboratory  we  have  followed 
photophosphorylation,  the  photoreduction  of  DPN  and  the  photo-oxidation  of 
ascorbate.  In  the  experiments  we  have  performed,  there  is  a  large  degree  of 
independence  between  these  reactions.  The  photoreduction  does  not  require  an 
associated  phosphorylation  and  the  photophosphorylation  does  not  require  an 
associated  photoreduction  of  DPN.  This  supports  your  idea  that  these  two 
reactions  are  separate  and  distinct. 

Frenkel  :  I  believe  in  your  paper  on  pyridine  nucleotide  reduction  you  men- 
tioned that  ADP  and  ATP  inhibited  reduction.  Did  you  find  that  simultaneous 
phosphorylation  will  inhibit  the  reduction  ? 

Vernon  :  No,  they  are  essentially  independent. 

Arnon  :  I  would  like  to  state  that  we  now  have  some  evidence  for  non-cyclic 
photophosphorylation  in  photosynthetic  bacteria,  that  is  coupled  with  the  reduc- 
tion of  DPN.  As  in  chloroplasts,  non-cyclic  photophosphorylation  in  bacteria, 
does  not  replace  cyclic  photophosphorylation  but  supplements  it. 

Frenkel:  In  the  work  of  Smith  and  Baltscheffsky  light-induced  phosphoryla- 
tion by  Rhodospirillum  chromatophores  was  shown  to  be  linked  to  the  oxidation  of 
cytochrome  c  and  possibly  also  to  the  reduction  of  a  6-type  cytochrome. 

Arnon:  I  am  speaking  of  new  evidence.  It  is  perhaps  premature  to  make  the 
comment  before  the  evidence  is  presented  but  I  wish  to  make  it  now  for  the  sake 
of  completing  the  record  of  this  discussion. 


The  Photosynthetic  Macromolecules  of 

Chlorobium  Thio sulfa tophilum^ 

J.  A.  Bergeron  and  R.  C.  FuLLER-f 

Biology  Department,  Brookhaven  National  Laboratory, 
Upton,  Xezc  York,  U.S.A. 

There  is  considerable  modern  evidence  that  in  higher  plants  the  entire 
process  of  photosynthesis  occurs  in  a  microscopic  but  complex  organelle — 
the  chloroplast  (see  review  by  Arnon  [4]).  The  study  of  simpler  systems 
offers  an  advantage  by  eliminating  variables  which  are  not  pertinent  for 
analysis  of  the  basic  light-dependent  phenomena.  This  consideration 
accounts,  in  large  measure,  for  the  current  interest  in  bacterial  photo- 
synthesis. 

Knowledge  of  the  submicroscopic  basis  of  bacterial  photosynthesis  is 
still  fragmentary  but  is  improving  rapidly.  It  was  assumed,  until  1952,  that 
the  photosynthetic  pigments  are  bound  to  protein  and  dispersed  through- 
out the  organism.  At  that  time  it  was  reported  by  Pardee  et  al.  [33]  that 
the  pigments  sediment  rapidly  in  crude  extracts  of  the  non-sulphur  purple 
bacterium,  Rhodospirilhon  ruhruni.  The  authors  isolated  the  pigmented 
component  and  applied  the  name  chromatophore  to  it.  The  electron 
micrographs  of  chromatophores  which  had  been  dried  and  shadowed  with 
metal  revealed  disks  about  iioo  A  in  diameter.  It  was  inferred  that  the 
disks  represented  spheres  with  a  diameter  of  about  600  A.  This  value 
agreed  roughly  with  the  diameter  of  400  A  which  had  been  calculated  by 
Stokes  relation  from  the  sedimentation  coefficient  (200  S)  of  the  purified 
preparation  [35].  At  the  same  time,  similar  electron  micrographs  also  were 
obtained  by  Thomas  [38]  with  crude  extracts  of  several  photosynthetic 
bacteria.  These  reports  provided  concrete  evidence  that  the  pigments  are 
localized  in  structures  which  are  several  orders  of  magnitude  larger  than 
soluble  proteins. 

The  first  evidence  that  this  order  of  structural  organization  could  be 
associated  with  a  relatively  high  degree  of  functional  capability  was  pro- 
vided in  the  report  by  Frenkel  [19]  of  the  light-dependent  phosphorylation 
of  ADP  by  subcellular  preparations  of  R.  ruhriun.  This  report  and  the 

*  Research  carried  out  at  Brookhaven  National  Laboratory  under  the  auspices 
of  the  U.S.  Atomic  Energy  Commission. 

t  Present  address :  Dartmouth  Medical  School,  Hanover,  Neiv  Hampshire. 


3o8  J.    A.    BERGERON    AND    R.    C.    FULLER 

description  of  photosynthetic  phosphorylation  in  chloroplast  preparations 
by  Arnon  et  a/.  [2,  3]  suppHed  direct  evidence  for  the  idea  of  Emerson  et  al. 
[17]  that  the  role  of  light  is  to  produce  energy-rich  bonds  and  also 
strengthened  the  belief  that  the  fundamental  photochemical  events  are  the 
same  in  all  photosynthetic  organisms.  Since  that  time  considerable 
information  has  accumulated  about  various  properties  of  pigmented 
preparations  of  photosynthetic  bacteria  (see  papers  in  this  symposium  and 
review  by  Frenkel  [21]).  However,  only  two  organisms  have  been  studied 
in  any  real  detail,  R.  rubrinn  and  Chromatium,  the  purple  sulphur  bac- 
terium. The  chromatophore  of  Chromatium  represents  the  simplest  level 
of  structural  organization  which  is  known  to  support  photophosphoryla- 
tion.  We  have  considered  the  structure  and  function  of  this  chromatophore 
in  some  detail  previously  [6,  7].  This  system  provides  the  perspective  for 
a  study  which  is  in  progress  of  the  photochemical  apparatus  of  the  green 
sulphur  bacterium,  Chlorobiiim  thiosulfatophilum.  In  the  interest  of 
clarity,  the  data,  of  several  kinds,  are  considered  at  successive  levels  of 
organization;  the  organism,  the  crude  extracts,  and  the  purified  pigmented 
component. 

Results 

THE    ORGANISM 

The  green  sulphur  bacterium,  Chlurobiuni  thiosulfatophilum,  is  a  strict 
anaerobe  and  an  obligate  phototroph.  It  can  use  hydrogen  sulphide,  thio- 
sulphate,  tetrathionite,  elementary  sulphur,  or  molecular  hydrogen  as  the 
electron  donor  for  carbon  dioxide  assimilation  [29].  The  quantum  require- 
ment of  the  light  dependent  process  is  8  10  quanta  for  four  hydrogen 
atoms  (or  four  electrons)  moved  with  molecular  hydrogen,  thiosulphate  or 
tetrathionite  as  the  reducing  agent  [30].  This  agreement  with  the  value 
obtained  with  the  other  photosynthetic  organisms  implies  a  fundamental 
similarity  in  the  basic  light-dependent  events  (cf.  [34]).  In  the  laboratory 
the  organism  is  cultured  in  an  inorganic  medium  containing  carbonate, 
sulphide  and  thiosulphate  [31].  Depending  upon  growth  conditions  and 
age,  the  in  vivo  absorption  spectrum  shows  differences  due  to  variation  in 
the  content  of  accessory  pigment  (Fig.  i).  All  the  data  reported  here  are 
based  upon  cultures  with  a  moderate  amount  of  accessory  pigment.  It  has 
recently  been  demonstrated  [37]  that  contrary  to  previous  assumptions,  at 
least  two  different  molecular  species  of  chlorophyll  exist  among  the  green 
bacteria.  The  organism  used  in  this  study  contains  Chlorohium  chlorophyll- 
650  and  is  strain  L. 

A  representation  of  the  submicroscopic  morphology  of  this  organism 
can  be  obtained  by  electron  microscopy  of  ultra-thin  sections.  Typically, 
the  organisms  are  fixed  by  exposure  to  osmium  tetroxide  at  a  concentration 


PHOTOSYNTHETIC  MACROMOLECULES  OF  CHLOROBIUM  THIOSUI.FATOPHILUM  309 

of  2"o  in  a  medium  buffered  at  pH  7-4  and  corresponding  to  the  culture 
medium  except  for  the  omission  of  reducing  substances.  The  specimens 
are  dehydrated  in  a  graded  series  of  alcohols  with  the  temperature  pro- 
gressively reduced  to  —  50  \  then  the  specimens  are  infiltrated  with  butyl 
methacrylate  monomer  and  polymerized  by  gamma  radiation  from  a 
cobalt-60  source  at  -50.  In  sections  through  an  axial  plane  (Fig.  2)  the 
peripheral  envelope,  or  cell  wall,  is  distinct  from  the  cytoplasmic  mem- 
brane. The  "nucleus"  is  represented  by  the  axial  region  of  low  electron 
densitv  which  contains  ramifying  spiral  filaments.  Typically,  there  are  one 
or  more   relatively  large  circular  areas  of  high   electron   density  which 


300 


400 


500 


600 


700 


800 


900 


Fig.  I.  Illustration  of  the  range  of  variation  in  content  of  accessory  pigments 
in  cultures  of  Chlorohiiim  thiosulfatophilum.  The  absorption  spectra  were  measured 
(Gary  Model  14)  through  opal  glass  to  reduce  the  effect  of  light  scattering. 


represent  sections  through  inclusions  rich  in  polyphosphates.  The  cyto- 
plasmic region  has  a  stippled  appearance  owing  to  the  presence  of  large 
numbers  of  small  particles.  The  lack  of  contrast  between  the  particles  and 
the  background  prevents  accurate  determinations  of  size  and  form  but  it 
is  clear  that  the  images  are  circular  rather  than  elongated  and  have  a 
maximum  extension  of  about  150  A.  There  is  no  indication  ot  the  vescular 
chromatophores  (Fig.  3)  which  characterize  Chromatiiim  [6,  7,  39]), 
RliodospiriUiim  rubrum  [22,  25,  39],  Rhodopseiidomonas  sphevoides  [39],  and 
Chlorohiiim  Umicola  [39]  of  the  peripheral  lamellae  which  have  been  or, 
described  in  Rliodomicrohium  vaniiielii  [40],  or  of  the  lamellated  inclusions 
of   Rhodospiyillum    moUsdiianiim    [16].    In    fact,    the    above    description, 


M 


W 


1/^ 


Fig.  2.  Electron  micrograph  (72  000  x  )  of  a  thin  section  in  the  axial  plane  of 
Chlorobimn  thiosulfatophilmn.  The  cell  wall  (W),  which  tends  to  appear  as  two 
layers,  is  distinct  from  the  cytoplasmic  boundary.  The  "nuclear"  region  has  a 
low  electron  density  and  contains  filaments  (F)  which  ramify.  Metaphosphate 
inclusions  (M)  appear  as  circular  areas  of  high  electron  density.  The  cytoplasm  is 
filled  with  small  particles  which  have  a  maximum  diameter  of  about  150  A. 


Fig.  3.  Electron  micrograph  {65  000  x  )  of  a  thin  section  of  the  purple  sulphur 
bacterium,  Chromatium.  The  organism  is  smooth  contoured  and  is  bounded  by  a 
dual  membrane  which  can  separate  into  two  distinct  structures,  the  cell  wall  (W) 
and  the  plasma  membrane.  The  cell  is  filled  with  the  chromatophores  (Ch)  which 
are  minute  vesicles.  These  appear  as  annular  images  with  an  outer  diameter  of 
about  300  A  and  a  cortical  thickness  of  about  70  A.  Large  vesicles  (V)  about  1000  A 
in  diameter  are  also  visible.  The  irregular  areas  of  low  density  (N)  are  considered 
to  be  parts  of  an  irregularly  shaped  nuclear  compartment.  The  closely  packed 
chromatophores  conceal  the  small  particles  (Sp)  which  are  observed  in  the  fractions. 


Fig.  4.  Electron  micrograph  (72  000  x  )  of  thin  sections  of  the  non-photo- 
synthetic  bacterium  Eschericliia  coli.  The  cell  wall  (W)  is  distinct  in  suitably 
oriented  sections.  The  "nuclear"  region  (N)  has  a  low  electron  density  and  con- 
tains very  fine  (~40  A)  filaments.  The  cytoplasmic  region  contains  minute  particles 
which  are  well  defined. 


PHOTOSYNTHETIC  MACROMOLECULES  OF  CHLOROBIUM  THIOSULFATOPHILUM 


313 


excepting    the    inclusions,    could    apply    to    typical    non-photosynthetic 
bacteria  [9,  11,  12,  13,  28,  32]  such  as  Escherichia  coli  (Fig.  4). 


CRUDE    EXTRACTS 

It  is  rather  difficult  to  rupture  this  organism  but  two  methods  have 
been  used  successfully:  breaking  frozen  cells  in  the  Hughes  pressor 
exposing  a  suspension  of  2  g.  of  cells  (wet  weight)  and  i  g.  of  very  fine 
synthetic  sapphire  abrasive  (Linde  B)  in  40  ml.  of  o-i  m  tris  (hydroxy- 
methylamino  methane)  buffer  at  pH  7-8  to  sonic  oscillation  for  2  min.  at 
0-5°  in  a  10  K.C.  Ra\i;heon  oscillator. 


0.6 


0.5 


-| — [ — 1 — I — 1 1 — I — \ — I — I r 


IN    VIVO 

CLEARED    EXTRACT 


"T 1 1 1 1 ! 1 T" 


400 


500 


700 


800 


900 


600 
X  m  ^ 

Fig.  5.  Illustration  of  the  correspondence  between  the  absorption  spectrum 
of  the  photosynthetic  pigments  in  vivo  and  in  the  cell-free  extracts.  The  divergence 
at  lower  wavelengths  is  attributable  to  differences  in  light  scattering  which  were 
not  completely  compensated  by  the  use  of  opal  glass. 

Cells  and  debris  are  remo\ed  from  the  crude  extract  by  two  successive 
centrifugations  for  30  min.  with  refrigeration  (5  )  at  26000  g.  The 
"cleared"  extract  has  an  absorption  spectrum  which  corresponds  with  the 
in  vivo  spectrum  (Fig.  5).  The  small  differences  which  are  observed  are 
attributable  to  differences  in  light  scattering.  This  agreement  provides 
some  assurance  that  the  phvsicochemical  characteristics  of  the  pigment 
bearer  have  not  been  disturbed  greatly  during  the  process  of  cell 
disintegration. 

A  pigmented  fraction,  free  from  other  macromolecular  constituents, 
can  be  prepared  by  repetitive  centrifugation  for  2  hr.  at  144  000  g  under 
refrigeration  {^  ).  The  progress  of  the  fractionation  is  indicated  by  changes 
in   the   components   observed   in   the   analytical   ultracentrifuge   and   by 


3H 


J.    A.    BERGERON    AND    R.    C.    FULLER 


changes  in  the  absorption  spectrum  (Fig.  6).  The  sedimentation  diagram 
of  the  "cleared"  extract  reveals  three  major  components  with  sedimenta- 
tion coefficients  of  about  5,  30,  and  50  Svedberg  units  (S)  respectively. 
The  colour  due  to  the  photosynthetic  pigments  is  related  to  the  most 
rapidly  sedimenting  component.  It  is  easy  to  eliminate  the  slowest  com- 
ponent (5  S)  by  repetitive  centrifugation  but  it  is  rather  difficult  to  elimin- 


200 


300 


400 


500         600 


700 


800 


900 


Fig.  6.  Record  of  the  changes  in  the  sedimentation  diagram  (Spinco  Model  E) 
and  absorption  spectrum  (Gary  Model  14)  during  the  isolation  of  the  pigmented 
component.  There  are  three  major  components  in  the  crude  extract.  The  colour 
due  to  the  photosynthetic  pigments  is  associated  with  the  component  (50  S)  which 
sediments  the  most  rapidly.  The  absorption  at  260  m//  due  to  nucleic  acid  is  greatly 
reduced  as  the  slower  components  are  eliminated. 


ate  completely  the  30  S  component  under  conditions  which  recover  the 
pigmented  component  (50  S)  in  high  yield.  The  progressive  elimination  of 
the  two  slower  components  is  reflected  in  the  absorption  spectrum  by  the 
drastic  reduction  in  nucleic  acid  absorption  at  260  m/i. 

If  a  crude  extract  is  placed  upon  a  linear  sucrose  gradient  (10%  to 
50%)  and  centrifuged  for  several  hours  in  a  swinging  bucket  head  at 
156000  g  two  coloured  zones  develop,  the  "green  "zone  with  a  sharply 
defined  leading  edge  is  followed  by  a  less  distinct  "yellow"  zone.  Com- 


PHOTOSYNTHETIC  MACROMOLECULES  OF  CHLOROBIUM  THIOsULFaTOPHILUM 


315 


parison  of  the  absorption  spectra  of  these  zones  with  the  original  extract 
reveals  (Fig.  7)  that  the  "yellow"  zone  is  rich  in  carotenoids  and  deficient 
in  chlorophyll.  The  "green"  zone,  which  accounts  for  the  bulk  of  the 
material,  shows  an  appreciable  decrease  in  the  absorption  due  to  caro- 
tenoids. Two  alternative  explanations  of  this  phenomenon  are  either  that 
a  considerable  fraction  of  the  intracellular  carotenoids  are  not  incor- 
porated into  the  pigmented  particle  or  that  these  pigments  are  exposed  and 
tend  to  strip  off  during  the  progressive  movement  into  more  concentrated 


i  I  I  I  I  I  I  I  I  I  I  I  I  I  { 


200 


300 


400 


600 


700 


800 


900 


500 
X  m^ 

Fig.  7.  Effect  of  centrifuging  the  crude  extract  in  a  linear  gradient  of  sucrose 
(io°o~5o"o).  The  pigmented  particles  are  recovered  in  a  "green"  zone  which  is 
followed  by  a  "  yellow  "  zone.  By  comparison  with  the  original  absorption  spectrum, 
the  "yellow"  zone  is  enriched  in  carotenoids  and  deficient  in  chlorophyll.  The 
"green"  zone  shows  a  decline  in  the  absorption  due  to  carotenoids  and  nucleic 
acids. 

sucrose  solutions.  The  latter  alternative  seems  to  be  favoured  by  the 
observation  that  these  pigments  are  not  left  in  the  original  layer  but  are 
recovered  in  the  sucrose  gradient  behind  the  green  zone;  then  too,  this 
tendency  is  well  defined  only  when  gradients  are  employed.  The  spectra 
also  show  disturbances  in  the  800  m/t  maximum  which  are  reminiscent  of 
the  effect  of  carotenoids  upon  the  infra-red  absorption  spectrimi  in 
Chroiuatium  [i,  6,  8,  23,  24]. 

The  components  present  in  these  extracts  resemble  in  number  and 
sedimentation  characteristics  the  extracts  of  non-photosynthetic  bacteria. 
In  the  photosvnthetic  bacteria  which  have  been  investigated  previously 
\j,^^  the  chromatophores  have  been  present  as  a  component  in  addition  to 


"M 


B 


B 


Fig.  8.  Electron  micrograph  (72  000  x  )  of  a  thin  section  of  a  pellet  of  a  pig- 
mented fraction  which  sedimented  previously  as  one  component  (50  S)  in  the 
analytical  ultracentrifuge.  The  preparation  consists  predominantly  of  small 
particles  with  a  diameter  of  about  150  A.  There  are  also  occasional  elongated 
figures  (B)  which  are  either  contaminants  or  aggregates. 


PHOTOSYNTHETIC  MACROMOLECULES  OF  CHLOROBIUM  THIOSULFATOPHILUM  317 

three  such  slower  components.  Thus,  the  resemblance  of  Chlorobium 
thiosulfatophiliim  to  E.  coli  which  was  observed  in  the  electron  micro- 
graphs is  carried  over  into  the  components  present  in  extracts.  The 
obvious  distinction  is  in  composition.  In  these  extracts  the  50  S  component 
bears  photosynthetic  pigments  and  appears  to  be  free  of  nucleic  acids. 

THE    PURIFIED    PIGMENTED    COMPONENT 

Characterization  of  the  physiochemical  properties,  composition  and 
photochemical  activity  of  the  pigmented  component  is  incomplete  but 
sufficies  to  place  limits  on  a  number  of  pertinent  variables.  We  have  two 
indices  of  the  homogeneity  of  the  fraction ;  sedimentation  behaviour  and 
electron  microscopical  observation.  Although  refined  analysis  may  reveal 
complexities,  it  is  clear  that  the  photosynthetic  pigments  do  sediment  with 
one  component  which  exhibits  a  well-defined  spike.  To  this  extent,  the 
preparation  appears  to  be  monodisperse  with  regard  to  the  photosynthetic 
pigment.  When  the  pellet  of  a  pigmented  fraction  that  sedimented  essen- 
tially as  a  single  component  is  removed  from  the  analytical  ultracentrifuge 
and  processed  for  electron  microscopy,  the  thin  sections  (Fig.  8)  reveal  a 
rather  uniform  population  of  particles.  These  particles  resemble  the 
particles  seen  in  the  cytoplasm  of  the  cell ;  that  is,  the  maximum  extension 
of  the  image  in  any  direction  is  about  150  A.  If  these  particles  are  slightly 
elongated  the  range  of  deviation  is  probably  between  100  and  150  A.  The 
sedimentation  of  the  pigmented  fraction  depends  to  a  considerable  degree 
upon  concentration  and  the  data  are  still  inadequate  for  an  accurate  cal- 
culation of  the  sedimentation  constant;  however,  the  maximum  value 
obtained  from  the  purest  preparations  at  high  dilution  is  50  Svedberg 
units  when  converted  to  20'  in  water.  It  is  of  interest  to  compare  the 
direct  and  indirect  data  on  particle  size  and  obtain  some  idea  of  the  agree- 
ment. If  we  use  a  sedimentation  constant  of  50  S  and  the  conventional 
assumption  of  a  density  of  about  1-2  g./ml.,  then  the  particle  diameter 
calculated  from  Stokes  relation  is  173  A.  This  degree  of  agreement  with 
the  electron  microscopical  observation  is  reassuring.  A  spherical  particle 
ot  this  size  and  density  would  have  a  molecular  weight  between  i  -3  and 
I  -6  million.  This  value  can  be  used  as  a  first  estimate  of  the  molecular 
weight. 

An  interesting  but  troublesome  property  of  the  50  S  particle  is  the 
tendency  to  aggregate  into  a  series  of  more  rapidly  sedimenting  com- 
ponents as  the  degree  of  purification  becomes  relatively  high.  Electron 
microscopic  examination  of  thin  sections  of  pellets  of  these  aggregates 
(Fig.  9)  reveal  elongated  profiles  with  diameters  up  to  400  A  and  lengths 
of  thousands  of  A.  When  such  preparations  are  sprayed  upon  on  specimen 
supports  and  shadowed  with  metal  the  electron  micrographs  reveal  rigid 


"^p^id!*-     .s*^^ 


# 


%^ 


^ 


*. 


<f 


1^ 


S    - 


I 


^ 


^^ 


W^ 


i^A 


¥■ 


Fig.  9.  Electron  micrograph  (72  000  x  )  of  a  pigmented  fraction  which  changed 
into  a  series  of  more  rapidly  sedimenting  components  during  purification.  There  is 
a  preponderance  of  elongated  figures  which  tend  to  pair  (P).  The  size  is  not  uniform. 
The  widths  appear  to  be  multiples  of  a  minimum  value  of  about  150  A.  The  length 
also  varies  but  cannot  be  established  in  thin  sections.  The  maximum  values 
observed  are  in  the  1000  A  range. 


PHOTOSYNTHETIC  MACROMOLECULES  OF  CHLOROBIUM  THIOSULFATOPHILUM 


319 


rods.  This  tendency  of  the  macromolecules  to  organize  into  structures  with 
a  higher  degree  of  order  than  is  observed  in  the  organism  is  unusual. 

In  the  initial  phase  of  this  study,  observations  on  the  chemical  composi- 
tion of  the  pigmented  fractions  have  dealt  primarily  with  chlorophyll, 
protein  and  nucleic  acids.  The  spectrophotometric  assay  of  chlorophyll 
content  is  based  on  the  specific  absorption  coefficient  of  this  chlorophyll 
in  acetone  [37]  and  a  recent  estimate  of  the  molecular  extinction  coefficient 
[14].  The  quantitative  relationship  between  the  723  m/x  maximum  in  the 
in  vivo  spectrum  and  the  652  m/z  maximum  of  the  chlorophyll  in  acetone 
has  been  determined  in  order  to  allow  direct  measurement  of  chlorophyll 


0.400 


Q300 


0200 


O.IOO 


1    M    I    I    M    I   I    [    I   I    I    I    I    I   I    i  T  I   I    M    ;    I    1    I    i    I    ]    M    I    I    I    I    I    M    I 


-EXTRACTED  IN  HjS  SATURATED 
ACETONE,  652.0  m^ 

ON  BEARER  IN  0.1  M  TRIS, 
pH  78,  723.5  m^ 


M  I  I   I   I  I   M 


2.00  3.00 

CONCENTRATION 


4.00 


Fig.  io.  Quantitative  determination  of  the  relationship  of  the  absorption  at 
652  ni/tt  of  the  chlorophyll  in  H.^S-saturated  acetone  and  the  absorption  at  723-5 
m^  of  the  chlorophyll  bound  to  the  pigmented  particle. 


content  in  the  extract  during  fractionation  (Figs.  lo  and  ii).  For  the  same 
reason  the  nucleic  acid  and  protein  estimates  have  been  limited  to  ultra- 
violet absorption  measurements.  During  the  course  of  fractionation  the 
absorption  maximum  at  260  m^u  decreases  from  an  initial  value  w^hich  is 
two  or  three  times  greater  than  the  chlorophyll  maximum  at  723  m/x  to  a 
limiting  value  which  is  13  of  the  chlorophyll  absorption.  The  initial  280/ 
260  m/i  absorption  ratio  is  about  0-5  and  increases  to  0-9;  thus,  the  50  S 
fraction  is  quite  free  of  nucleic  acids.  On  a  mass  basis,  the  chlorophyll/ 
protein  ratio  in  the  more  highly  purified  preparations  is  1/13.  Assuming 
that  these  two  components  account  for  the  bulk  of  the  mass  of  the  particle, 
this  ratio  represents  about  100  chlorophyll  molecules  for  each  particle  with 
a   molecular   weight   of   1-5    million.    The   cytochromes   present   in   the 


320 


J.    A.    BERGERON    AND    R.    C.    FULLER 


purified  fraction  have  been  studied  in  some  detail  [26].  On  the  basis  of  the 
above  estimate  of  particle  size,  the  cytochrome  content  approaches  limiting 
proportions,  that  is,  i  or  2  cytochromes  per  particle. 

Several  years  ago  it  was  reported  by  Williams  [41]  that  photophos- 
phorylation  might  have  occurred  in  experiments  with  crude  extracts  of  a 
green  sulphur  bacterium.  The  observations  in  our  laboratory  indicate  that 
light-dependent  uptake  of  phosphate  occurs  in  the  "cleared"  extract  but 
disappears  or  is  greatly  reduced  during  the  course  of  fractionation.  Further 


1.0 
0.9 
0.8 

0.7 

>- 

^    0.6 

UJ 
Q 

-I    0.5 
< 

%  0.4 
0.3 
0.2 
0.1 
0.0 


-y-r-r-t-^^ 


IN    H2S     SATURATED    ACETONE 
■IN   0.1  M    TRIS    BUFFER,  PH  7.5 


Fig.  II.  Quantitative  comparison  of  the  spectral  change  produced  by  trans- 
ferring the  photosynthetic  pigments  from  the  physiological  environment  of  the 
pigmented  particle  into  solution  in  HoS-saturated  acetone. 

w'ork  is  needed  to  establish  whether  or  not  the  phosphate  uptake  in  the 
crude  extracts  really  represents  ATP  formation  and  what  relationship  the 
particles  have  to  this  activity. 


Discussion 

The  wide  range  of  opinion  which  exists  concerning  the  importance  of 
structural  organization  for  photosynthesis  is  illustrated  in  a  recent  sym- 
posium entitled  "The  Photochemical  Apparatus — Its  Structure  and 
Function"  {BrookJiaven  Symp.  Biol,  ii,  1958).  Structural  organization 
above  the  molecular  level  was  regarded  as  essential,  or  important  for 
efficiency,  or  irrelevant  depending  upon  the  type  of  data  and  particular 
aspect  of  photosynthesis  under  consideration.  Most  of  the  participants, 


PHOTOSYNTHETIC  MACROMOLECULES  OF  CHLOROBIUM  THIOSULFATOPHILUM         32 1 

however,  were  prepared  to  assume  that  structural  organization  probably 
contributes  to  efficiency  and  might  even  be  essential  for  the  primary 
process.  Those  so  inclined  inferred  that  the  submicroscopic  lamella, 
present  in  rudimentary  form  as  the  cortex  of  the  chromatophore  and 
elaborated  in  the  grana  and  stroma  of  the  chloroplast,  is  a  universal 
architectural  characteristic  of  photosynthetic  systems.  Since  the  properties 
of  a  lamella  fulfill  the  requirements  for  the  separation  of  primary  reducing 
and  oxidizing  components  in  a  photolysis  scheme  as  well  as  for  the  con- 
densed state  required  in  the  semiconductor  concept  of  the  primary  photo- 
synthetic  process,  a  theoretical  basis  could  be  proposed  to  account  for  the 
universality  of  this  characteristic. 

Our  approach  to  the  problem  has  been  to  try  to  characterize  both 
structure  and  function  at  the  lowest  level  of  structural  organization  capable 
of  supporting  photosynthesis.  The  present  study  of  the  structure  and 
function  of  the  photochemical  apparatus  of  Chlorohium  thiosiilfatophilum 
is  raising  some  questions.  The  data  lead  tentatively  to  the  following  con- 
cept. The  structural  unit  is  a  particle  which  is  spherical  rather  than 
elongated,  has  a  maximum  dimension  of  about  150  A  and  a  molecular 
weight  of  about  i  -5  million.  Such  a  macromolecule  is  about  one  order  of 
magnitude  smaller  in  mass  than  the  simplest  photosynthetic  unit  studied 
to  date;  namely,  the  Chromafiuni  chromatophore.  Does  this  particle 
represent  the  limiting  size  of  chromatophore  or  a  lower  level  of  organiza- 
tion ?  Although  it  is  possible  to  construct  a  sphere  150  A  in  diameter  with 
a  60  A  thick  cortex,  the  area  available  at  the  inner  surface  could  accommo- 
date onlv  about  one-tenth  of  the  chlorophyll  contained  in  the  particle; 
thus,  the  type  of  architecture  postulated  for  the  Chnmatiuiu  chromatophore 
[6]  is  not  applicable  to  this  system.  In  addition,  in  some  properties,  the 
pigmented  particle  ditfers  rather  sharply  from  the  chromatophores  which 
have  been  studied ;  for  example,  in  the  degree  to  which  accessory  pigments 
separate  from  the  particle  and  the  tendency  of  the  particles  to  aggregate 
into  progressively  larger  rod-like  structures.  For  these  reasons,  we  prefer, 
for  the  time  being,  to  regard  this  system  as  less  highly  organized  than  the 
chromatophore  and  refer  to  the  pigmented  component  either  as  photo- 
svnthin,  a  name  employed  for  the  pigmented  extracts  prior  to  the  advent 
of  the  chromatophore  concept  [18]  or  as  holochrome,  the  term  used  to 
designate  "a  colored  substance  as  it  exists  in  its  natural  state  within  an 
organism,  where  the  colored  group  is  combined  or  associated  with  a 
carrier  which  alters  the  physical  or  physiological  properties  of  the 
prosthetic  group"  [36]. 

If  we  assume  that  the  lamella  is  not  an  architectural  feature  in  this 
system,  then  the  idea  that  lamellar  organization  is  essential  for  photo- 
synthesis is  brought  into  question.  One  solution  to  this  dilemma  is  to 
consider  the  lamella  as  the  expression  of  a  more  fundamental  characteristic 

VOL.   II. Y 


322  J.    A.    BERGERON   AND   R.    C.    FULLER 

which  can  be  extrapolated  to  the  molecular  level.  An  application  of  this 
concept  is  illustrated  in  the  model  proposed  for  the  ultrastructure  of  the 
Chromatium  chromatophore  [6].  In  this  instance  (Fig.  12),  the  submicro- 
scopic  architecture  is  generated  by  the  juxtaposition  of  molecular  units. 
As  in  crystallization,  it  is  the  properties  of  the  building  block,  a  pigmented 
protein  with  hydrophilic  and  hydrophobic  poles,  which  determine  the 


Fig.  12.  Working  hypothesis  of  the  ultrastrvicture  of  the  chromatophore.  The 
Chromatiimi  chromatophore  is  described  as  a  hollow  sphere  about  320  A  in  diameter 
with  a  cortex  about  90  A  thick.  The  pigment  molecules  (B)  aligned  in  a  monolayer 
are  bounded  internally  by  a  phospholipid  (A)  monolayer  and  externally  by  a 
60  A  thick  protein  layer.  The  "minimal  unit"  of  composition  has  been  used  as  a 
structural  subunit.  The  protein  has  been  folded  and  is  related  directly  to  two 
chlorophyll  molecules.  On  the  average  the  protein  is  related  indirectly  to  one 
carotenoid  molecule  and  ten  phospholipid  molecules. 


form  of  the  assemblage.  Such  a  scheme  provides  for  specificity  in  a  protein 
of  conventional  dimensions,  requires  no  assumptions  beyond  the  principles 
of  molecular  interaction  for  obtaining  a  higher  level  of  organization,  and 
also  allows  for  great  flexibility  in  the  composition  of  the  lipid  phase. 

If  structural  organization,  above  the  molecular  level,  is  a  prerequisite 
for  photosynthesis,  then  the  Chlorobiiim  holochrome  must  approach  the 
limiting  conditions.  This  particle  is  already  within  the  physical  range  of 


PHOTOSYNTHETIC  MACROMOLECULES  OF  CHLOROBIUM  THIOSULFATOPHILIUM      323 

materials,  such  as  the  haemocyanins,  which  are  classed  as  respiratory 
proteins.  Such  considerations  as  these  add  interest  to  the  study  of  the 
photochemical  activity  and  composition  of  this  holochrome. 

Unequivocal  evidence  of  photophosphorylation  has  not  been  obtained 
with  the  isolated  holochrome,  but  many  reasons  can  be  advanced  to  explain 
failure.  There  is  no  indication,  so  far,  in  the  composition  data  that  this 
particle  is  fundamentally  different  from  the  other  photosynthetic  systems. 
There  are  two  pigment  types,  the  chlorophyll  and  the  carotenoids.  There 
are  enough  chlorophyll  molecules  present  [100]  to  meet  the  requirements 
of  the  semiconductor  hypothesis.  The  cytochromes,  which  figure  promi- 
nently in  current  concepts  of  the  primary  events  [5,  27]  are  present  albeit 
in  near-limiting  quantities.  It  is  not  known,  however,  whether  the  import- 
ant [10,  15]  lipid-soluble  quinone  compounds  are  present. 

It  seems  reasonable  to  expect  that  continued  study  of  this  system  will 
help  to  define  fundamental  relationships  between  structure  and  function 
in  photosynthesis. 

Acknowledgments 

We  wish  to  thank  Dr.  S.  Conti  and  Aliss  H.  Kelly  for  culturing  the 
organisms  employed  in  this  study.  The  skilful  technical  assistance  of 
M.  Gettner  and  W.  Geisbusch  is  also  gratefully  acknowledged. 


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324  J-    A.    BERGERON   AND   R,    C.    FULLER 

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21.  Frenkel,  A.  W.,  Amer.  Rev.  Plant  Physiol.  10,  53  (1959). 

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24.  Fuller,  R.  C,  Bergeron,  J.  B.,  and  Anderson,  I.  C,  Arch.  Biochem.  Biophys. 
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Discussion 

Chance  :  Was  there  more  than  one  kind  of  cytochrome  present  in  the  purified 
particles  ? 

Bergeron  :  I  am  not  speaking  from  my  own  data  now,  I  am  speaking  for 
Hulcher  and  Conti  [26];  there  appears  to  be  an  /-type  and  an  (7-type. 


Some  Physical  and  Chemical  Properties 
of  the  Protochlorophyll  Holochrome 

James  H.  C.  Smith 

Carnegie  Institution  of  Washington,  Department  of  Plant  Biology, 
Stanford,  Calif.,  U.S.A. 

Protochlorophyll,  the  chlorophyll  precursor  of  dark-grown  seedlings, 
has  been  isolated  in  active  form.  Even  though  this  material  is  separated 
from  the  plant  it  is  transformed  by  light  to  chlorophyll.  Since  the  active 
material  is  proteinaceous,  it  can  be  extracted  and  fractionated  by  the  pro- 
cedures of  protein  chemistry.  The  active  material  is  particulate  and  shows 
a  distinct  sedimentation  peak  in  the  ultracentrifuge  diagram  [i]  with  sedi- 
mentation constant  of  about  i6  to  17  Svedberg  units.  If  it  is  assumed  to 
have  the  same  density,  i  •  2^2>^  ^^  many  other  proteins  with  the  same  sedi- 
mentation constant,  its  molecular  weight  would  be  about  400  000.  Later 
experiments  have  shown  the  particle  to  have  a  density  in  solution  of 
approximately  i  •  16.  Based  on  this  density  the  molecular  weight  would  be 
about  700  000  [2].  This  agrees  fairly  well  with  a  molecular  weight  of 
900  000  calculated  from  the  ratio  of  protochlorophyll  to  protein  obtained 
by  analysis  [2]  when  a  molecular  ratio  of  one,  for  pigment  to  protein,  was 
assumed.  Conversely,  the  agreement  of  the  results  obtained  bv  analysis 
and  by  centrifugation  supports  the  assumption  that  the  pigment-protein 
complex  contains  only  one  protochlorophyll  component. 

Carotenoids  in  protochlorophyll  holochrome 

Although  the  centrifugation  pattern  indicates  a  fairly  homogeneous 
molecular  species  in  respect  to  molecular  weight,  the  carotenoid  content 
of  the  isolated  material  shows  that  all  the  particles  cannot  be  of  exactlv  the 
same  composition.  The  caroienoid-protochlorophvH  ratio  varies  from 
preparation  to  preparation.  Furthermore,  on  occasion,  the  molecular  ratio 
of  total  carotenoid  to  protochlorophyll  is  about  one-half  [3].  If  each  holo- 
chrome particle  contains  one  protochlorophyll,  then  some  particles  lack 
carotenoids  entirely.  But  the  carotenoid  fraction  is  made  up  of  several 
carotenoids,  as  reference  to  Fig.  i  shows,  and  this  complicates  the 
stoicheiometry  of  the  holochrome  still  further. 

The   ratios   of  carotenoid   to   protochlorophyll    imply   either   of  two 


326 


JAMES    H.    C.    SMITH 


possible  situations.  One  is  that  on  any  individual  holochrome  particle  no 
fixed  ratio  of  yellow  pigments  to  protochlorophyll  exists.  This  means, 
therefore,  that  the  holochrome  cannot  be  a  definite  compound.  The  other 
situation  is  that  lack  in  uniformity  of  composition  results  from  con- 
tamination of  the  protochlorophyll  holochrome  by  carotenoid  holochromes. 
If  this  is  true,  the  carotenoid  and  protochlorophyll  holochromes  must  have 
very  similar  sedimentation  and  precipitation  properties. 


u  o 

446 

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0-3 

- 

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Ai7     /      \     /    \ 

/    1            /      443    \         /          \ 

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0-2 

//              /   \\y    A  \ 
^Z/     418        /          \       /    \\ 

/    J       A -^           448  ^^           M 

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\ 

0-1 

-     1 

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^^ 

■^r-^ 

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00 

M 

3 

1                               1 

N^,^-^ 

400 


500 


450 
Wavelength   (m/i) 

Fig.    I.  Absorption    spectra    of   carotenoid    fractions    from    protochlorophyll 
holochrome  isolated  by  chromatography  on  columns  of  powdered  cellulose. 


The  carotenoids  were  extracted  from  the  pigment-protein  complex, 
named  protochlorophyll  holochrome,  with  8o",,  acetone.  They  were  then 
transferred  to  petroleum  ether  and  chromatographed  on  powdered 
cellulose.  The  chromatograms  were  developed  by  various  mixtures  of 
acetone  in  petroleum  ether. 

Positive  identification  of  the  individual  carotenoids  has  not  been  made, 
but  from  the  shapes  of  the  absorption  curves,  the  positions  of  the  maxima, 
and  certain  colour  reactions  the  pigments  have  been  tentatively  identified 
as  lutein  epoxide  ester  (curve  i),  lutein  (curve  2),  isolutein  (curve  3),  and 
violaxanthin  h  (curve  4).  These  assignments  dilTer  from  those  given  the 
carotenoids  from  etiolated  bean  leaves  {Phaseolus  vulgaris)  by  Goodwin 
and  Phagpolngarm  [4]  who  have  identified  the  following:  /3-carotene, 
10-8%  of  the  carotenoid  pigments  present,  lutein,  38 •4",,,  neoxanthin, 
50-7%,  and  a  trace  of  an  unknown  yellow  pigment.  At  present  it  is  im- 
possible to  evaluate  the  cause  of  the  discrepancies. 

The  function  of  the  carotenoids  in  the  holochrome  is  still  unknown. 
Previously,  it  was  intimated  that  these  pigments  played  an  obscure  role  in 


PHYSICAL    AND    CHEMICAL    PROPERTIES — PROTOCHLOROPHYLL    HOLOCHROME       327 

the  protochlorophyll  conversion  since  the  amount  of  chlorophyll  formed 
by  illumination  was  found  to  be  statistically  related  molecule-for-molecule 
to  the  carotenoid  present  [3].  Now  that  we  have  found  the  carotenoid 
fraction  to  be  made  up  of  several  constituents  it  would  be  necessary  to 
assume  that  each  constituent  was  equally  effective  in  the  conversion  of 
protochlorophyll  in  order  to  account  for  the  stoicheiometry  of  the  reaction. 
This  is  very  unlikely,  and  militates  against  such  an  hypothesis.  A  further 
argument  against  such  an  assumption  is  that  in  etiolated  albino  leaves 
nearly  complete  conversion  occurs  in  the  absence  of  carotenoids  [3].  It  is 
improbable,  therefore,  that  carotenoids  are  involved  in  the  protochloro- 
phyll transformation. 

It  has  frequently  been  proposed  that  carotenoids  function  as  inhibitors 
of  the  photo-oxidation  of  chlorophyll.  The  isolated  carotenoid-containing 
holochrome,  however,  loses  its  chlorophyll  by  extended  illumination.  In 
this  system,  little  protection  against  bleaching  is  afforded  by  the  presence 
of  yellow  pigments. 

Fluorescence  polarization  of  protochlorophyll  holochrome 

When  the  fluorescence  of  a  molecule  is  excited  with  plane  polarized 
monochromatic  light,  the  fluorescence  emitted  under  certain  circum- 
stances may  also  be  partly  polarized.  In  principle,  if  a  fluorescing  molecule 
remains  stationary  and  retains  its  absorbed  energy  during  the  interval 
between  absorption  of  the  exciting  light  and  emission  of  the  fiuorescence, 
it  emits  fluorescence  having  a  certain  maximum  degree  of  polarization. 
Polarization  values  lower  than  this  indicate  that  the  molecule  has  either 
rotated  or  else  transferred  its  energy  to  like  molecules  during  the  period  of 
excitation  [5,  6].  Much  can  be  learned  about  the  state  of  fluorescent  sub- 
stances from  measurement  of  this  property.  Because  of  this,  the  fluorescent 
properties  of  the  protochlorophyll  holochrome  have  been  studied  by  this 
technique  [7,  8]. 

The  fluorescence  polarization  of  the  protochlorophyll  holochrome  was 
measured  in  an  apparatus  similar  to  that  used  previously  by  Goedheer  [5]. 
To  test  the  operation  of  the  apparatus,  the  fluorescence  polarization  of 
chlorophyll  in  castor  oil  was  determined.  The  value,  28-9,  was  found 
which  agrees  well  with  Goedheer's  former  measurement,  viz.  28.  Light 
from  the  cadmium  arc,  wavelength  644  m/z,  was  used  for  exciting  the 
fluorescence. 

Preliminary  experiments  in  collaboration  with  Dr.  Paul  Latimer  [7] 
gave  polarization  of  fluorescence  values  for  protochlorophyll  holochrome 
lower  than  for  chlorophyll  in  castor  oil.  This  observation  was  corroborated 
by  Goedheer  and  Smith  [9],  who  obtained  a  value  of  15  for  a  glycerine 
extract  of  protochlorophyll  holochrome  from  etiolated  bean  leaves,  and 


328  JAMES    H.    C.    SMITH 

18  for  a  glycine  buffer  extract  (pH  9-5).  Because  of  the  irradiation 
necessary  for  measuring  fluorescence  polarization,  the  protochlorophyll  is 
largely  converted  into  chlorophyll,  consequently,  the  fluorescence  obtained 
is  mostly  irom  chlorophyll.  These  values,  15  and  18,  are  lower  than  the 
value  for  chlorophyll  in  castor  oil,  28-9.  Three  possibilities  suggest  them- 
selves to  account  for  the  lowered  polarization:  one,  that  the  holochrome 
rotates  more  freely  than  chlorophyll  immobilized  in  castor  oil;  two,  that 
it  transfers  its  energy  to  other  chlorophyll  molecules;  or  three,  that  the 
pigment  is  free  to  rotate  within  the  holochrome. 

Because  the  holochrome  is  so  large,  it  cannot  conceivably  rotate  fast 
enough  to  depolarize  its  fluorescence.  The  second  suggestion  of  energy 


0-4  08  1-2 

Chlorophyll  absorbance 

Fig.  2.  The  reciprocal  of  the  fluorescence  polarization  of  the  protochlorophyll- 
chlorophyll  holochrome  plotted  against  the  optical  density  of  the  chlorophyll 
maximum  ( ~  670  m^u)  at  different  stages  of  greening. 


transfer  between  chlorophyll  molecules  also  seems  improbable  in  view  of 
the  small  number  of  pigment  molecules  per  holochromatic  particle.  This 
leaves  only  the  third  alternative  as  likely. 

An  estimate  of  the  limiting  fluorescence  polarization  value  when  no 
energy  transfer  exists  can  be  obtained  by  extrapolating  the  fluorescence  to 
zero  pigment  concentration.  This  was  done  by  extracting  the  holochrome 
from  leaves  at  different  stages  of  greening,  and  by  relating  the  fluorescence 
polarization  with  chlorophyll  content  through  the  expression 

i/P  =  iIPo  +  ACt  (8) 

in  which  P  is  the  polarization  of  fluorescence  measured ;  A  is  a  constant ; 
C  is  the  optical  density  of  the  chlorophyll  peak  at  about  670  rufx,  which  is 
proportional  to  the  chlorophyll  content;  r  is  the  lifetime  of  the  activated 
state;  and  Pq  is  the  polarization  when  C  is  zero.  A  plot  of  ijP  against 


PHYSICAL    AND    CHEMICAL    PROPERTIES — PROTOCHLOROPHYLL    HOLOCHROME       329 

chlorophyll  absorbance,  Fig.  2,  gave  a  straight  line  within  the  experi- 
mental error  which  extrapolated  to  a  value  of  15-4  for  Pq.  This  is  in  good 
agreement  with  the  \alue  of  15%  obtained  by  direct  measurement  on  the 
protochlorophvll  holochrome.  This  indicates  that  the  lower  value  of 
fluorescence  polarization  in  the  original  protochlorophyll  holochromes  is 
not  due  to  energy  transfer  between  chlorophyllous  pigments. 

The  third  alternative  put  forward  to  account  for  the  definite  but  sub- 
maximal  polarization  of  the  holochromatic  pigment  is  that  the  pigment 
exists  in  the  holochrome  in  such  a  way  as  to  have  partial  freedom  of 
rotation.  This  could  be  accomplished  if  the  pigment  were  attached  to  the 
amino-acid  "tails"  of  the  holochrome  protein  similarly  to  haem  in  haemo- 
globin and  myoglobin  [8,  10]. 

Alkaline  inhibition  of  protochlorophyll  transformation 

If  the  binding  of  haem  in  haemoglobin  and  of  protochlorophyll  in  its 
holochrome  are  analogous,  then  the  bonding  of  protochlorophyll  to 
protein  in  the  holochrome  should  be  influenced  by  treatment  with  alkali 
at  specific  pH  values  [n].  The  eftectiveness  of  the  various  pH  values  for 
disrupting  the  pigment-protein  complex  should  depend  upon  the  acid 
dissociation  constants  of  the  amino-acid  groups  binding  the  pigment. 

Inasmuch  as  the  transformation  of  protochlorophyll  to  chlorophyll  is 
stopped  when  the  protochlorophyll  is  separated  from  the  protein,  a  dis- 
sociation by  treatment  with  alkali  should  stop  the  transformation.  The  pH 
at  which  the  transformation  is  stopped  should  be  characteristic  of  the 
ionization  constant  of  the  protochlorophyll-amino  acid  complex  involved. 
Conversely,  the  pH  values  at  which  the  transformation  is  stopped  should 
indicate  what  amino  acid  groups  hold  the  pigment.  For  this  reason,  a 
detailed  studv  of  the  efi"ect  of  alkali  on  the  protochlorophyll  holochrome 
and  on  the  inhibition  of  the  transformation  has  been  undertaken. 


Effect  of  pH  on  the  protochlorophyll-chlorophyll  transformation 

The  various  degrees  of  inhibition  of  the  protochlorophyll-chlorophyll 
conversion  caused  by  treatment  of  the  protochlorophyll  holochrome  at 
diflFerent  alkalinities  are  shown  in  Fig.  3.  The  protochlorophyll  holochrome 
was  suspended  in  solutions  of  various  pH  values  for  different  lengths  of 
time.  At  stated  intervals,  samples  were  removed,  neutralized  with  glycine, 
and  spectrophotometered  before  and  after  being  illuminated  for  3  min. 
The  optical  densities  of  the  chlorophyll  formed  were  measured  at  the 
chlorophyll  absorption  maximum,  ~  678  m/t.  In  Fig.  3,  thev  are  plotted 
as  ordinate  against  the  time  of  standing  in  the  alkaline  medium. 

As  is  evident  from  Fig.  3,  pH  values  between  7-20  and  9- 16  have  little 


330 


JAMES   H.    r.    SMITH 


if  anv  effect  on  the  transformation.  At  pH  9-70,  the  alkahnity  partly 
inhibits  the  transformation  and  as  the  alkahnity  is  increased  to  10-27, 
ID -88,  and  11-92  the  inhibition  is  intensified.  In  order  to  estimate  the 
maximum  degree  of  inhibition  at  each  pH,  an  algebraic  equation  was 
sought  which  permitted  the  limit  of  the  transformation  to  be  calculated 
from  the  experimental  data.  The  third  order  reaction  velocity  equation 
did  this  as  the  concordance  between  experimental  points,  marked  with 
circles,  and  calculated  values,  depicted  by  solid  lines,  demonstrates. 


0-25 


0-20 


015 


010 


005 


000 


Equation-jY  =  f^^C  'C^) 


7  20 
916 


Fk;.   3.   Comparison  of  the  rates  of  inactivation  and  limits  of  the  transformation 
of  protochlorophyll  holochrome  to  chlorophyll  holochrome  at  various  alkalinities. 


Between  pH  9-70  and  10 -88,  the  limiting  ^alue,  6\^„  of  the  chlorophyll 
formed  decreases  with  increase  in  pH.  The  rate  constants,  R,  do  not 
increase  markedly  in  this  range.  The  amount  of  pigment  that  can  be 
inhibited  from  transforming  at  each  pH,  C„,  increases  with  increase  in  pH. 
It  is  this  increase  of  C^,  rather  than  the  change  in  the  velocity  constant, 
R,  that  causes  the  greater  initial  velocity  of  inhibition  at  higher  pH.  In 
fact,  the  initial  velocity  of  inhibition  at  9-70,  10-27,  '^^^^  10-88  are  directly 
proportional  to  the  hvdroxyl  ion  concentrations.  At  higher  pH  values  the 
increased  rate  of  inactivation  must  be  due  to  a  greater  velocity  constant 
rather  than  to  an  increase  of  inactivatible  material,  which  has  already 
reached  its  limit  at  10-88. 

The  facts  presented  in  Fig.  3  indicate  that  the  inactivation  reaches 
different  limits  depending  on  pH.  One  interpretation  of  this  result  is  that 


PHYSICAL    AND    CHEMICAL    PROPERTIKS — PROTOCHLOROPHYLL    HOLOCHROME       33 1 

the  pigment  attaches  itself  to  protein  through  a  bonding  that  is  sensitive 
to  hydroxy!  ions,  such  as  amino  groups  that  form  ammonium  compounds, 
or  phenohc  groups  that  act  through  hydrogen  bonding.  This  may  be 
iUustrated  as  follows: 

R— NH2  +  HOR'  =  R^NHa      H+      OR' 

When  the  acidic  hydrogen  ion  is  neutralized  the  addition  compound 
dissociates 

R— NH.,     H+      -0R'+    OH  =  R— NH0  +  HOH+  -OR' 

In  the  case  of  the  protochlorophyll-protein  complex,  when  it  is  dis- 
sociated by  hydroxyl  ions,  the  protochlorophyll  could  no  longer  be 
transformed  to  chlorophyll  by  light.  This  is  the  proposed  explanation  for 
the  inhibition  of  the  transformation  by  action  of  hydroxyl  ions. 

The  pH  values  that  inhibit  the  photochemical  transformation  corre- 
spond to  the  dissociation  constants  of  certain  amino  acids  which  may  be 
implicated  in  the  bonding  of  protochlorophyll.  These  amino  acids  with 
their  approximate  pK  values  [12]  are  e-amino  of  lysine,  9 -410  10 -6,  phenolic 
hydroxyl  of  tyrosine,  9-8  to  10-4,  and  the  sulphydryl  of  cysteine,  9-1  to 
10 -8.  These  values  may  differ  considerably  from  one  protein  to  another, 
and  even  in  the  same  protein.  For  example,  Stracher  [13]  found  in  the 
spectrophotometric  titration  of  myosin  two  groups  ol  tyrosine  residues 
with  pK  values  of  10-5  and  12-2  respectively.  For  this  reason,  no  precise 
values  for  the  pK  values  of  the  amino  acids  in  the  protochlorophyll 
holochrome  can  be  assigned  a  priori. 

IONIZATION    CONSTANTS    FROM    TITRATION    CURVES 

'Fhe  titration  cur\e  of  protochlorophyll  holochrome  is  shown  in  Fig.  4. 
From  this  curve  it  is  obvious  that  two  titration  steps  exist  within  the  pH 
range  effectively  inhibiting  transformation  of  protochlorophyll.  The 
inflection  points  are  at  pH  values  of  about  10-2  and  11  "3.  For  groups  with 
these  pK  values,  ionization  would  be  about  10",,  complete  at  pH  9-2  and 
10-3.  Thus  it  appears  that  the  coincidence  of  the  pK  values  from  the 
titration  curve  and  the  pH  values  efl'ectively  inhibiting  protochlorophyll 
conversion  makes  the  assumption  reasonable  that  the  pigment  is  bound 
to  protein  through  the  amino  acid  residues — the  most  likely  candidates 
being  lysine,  cysteine,  and  tyrosine. 

IONIZATION    CONSTANTS    FROM    SPECTROSCOPIC    MEASUREMENTS 

Changes  in  pH  profoundly  modify  the  protein  part  of  the  proto- 
chlorophyll holochrome  as  variations  of  the  ultraviolet  absorption  spectrum 
show.  In  Fig.  5  is  pictured  the  absorption  of  protochlorophyll  holochrome 


332 


JAMES    H.    C.    SMITH 


in  the  visible  and  ultraviolet  regions  of  the  spectrum.  The  absorption  in 
the  ultraviolet  is  due  very  largely  to  the  protein  part  of  the  holochrome. 
In  Fig.  6  is  shown  the  effect  of  pH  on  the  ultraviolet  absorption  of  the 


0-2  04  0-6 

Volume  004N  KOH 

Fig.  4.  The  alkaline  titration  curve  of  protochlorophyll  holochrome. 

holochrome.  At  about  pH  9-7,  spectral  changes  become  obvious.  Perhaps 
it  is  noteworthy  that  at  this  alkalinity  the  inhibition  of  the  protochlorophyll 
conversion  begins  to  intensify  significantly. 


1-8  _ 


300 


600 


700 


400  500 

Wavelength  (m//) 

Fig.   5.  The  absorbance  of  protochlorophyll  holochrome  in  the  visible  and 
ultraviolet  regions  of  the  spectrum. 


PHYSICAL    AXD    CHEMICAL    PROPERTIES — PROTOCHLOROPHYLL    HOLOCHROME      333 

From  the  changes  in  absorption  with  changes  in  pH  it  is  possible  to 
calculate  pK  values  for  the  components  undergoing  change.  The  equation 
for  this  calculation  is 

pK  =  pH-log.4  +  log(.4o-^) 
Here  pK  and  pH  have  their  usual  meaning,  and  A  is  the  absorbance 
change  at  a  particular  pH  while  ^o  ^^  the  maximum  change  in  absorbance 
produced  by  increase  of  pH.  The  change  in  absorbance  is  measured  from 
a  reference  absorbance  which  is  constant  over  a  considerable  pH  range  at 
the  lower  pH  values.  In  order  to  make  the  calculations  consistent  among 
themselves,  the  changes  in  absorption  were  always  related  to  the  maximum 
absorption  of  the  corresponding  curves.  From  manv  ultraviolet  absorption 


Wavelength  {m/.i) 


Fig.  6.  The  variation  in  the  ultraviolet  absorption  spectrvim  of  protochlorophyll 
holochronie  with  pH. 

curves,  such  as  those  presented  in  Fig.  6,  pK  values  were  calculated  from 
changes  in  the  absorbancies  at  ^oo  m/x  and  at  the  minimum  near  2^0  m/Lt 
prominent  in  the  left-hand  cur\e.  The  two  values  obtained  were  11  -o  and 
10-4,  respectivelv. 

The  changes  in  absorption  at  ^00  m/t  and  the  pK  \alue  obtained 
certainly  implicate  tyrosine  as  one  of  the  amino  acids  undergoing  ionization 
in  the  holochrome.  The  changes  at  the  absorption  minimum  could  possibly 
be  ascribed  to  cysteine  [14]  although  this  is  by  no  means  certain.  The  pK 
value  of  10-4  ajiproximates  to  that  reported  for  cysteine,  9- 1  to  10 -8  [12]. 


ULTRAVIOLET    IRRADIATION    AND    PROTOCHLOROPHYLL    TRANSFORMATION 

A  further  reason  for  assuming  that  protochlorophyll  is  attached  to 
more  than  one  amino  acid  is  the  effect  of  exposing  the  holochrome  to 


334 


JAMES   H.    C.    SMITH 


ultraviolet  radiation.  Mr.  G.  C.  McLeod  and  Miss  J.  Coomber,  in  our 
laboratory  [15],  discovered  that  protochlorophyll  holochrome  irradiated 
with  various  ultraviolet  wavelengths  between  250  and  330  myu,  converted 
only  25  to  30%  of  the  protochlorophyll  transformed  at  366,  436  m^ 
(Fig.  7),  or  with  visible  light  from  an  electric  lamp.  If  after  ultraviolet 
irradiation,  however,  the  holochrome  solution  was  placed  in  visible  light, 
the  same  degree  of  transformation  was  achieved  as  if  no  previous  con- 
version with  ultraviolet  had  occurred.  Wherefore,  the  ultraviolet  at  the 
intensities  used  had  no  ill  effect  on  the  transformation. 


300  350  400 

Wavelength  (my/) 


450 


Fig.  7.  The    maximum    conversion    of    holochromatic    protochlorophyll    to 
chlorophyll  in  the  range  436  to  250  m/j. 


The  conversion  with  ultraviolet  light  could  not  be  explained  by  proto- 
chlorophyll absorption  else  the  conversion  would  have  been  augmented 
with  longer  exposures.  But  the  exposures  given  were  two  or  three  times 
those  necessary  to  achieve  maximum  conversion  in  the  250-330  mn  range. 

The  limited  action  of  ultraviolet  light  may  be  reasonably  explained  by 
assuming  the  protochlorophyll  to  be  activated  through  transfer  of  the 
energy  absorbed  by  a  closely  associated  amino  acid.  Only  the  aromatic 
amino  acids  absorb  appreciably  throughout  this  range,  and  of  these  acids 
only  tyrosine  has  the  proper  pK  value  to  correspond  with  the  alkalinities 
effective  in  the  inhibition  of  the  transformation.  From  these  considerations 
it  is  concluded  that  about  25  to  30%  of  the  protochlorophvll  is  attached 
to  protein  in  the  holochrome  through  the  tyrosinyl  group. 


PHYSICAL    AND    CHEMICAL    PROPERTIES — PROTOCHLOROPHYLL    HOLOCHROME      335 

INFLUENCE    OF    PH    ON    THE    SPECTRAL    ABSORPTION    OF    PROTO- 
CHLOROPHYLL   IN   THE    HOLOCHROME 

If  alkalinity  affects  the  association  of  protochlorophyll  with  protein, 
the  absorption  spectrum  of  protochlorophyll  in  the  holochrome  should 
vary  with  pH.  This  deduction  comes  from  the  fact  that  the  absorption 
spectra  of  protochlorophyll  in  the  free  and  holochromatic  states  differ. 

The  absorption  spectrum  of  protochlorophyll  does  vary  with  pH.  It 
shifts  to  shorter  wavelengths  with  higher  pH  as  the  results  of  Table  I  show. 
This  is  what  would  be  expected  if  dissociation  were  greater  at  higher 
alkalinities. 

TABLE  I 

Effect  of  pH  Values  on  the  Absorption  Maximum 
OF  Protochlorophyll  Holochrome 


pH 

Wavelength  of 

absorption  max. 

9- 16 

639-5 

9-70 

637-5 

/                        10-27 

637-5 

10 -88 

634-5 

1 1  -92 

633-5 

DISRUPTION    OF    THE    CYCLOPENTANONE    RING 

When  protochlorophyll  holochrome  is  treated  at  alkalinities  near  pH  1 1, 
the  absorption  spectrum  of  the  protochlorophyll  changes  drastically  in  the 
blue  region  of  the  spectrum.  The  absorption  band  at  421  m/z  is  increased 
in  height  at  the  expense  of  the  440  m^u.  band.  The  spectrum  obtained, 
Fig.  8,  is  similar  to  that  obtained  by  Granick  [16]  for  protoporphyrin  and 
magnesium  protoporphyrm  (cf.  insert  Fig.  8),  which  indicates  the  con- 
version of  pheoporphyrin  to  porphyrin.  Whether  this  disruption  of  the 
cyclopentanone  ring  takes  place  before  or  after  the  splitting  of  the  pigment- 
protein  complex  is  being  examined  at  the  present  time. 

Summary 

Measurements  on  the  fluorescence  polarization  of  protochlorophyll 
holochrome  have  led  to  the  supposition  that  protochlorophyll  is  attached 
to  amino  acid  "tails"  of  the  protein  in  the  holochrome.  This  supposition 
has  been  strengthened  by  determinations  on  the  inhibition  of  the  trans- 
formation by  alkali.  The  results  of  these  determinations  indicate  the 
involvement  of  several  amino  acids  in  this  pigment-protein  binding. 
Comparison  of  the  pH  values  effective  in  preventing  the  transformation 


336  JAMES   H.    C,    SMITH 

with  the  pK  values  of  various  amino  acids  suggests  the  participation  of 
lysine,  cysteine,  and  tyrosine.  Changes  in  ultraviolet  absorption  of  the 
holochrome  with  pH  implicates  cysteine  and  tyrosine.  Furthermore,  the 
limited  transformation  produced  by  ultraviolet  radiation  points  strongly 


Chlorella  Vulgaris 
Mutant  60 


pH  =1133 
55  min 


pH  =  ll-54 
2  _  135  min 

pH=ll-56 
2    25  min 


400        450      500      550      600      650       700      750 
Wavelength  (m/x) 

Fig.   8.  The  effects  on  the   absorption  spectrum  and  transformation   of  the 
protochlorophyll  holochrome  caused  by  extended  treatment  at  high  pH. 


to  tyrosine  as  binding  from  25  to  30",,  of  the  protochlorophyll.  The  shift 
of  the  absorption  spectrum  of  protochlorophyll  holochrome  in  the  visible 
with  increased  pH  values  also  implies  a  disturbance  of  the  linkage  between 
pigment  and  protein.  pH  values  of  11  and  above  cause  rapid  splitting  of  the 
cyclopentanone  ring.  How  far  this  controls  the  inhibition  of  transformation 
is  yet  to  be  determined. 

References 

1.  Smith,  J.  H.  C,  and  Kupke,  D.  W.,  Nature,  Loud.  178,  751-752  (1956). 

2.  Smith,  J.  H.  C,  and  Coomber,  J.,   Yearb.  Carneg.  lustu.  58,  333  (1959). 

3.  Smith,  J.  H.  C,  Yearb.  Carneg.  Instil.  57,  289  (1958). 

4.  Goodwin,  T.  W.,  and  Phagpolngarm,  S.,  Biochem.  jf.  76,  197-199  (i960). 


PHYSICAL    AND    CHEMICAL    PROPERTIES PROTOCHLOROPHYLL    HOLOCHROME       337 

5.  Goedheer,   J.    C,   "Optical   Properties   and  In    Vivo   Orientation   of  Photo- 
synthetic  Pigments".  Drukkerij  Gebr.  Janssen-Nijmegen,  Utrecht  (1957)- 

6.  Forster,  Th.,  "  Fluoreszenz  Organischer  Verbindungen".  Vandenhoeck  and 
Ruprecht,  Gottingen  (195 1). 

7.  Latimer,  Paul,  and  Smith,  J.  H.  C,  Yearb.  Carneg.  Instn.  57,  293-295  (1958)- 

8.  Goedheer,  J.  C,  and  Smith  J.  H.  C,  Yearb.  Carneg.  Instn.  58,  334-336  (i959)- 

9.  Goedheer,  J.  C,  and  Smith,  J.  H.  C,  unpublished. 

10.  Kendrew,  J.  C,  et  al.  Xatiire,  Lond.  185,  422-427  (i960). 

11.  Wyman,  Jeffries,  Jr.,  Advanc.  Protein  Chem.  4,  410-531  (1948). 

12.  Cohn,   E.   J.,   and   Edsall,  J.   T.,   "Proteins,   Amino  Acids,   and   Peptides". 
Reinhold  Publishing  Corp.  445  (i943)- 

13.  Stracher,  X.,J.  biol.  Chem.  235,  2302-2306  (i960). 

14.  Fromageot,  Claude,  and  Schnek,  Georges,  Bioclmn.  biopliys.  Acta  6,  114-122 

(1950)- 

15.  McLeod,  G.  C,  and  Coomber,  J.,   Yearb.  Carneg.  Instn.  59,  324.  (i960). 

16.  Granick,  S.,  J.  bin/.  Chem.  175,  333-342  (1948). 


Discussion 

Goodwin:  It  doesn't  appear  that  carotenoids  play  any  important  part  in  the 
normal  transformation  of  protochlorophyll  into  chlorophyll.  I  was  wondering  if 
thev  can  play  a  part  if  required.  In  other  words  have  you  run  an  action  spectrum 
for  this  transformation  and  can  the  carotene-absorbed  light  be  used  ? 

Smith  :  Actually  the  carotene-absorbed  light  is  a  hindrance,  because  it  acts  as  a 
screen.  When  you  run  the  action  spectrum  you  find  that  it  is  exactly  the  absorption 
spectrum  of  protochlorophyll  in  the  albino  leaf,  and  in  a  normal  leaf  which  contains 
large  amounts  of  carotenoids  the  peak  in  the  violet  is  ver\'  low  compared  to  the 
peak  in  the  red.  But  the  action  spectrum  in  an  albino  plant  is  very  high  in  the 
violet  as  compared  to  that  in  the  red,  so  consequently  the  carotenoids  actually  act 
as  a  screen. 

Chanxe:  Since  you  appear  to  have  one  chlorophyll  per  particle  do  you  then 
consider  that  you  have  a  heterogeneous  distribution  of  particles  or  two  separate 
bonds  on  a  single  particle  ? 

Smith:  We  presume  a  distribution  of  particles  and  this  is  probably  right 
because  otherwise  you  would  expect  the  transfer  of  energy,  once  it  is  absorbed, 
through  the  whole  protein  to  carry  on  the  transformation,  but  since  there  are 
discrete  particles  and  these  particles  are  80  to  100  A  in  diameter  you  can't  get 
energy  transfer  very  well  between  the  particles. 

K.^MEN :  How  do  you  conceive  the  process  of  chlorophyll  formation  ? 

Smith:  I  wish  we  knew  the  answer  to  that  question.  I  pointed  out  that  you 
have  a  change  in  the  absorption  spectrum  of  chlorophyll  in  the  plant  after  it  is 
formed.  We  are  thinking  that  perhaps  it  is  formed  on  one  protein  and  transferred 
to  another,  but  it  must  go  onto  the  same  protein  molecule  or  else  you  would  not 
get  the  increase  in  depolarization  that  you  do.  If  it  went  onto  separate  molecules 
you  wouldn't  get  this  depolarization  linear  with  concentration,  but  since  you  do 
get  that  you  are  piling  them  up  on  the  same  protein  molecule,  and  the  explanation 
that  we  have  of  this  is  that  owing  to  the  change  in  absorption  spectrum  and  owing 

VOL.  II. — z 


338  JAMES   H.    C.    SMITH 

to  this  polarization  effect  you  are  actually  making  them  on  one  enzymic  particle 
and  moving  them  over  to  another.  We  have  no  experimental  evidence  on  this. 

Frenkel  :  Are  there  two  types  of  protochlorophyll  on  your  particles,  one 
phytylated  and  one  non-phytylated  ? 

Smith  :  There  are  no  phytylated  compounds  present.  All  we  have  is  the  non- 
phytylated. 

Frenkel:  I  wonder  if  anyone  has  carried  out  an  experiment  yet  to  ascertain 
whether  the  hydrogens  in  the  transformation  of  protochlorophyll  to  chlorophyll  a 
come  from  water  or  from  some  non-exchangeable  hydrogens  on  the  protein  ? 

Smith  :  We  did  do  this  a  number  of  years  ago  when  we  had  high  hopes  that 
protochlorophyll  would  be  the  photosynthetic  hydrogen  acceptor.  We  did  this  by 
the  Fringsheim  method  of  quenching  of  phosphorescence  of  tryptoflavin.  We  put 
etiolated  leaves  into  the  apparatus  and  pumped  off  all  the  oxygen  so  that  we  had 
no  quenching  of  the  phosphorescence.  Then  we  illuminated  the  leaves  and  although 
the  transformation  of  the  protochlorophyll  was  80%  complete,  we  got  off  the  leaves 
only  I  or  2%  of  the  theoretical  amount  of  oxygen.  In  other  words  the  hydrogen 
did  not  seem  to  be  pulled  away  from  the  water.  Now  if  you  went  ahead  with  this 
and  then  had  the  chlorophyll  already  formed  and  put  those  leaves  in,  then  on 
illumination  the  oxygen  just  rolled  off,  so  there  is  a  time  factor  involved,  so  the 
failure  to  produce  oxygen  by  the  initial  illumination  can't  be  just  the  question  of 
utilization  of  that  oxygen  by  respiration. 


Photosynthetic  Phosphorylation  and  the  Energy 
Conversion  Process  in  Photosynthesis* 

Daniel  I.  ARxoxf 

Laboratory  of  Cell  Physiology,  University  of  California, 
Berkeley,  Calif.,  U.S.A. 

I.  Photosynthesis  outside  the  Hving  cell 

It  is  fitting  to  recall  in  a  symposium  devoted  to  biological  structure  and 
function,  that  understanding  of  life  phenomena  at  a  molecular  level  was 
always  advanced  by  the  separation  of  a  physiological  process  from  the 
structural  complexity  of  the  living  cell.  This  happened  first  for  fermenta- 
tion when  Biichner  in  1897  prepared  from  yeast  a  cell-free  juice  that 
fermented  sugar  [2].  The  most  recent  example  is  the  cell-free  synthesis  of 
DNA  by  Romberg  [3],  a  development  which  demonstrated  that  the  key 
events  in  reproduction  can  be  investigated  with  isolated  enzyme  systems. 

With  regard  to  photosynthesis,  in  1953  Rabinowitch  wrote  that  "the 
task  of  separating  it  from  other  life  processes  in  the  cell  and  analyzing  it 
into  its  essential  chemical  reactions  has  proved  to  be  more  difficult  than 
was  anticipated.  The  photosynthetic  process,  like  certain  other  groups  of 
reactions  in  living  cells,  seems  to  be  bound  to  the  structure  of  the  cell;  it 
cannot  be  repeated  outside  that  structure"  [4]. 

There  was  no  special  reason  why,  at  that  late  date  in  the  development 
of  biochemistry,  photosynthesis  could  not  be  reconstructed  outside  the 
living  cell.  The  simplest  explanation  for  the  repeated  failures  was  that 
inappropriate  experimental  methods  had  been  used  for  this  task  in  different 
laboratories,  including  our  own  [5].  A  continuing  search  for  improved 
experimental  methods  appeared  therefore  woith  while. 

The  most  hopeful  possibility  for  isolating  photosynthesis  from  the 
structural  complexity  of  the  whole  cell  seemed  to  lie  in  chloroplasts.  Few- 
physiological  processes  have  such  an  obvious  relation  to  a  distinct  cellular 
particle  as  photosynthesis  has  to  chloroplasts.  In  all  plants  which  have 
chloroplasts,  not  only  do  these  particles  contain  all  the  chlorophvll  (and 
the  accessory  pigments)  without  which  photosynthesis  cannot  proceed, 

*  This  article  is  based  on  a  paper  presented  at  the  Symposiuni  on  "Light 
and  Life",  at  Johns  Hopkins  LTniversity,  March  28-31,  i960  [i]. 

t  Aided  by  grants  from  the  National  Institutes  of  Health  and  the  Office  of 
Naval  Research. 


340  DANIEL    I.    ARNON 

but  also,  the  final  products  of  this  process,  starch  and  molecular  oxygen, 
are  formed  in  or  at  the  surface  of  illuminated  chloroplasts. 

Chloroplasts  were  once  widely  believed  to  be  the  site  of  complete 
photosynthesis,  that  is,  of  oxygen  evolution  coupled  with  carbon  dioxide 
assimilation  [6,  7].  But  this  view  was  not  supported  by  critical  experi- 
mental evidence  and  was  later  abandoned  when  Hill  found  in  1937  that 
isolated  chloroplasts  produce  oxygen  in  light  but  cannot  assimilate  CO., 
[8-12].  Investigators  who  followed  Hill  corroborated  his  statement  that 
"  if  we  break  the  green  cell,  it  is  possible  to  separate  the  fluid  containing 
the  chloroplast  and  chloroplast  fragments  from  the  tissue  residue.  This 
green  juice  can  no  longer  assimilate  CO^  but  in  the  case  of  many  plants 
the  insoluble  green  material,  for  a  time  at  least,  is  still  capable  of  giving 
oxygen  in  light"  [8]. 

In  1954  we  found  that  previous  difficulties  in  obtaining  CO2  fixation 
by  isolated  chloroplasts  were  indeed  methodological.  By  using  gentler 
techniques  of  isolating  chloroplasts  from  leaves,  we  prepared  spinach 
chloroplasts  that  were  capable  not  only  of  giving  the  expected  Hill  reaction, 
i.e.  oxygen  evolution,  but  also  of  converting  COo  to  starch  and  sugar  at 
physiological  temperatures  and  with  no  energy  supply  except  visible 
light  [13-15]. 

Under  the  new  experimental  conditions,  COo  assimilation  by  isolated 
chloroplasts  was  strictly  light-dependent  and  proceeded  at  an  almost 
constant  rate  for  at  least  an  hour.  There  was  approximate  correspondence 
between  the  oxygen  evolved  and  the  COo  fixed,  as  would  be  expected  from 
the  well-known  photosynthetic  quotient  in  green  plants,  O2/CO2  =  i.  The 
products  of  CO2  assimilation  were  found  to  be  the  same  as  in  photosyn- 
thesis by  whole  cells.  The  insoluble  product  of  CO2  fixation  by  chloro- 
plasts was  identified  as  starch.  Among  the  soluble  products  the  following 
were  found :  phosphate  esters  of  fructose,  glucose,  ribulose,  sedoheptulose, 
dihydroxyacetone,  and  glyceric  acid;  glycolic,  malic,  aspartic  acids, 
alanine,  glycine  and  free  dihydroxyacetone  and  glucose  [14,  15].  Using 
similar  techniques,  investigators  in  several  different  laboratories  have 
confirmed  the  ability  of  illuminated  chloroplasts  to  form  starch  and  sugars 
from  CO2  and  water  [cf.  16-21]. 

Most  of  the  early  work  on  extracellular  photosynthesis  was  done  with 
spinach  chloroplasts.  But  more  recently,  the  same  products  of  CO2 
assimilation  in  light  were  also  obtamed  with  isolated  chloroplasts  from 
several  diflerent  species :  sugar  beet,  sunflower,  Phytolacca  americana  and 
Tetragonia  expansa  [22,  23]. 

There  was  thus  finally  a  firm  experimental  basis  for  concluding  that 
chloroplasts  are  indeed  the  sites  of  complete  photosynthesis  in  green 
plants.  In  the  light  of  the  new  evidence,  chloroplasts  emerged  as  remark- 
ably  complete    and    autonomous   cellular   structures   that   have    become 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       341 

specialized  for  carrying  out  the  complete  process  of  photosynthesis  in 
green  plants.  It  seemed  legitimate  therefore  to  explore  the  component 
photochemical  reactions  in  isolated  chloroplasts  with  the  expectation  that 
they  would  also  be  relevant  to  photosynthetic  events  in  intact  cells. 

2.  The  role  of  light  in  COo  assimilation 

From  a  biochemical  point  of  view,  the  central  problem  of  photo- 
synthesis was  the  identification  of  those  photochemical  reactions  that 
provide  the  energy  required  for  the  conversion  of  COo  to  carbohydrates. 
As  for  COo  assimilation  proper,  it  became  evident  by  1956  that  the  early 
proposals  of  Thimann  [24],  IJpmann  [25],  and  Ruben  [26]  about  it  being 
a  dark  process,  were  correct.  Their  hypotheses  that  CO^  reduction  in 
photosynthesis  is  a  dark  reaction,  a  reversal  of  the  well-known  oxidative 
reaction  of  glycolysis,  received  experimental  support  mainly  from  the  work 
of  Calvin  and  his  associates  [27],  who  identified  phosphoglyceric  acid  and 
other  well-known  products  of  glycolysis  among  the  early  products  of 
photosynthesis. 

A  special  feature  of  CO.,  assimilation  in  photosynthesis  was  found  to 
be  the  carboxylation  reaction  that  accounted  for  the  appearance  of  phos- 
phoglyceric acid  as  the  first  stable  product  of  COo  fixation.  Work  in  the 
laboratories  of  Calvin  [zH],  Horecker  [29],  Ochoa  [30],  and  Racker  [31] 
established  the  presence  in  photosynthetic  tissues  of  two  special  enzymes, 
carboxydismutase  and  phosphoribulokinase,  which  accounted  for  the 
entry  of  COo  into  the  metabolism  of  photosynthetic  cells  by  way  of  a  five- 
carbon  phosphorylated  sugar,  ribulose  diphosphate.  Ribulose  diphosphate  on 
combining  with  COo  is  split  to  gi\e  two  molecules  of  phosphoglyceric  acid. 

However,  even  this  special  feature  of  carbon  assimilation  was  soon 
found  in  non-photosynthetic  bacteria  as  well.  In  fact,  Trudinger  [32]  and 
Aubert  ef  a/.  [t,t,]  found  the  entire  "photosynthetic  carbon  cycle"  in  the 
non-photosynthetic  sulphur  bacterium  Thiolnicilhis  denitrificans.  It  thus 
became  clear  that  COo  assimilation  is  fundamentally  extraneous  to  the 
photosynthetic  process.  All  the  reactions  of  COo  assimilation  in  photo- 
synthesis occur  also  in  non-chlorophyllous  cells. 

The  carboxylation  reaction  resulting  in  the  formation  of  phospho- 
glyceric acid  (PGA)  requires  ATP,  and  the  reduction  of  PGA  to  the  level 
of  carbohydrate  requires  both  ATP  and  reduced  pyridine  nucleotide.  The 
distinction  between  photosynthetic  and  non-photosynthetic  cells  seems  to 
lie,  therefore,  in  the  manner  in  ^\hich  ATP  and  reduced  pyridine  nucleo- 
tide are  formed.  Photosynthetic  cells  form  these  compounds  at  the  expense 
of  light  energy  whereas  non-photosynthetic  cells  form  them  at  the  expense 
of  energy  released  by  dark  reactions. 

Before  this  biochemical  interpretation  of  photosynthesis  could  be 
accepted  with   confidence,   it   was   necessary   to   determine   whether  the 


342  DANIEL    I.    ARNON 

process  of  CO2  assimilation  by  isolated  chloroplasts  followed  the  same 
pathway  as  in  algal  cells  and  leaves.  This  was  done  by  subdividing  the 
chloroplasts  into  component  parts  and  identifying  in  them,  or  isolating 
from  them,  the  individual  enzyme  systems  that  account  for  the  conversion 
of  CO2  to  carbohydrate  [34-36].  The  results  have  established  that  in 
isolated  chloroplasts,  as  in  whole  cells,  the  conversion  of  CO2  to  carbo- 
hydrate proceeds  by  the  same  series  of  dark  reactions  that  are  driven  by 
ATP   and   TPNH2   (TPNH2,   not   DPNHo,   was   the   reduced   pyridine 


Triose 
phosphate ' 


,    Hexose   , 
•^phosphate 


STARCH 


Carbohydrate    synthesis    by    isolated    chloroplasts. 


Fig.  I .  Condensed  diagram  of  the  reductive  carbohydrate  cycle  in  chloroplasts. 
The  cycle  consists  of  three  phases.  In  the  carboxylative  phase  (I),  ribulose-5-phos- 
phate  (Ru-5-P)  is  phosphorylated  to  ribulose  diphosphate  (RuDP)  which  then 
accepts  a  molecule  of  CO.,  and  is  cleaved  to  2  molecules  of  phosphoglyceric  acid 
(PGA);  in  the  reductive  phase  (II)  PGA  is  reduced  to  triose  phosphate;  in  the 
regenerative  phase  (III)  triose  phosphate  is  partly  converted  into  Ru-5-P  and  partly 
into  hexose  phosphate  and  starch.  All  the  reactions  of  the  cycle  occur  in  the  dark. 
The  reactions  of  the  carboxylative  and  reductive  phases  are  driven  by  ATP  and 
TPNH.,  formed  in  the  light.  One  complete  turn  of  the  cycle  results  in  the  assimila- 
tion of  I  mole  of  CO2  at  the  expense  of  3  moles  of  ATP  and  2  moles  of  TPNH2. 


nucleotide  formed  by  illuminated  chloroplasts).  The  general  scheme  for 
CO2  assimilation  by  isolated  chloroplasts  is  summarized  in  Fig.  i. 

The  validity  of  the  scheme  shown  in  Fig.  i  was  supported  by  a  physical 
separation  of  the  light  and  dark  reactions  of  photosynthesis  in  chloroplasts 
[37].  The  light  phase  was  carried  out  first  by  the  complete  chloroplast 
system  in  the  absence  of  COo  and  resulted  in  an  evolution  of  oxygen  accom- 
panied by  an  accumulation  of  TPNHo  and  ATP  in  the  reaction  mixture. 
The  green  portion  of  the  chloroplasts  (grana ;  cf.  Fig.  2)  was  then  discarded 
and  when  COo  was  next  supplied  to  the  remaining  non-green  portion  of  the 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      343 


/ 


Fig.  2.   Electron  micrograph   of  a  section   of  a  maize   chloroplast   showing 
details  of  structure.  The  dense  areas  that  resemble  stacks  of  coins  are  the  grana. 
The  layers  within  each  granum  are  called  grana  lamellae.  The  grana  lamellae  of 
different  grana  are  inter-connected  by  stroma  lamellae.   Magnification  35  000  x 
(courtesy  of  Dr.  A.  E.  Vatter). 


chloroplasts  [38,  39]  in  the  dark,  it  was  converted  to  sugar  phosphates.  The 
hght  and  dark  phases  when  carried  out  separately,  yielded  essentially  the 
same  final  photosynthetic  products  as  the  continuously  illuminated 
chloroplast  system.  The  products  included  hexose  and  pentose  mono-  and 
diphosphates,  phosphoglyceric  acid,  dihydroxyacetone  phosphate,  and 
small  amounts  of  phosphoenolpyruvate  and  malate  [37]. 


344  DANIEL    I.    ARNON 

The  same  products  of  COo  assimilation  by  chlorophyll-free  chloroplast 
extracts,  including  phosphorylated  sugars,  were  also  obtained  in  a  total 
dark  chemosynthesis  where  TPNHg  and  ATP  were  not  supplied  by  a 
preceding  photochemical  reaction  but  were  prepared  either  chemically 
or  enzymically,  or  were  derived  from  animal  material  [37].  (Similar 
experiments  were  carried  out  earlier  by  Racker  with  a  multi-enzyme 
system  that  included  enzymes  from  rabbit  muscle,  yeast,  and  spinach 
leaves  [40,  41]). 

3.  Photosynthetic  phosphorylation 

The  experiments  with  isolated  chloroplasts  have  thus  underlined  the 
essence  of  photosynthesis  in  green  plants,  i.e.  the  energy  conversion 
problem,  as  comprising  those  chloroplast  reactions  in  which  TPNHo  and 
ATP  are  formed  by  light.  With  respect  to  TPNH.,  it  has  already  been 
shown  by  several  laboratories  that  isolated  chloroplasts  were  capable  of 
reducing  TPN  to  TPNHo  in  light,  with  a  simultaneous  evolution  of 
oxygen  [42,  43,  5].  What  remained  to  be  determined  was  the  source  of 
ATP  in  photosynthesis,  or  more  specifically,  the  cellular  site  and  the 
mechanism  by  which  ATP  is  being  formed  during  photosynthesis.  From 
the  standpoint  of  cellular  physiology,  the  important  question  is  whether  the 
ATP  used  in  photosynthesis  is  supplied  by  some  light-driven  assimilation 
of  inorganic  phosphate  that  is  peculiar  to  photosynthesis,  or  whether  the 
ATP  used  in  photosynthesis  is  supplied  by  respiration. 

Before  the  recent  investigations  with  isolated  chloroplasts  the  only 
cytoplasmic  particles  known  to  form  ATP  were  mitochondria,  by  the 
process  of  oxidative  phosphorylation  [44].  Oxidative  phosphorylation  by 
mitochondria  has  therefore  usually  been  invoked  in  explaining  the  source 
of  ATP  used  in  photosynthesis  (see,  for  example.  Fig.  7  in  ref.  [45] ;  also 
review,  ref.  [46]).  In  early  models  of  ATP  formation  in  photosynthesis  it 
was  proposed  that  the  reduction  of  pyridine  nucleotide  was  carried  out  by 
illuminated  chloroplasts  and  the  resulting  reduced  pyridine  nucleotide  was 
re-oxidized  with  molecular  oxygen  by  mitochondria  [47].  This  coupled 
chloroplast-mitochondrial  system  differed  from  conventional  oxidative 
phosphorylation  only  in  the  source  of  the  reduced  pyridine  nucleotide.  In 
one  case  the  pyridine  nucleotide  was  reduced  by  light  and  in  the  other  by 
a  respiratory  substrate.  The  phosphorylation  reactions  proper  leading  to 
the  synthesis  of  ATP  were  in  both  cases  dependent  on  enzymes  localized 
in  mitochondria. 

This  model  for  the  generation  of  ATP  in  photosynthesis  posed  a 
serious  difficulty.  The  most  specialized  photosynthetic  tissue,  the  meso- 
phyll  of  leaves,  is  noted  for  its  paucity  of  mitochondria.  Within  the 
mesophyll  cells,  especially  in  the  palisade  parenchyma,  chloroplasts  are  the 
dominant  cytoplasmic  bodies;  mitochondria  are  few  [48,  49].  It  was  diffi- 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       345 

cult,  therefore,  to  visualize  how  oxidative  phosphorylation  by  mitochondria 
could  generate  enough  ATP  in  leaf  tissues  that  are  noted  for  their  vigorous 
photosynthetic  activity. 

The  difficulty  was  removed  in  1954,  when  isolated  chloroplasts  were 
found  to  synthesize  ATP  in  light  without  the  aid  of  mitochondria  [13]. 
When  conditions  were  so  arranged  that  CO2  assimilation  was  excluded, 
isolated  chloroplasts  used  light  energy  for  the  esterification  of  inorganic 
phosphate  in  accord  with  the  overall  reaction : 

n-P  +  n-ADP-^^n-ATP  (i) 

Light-induced  ATP  formation  in  chloroplasts  raised  at  once  the 
question  whether  this  process  is  analogous  to  oxidative  phosphorylation  by 
mitochondria.  At  least  two  fundamental  differences  were  apparent.  ATP 
formation  by  illuminated  chloroplasts  occurred  without  the  consumption 
of  molecular  oxvgen  and  without  the  addition  of  a  chemical  substrate  to 
supplv  free  energy  needed  for  the  formation  of  the  pyrophosphate  bonds 
of  ATP.  The  term  photosynthetic  phosphorylation  [13,  14]  was  therefore 
given  to  the  light-induced  ATP  formation  by  chloroplasts  to  distinguish 
it  from  oxidative  (respiratory)  phosphorylation  by  mitochondria  and  the 
anaerobic  phosphorylations  at  substrate  level  that  occur  in  glycolysis.  In  both 
of  these  processes  ATP  formation  occurs  at  the  expense  of  energy  liberated 
bv  the  oxidation  of  a  chemical  substrate,  whereas  the  only  "substrate" 
which  is  being  consumed  in  photosynthetic  phosphorylation  is  light. 

4.  Photosynthetic  phosphorylation  in  chloroplasts  and  bacteria 

Although  there  was  no  net  consumption  (as  measured  by  manometric 
pressure  change)  of  molecular  oxygen  in  photosynthetic  phosphorylation, 
the  process  when  first  discovered,  proceeded  at  a  sustained  rate  only  in  the 
presence  of  oxygen  [13,  Fig.  2  (b)].  Oxvgen  seemed  to  act  as  a  catalyst  in 
photosynthetic  phosphorylation,  not  as  a  substrate,  as  it  does  in  oxidative 
phosphorylation.  A  decisive  difference  between  photosynthetic  and 
oxidative  phosphorvlation  was  the  inability  of  chloroplasts  to  form  ATP 
in  the  dark  by  oxidizing  hvdrogen  donors  of  oxidative  phosphorylation 
with  molecular  oxvgen  [50]. 

Further  investigation  of  photosynthetic  phosphorylation  by  spinach 
chloroplasts  soon  resulted  in  the  identification  of  FMX  and  ^-itamin  K  as 
catalysts  in  the  process  [51,  p.  6326;  52,  53].  At  optimal  (but  still  catalytic) 
concentrations  of  either  F^MX  [>,!,]  or  vitamin  K  (Fig.  3),  photosynthetic 
phosphorylation  became  independent  of  external  oxygen  and  proceeded 
vigorously  in  an  atmosphere  of  nitrogen  or  argon.  At  a  much  lower, 
"  microcatalytic ",  concentration  of  the  added  cofactors,  photosynthetic 
phosphorylation  remained  dependent  on  oxvgen,  although  still  showing  no 
net  oxygen  consumption. 


346  DANIEL    I.    ARNON 

These  findings  are  in  agreement  with  the  recent  resuhs  of  Wessels  [54], 
Jagendorf  and  Avron  [55]  and  Nakamoto,  Krogmann,  and  Vennesland 
[56],  that  photosynthetic  phosphorylation  with  suboptimal  amounts  of 
cofactors  is  oxygen-dependent  but  becomes  oxygen-independent  at  higher 
concentrations  of  cofactors. 

In  charting  their  subsequent  investigations  Arnon  and  his  associates 
laid  special  stress  on  the  anaerobic  photosynthetic  phosphorylation  which 
proceeds  in  isolated  chloroplasts  at  optimal  catalytic  concentrations  of 
FMN  and  vitamin  K.  They  considered  this  type  more  fundamental  to 
photosynthesis  in  general  than  the  oxygen-catalyzed  type  because  it  would 
also  apply  to  bacterial  photosynthesis,  in  which  oxygen  cannot  be  involved. 

INTENSE    LIOIT  ond   HIGH   CHLOROPHYLL 


0-003  001       0-OS  0-1     0-3      10 
//moles  vit.  K3  added 


0-003  001       0-OS    0-1     0-3    10 
//moles  FMN  added 


Fig.  3.  Effect  of  vitamin  K3  (2-methyl-i,4-naphthoquinone)  and  FMN  con- 
centration on  cyclic  photophosphorylation  by  spinach  chloroplasts  in  nitrogen  and 
air  at  high  light  intensity.  The  reaction  mixture  (3  ml.  final  volume)  included 
chloroplast  fragments  (Cij)  containing  i  ■  5  mg.  chlorophyll ;  and  in  micromoles : 
tris  buffer  pH  8  •  3,  80 ;  K2H32PO4,  20 ;  ADP,  20 ;  MgS04,  5  ;  and  TPN,  o  •  3  (only 
in  the  FMN  series).  FMN  and  vitamin  K3  were  added  as  indicated.  The  reaction 
was  run  for  5  min.  at  an  illumination  of  50  000  Lux  (Tsujimoto,  Hall,  and  Arnon 
[92];  Arnon,  Whatley,  and  Allen,  unpublished  data,  1954). 

Soon  after  the  discovery  of  photosynthetic  phosphorylation  in  isolated 
chloroplasts,  Frenkel  [57]  reported  a  similar  phenomenon  in  the  photo- 
synthetic bacterium  Rhodospirilhnn  ruhrum.  Although  Frenkel  suggested 
that  the  light-induced  ATP  formation  in  bacterial  preparations  was 
similar  to  that  in  chloroplasts,  the  similarity  seemed  uncertain  at  first, 
because  Frenkel's  photophosphorylation  system,  which  was  a  sonic 
macerate  of  R.  rubrum  cells,  differed  in  several  respects  from  its  counter- 
part in  isolated  chloroplasts  [13].  Frenkel's  preparations  became  substrate- 
dependent  after  washing;  the  rate  of  phosphorylation  was  doubled  on 
adding  a-ketoglutarate  [57].  But  in  later  experiments  he  ruled  out  the 
dependence  on  an  added  chemical  substrate  [58]  and  the  equivalence  of 
chloroplast  and  bacterial  photophosphorylation  seemed  probable. 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       347 

Frenkel's  findings  were  followed  by  those  of  Williams  [59]  who 
demonstrated  photosynthetic  phosphorylation  in  cell-free  preparations  of 
the  obligately  anaerobic  photosynthetic  bacteria,  Chromatium  and  Chloro- 
bitim.  It  thus  became  clear  that  a  common  anaerobic  mechanism  for  a  light- 
induced  phosphorylation,  that  does  not  depend  on  an  exogenous  chemical 
substrate  or  on  oxygen  consumption,  is  shared  by  both  green  plants  and 
photosynthetic  bacteria.  The  energy  conversion  process  proper  seemed  to 
be  fundamentally  independent  of  oxygen  although  it  was  still  possible 
that  details  of  mechanisms  were  diiferent  in  green  plants  and  photo- 
synthetic bacteria. 

The  discovery  of  photosynthetic  phosphorylation  in  chloroplasts  by 
Arnon  et  al.  [13]  and  in  bacterial  particles  by  Frenkel  [57]  was  confirmed 
and  extended  in  a  number  of  laboratories.  Photosynthetic  phosphorylation 
in  isolated  chloroplasts  was  observed  by  Avron  and  Jagendorf  [60,  61], 
Wessels  [62],  and  Vennesland  and  her  associates  [63,  56];  in  algae  by 
Thomas  and  Haans  [64]  and  Petrack  [65] ;  and  in  photosynthetic  bacteria 
by  Geller  [66],  Kamen,  and  Newton  [67]  and  Anderson  and  Fuller  [68]. 
In  later  experiments  Whatley  et  al.  [22,  23]  have  shown  that  photo- 
synthetic phosphorylation  by  chloroplasts,  which  had  previously  been 
almost  entirely  limited  to  observations  on  chloroplasts  isolated  from  one 
species,  viz.  spinach,  is  also  operating  in  chloroplasts  isolated  from  several 
other  species  of  higher  plants.*  It  now  seems  well  established,  therefore, 
that  all  photosynthetic  organisms  contain  a  phosphorylating  system  that  is 
intimately  associated  with,  and  structurallv  bound  to,  the  chlorophyll 
pigments. 

Soon  after  the  demonstration  of  photosvnthetic  phosphorylation  in 
isolated  chloroplasts  attempts  were  made  to  compare  its  rate  with  that  of 
COo  assimilation  bv  illuminated  whole  cells.  Since,  as  with  most  newly 
isolated  processes  in  cell-free  systems, f  the  rates  of  photosynthetic 
phosphorylation  were  rather  low,  there  was  little  inclination  at  first  to 
accord  this  process  quantitative  importance  [72,  pp.  292,  345]  as  a 
mechanism  for  converting  light  into  chemical  energv. 

With  further  impro\ement  in  experimental  methods  we  obtained  rates 
of  photosynthetic  phosphorylation  up  to  170  times  higher  [73]  than  those 

*  Other  accounts  of  the  discovery  of  COo  assimilation  and  photosynthetic 
phosphorylation  by  isolated  chloroplasts  are  given  by  Calvin.  In  1956  he  ascribed 
(69,  p.  31)  the  discovery  of  COo  assimilation  by  isolated  chloroplasts  to  Boychenko 
and  Baranov  (70)  and  in  1959  he  ascribed  the  discovery  of  both  CO.,  assimilation 
by  isolated  chloroplasts  and  photosynthetic  phosphorylation  to  his  own  laboratory 
(71,  p.  152). 

t  The  most  recent  instance  of  this  kind  is  the  cell-free  synthesis  of  DN.A 
investigated  by  Kornberg.  "The  first  positive  results  represented  the  conversion 
of  only  a  very  small  fraction  of  the  acid-soluble  substrate  into  an  acid-insoluble 
fraction  (50  or  so  covmts  out  of  a  million  added)"  [3]. 


348  DANIEL   I.    ARNON 

originally  described  [13]  and  even  these  high  rates  were  exceeded  by 
Jagendorf  and  Avron  [74].  The  improved  rates  of  photosynthetic  phos- 
phorylation were  equal  or  greater  than  the  maximum  known  rates  of  carbon 
assimilation  in  intact  leaves.  It  appeared,  therefore,  that  the  enzymic 
apparatus  for  photosynthetic  phosphorylation  that  is  present  in  chloro- 
plasts,  can  under  appropriate  experimental  conditions,  function  outside 
the  organized  cell  without  substantial  loss  of  activity. 

Unlike  the  phosphorylating  system,  the  enzymes  catalyzing  CO2 
assimilation  are  water-soluble  [38,  39,  37]  and  are  therefore  partly  lost 
during  the  isolation  of  chloroplasts.  This  results  in  lower  rates  of  COg 
assimilation  by  isolated  chloroplasts  than  by  the  intact  parent  leaves.  The 
difference  between  the  rate  of  CO.,  assimilation  by  isolated  chloroplasts 
and  that  of  intact  leaves  may  be  made  to  appear  greater,  though  less 
relevant,  if  the  comparison  is  made  not  between  isolated  chloroplasts 
and  their  parent  leaf  tissue,  but  between  isolated  chloroplasts  and  un- 
related leaf  material  that  gave  maximum  rates  of  CO2  assimilation  under 
different  experimental  conditions.  Nevertheless,  the  now  known  rates  of 
CO.,  assimilation  in  isolated  chloroplasts  (10  to  20'^  ^  of  that  in  parent  leaf 
tissue  [20,  35])  are  substantial  enough  to  strengthen  the  conclusion  that 
photosynthesis  by  isolated  chloroplasts  mirrors  that  in  the  intact  leaf.  This 
conclusion  is  fortified  by  the  identity  of  the  photosynthetic  products  found 
in  both  cases. 

5.  Catalysts  of  photosynthetic  phosphorylation 

Photosynthetic  phosphorylation  emerged  as  a  major  mechanism  for 
converting  light  into  useful  chemical  energy  independently  of  CO2 
assimilation.  It  became  important  therefore  to  investigate  systematically 
the  mechanism  of  this  direct  conversion  of  light  into  pyrophosphate  bond 
energy.  The  first  question  that  received  attention  was  the  identity  of  the 
catalysts. 

In  searching  for  catalysts  of  photosynthetic  phosphorylation  by 
isolated  chloroplasts  special  attention  was  given  to  normal  constituents  of 
chloroplasts  and  green  leaves.  The  first  factors  which  were  found  to 
stimulate  cyclic  photophosphorylation  without  themselves  being  con- 
sumed in  the  reaction  were  magnesium  ions  and  ascorbate  [13,  51];  the 
next  to  be  recognized  were,  as  already  mentioned,  FMN  and  vitamin  K 
compounds  [53,  52].  Magnesium  and  ascorbate  have  long  been  known  to 
be  present  in  chloroplasts  [75].  FMN  is  widely  distributed  in  green 
leaves  [76].  Ohta  and  Losada  in  our  laboratory  (unpublished  data)  have 
found  FMN  to  be  a  regular  constituent  of  chloroplasts.  Of  unusual  interest, 
however,  was  the  antihaemorrhagic  factor,  vitamin  K,  which  occupied, 
since  its  discovery  in  plants,  a  unique  position  among  other  vitamins  in 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       349 

being  specifically  associated  with  chloroplasts*  [77].  Moreover,  Martius 
and  others  have  recently  assigned  a  role  to  vitamin  K  in  oxidative  phos- 
phorylation [82,  83;  cf.  84,  review;  85]. 

Apart  from  the  catalytic  efl:ect  of  FMN  and  vitamin  K  (and  TPN ;  cf. 
[86,  87]),  photosynthetic  phosphorylation  may  also  be  increased  by  the 
addition  of  non-physiological  cofactors  [74;  cf.  [88,  89].  Among  these  of 
particular  interest  is  phenazine  methosulphate,  since  this  dye  is  known  to 
be  a  strong  reducing  agent  for  cytochromes  [90].  Phenazine  methosulphate 
was  found  to  stimulate  photosynthetic  phosphorylation  in  bacterial 
preparations  by  Geller  [66]  and  Kamen  and  Newton  [67]  and  in  spinach 
chloroplasts  by  Jagendorf  and  Avron  [74]. 

COFACTORS    OF    BACTERIAL    PHOTOPHOSPHORYLATION 

Of  the  cofactors  of  photosynthetic  phosphorylation  discussed  so  far, 
ascoibate  and  phenazine  methosulphate  were  found  to  be  effective  in 
photosynthetic  phosphorylation  by  cell-free  preparations  from  RJiodo- 
spirillum  ritbntm  [66]  and  C/iroiiuitiiint  [67].  In  addition,  Geller  [66]  has 
also  found  a  stimulatory  effect  of  vitamin  K3. 

Under  our  experimental  conditions  photosynthetic  phosphorylation  by 
cell-free  preparations  of  C/ironiatiiini  showed  no  response  to  added  co- 
factors  when  the  particles  were  freshly  prepared  under  anaerobic  conditions. 
On  ageing,  however,  an  effect  of  added  vitamin  K  and  phenazine  metho- 
sulphate was  observed  (Table  I) ;  the  joint  addition  of  these  two  cofactors 
gave  a  greater  increase  of  phosphorylation  than  when  they  were  added 
singly.  The  addition  of  FMN  gave  no  increase  [66,  67]  and  in  fact,  under 
our  experimental  conditions  often  inhibited  photosynthetic  phosphoryla- 
tion by  Chromatium  particles. 

Table  II  shows  that  photosynthetic  phosphorylation  by  Chromatium 
particles  also  resembled  that  of  chloroplasts  in  its  resistance  to  inhibition  by 
dinitrophenol,  o-phenanthroline  and  antimycin  A  (when  phenazine  metho- 
sulphate was  present  in  the  reaction  mixture  [cf.  66])  and  its  sensitivity 

*  Bishop,  who  earlier  presented  evidence  that  vitamin  K  is  an  essential  factor 
for  the  photochemical  activity  of  isolated  chloroplasts  [78],  has  reported  in  a  more 
recent  publication  [79]  that  spinach  chloroplasts  do  not  contain  naphthoquinones 
of  the  vitamin  K  type  but  contain  instead  the  benzoquinone  Q-255  ("plastoqui- 
none "),  which  Crane  [80]  and  Folkers  and  his  associates  [81]  found  in  green 
tissues  and  which  Crane  also  found  to  be  specifically  concentrated  in  chloroplasts 
[80].  Bishop  [79]  has  reported  that  Q-255  activ'ates  the  Hill  reaction.  The  role  of 
Q-255  in  photosynthetic  phosphorylation  is  still  unknown,  but  it  should  be  noted 
that  from  the  standpoint  of  the  mechanism  of  photosynthetic  phosphorylation 
(see  next  Section),  either  a  naphthoquinone  of  the  vitamin  K  type  or  a  benzoqui- 
none would  appear  suitable  as  an  electron  carrier  in  the  process.  A  clarification  of 
the  disagreement  between  the  earlier  reports  of  vitamin  K  distribution  in  chloro- 
plast  [77]  and  the  recent  reports  on  Q-255  will  be  awaitetl. 


35°  DANIEL   I,    ARNON 

TABLE  I 

Effect  of  Vitamin  K3  and  Phenazine  Methosulphate  (PMS) 

ON  Photophosphorylation  by  Aged  Cell-Free  Preparations  of  Chromatium 

[Chromatophores  (P)  and  Supernatant  Fluid  (S)] 

(Ogata,  Nozaki,  and  Arnon  [91]) 


Treatment* 

/xmoles  P  este rifled /mg 

.  chlorophyll /hr. 

Ageing  time 

(days) 

0 

I 

2 

4 

8 

I. 

P  + 

S 

83 

55 

42 

40 

26 

2. 

P  + 

s, 

vit. 

K,,  PMS 

99 

98 

91 

80 

66 

3- 

PS 

87 

30 

27 

21 

15 

4- 

PS, 

Vlt. 

K, 

PMS 

96 

1 06 

75 

6S 

45 

*  P  and  S  were  stored  separately  in  Treatments  i  and  2  and  together  in  Treat- 
n\ents  3  and  4. 

Each  vessel  contained,  in  a  final  volume  of  3-0  ml.,  cell-free  preparation 
containing  0-3  mg.  bacteriochlorophyll,  and  the  following  in  micromoles :  tris 
buflFer,  pH  7-8,  80;  MgCL,  5;  K,H^^PO,,  10;  and  ADP,  10.  o-i  ^mole  each  of 
vitamin  K3  (2-methyl-4-amino-i-naphthol  hydrochloride)  and  PMS  were  added 
as  indicated.  Gas  phase  was  argon.  The  reaction  was  carried  out  at  20°  for  30  min. 
and  stopped  bv  adding  0-3  ml.  of  20^',,  TCA  to  each  vessel.  Illumination  35  000 
Lux. 


TABLE  II 

Effect  of   Inhibitors   on   Photophosphorylation   by  Chromatium   Particles 

(Ogata,  Nozaki,  and  Arnon  [91]) 

rp       ^         ^  umoles  P  esterified/ 

ireatment  '^         ,  ,         .    ,,/u 

mg.  chlorophyll /hr. 

Control  75 

10^  M  dinitrophenol  52 

5    X    10  ^  M  o-phenanthroline  50 

Control  126 

Antimycin  A,  10 /ug.  119 

Gramicidin,  40  ju,g.  119 

10^  M  methylene  blue  100 

10  ^  M  /)-chloromercuribenzoate  14 

Each  vessel  contained,  in  a  final  volume  of  3-0  ml.,  cell-free  preparation  (PS) 
containing  o  •  2  mg.  bacteriochlorophyll  and  the  following,  in  /xmoles :  tris  buffer, 
pH  7-8,  80;  MgClo,  5;  KoH^-POj,"  15;  ADP,  15;  vitamin  K3  (2-methyl-i,4- 
naphthoquinone),  0-3;  and  phenazine  methosulphate,  o-i.  Inhibitors  were  added 
as  indicated.  Other  conditions  were  the  same  as  described  for  Table  I. 


PHOTOSYXTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       35  I 

to  /)-chloromercuribenzoate  [cf.  89].  However,  unlike  chloroplasts  [50, 
88,  89],  photophosphorylation  by  Chromatium  particles  was,  under 
our  experimental  conditions,  resistant  to  inhibition  bv  methvlene  blue 
[cf.  66]. 

6.  The  electron  flow  mechanism  of  photosynthetic 
phosphorylation 

Photosynthetic  phosphorylation  has  provided  direct  experimental 
evidence  for  the  view  that  the  key  event  in  photosynthesis,  the  conversion 
of  light  into  chemical  energy,  is  independent  of  the  classical  manifestations 
of  this  process  in  green  plants;  oxygen  evolution  and  COo  assimilation.  If 
it  is  accepted  that  photosynthetic  phosphorylation  represents  the  simplest 
common  denominator  of  photosynthesis  in  green  plants  and  bacteria,  then 
a  mechanism  for  this  process  would  be  expected  to  provide  a  basic  pattern 
for  the  conversion  of  light  into  chemical  energy.  The  salient  facts  which 
must  be  explained  are  that  a  "  high  energy"  pyrophosphate  bond  is  formed 
at  the  expense  of  absorbed  light  energy.  There  is  no  need,  a  priori,  to 
connect  this  reaction  either  with  photolysis  of  water  or  with  reduction  of 
COg.  Photosynthetic  phosphorylation  catalyzed,  for  example,  by  phenazine 
methosulphate  or  vitamin  K,  produces  neither  a  reductant  for  COo 
assimilation  nor  molecular  oxygen ;  the  sole  product  is  ATP. 

The  simplest  hypothesis  to  account  for  the  formation  of  ATP  in  photo- 
synthetic phosphorylation  is  to  assume  that,  as  in  the  dark  phosphorvla- 
tions  of  glycolysis  and  respiration,  the  formation  of  a  pvrophosphate  bond 
is  also  coupled  with  a  release  of  free  energy  which  occurs  during  electron 
transport,  i.e.  when  an  electron  drops  from  the  higher  energv  level  (that 
it  has  when  it  resides  in  the  electron  donor  molecule)  to  the  lower  energy 
level  that  it  assumes  on  joining  the  electron  acceptor  molecule.  But  a 
mechanism  for  photosynthetic  phosphorylation  must  also  account  for  its 
unique  features :  ATP  is  formed  without  the  consumption  of  an  exogenous 
electron  donor  and  electron  acceptor.  Unlike  oxidative  phosphorylation, 
photosynthetic  phosphorylation  consumes  neither  exogenous  substrate 
nor  molecular  oxygen,  only  light  energy. 

A  mechanism  for  photosynthetic  phosphorylation  must,  therefore, 
provide  for  the  generation  of  both  an  electron  donor  and  an  electron 
acceptor  in  the  primary  photochemical  act  when  radiant  energv  is 
absorbed  by  chlorophyll.  Investigations  of  photosynthesis  at  the  cellular 
level,  in  which  the  main  preoccupation  has  usually  been  with  CO2  assimila- 
tion and  oxygen  evolution,  led  to  no  cogent  theory  of  the  primarv  act  of 
photosynthesis  that  would  fit  the  experimental  facts  of  photosynthetic 
phosphorylation.  As  summed  up  recently  by  Livingston  "physiologists 
and  biochemists  appear  to  believe  that  this  question  (the  primary  act  of 


352  DANIEL   I.    ARNON 

photosynthesis)  was  answered  long  ago  by  physicists  while  physicists  find 
the  problem  distressingly  complicated  and  therefore  uninterestmg"  [93]. 

The  mechanism  of  photosynthetic  phosphorylation  that  we  have 
proposed  [94]  regards  the  photosynthetic  particle,  chloroplast  or  bacterial 
chromatophore,  as  a  "closed"  catalytic  system.  We  have  suggested  that 
during  the  primary  photochemical  act,  one  component  of  the  "closed" 
system,  chlorophyll  (bound  to  protein),  becomes  excited  on  absorbing  a 
photon  and  "expels"  one  of  its  electrons  that  has  been  raised  to  a  higher 
energy  level.  The  excited  chlorophyll  thus  becomes  the  electron  donor. 
On  losing  an  electron,  chlorophyll  assumes  a  positive  charge,  and  in  this 
way  also  becomes  the  electron  acceptor  in  photosynthetic  phosphorylation. 

The  "expelled"  electron  returns  in  a  stepwise  manner  to  the  oxidized 
chlorophyll  molecule  which  thereupon  resumes  its  normal  ground  state.  On 
its  return  "downhill"  path,  the  expelled  electron  releases  free  energy  as  it 
passes  through  several  electron  carriers.  These  intermediate  electron 
carriers  are  coupled  w  ith  enzyme  systems  that  catalyze  the  phosphorylation 
process  during  which  electron  energy  is  converted  into  pyrophosphate 
bond  energy.  After  returning  to  chlorophyll,  the  cyclic  journey  of  the 
electrons  begins  once  more  as  chlorophyll  molecules  acquire  fresh  excita- 
tion energy  by  recurrent  absorption  of  photons.  The  stepwise  interaction 
of  the  "activated"  electron  with  the  intermediate  electron  acceptors 
constitutes  the  energy  conversion  process  in  photosynthetic  phosphoryla- 
tion. Because  of  the  cyclic  path  travelled  by  electrons  that  are  activated  by 
light,  this  type  of  photosynthetic  phosphorylation  has  been  called  cyclic 
photophosphorylation  [95,  94]. 

Chlorophyll  can,  of  course,  also  be  restored  to  the  ground  state  without 
the  excited  electron  going  through  the  enzymic  "energy  transformer 
stations",  but  in  that  case  electron  energy  has  not  been  converted  into 
chemical  energy  and  hence  photosynthesis  has  not  occurred.  Instead,  the 
energy  of  electronic  excitation  is  emitted  as  a  light  quantum  and  the 
characteristic  fluorescence  of  chlorophyll  is  observed. 

The  primary  photochemical  reaction  in  which  an  absorbed  light  quan- 
tum "excites"  a  chlorophyll  molecule  and  "expels"  an  electron,  is 
represented  by  equations  (2)  and  (2a).  The  symbol  [Chi]  +  is  intended  to 
denote  that  the  chlorophyll  molecule  as  it  loses  an  electron,  acquires  a 
positive  charge,  i.e.  becomes  "oxidized"  or  forms  a  "hole"  ("odd  ion", 
[96])  that  is  ready  to  accept  another  electron  and  to  return  in  this  way  to 
its  normal  ground  state. 

Chl  +  //v^Chl*  (2) 

Chi*  =  [Chl]+  +  e-  (2a) 

In  the  proposed  mechanism  of  cyclic  photophosphorylation  [Chi]  +  is 
restored  to  its  ground  state  by  accepting  an  electron  from  a  cytochrome 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      353 

present  in  the  photosynthetic  particle  [94].  This  "terminal"  cytochrome 
component,  i.e.  a  cytochrome  that  is  adjacent  to,  and  interacts  with,  the 
excited  chlorophyll  molecule,  becomes  oxidized  after  donating  an  electron 
to  chlorophyll.  We  have  visualized  [94]  that  phosphorylation  is  coupled 
with  the  oxidation  of  the  terminal  cytochrome,  in  a  manner  analogous  to 
the  phosphorylations  which  accompany  the  oxidation  of  cytochromes  by 
oxygen  in  mitochondria  [44].  Thus,  chlorophyll  (with  the  aid  of  light)  is  the 
ultimate  oxidant  in  photosynthetic  phosphorylation  and  plays  a  part  which 
corresponds  to  that  of  molecular  oxygen  in  oxidative  phosphorylation 
[cf.  97].  The  terminal  phosphorylation  reaction  is  represented  by 
equation  (3). 

2[Chl]  +  +  zFe'  -cyt  +  ADP  +  H3PO4  ->  ATP  +  zChl  +  2Fe'^  +cyt     (3) 


(^-) 


i^PMS 


Chi    ff^*)  t ^  M^       Cyt 

LIGHT   -f-2DP "Xz) 

Cyclic    photophosphorylation    (PMS  type) 

Fig.   4.   Scheme    for    anaerobic    cyclic    photophosphorylation    catalyzed    by 
phenazine  methosulphate  (PMS).  Details  in  the  text. 


The  photophosphorylation  reaction  would  leave  the  cytochrome  in  the 
oxidized  state.  Since  cvtochromes  are  present  in  catalytic  amounts,  cyclic 
photophosphorylation  would  soon  cease  unless  the  cytochrome  could 
become  reduced  again.  Our  theory  provides  that  in  cyclic  photophos- 
phorylation the  reduction  of  cytochrome  occurs  by  the  return  of  the  elec- 
tron originally  "expelled"  from  chlorophyll  in  the  primary  photochemical 
reaction  (equations  (2)  and  (2a)). 

In  isolated  chloroplasts,  the  reduction  of  cytochrome  by  the  electron 
expelled  from  chlorophvU  requires  an  added  catalyst,  i.e.  an  intermediary 
electron  carrier.  In  the  simplest  case,  as  shown  in  the  scheme  in  Fig.  4, 
the  part  of  the  electron  carrier  is  played  by  a  non-physiological  catalyst, 
phenazine  methosulphate.  Phenazine  methosulphate  is  known  to  be  a  very 


354  DANIEL   I.    ARNON 

effective  electron  carrier  in  reactions  involving  cytochromes.  For  example, 
Massey  [90]  has  found  that  cytochrome  c  is  rapidly  reduced  in  a  non- 
enzymic  reaction  with  reduced  phenazine  methosulphate.  In  Fig.  5,  the 
intermediary  electron  carrier  is  vitamin  K  or  FMN. 

The  cyclic  electron  flow  diagrams,  illustrated  by  Figs.  4  and  5,  are 
components  of  the  scheme  presented  earlier  [94].  The  key  reaction  in  the 
proposed  mechanism,  the  photo-oxidation  of  chlorophyll  by  the  loss  of  an 
electron,  is  based  on  a  type  of  reaction  in  photochemistry  that  was  experi- 
mentally documented  by  Lew'is  and  Lipkin  [96].  They  found,  by 
illuminating  a  variety  of  substances  in  rigid  media,  "that  one  of  the 
commonest  photochemical  processes  is  the  mere  loss  of  an  electron  by  an 
activated  molecule"  [96].  The  evidence  for  the  then  (in  1942)  "new  and 


■^  Cof  actor 


'~P 


ADP 


Chi     '.  V  <  ^>^     Cyt 


LIGHT  ~P— ADP — Katp, 


Anaerobic    cyclic  photophosphorylation 

Fig.  5.  Scheme  for  anaerobic  cyclic  photophosphorylation  catalyzed  by  vitamin 
K,  or  FMN.  Details  in  the  text. 


somewhat  surprising  phenomenon"  [96]  was,  for  example,  "that  chemical 
oxidation  at  room  temperature  and  photo-oxidation  at  liquid  air  tempera- 
ture (of  tri-/)-tolylamine)  have  given  the  same  substance,  namely,  the 
positive  ion  left,  (/>-CH3C6H4)3N +,  when  one  electron  has  been 
removed"  [96]. 

In  the  reactions  studied  by  Lewis  and  Lipkin  the  fate  of  the  ejected 
electron  was  uncertain  but,  as  they  pointed  out,  "the  electron  must  lie  in 
a  potential  hole  which  is  deep  enough  so  that  the  large  electrostatic  field 
of  the  ion  is  unable  to  dislodge  it"  for  considerable  periods  of  time.  This 
was  indicated  by  the  fact  that  "the  (blue)  color  (  of  the  'odd  ion'  formed) 
persists  at  liquid  air  temperature  for  several  days,  but  at  only  slightly 
higher  temperatures  the  color  disappears.  Then  presumably  the  electron 


PHOTOS YNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      355 

has  returned  to  the  ion"  [96].  In  cycHc  photophosphorylation  the  electron 
expelled  from  chlorophyll  is  visualized  as  being  transferred  [98]  to  the 
first  intermediary  acceptor  in  the  photosynthetic  electron  transport  chain 
and  thus  initiating  the  electron  transfer  process  that  makes  cyclic  photo- 
phosphorylation possible  [94]. 

To  summarize,  then,  the  simplest  experimentally  demonstrable  case  of 
conversion  of  light  energy  into  chemical  energy,  a  case  that  is  common  to 
all  chlorophvU-containing  particles,  is  cyclic  photophosphorylation.  We 
visualize  that  in  cyclic  photophosphorylation  electrons  flow  from  chloro- 
phyll that  becomes  excited  by  light,  to  a  cofactor  (Figs.  4  and  5),  from  the 
cofactor  to  cytochromes  and  from  cytochromes  back  to  chlorophyll. 
During  this  cyclic  flow  of  electrons  the  cofactor  and  cytochromes  present 
in  the  photosvnthetic  particles  undergo  oxidation-reductions  which  are 
believed  to  be  coupled  to  phosphorylation  reactions  that  produce  ATP. 

The  proposed  mechanism  for  this  process  may  be  divided  into  three 
phases :  (a)  the  primary  photochemical  act  that  results  in  the  generation  by 
the  excited  chlorophvU  molecule  of  a  high  energy  electron  and  of  the 
ultimate  electron  acceptor  [Chi  "^],  (b)  transport  of  the  high  energy  electron 
through  a  photosynthetic  electron  transport  system,  and  (r)  phosphorylation 
reactions  coupled  to  electron  transport.  Phases  (b)  and  (c)  are  analogous 
and  possibly  identical  in  some  respects  with  their  counterparts  in  oxidative 
phosphorylation,  whereas  phase  (a)  is  peculiar  to  photosynthetic  phos- 
phorylation. 

7.  Evidence  for  electron  flow  mechanism  in  cyclic 
photophosphorylation 

The  validitv  of  the  proposed  mechanisms  for  cyclic  photophosphoryla- 
tion is  supported  bv  several  lines  of  evidence.  These  include  recent 
experiments  on  the  eflfect  of  chloride  and  ferricyanide  on  photosynthetic 
phosphorylation  in  isolated  chloroplasts  and  chromatophores,  and  experi- 
ments on  the  effect  of  light  and  vitamin  K  on  cytochromes  of  chloro- 
phvllous  particles.  This  evidence  will  now  be  discussed  in  more  detail. 

EFFECT    OF    CHLORIDE 

The  role  of  chloride  in  photosynthesis  was  discovered  by  Warburg  [99], 
who  found  that  chloride,  replaceable  by  bromide  but  not  by  other  anions, 
was  essential  for  oxvgen  evolution  by  isolated  chloroplasts.  This  discovery 
was  fully  confirmed  by  Arnon  and  Whatley  [100],  but  they  were  dis- 
inclined to  accept,  at  that  time,  Warburg's  conclusion  that  chloride  is  a 
coenzvme  of  photosvnthesis,  because  this  conclusion  would  have  conferred 
on  chloride  the  then  unwarranted  status  of  an  essential  element  for  green 


356  DANIEL    I.    ARNON 

plants.  However,  they  envisaged  the  possibiHty,  which  has  since  been 
documented  by  Broyer  and  associates  [loi]  and  Martin  and  Lavollay  [102]. 
that  chloride  may  prove  to  be  an  essential  micronutrient  for  green  plants, 
A  reinvestigation  by  Bove  et  a/.  [103]  of  the  role  of  chloride  in  the 
photochemical  reactions  of  chloroplasts  confirmed  Warburg's  conclusion 
that  chloride  is  essential  for  those  photosynthetic  reactions  in  which  oxygen 
is  liberated.  Chloride  was  not  required,  however,  for  the  anaerobic  cyclic 
photophosphorylation  that  is  shared  by  bacterial  particles  and  chloroplasts. 
Thus,  in  the  absence  of  chloride,  chloroplasts  behaved  like  bacterial 
chromatophores.  They  were  able  to  carry  out  the  anaerobic  cyclic  photo- 
phosphorylation but  were  unable  to  evolve  oxygen.  Oxygen  evolution, 
not  included  in  the  mechanism  of  cyclic  photophosphorylation,  appeared 
therefore  as  an  additional  secondary  feature  of  photosynthesis,  not  essen- 
tial to  the  primary  conversion  of  light  energy  into  the  pyrophosphate  bonds 
of  ATP,  and  peculiar  to  green  plants, 

EFFECT    OF    FERRICYANIDE 

The  key  premise  in  the  proposed  mechanisms  for  cyclic  photophos- 
phorylation is  that  the  electron  expelled  from  the  chlorophyll  molecule  in 
the  primary  photochemical  act  is  not  removed  from  the  "closed  circuit" 
within  which  it  travels  before  it  returns  to  the  chlorophyll.  If  this  basic 
postulation  is  correct  it  follows  that  cyclic  photophosphorylation  should  be 
abolished  if  the  electrons  are  prevented  from  completing  the  cycle  because 
of  capture  by  an  external  electron  acceptor.  To  be  convincing,  such  an 
experiment  should  be  carried  out  with  an  electron  acceptor  which  would 
be  free  from  the  suspicion  that  it  prevented  phosphorylation  by  acting  as 
an  uncoupler,  or  in  some  toxic  manner. 

An  electron  acceptor  that  fulfills  these  requirements  is  ferricyanide. 
As  shown  by  Jagendorf  [104],  and  confirmed  in  this  laboratory,  ferri- 
cyanide has  a  great  affinity  for  trapping  electrons  during  photophosphoryla- 
tion. Thus,  by  adding  ferricyanide,  in  the  absence  of  chloride,  cyclic 
photophosphorylation  in  both  chloroplasts  and  chromatophores  should  be 
inhibited,  if  the  proposed  hypothesis  is  correct.  The  cyclic  flow  of  electrons 
in  the  closed  circuit  would  be  interrupted  when  the  electrons  are  trapped 
by,  and  used  in,  the  reduction  of  ferricyanide. 

Table  III  shows  that  this  theoretical  prediction  has  been  experi- 
mentally verified.  The  addition  of  ferricyanide  abolished  cyclic  photo- 
phosphorylation both  in  chloroplasts  and  in  chromatophores.  Adding  this 
ion  in  its  reduced  form  as  ferrocyanide,  was  without  efi^ect.  The  reduction 
of  ferricyanide  with  ascorbate  either  prior  to,  or  during  illumination  of  the 
photosynthetic  particles,  restored  in  full  their  capacity  for  cyclic  photo- 
phosphorylation.   The    conclusion    seemed   justified    therefore    that    the 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       357 

TABLE  III 

Influence  of  Ferricyanide  (in  the  Absence  of  Chloride)  on  Cyclic  Photo- 

PHOSPHORYLATION   BY   SPINACH   ChLOROPLASTS   AND   BACTERIAL   ChROMATOPHORES 

{Chromatiiim)  (/iMOLES  Phosphate  Esterified  in  30  min.) 
(Bove,  Bove,  Whatley,  and  Arnon  [103]) 

Treatment  Chloroplasts         Chromatophores 


Control  9-2  4-9 

Ferricyanide,  i  /xmole  0-5  0-4 


Chloroplasts 

9 

2 

0 

5 

0 

5 

0 

5 

7 

^ 

9 

4 

Ferricyanide,  2  /itmoles  0-5  o  ■ 

Ferricyanide,  3  /^moles  0-5  0-4 
Ferricyanide,  5  /^imoles,  reduced  by 

ascorbate*  7-2  6-2 

Ferrocyanide,  5  /(moles  9-4  5-4 

*  Sodium  ascorbate  (5  /xmoles)  was  tipped  in  from  a  sidearm  15  min.  after 
the  beginning  of  the  experiment,  and  illumination  (35  000  Lux)  was  then  continued 
for  30  min. 

inhibitory  effect  of  ferricyanide  resulted  from  the  capture  by  this  ion  (in 
its  oxidized  form)  of  electrons  which  would  have  normally  travelled  the 
cyclic  electron  transport  route  (fig.  5).  This  conclusion  was  strengthened 
by  the  finding  that  the  inhibition  was  produced  by  very  low  concentrations 
of  ferricyanide.  This  would  be  expected  if,  as  demanded  by  the  hypothesis, 
the  quantity  of  ferricyanide  needed  to  capture  electrons  from  the  cyclic 
system  needs  only  to  be  sufficient  to  leave  the  catalytic  components  of  the 
svstem  in  an  oxidized  form. 


LIGHT-INDUCED    OXIDATIONS    OF    CYTOCHROMES 

Our  theory  assigns  to  cytochromes  the  role  of  electron  carriers  in 
photosynthetic  phosphorylation.  The  initial  suggestion  [13]  that  cyto- 
chromes oxidized  by  hght  may  act  as  electron  carriers  in  the  electron 
transport  chain  of  photosvnthetic  phosphorylation  was  based  on  the 
observation  of  Lundegardh  [105]  that  the  cytochrome  peculiar  to  chloro- 
plasts, cytochrome  /  [97],  is  oxidized  on  illumination.  The  oxidation  of 
cytochromes  on  illumination  has  also  been  observed  in  intact  algae  and 
photosynthetic  bacteria  by  Duysens  [106,  107]  and  by  Olson  and  Chance 
[108].  Of  special  relevance  is  the  recent  finding  of  Chance  and  Nishimura 
[109]  that,  in  whole  Chromatium  cells,  a  light-induced  oxidation  of  cyto- 
chrome C.2  is  independent  of  temperature.  This  accords  with  the  main 
postulate  of  our  theory  [94]  that  the  primary  photochemical  act  in  photo- 
synthesis consists  of  electronic  excitation  and  is  thus  independent  of  a 
thermal  reaction. 


358  DANIEL    I.    ARNON 

In  illuminated  cell-free  preparations  of  R.  rubrimi,  Smith  and  Ramirez 
[no]  and  Smith  and  Baltscheffsky  [in]  have  observed  changes  in  the 
absorption  spectrum  of  cytochrome  f.,  that  were  associated  with  phos- 
phorylation. Their  conclusions  [m],  that  in  the  facultative  anaerobe  R. 
rubrum  the  "  photosynthetic  "  cytochrome  Co  is  not  a  part  of  the  respiratory 
chain,  and  that  "two  different  enzyme  systems  mediate  the  oxidation  of 
substrates  by  oxygen  and  the  phosphorylation  of  ADP  by  illumination", 


400 


500     600 
A  {mil) 


Fig.  6.   Difference  spectrum  (reduced  minus  oxidized)  of  purified  cytochrome 
^2  oi  Chromatiimi  (Nozaki,  Ogata,  and  Arnon  [114]). 


are  concordant  with  our  distinction  between  photosynthetic  and  oxidative 
phosphorylation  in  green  cells  [14,  46,  112,  50]. 

A  reversible  light-induced  oxidation  of  cytochrome  r.,  i^i  cell-free 
preparations  of  Chromatium—z  cytochrome  that  has  been  isolated  and 
purified  by  Bartsch  and  Kamen  [113] — was  measured  by  Nozaki  et  al. 
[114].  The  absorption  spectrum  of  the  reduced  form  of  a  purified  cyto- 
chrome ^2  from  our  preparations  (Fig.  6)  is  the  same  as  that  described  by 
Bartsch  and  Kamen  [113]. 

The  effect  of  light  on  the  absorption  spectrum  (difference  spectrum, 
light  minus  dark)  of  cytochromes  in  cell-free  preparations  of  Chromatiutn 
is  shown  in  Fig.  7.  On  illumination,  the  absorption  spectrum  of  cyto- 
chromes shows  oxidation  followed  by  a  reduction  in  the  dark  and  re- 
oxidation  on  repeated  illumination.  Under  the  experimental  conditions  in 
which  the  C/iromatinm  cell-free  system  was  investigated,  the  light-dark 
reversible  oxidation-reduction  reactions  were  sufficiently  decelerated  to  be 
conveniently  measurable  with  a  recording  spectrophotometer,  at  room 
temperature. 


FHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      359 


Reversible  oxidation  of  Chromatium  cytochromes  by  light 


LIGHT 
t 


400  450  500  550  600  , 

Fig.  7.  Successive  oxidation  by  light  and  reduction  in  the  dark  of  cytochromes 
in  cell-free  preparations  of  Chromatium.  The  reaction  mixture  included  in  a  final 
volume  of  3-0  ml.,  chromatophores  (P)  containing  o-o6  mg.  bacteriochlorophyll 
and  supernatant  fluid  (S)  corresponding  to  0-5  mg.  bacteriochlorophyll.  A  small 
amount  of  Xa.,So04  was  previously  added  to  S,  which  was  then  dialyzed  against 
0-2  M  tris  buffer,  pH  7-8  prior  to  use.  The  reaction  was  carried  out  at  room 
temperature.  Gas  phase  argon.  Difference  spectra,  using  the  dark  treatment  as 
control,  were  made  in  Thunberg  type  cuvettes,  with  a  Gary  recording  spectro- 
photometer. Illumination  was  by  a  tungsten  lamp  (35  000  Lux).  (Nozaki,  Ogata, 
and  Arnon  [i  14]). 

EFFECT   OF    VITAMIN    K 

In  fresh  preparations  of  chromatophores  the  reduction  of  oxidized 
cytochrome  in  the  dark  was  not  influenced  by  the  additions  of  added 
cofactors  (compare  Table  I).  However,  as  shown  in  Fig.  8,  in  aged  prepara- 
tions, the  reduction  of  the  oxidized  cytochromes  was  greatly  accelerated  by 
the  addition  of  vitamin  K,  either  in  the  oxidized  form  (vitamin  K3)  or  in 
the  reduced  form  (vitamin  K5).  The  effect  of  vitamin  K,  as  an  electron 
carrier  in  accelerating  the  reduction  of  oxidized  cytochrome  depended  on 
the  presence  of  chromatophores.  Without  chromatophores,  using  a 
purified  cytochrome  Co,  a  hundred-fold  greater  concentration  of  reduced 
vitamin  K  was  required  to  reduce  the  oxidized  cytochrome. 

The  observed  effects  of  vitamin  K  in  catalyzing  the  reduction  of  cyto- 
chromes which  had  been  oxidized  in  light,  support  the  electron  flow- 
theory  [94]  for  cyclic  photophosphorylation.  This  theory  assigns  to- 
vitamin  K,  or  some  analogous  quinone,  a  role  of  an  intermediate  electron 
carrier  in  the  electron  transport  chain  associated  with  photophosphory- 
lation. 


360 


DANIEL    1.    ARNON 


FRESH    PREPARATION 
(ILL.-D) 


AGED   PREPARATION 
(ILL.-D) 


-0  2 


Light  on 


40 
min 


y^  ^^  Vit.   K5 

»  -.-   Vlt.    K3 


Control 
DPN 


Light  off 


Light  off 


Fig.  8.  Effect  of  vitamin  K  and  other  cofactors  on  the  reduction  of  cytochrome 
C2  in  cell-free  preparations  of  Chronuitiinn.  The  cytochromes  were  oxidized  by 
previous  illumination  (cf.  Fig.  7).  The  reaction  mixture  included  in  a  final  volume 
of  3  -o  ml.,  dialyzed  cell-free  suspension  (PS)  containing  o-o6  mg.  bacteriochloro- 
phyll,  0-02  /imole  of  purified  cytochrome  To  and  0-03  /xmole  of  the  respective 
cofactors.  Difference  in  optical  density  was  measured  on  a  Beckman  DU  spectro- 
photometer with  an  attached  photomultiplier  using  cuvettes  with  the  respective 
cofactor  omitted  as  controls  in  each  case  (Nozaki,  Ogata,  and  Arnon,  [114]). 


LIGHT-INDUCED    CHANGES    IN    CHLOROPHYLL 

New  experimental  evidence  for  the  electron  flow  theory  has  come  from 
the  recent  work  of  Arnold  and  Clayton  [115]  who,  on  illuminating  bacterial 
chromatophores,  observed  temperature-independent  (i  K  to  300°K) 
reversible  spectral  changes  in  the  absorption  bands  of  bacteriochlorophyll. 
These  spectral  changes  in  chlorophyll  that  are  independent  of  temperature 
are  consistent  with  the  proposed  electron  shift  that  would  result  from  the 
primary  photochemical  act  (compare  equations  (2)  and  (2a)). 


8.  Multiple  sites  in  cyclic  photophosphorylation 

As  already  discussed,  cyclic  photophosphorylation  is  catalyzed  by 
vitamin  K  and  FMN  and  also  by  non-physiological  factors  such  as 
phenazine  methosulphate.  This  latter  fact  has  given  rise  to  questions 
whether  vitamin  K  and  FMN,  or  equivalent  quinone  and  flavin  consti- 
tuents of  photosynthetic  tissues,  are  to  be  considered  the  physiological 
catalysts  of  cyclic  photophosphorylation  or  whether  they  are  to  be  regarded 
as  non-specific  agents  in  no  way  distinguishable  from  non-physiological 
catalysts.  The  question  was  of  special  interest  in  connection  with  phenazine 
methosulphate  because  this  dye  has  given  rates  of  photosynthetic  phos- 
phorylation higher  than  either  vitamin  K  or  FMN  [cf.  74]. 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      36 1 

The  marked  effectiveness  of  phenazine  methosulphate  could  be  ex- 
plained by  its  acting  as  an  electron  carrier  that  bypasses  a  rate-limiting  step 
in  photosynthetic  phosphorylation  [66].  This  is  suggested  by  the  observa- 
tion of  Geller  [66]  that  the  severe  inhibition  of  photophosphorylation  in 
R.  riibrian  by  antimycin  A  does  not  occur  in  the  presence  of  phenazine 
methosulphate.  On  this  hypothesis  phenazine  methosulphate  might  give 
higher  rates  of  phosphorylation  at  high  light  intensity  when  there  is  a  rapid 
flux  of  electrons  from  excited  chlorophyll.  Assuming,  however,  several 
sites  of  phosphorylation  in  the  cyclic  pathway,  the  advantage  of  phenazine 
methosulphate  might  disappear  at  loic  light  intensity  when  the  overall  rate 


Low  light  Intensity 
(3000  Lux) 


vit.  K, 


PMS 


10       15        20      25 
minutes 

Fig.  9.  Effect  of  vitamin  K^  and  phenazine  methosulphate  (PMS)  on  anaerobic 
cyclic  photophosphorylation  in  spinach  chloroplasts  at  a  limiting  light  intensity 
(3000  Lux).  Reaction  mixture  included  chloroplast  fragments  (C,,)  containing  i  mg. 
chlorophyll  and  in  micromoles :  tris  buffer,  pH  8-3,  80;  K2H^'-P04,  15;  MgS04, 
5;  ADP,  15;  vitamin  K:,  or  PMS,  03;  gas  phase  nitrogen  (Tsujimoto,  Hall, 
and  Arnon  [92]). 


of  the  process  is  limited  by  the  electron  flux.  Under  such  conditions  the 
highest  rate  of  photophosphorylation  would  be  observed  in  a  system  in 
which  none  of  the  phosphorylation  sites  was  bypassed.  Thus,  a  com- 
parison of  photosynthetic  phosphorylation,  catalyzed  by  vitamin  K  and 
phenazine  methosulphate  under  conditions  of  limiting  light,  seemed 
desirable. 

The  results  of  such  a  comparison  are  shown  in  Y'\<g.  9.  At  low  light 
intensity  photophosphorylation  catalyzed  by  vitamin  Kg  was  markedly 
greater  than  that  catalyzed  by  phenazine  methosulphate  (or  pyocyanin). 
This  diflFerence  was  persistent  and  gave  a  straight-line  relationship  for  a 
considerable  period  of  time. 


362  DANIEL   I,    ARNON 

These  experiments  were  extended  by  comparing  the  rates  of  photo- 
synthetic  phosphorylation  at  different  hght  intensities.  The  results  shown 
in  Fig.  10  confirm  and  extend  those  illustrated  in  Fig.  9.  At  low  intensity 
cyclic  photophosphorylation  catalyzed  by  either  vitamin  K3  or  FMN 
proceeded  at  a  much  higher  rate  than  that  catalyzed  by  phenazine  metho- 
sulphate.  However,  at  higher  light  intensities  the  phenazine  methosulphate 
system  gave  much  greater  rates  of  phosphorylation  [92]. 

These  results  suggest  that  at  high  light  intensity  the  vitamin  K  and 
FMN  systems  became  limited  by  enzymic  reactions  which  were  unable  to 


10 

- 

/PMS 

Ǥ    5 

C 

■a;  8 

- 

/ 

<o 

>/ 

<i) 

/ 

.  7 

- 

/ 

•5 

ta 

■c   6 

/ 

% 

/_ 

FMN*TPN 

^5 

- 

J^'^^^'a 

vit    K,          ° 

<5l. 

^^  /i 

<o    4 

^r 

/ 

-S? 

//^ 

/ 

// 

/ 

0 

<    2 

-  If       y^ 

u 

^    / 

Y           1 

1               1 

1                 1 

10000     20000    30000   40000    50000 
light   intensity  (Lux) 

Fig.  io.  Effect  of  light  intensity  on  anaerobic  cyclic  photophosphorylation. 
Gas  phase  nitrogen.  Illumination  period  30  min.  The  reaction  mixture  included 
chloroplast  fragments  (C,,)  containing  01  mg.  chlorophyll  and  chloroplast 
extract  equivalent  to  i  mg.  chlorophyll.  0-3  /xmole  TPN  and  0-3  /xmole  FMN 
were  included  in  the  FMN  system.  Other  conditions  as  given  for  Fig.  9  (Tsujimoto, 
Hall,  and  Arnon  [92]). 


keep  pace  with  the  rapid  electron  flux.  The  increasing  rates  of  phos- 
phorylation obtained  at  high  light  intensity  with  the  phenazine  metho- 
sulphate system  are  consistent  with  the  explanation  that  this  agent  does 
indeed  serve  as  a  bypass  around  some  rate-limiting  step,  probably  by 
catalyzing  the  reduction  of  cytochromes  [90].  These  findings  are  inter- 
preted as  an  indication  of  enzymic  steps  that  may  limit  cyclic  photophos- 
phorylation at  high  light  intensities,  when  physiological  catalysts  such  as 
vitamin  K  or  FMN  (or  their  analogues)  are  involved. 

The  findings  that,  when  light  is  limiting,  vitamin  K  and  FMN  catalyze 
higher  rates  of  photophosphorylation  than  phenazine  methosulphate 
(Fig.  10),  suggest  the  involvement  of  at  least  two  phosphorylation  sites  in 
the  vitamin  K  and  FMN  pathways.  A  diagrammatic  representation  of  this 
mechanism  is  given  in  Fig.  5. 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       363 

A  primary  phosphorylation  reaction,  common  to  all  pathways  of  cyclic 
photophosphorylation,  is  en\'isaged  as  being  coupled  with  the  oxidation 
of  the  terminal  cytochrome  in  the  photosynthetic  particle,  i.e.  the  cyto- 
chrome that  reacts  with  the  excited  chlorophyll  molecule  (compare  Figs. 
4  and  5).  A  second  site  of  phosphorylation  in  the  vitamin  K  pathway  is 
likely  to  occur  on  oxidation  of  reduced  vitamin  K  (or  its  analogue)  by 
cytochromes,  as  was  suggested  by  the  model  reactions  proposed  by 
Wessels  [85],  Harrison  [116]  and  Clark  et  al.  [117].  In  the  FMN  pathway, 
an  additional  phosphorylating  site  can  be  readily  envisaged  in  the  span 
between  TPN  and  cytochromes  [94]. 

Further  evidence,  derived  from  fractionating  chloroplasts,  will  be 
given  in  the  next  Section  for  the  conclusion  that  cyclic  photophosphoryla- 
tion catalyzed  by  phenazine  methosulphate  probably  proceeds  by  way  of  a 
"bypass"  and  is  less  dependent  on  enzymic  chloroplast  constituents  than 
photosynthetic  phosphorylation  catalyzed  by  either  vitamin  K  or  FMN. 

9.  Structural  association  of  chlorophyll  with  the 
photophosphorylating  system 

In  photosynthetic  bacteria  the  photophosphorylating  system  is 
structurally  bound  to  chlorophyll  in  the  smallest  particles  that  function  as 
units  in  the  absorption  of  light  energy,  the  chromatophores.  Their 
analogues  in  green  plants  are  the  grana  and  it  was  of  interest,  therefore, 
to  determine  whether  in  chloroplasts  photophosphorylation  is  indeed 
localized  in  the  grana. 

Photosynthetic  phosphorylation  was  tirst  observed  in  intact  chloro- 
plasts (Fig.  2)  but  experiments  with  disrupted  chloroplasts  soon  demon- 
strated that  structural  integrity  was  not  essential  for  this  process.  When 
whole  chloroplasts  were  broken,  active  photophosphorylation  systems 
were  reconstituted  by  a  recombination  of  chloroplast  fractions  and  added 
cofactors  [38,  39].  This  technique  proved  effective  in  investigating  the 
mechanism  of  photophosphorylation  but  provided  no  rigid  e\idence  that 
the  site  of  photophosphorylation  is  in  the  grana. 

Direct  evidence  for  the  localization  of  photosynthetic  phosphorylation 
in  grana,  freed  from  other  chloroplast  fractions,  was  obtained  by  Miiller 
et  al.  [118]  who  prepared  purified  grana  by  sonication  of  isolated  whole 
chloroplasts  followed  by  a  density  gradient  centrifugation  technique.  The 
purity  of  the  grana  obtained  by  these  methods  was  determined  by  exami- 
nation of  electron  micrographs  of  freeze-dried  and  air-dried  grana  prepara- 
tions (Figs.  II  and  12).  The  freeze-drying  technique  avoids  artifacts 
resulting  from  chemical  fixation  and  retains  the  natural  shape  of  the 
particles  [119].  Cyclic  photophosphorvlation  bv  purified  grana  is  shown  in 
Table  IV. 


364  DANIEL   I.   ARNON 

Table  IV  shows  that,  at  the  high  Hght  intensity  at  which  cycHc  photo- 
phosphorylation  by  purified  grana  was  measured,  the  highest  rates  were 
obtained  in  the  system  catalyzed  by  phenazine  methosulphate.  Photo- 
phosphorylation  in  this  system  was  not  increased  by  the  addition  of  an 
aqueous  chloroplast  extract.  In  contrast,  photophosphorylation  catalyzed 


Fig.  II.  Electron  micrograph  of  isolated  spinach  grana  prepared  for  electron 
microscopy  by  a  freeze-drying  technique,  i  cm.-  of  leaf  surface  is  estimated  to  have 
about  50  million  chloroplasts.  Whole  chloroplasts  were  disrupted  by  a  sonic  vibra- 
tion treatment  for  10  sec.  The  grana  were  isolated  in  sucrose  by  a  density  gradient 
centrifugation  technique  and  the  sucrose  removed  by  washing  with  a",,  NaCl. 
The  grana  were  used  in  electron  microscopy  involving  a  modified  freeze-drying 
technique  [119]  that  avoids  possible  artifacts  resulting  from  chemical  fixation  and 
retains  the  natural  shape  of  particles.  Magnification :  59  000  x  (Miiller,  Steere, 
and  Arnon,  [118]). 

by  vitamin  K  or  FMN  proceeded  at  a  lower  rate  and  was  markedly 
increased  by  the  addition  of  chloroplast  extract ;  however,  even  with  this 
increase,  it  failed  to  reach  the  rate  of  photophosphorylation  in  the  phen- 
azine methosulphate  system. 

These  results  support  the  conclusion  that  grana  are  the  site  of  the 
"primary"  photophosphorylation  reaction  (equation  (3)),  the  one  that  is 


PHOTOSYXTHETIC  PHOSPHORYLATION  AXD  THE  ENERGY  CONVERSION  PROCESS      365 

visualized  as  occurring  between  the  "terminal"  cytochrome  and  chloro- 
phvU  and  is  shown  in  Fig.  4  as  being  catalyzed  by  phenazine  methosul- 
phate.  (Significant  in  this  connection  are  the  recent  findings  of  James  and 
Leech    that,    like    the    bacterial    cytochromes    in    chromatophores,    the 


Whole  chloroplasts  were  disrupted  by  sonic  vibration  treatment  for  10  sec.  The 
grana  were  isolated  by  differential  centrifugation  and  filtration  through  a  double 
layer  of  Whatman  No.  2  filter  paper.  The  isolated  grana  were  fixed  for  2  hr.  in 
6"o  formaldehyde  at  pH  6-5.  A  comparison  with  similar  particles  prepared  by  the 
freeze-drying  technique  (Fig.  11)  shows  that  they  collapsed  in  the  formaldehyde 
treatment.  The  background  in  the  formaldehyde  treatment  is  free  from  salt 
(compare  with  Fig.  11).  Magnification  :  10  000  x  (Muller,  Steere,  and  Arnon  [i  18]). 


chloroplast  cytochromes,  /  and  b,  are  "entirely  confined  to  the  grana" 
[120]).  At  high  light  intensity  the  "bypass"  pathway  catalyzed  by  phen- 
azine methosulphate  gives  high  rates  of  photophosphorylation  because  it 
is  not  limited  by  the  absence  of  chloroplast  constituents  that  lie  outside 
the  grana.  The  beneficial  effect  of  added  chloroplast  extract  on  the  FMN 


366  DANIEL   I.    ARNON 

TABLE  IV 

Cyclic  Photophosphorylation  by  Purified  Ghana  with  and  without  added 
Chloroplast  Extract.   Illumination  35  000  Lux 

(Miiller,  Steere,  and  Arnon  [118]) 

Treatment  Q,?''* 


Phenazine  methosulphate  157 

Phenazine  methosulphate  +  chloroplast  extract  145 

Vitamin  Kg  39 

Vitamin  K3  +  chloroplast  extract  78 

FMN  46 

FMN  +  chloroplast  extract  6g 

*  Micromoles  orthophosphate  esterified  per  mg.  chlorophyll  per  hour. 

and  vitamin  K  systems  (Table  IV)  suggests  that  the  extract  contains  some 
chloroplast  constituents  that  are  involved  in  these  pathways  but  not  in  the 
pathway  catalyzed  by  phenazine  methosulphate. 

The  close  structural  association,  in  both  chloroplasts  and  bacterial 
chromatophores,  of  the  phosphorylating  activity  with  the  chlorophyll 
pigments  suggests  that  the  harnessing  of  light  energy  in  photosynthesis  is 
more  closely  associated  with  ATP  formation  than  w'ith  CO2  assimilation. 
The  enzymes  responsible  for  COo  assimilation  are  easily  dissociable  from 
granaf  in  the  case  of  chloroplasts  [38,  39,  37],  and  not  even  structurally 
joined  together  in  the  case  of  bacterial  chromatophores  [68,  121].  These 
facts  are  in  agreement  with  the  view  [94,  95]  that  in  the  course  of  bio- 
chemical evolution,  photosynthesis  first  emerged  as  a  process  for  con- 
verting light  energy  into  ATP  and  this  "primitive"  photosynthesis 
became  only  later  a  process  linked  to  CO.,  reduction. 

10.  Cyclic  photophosphorylation  as  primitive  photosynthesis 

In  the  conventional  view  of  photosynthesis,  the  chemical  energy 
obtained  by  the  conversion  of  absorbed  light  is  always  used  for  the  reduction 
of  CO.,.  The  case  that  cyclic  photophosphorylation  is  a  "primitive" 
photosynthesis  in  the  evolutionary  sense,  would  therefore  be  strengthened, 
if  examples  could  be  found  today  of  cases  in  which  the  contribution  of 
light  to  carbon  assimilation  could  be  experimentally  limited  to  the 
formation  of  ATP. 

t  Grana,  as  contrasted  with  whole  chloroplasts,  cannot  assimilate  CO.,  to  the 
level  of  carbohydrates  but  retain  a  capacity  for  photochemical  oxygen  evolution 
and  photosynthetic  phosphorylation.  These  findings  do  not  exclude  the  catalytic 
participation  of  CO. 2  in  the  mechanism  of  oxygen  evolution  as  has  recently  been 
proposed  by  Warburg  et  al.  (Z.  Natio[f.  14b,  712-724,  1959). 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVTIRSION  PROCESS      367 

Two  such  cases  of  photosynthesis  in  Chromatium  have  recently  been 
described  by  Losada  et  al.  [121].  In  one  case  the  sole  source  of  carbon  was 
acetate  and  in  the  other,  COo.  The  photoassimilation  of  acetate  occurred 
in  the  absence  of  an  external  hydrogen  donor  whereas  in  the  photo- 
assimilation  of  COo  the  reductant  was  exogenous  hydrogen  gas.  The  sole 
contribution  of  light  in  both  cases  was  the  formation  of  ATP. 

In  the  photoassimilation  of  CO.,,  ATP  was  required  for  the  formation 


ATP 

V 


ribulose-S-P 


ribulose  di-P 
CLUTAMATE 


triose-P 


itaconate  P-glycerate 

II 

P-enolpyruvate 


DPN  +  H; 
ATP  ASPARTATE 


citramalate 


acetate/    yhc-Cok-^ — |  ACETATE 


ATP 


_  .  [acetaldehyde]  +  CO2 

•  pyruvate  -"^  citrate 

^~^ --ALANINE 


Ac-CoA 


CO. 
ATP— ^^       TPNH 


ate-<—'socitrate-»-rt-ketogluta  rate-*- GLUT  A  MATE 


glyoxylate 


Fig.    13.  Reactions  of  carbon  assimilation  in  CJimmatinm.  Further  details  are 
given  by  Losada,  Trebst,  Ogata,  and  Arnon  [121]. 


of  an  activated  intermediate  (ribulose  diphosphate,  phosphoenolpyruvate, 
or  1,3-diphosphoglycerate)  for  a  subsequent  carboxylation  or  reduction, 
whereas  in  the  photoassimilation  of  acetate,  ATP  was  required  for  the 
activation  of  the  carbon  source  itself,  by  forming  acetyl-CoA  from  acetate 
and  coenzyme  A.  The  activated  compounds  then  become  ready  for 
participation  in  the  synthetic  reactions  that  are  catalyzed  by  specific 
enzyme  systems,  all  of  which  function  in  the  dark.  A  summary  of  the 
reactions  of  ATP,  that  have  now  been  experimentally  documented  [121] 
in  the  carbon  metabolism  oi  Chromatium,  is  given  in  Fig.  13. 

Evidence  that  the  sole  contribution  of  light  in  these  reactions  is  the 


368  DANIEL    I,    ARNON 

formation  of  ATP,  was  obtained  by  replacing  light  with  a  supply  of 
exogenous  ATP  and  finding  that  carbon  assimilation  would  then  proceed 
in  the  same  manner  in  the  dark  as  in  the  light  [121].  Other  evidence  for 
the  equivalence  of  light  and  ATP  is  given  in  Table  V.  Here  assimilation 
occurred  either  in  the  dark  with  added  ATP  or  in  the  light  when  ATP  was 
allowed  to  form  photosynthetically.  If,  however,  the  ATP  formed  in  light 
was  trapped  by  an  added  hexokinase-glucose  system  then  acetate  assimila- 
tion ceased.  The  addition  of  hexokinase  alone,  without  glucose  as  the  ATP 
acceptor,  was  not  inhibitory  (Table  V). 

TABLE  V 

Equivalence  of  ATP  and   Light  in  the  Assimilation  of   "C-Acetate  by 
Cell-Free  Preparations  of  Chromatinm 

(Losada,  Trebst,  Ogata,  and  Arnon  [121]) 

^''Carbon  fixed  in 
Treatment  soluble  compounds 

(Thousands  of  counts /min) 

1.  Dark,  control  27 

2.  Dark,  ATP  180 

3.  Dark,  ATP,  hexokinase  186 

4.  Dark,  ATP,  hexokinase,  glucose  6 

5.  Light,  control  414 

6.  Light,  hexokinase  348 

7.  Light,  hexokinase,  glucose  20 

Each  vessel  included,  in  a  final  volume  of  i  -5  ml.,  cell-free  extract,  containing 
o  •  3  mg.  bacteriochlorophyll  and  the  following  in  micromoles :  tris  buffer,  pH  7  •  8, 
80 ;  cysteine,  20 ;  magnesium  chloride,  5 ;  manganese  chloride,  2 ;  potassium 
chloride,  20;  coenzyme  A,  0-3;  oxalacetate,  10;  [i-^*C]-acetate,  3.  1-5  mg.  hexo- 
kinase, type  III  (Sigma  Chemical  Co.),  10  /xmoles  glucose,  and  4  /^imoles  ATP 
were  added  as  indicated.  In  treatment  5,  6  and  7  no  addition  of  ADP  was  necessary 
to  supplement  the  catalytic  amounts  present  in  the  cell-free  extracts. 


The  experimental  substitution  of  ATP  for  light  was  considered 
particularly  significant  because  it  was  found  in  photosynthetic  bacteria 
such  as  Chromatiiwi,  that  are  unique  in  the  living  world  in  being  strict 
phototrophs.  Chromotiiim,  unlike,  for  example,  Chlorella  or  photosynthetic 
bacteria  of  the  genus  Rhodospir ilium,  cannot  replace  its  light-dependent 
mode  of  life  by  a  heterotrophic,  aerobic  metabolism  in  the  dark  [122,  123, 
124].  Chromatinm  grows  only  in  the  light  [122,  123],  and  being  an  obligate 
anaerobe,  does  not  possess  an  alternative  way  for  forming  ATP  by  the 
mechanism  of  oxidative  phosphorylation. 

As  regards  the  photoassimilation  of  acetate  in  another  photosynthetic 
bacterium,  the  facultative  anaerobe  R.  nibrum,  a  similar  view  that  the 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      369 

contribution  of  light  is  limited  to  cyclic  photophosphorylation  was  recently 
expressed,    on    the   basis   of   independent    evidence,    by    Stanier    et    al. 

In  certain  circumstances,  ATP  formation  may  be  the  sole  contribution 
of  the  photosynthetic  process,  not  only  in  bacteria  but  also  in  higher  plants. 
We  have  suggested  elsewhere  [95]  that  in  green  plants  cyclic  photo- 
phosphorylation may  continue  forming  ATP  when  CO2  assimilation  is, 
for  one  reason  or  another,  reduced  or  even  stopped  altogether.  This  might 
arise  during  the  well-known  midday  closure  of  stomata  in  leaves  of  higher 
plants  [126,  127]  which  restricts  the  supply  of  CO.,.  The  closure  of  stomata 
often  coincides  with  an  abundance  of  starch  and  an  incipient  water  deficit 
in  the  photosynthesizing  cells.  Under  these  conditions  cyclic  photo- 
phosphorylation, which  consumes  neither  COo  nor  water,  would  be  a 
useful  device  for  generating  ATP  to  drive  the  many  ATP-dependent 
reactions,  notably  the  synthesis  of  polysaccharides,  proteins  and 
fats. 

These  theoretical  deductions  for  higher  plants  have  recently  received 
experimental  support  from  the  work  of  Maclachlan  and  Porter  [128].  They 
reported  the  first  known  instance  of  utilization  of  light  energy  in  leaf  tissue 
for  the  synthesis  of  starch  from  labelled  glucose,  under  conditions  when 
CO2  assimilation  was  excluded  but  cyclic  photophosphorylation  could 
proceed. 

II.  Pyridine  nucleotide  reduction  by  hydrogenase  in  the  dark 

In  the  examples  of  photosynthesis  in  which  the  contribution  of  light 
was  limited  to  ATP  formation,  no  reductant  was  needed  in  the  conversion 
of  glucose  to  starch  in  leaves.  In  the  assimilation  of  acetate  by  bacteria, 
hydrogen  is  released  for  metabolic  purposes  and  no  additional  hvdrogen 
donor  is  required  [121].  But  the  assimilation  of  COo  requires  in  addition 
to  ATP,  a  supply  of  a  reductant,  i.e.  reduced  pyridine  nucleotide.  It  was 
stated  earlier  that  in  photosynthesis  of  green  plants  both  of  these  com- 
ponents of  assimilatory  power  are  formed  at  the  expense  of  light  energy. 
It  is  necessary,  therefore,  to  trace  the  transition  from  a  primitive  photo- 
synthesis in  which  light  is  used  only  for  the  formation  of  ATP  to  the 
"advanced"  type  of  photosynthesis,  observed  in  green  plants,  in  which 
light  energy  is  used  not  only  for  ATP  formation  but  also  for  the 
reduction  of  pyridine  nucleotide  and  the  simultaneous  evolution  of 
oxygen. 

In  the  photoassimilation  of  CO.,  by  Chromatium  the  added  reductant 
was  hydrogen  gas  [129].  This  is  the  simplest  reductant  usable  by  living 
cells.  Cell-free  hydrogenases  from  non-photosynthetic  bacteria  are  known 
to  reduce  pyridine  nucleotides  with  molecular  hydrogen  [130,  131 ;  cf.  40]. 


37°  DANIEL    I.    ARNON 

From  the  standpoint  of  photosynthesis,  it  was  important  to  know  if  the 
hydrogenases  of  photosynthetic  bacteria  could  also  reduce  pyridine 
nucleotide  with  molecular  hydrogen  in  the  dark,  since  this  would  provide 
a  mechanism,  independent  of  light,  for  the  formation  of  the  reductant  for 
CO2  assimilation.  In  photosynthetic  bacteria,  the  only  cell-free  hydro- 
genase  tested  in  this  respect,  that  of  R.  riibriim,  was  reported  to  be  unable 
to  reduce  acceptors  with  potentials  less  than  o  volts  [132]  which  would 
thus  exclude  pyridine  nucleotides  (is^  =  —0-32  V.). 

TABLE  VI 

Pyridine  Nucleotide  Reduction  with  Molecular  Hydrogen  by  Cell-Free 
Preparations  of  Chroynatium 

(Ogata,  Nozaki,  and  Arnon  [91]) 


Treatment 

DPN  Series 

TPN  Series 

Light 

Dark 

Light 

Dark 

Pyridine  nucleotide  (PN) 
Benzyl  viologen  (BV) 
PN  +  BV 

o-o8 

—  o-o8 

o-8o 

0-09 

-o-o8 

0-86 

0-07 

—  o-o8 

0-33 

0-07 

-o-o8 

0-42 

Each  vessel  included,  in  a  final  volume  of  3  -o  ml.,  a  cell-free  preparation  (PS) 
containing  o  •  3  mg.  bacteriochlorophyll  and  the  following  in  micromoles :  tris 
buffer,  pH  7-8,  80;  MgCl.2,  5  ;  potassium  phosphate,  5  ;  KCl,  50;  and  when  added, 
DPN,  4;  TPN,  4;  and  benzyl  viologen,  o-i  (a  gift  of  Dr.  H.  Gest).  o-i  ml.  of 
20*^)0  KOH  was  present  in  the  centre  well.  The  reaction  was  carried  out  in  an 
atmosphere  of  hydrogen  at  25'^.  Illumination,  when  given,  was  35  000  Lux.  At 
the  end  of  the  reaction,  an  aliquot  of  the  reaction  mixture  was  precipitated  with 
saturated  (NH4)2S04,  pH  8,  centrifuged  and  the  optical  density  of  the  clear 
supernatant  fluid  was  measured  at  340  m/x. 

The  subject  was  reinvestigated  by  Ogata  et  al.  [91],  using  the  cell-free 
hydrogenase  of  Chromatium.  As  in  other  photosynthetic  bacteria  (for 
example,  R.  nibrum  [132],  an  active  hydrogenase  was  also  found  in 
Chromatium.  The  Chromatium  hydrogenase  reduced  DPN  and  TPN  with 
molecular  hydrogen  in  the  dark  in  the  presence  of  benzyl  viologen.  The 
enzyme  was  more  active  toward  DPN  than  TPN  (Table  VI). 

These  results  indicated  that  in  the  presence  of  hydrogen  gas,  Chro- 
matium cells  do  not  require  light  for  the  reduction  of  pyridine  nucleotides. 
The  role  of  light  is  then  limited  to  ATP  formation,  without  which  CO2 
assimilation  cannot  occur  [121].  Photosynthesis  by  Chromatiu?n  in  the 
presence  of  molecular  hydrogen  may,  therefore,  be  summarized  as 
follows : 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       37 1 

Light  phase 

Cyclic  photophos- 

phorylation:  n-ADP  +  n-P >n-ATP 

Dark  phase 

DPN  reduction :       2DPN  +  2H0 >  2DPNH.3 

COo  assimilation:     CO0  + aDPXHo  +  n-ATP >(CH,0)  +  HoO  + 

2DPN  +  nADP  +  nP 

Sum  :  CO,  +  2H,  ^^  (CH,,0)  +  H^O 

Several  algal  species  are  known  to  contain  hydrogenases  and  to  acquire, 
after  adaptation  to  hydrogen,  a  capacity  to  photoassimilate  CO,  with  the 
aid  of  molecular  hydrogen  [133,  134,  135]-  This  process,  which  Gaffron 
named  photoreduction  [134],  appears  to  be  the  same  type  of  photo- 
synthesis as  that  in  CJiromatiiim  when  it  is  supplied  with  hydrogen  gas.  It 
seems  likely  that  photoreduction  by  algae  is  a  case  of  reversion  to  a 
primitive  photosynthesis  of  an  earlier  epoch  when  hydrogen  gas  was 
present  in  the  environment  and  the  sole  contribution  of  light  was  the 
formation  of  ATP  by  cyclic  photophosphorylation. 


12.  The  photoreductant  in  bacteria 

Although  photosvnthetic  bacteria  when  supplied  with  hydrogen  gas  do 
not  require  light  energv  for  the  production  of  DPXH,  (or  TPNH,),  a 
different  situation  arises  when  photosynthetic  bacteria  are  grown  with 
such  hvdrogen  donors  as  succinate  or  thiosulphate  [122,  123].  Electrons 
donated  by  these  substances  have  an  insufficient  reducing  potential  for 
reducing  DPN  (or  TPX)  in  the  dark. 

Additional  energy  is  then  required  to  bring  about  the  reduction  of 
DPN  (or  TPN)  and,  in  a  photosvnthetic  mode  of  life  without  oxygen 
which  is  characteristic  of  photosynthetic  bacteria,  this  additional  energy 
must  come  from  light.  If  the  electron  flow  mechanism  is  fundamental  to 
the  conversion  of  light  into  chemical  energy,  how  can  it  apply  to  the 
photoreduction  of  pyridine  nucleotides  by  thiosulphate  or  succinate  ? 

An  attractive  hypothesis  was  to  consider  bacterial  photosynthesis  with 
thiosulphate  and  succinate  as  an  extension  of  bacterial  photosynthesis  with 
hydrogen  gas,  when  the  photochemical  events  proper  are  restricted  to  the 
formation  of  ATP  by  cvclic  photophosphorylation.  The  primary  photo- 
chemical act  that  results  in  the  generation  by  the  excited  chlorophyll  of  a 
high  energy  electron  and  of  the  ultimate  electron  acceptor,  [Chl+],  would 
be  the  same  in  both  cases.  But  in  the  thiosulphate  and  succinate  type  of 


372  DANIEL    I.    ARNON 

bacterial  photosynthesis,  not  all  of  the  high  energy  electrons  would  return 
via  the  cyclic  route  to  [Chl+].  Some  of  them  would  be  passed  on  to 
pyridine  nucleotide  and  used  for  CO2  assimilation. 

The  electrons  so  removed  from  the  photoreceptor  particle  would  be 
replaced  by  electrons  donated  by  thiosulphate  or  succinate.  This  electron 
transfer  would  be  mediated  by  cytochromes.  Thiosulphate  and  succinate 
would  thus  act  as  hydrogen  donors  that  reduce  bacterial  cytochromes  after 
these  are  oxidized  by  chlorophyll  in  light.  The  cytochrome  system  in 
photosynthetic  bacteria  would  be  a  gateway  for  the  entry  of  electrons  of  a 


004 

■     1              1 
420 

'    A 

— T    '             1 

1 

ol  density 
0            0 

8       S 

552 

- 

\ 

^^y^^    \^ 

a- 

°  -002 

xJ 

- 

-004 

1            1 

1               1 

1 

400 


500 
^  im/i) 


600 


Fig.  14.  Reduction  of  Chromatium  cytochromes  by  thiosulphate  in  a  cell-free 
system.  Reaction  mixture  included,  in  a  final  volume  of  3  -o  ml.  of  o  ■  i  M  tris  buffer, 
pH  7-8,  chromatophores  (P)  containing  o-i  mg.  bacteriochlorophyll  and  super- 
natant fluid  (S)  corresponding  to  0-3  mg.  bacteriochlorophyll.  20  ^umoles  of  thio- 
sulphate were  added  to  one  of  a  pair  of  Thunberg-type  cuvettes  and  the  resulting 
difference  spectrum  was  measured  in  a  Gary  spectrophotometer  after  20  min.  at 
room  temperature.  Gas  phase,  argon  (Losada,  Nozaki  and  Arnon  [136]). 


relatively  low  reducing  potential  and  for  their  transfer  to  chlorophyll, 
where  they  would  be  raised  to  a  higher  reducing  potential  at  the  expense 
of  the  energy  of  absorbed  light. 

The  proposed  sequence  of  reactions  in  photosynthetic  bacteria  will  be 
collectively  designated  as  the  non-cyclic  electron  flow  mechanism.  The 
three  components  of  the  non-cyclic  electron  flow  mechanism  are  {a)  an 
external  electron  donor  system  (represented  here  by  thiosulphate  or 
succinate),  {h)  the  photoreceptor  particle  which  raises  the  donated  electron 
to  a  higher  reducing  potential  at  the  expense  of  the  energy  of  light,  and 
[c)  the  electron  acceptor  system  (exemplified  by  DPN  or  TPN). 

Experimental  support  for  the  non-cyclic  electron  flow  mechanism  in 
bacterial    photosynthesis   has   recently   become   available.    First,    it  was 


PHOTOSYXTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      373 

established  that  Chromatium  particles  have  enzymes  catalyzing  the  transfer 
of  electrons  from  thiosulphate  and  succinate  to  cytochromes  [136,  i] :  the 
reduction  of  oxidized  Chromatium  cytochromes  by  thiosulphate  is  shown  in 
Fig.  14  and  by  succinate  in  Fig.  15.  Second,  illuminated  photosynthetic 
bacteria  reduce  pyridine  nucleotides  in  the  presence  of  succinate  or  some 
other  electron  donor  that  is  less  reduced  than  pyridine  nucleotides.  The 
photoreduction  of  DPN  was  observed  by  Frenkel  [146]  and  Vernon  and 


Reduction  of  Chromatium  (P+S) 
cytochromes   by  succinate 


0-2 


0-1 


0-0 


-0-1 


-0-2 


400 


450  500 

A  (m//) 


550 


600 


Fig.  15.  Reduction  of  Cliromathim  cytochromes  by  succinate  in  a  cell-free 
system.  Reaction  mixture  included,  in  a  final  volume  of  o  •  3  ml.  of  o  •  2  m  tris  buflfer, 
pH  7-8,  chromatophores  (P)  containing  o-o6  mg.  bacteriochlorophyll  and  super- 
natant fluid  (S)  corresponding  to  0-3  mg.  bacteriochlorophyll.  10  /j.moles  of 
succinate  was  added  to  one  of  a  pair  of  cuvettes  and  the  resulting  difference 
spectrum  was  measured  in  a  Gary  spectrophotometer  at  the  indicated  time 
intervals  (Nozaki,  Ogata,  and  Arnon  [114]). 


Ash  [147]  in  R.  riihnim  and  by  Ogata  et  al.  in  Chromatium  [91].  In  more 
recent  experiments  we  have  found  that  in  the  presence  of  succinate  and 
light,  unwashed  chromatophores  from  R.  rubriim,  unaided  by  enzymes 
from  chloroplasts  (cf.  [147])  reduce  both  di-  and  triphosphopyridine 
nucleotide. 

Additional  support  for  the  non-cyclic  electron  flow  mechanism  in 
bacterial  photosynthesis  has  come  from  recent  experiments  on  the  photo- 
production  of  hydrogen  gas  and  photofixation  of  nitrogen  gas.  We  found 


374  DANIEL    I.    ARNON 

in  Chroniatiiim  a  light-dependent  transfer  of  electrons  from  thiosulphate 
or  succinate  not  only  to  pyridine  nucleotides  but  also  to  H  +  and  N.,.  The 
transfer  of  electrons  to  H  +,  a  reaction  that  is  catalyzed  by  hydrogenase, 
results  in  photoproduction  of  hydrogen  gas.  The  transfer  of  electrons  to 
N2  constitutes  photofixation  of  N.,.  These  light  reactions  will  now  be 
discussed  in  more  detail. 


PHOTOPRODUCTION  OF  HYDROGEN  GAS 

P'igure  16  illustrates  a  vigorous  photoproduction  of  molecular  hydrogen 
from  thiosulphate  [136]  and  Fig.  17  shows  photoproduction  of  molecular 


1 

I 

6 

- 

: 

?     ^ 

-0 

0) 

^   5 

■0 

0 

Light 

i 

■DarkJ 

Light  y 

/ 

0.  4 

- 

/ 

- 

x* 

/ 

-0  ' 

^ 

/ 

-~ 

in 

-S   2 

-/ 

^ 

- 

0   '■ 
E 
^    1 

/  , 

;f „.„{..,, 

1         1 

1 

0         10      20      30     40      50 
minutes 

Fig.  16.  Light-dependent  evolution  of  hydrogen  gas  from  thiosulphate  by 
Chromotiinn  cells.  The  reaction  mixture  included  o-  i  g.  of  washed  cells,  suspended 
in  2-6  ml.  of  a  modified  nutrient  solution  from  which  nitrogen  compounds  were 
omitted,  0-3  ml.  of  0-5  M  tris  buffer,  pH  7-2,  and  o-i  ml.  of  0-2  M  sodium  thio- 
sulphate. o-i  ml.  of  2o"o  KOH  was  placed  in  the  centre  wells  of  the  Warburg 
nianometer  flasks.  The  reaction  was  run  at  30  .  Gas  phase  argon.  Illumination 
50  000  Lux  (Losda,  Nozaki,  and  Arnon  [136]). 


hydrogen  from  succinate  [i].  In  both  cases  the  evolution  of  hydrogen 
occurred  in  the  presence  of  KOH  and  seemed  to  be  independent  of  COg 
assimilation.  Gas  evolution  ceased  when  light  was  turned  off  and  resumed 
when  light  was  turned  on  again.  The  evolved  gas  was  identified  as  hydrogen 
(Table  VII)  by  adsorption  on  palladium  asbestos  [137].  Photoproduction 
of  H2  was  inhibited  by  carbon  monoxide  (Table  VII). 

The  evolution  of  hydrogen  by  illuminated  Chromatium  cells  showed  a 
marked  pH  dependence  (Fig.  18).  The  reaction  was  most  vigorous  at  the 
more  acid  pH.  Little  hydrogen  was  evolved  at  pH  S-o. 

Similar  to  the  inhibition  of  hydrogen  evolution  in  the  presence  of 
organic  hydrogen  donors  [138,  139],  photoproduction  of  hydrogen  from 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION 'PROCESS      375 

TABLE  VII 

Photoproduction  of  Hydrogen  from  Succinate  by  Chromatium 
(Ogata,  Nozaki,  and  Arnon  [91]) 


Treatment 


1.  Complete 

2.  Complete,  KOH  omitted 

3.  Complete,  dark 

4.  Complete,  succinate  omitted 

1 .  Complete 

2.  Complete,  succinate  omitted 

3.  Complete,  plus  palladium  asbestos 

4.  Complete,  plus  carbon  monoxide 


ftmoles  H; 
evolved 


8-6 


The  complete  system  contained,  in  a  final  volume  of  3-0  ml.,  100  mg.  wet 
cells  that  were  suspended  in  a  modified  nutrient  solution  with  nitrogen  omitted, 
and  the  following  in  micromoles ;  tris  buffer,  pH  7-2,  80;  AlgCl,,  5  ;  succinate,  20. 
o-  I  ml.  of  20",,  KOH  was  present  in  the  centre  well.  The  reaction  was  carried  out 
for  2  hr.  at  30    in  argon.  Illumination  35  000  Lux. 


thiosulphate  was  also  inhibited  by  molecular  nitrogen  and  ammonium  ions. 
The  results  are  shown  in  Fig.  19. 

The  photoproduction  of  hydrogen  was  dependent  on  the  concentration 
of  thiosulphate  and  was  abolished  by  heating  the  cells  (Fig.  20).  Growing 
Chromatium  cells  were  found,  by  analysis  to  have  oxidized  in  4  days  27 
millimoles  of  added  thiosulphate  into  54  millimoles  of  sulphate.  During 


30  60  90  120         150 

minutes 

Fig.  17.  Photoproduction  of  hydrogen  gas  from  succinate  by  Chromatium 
cells.  Experimental  conditions  as  described  in  Table  \ll  (Ogata,  Xozaki,  and 
Arnon  [91]). 


376 


DANIEL    I.    ARNON 


PHOTO  PRODUCTION     OF  H2     FROM    THIOSULFATE 
7  \ 1 1 1 1 r 


Fig.  18.  Effect  of  pH  on  photoproduction  of  hydrogen  gas  from  thiosulphate 
by  Chromatmm  cells.  Experimental  conditions  as  described  for  Fig.  16.  Phosphate 
buffer  was  used  at  pH  6-5  and  7-0  and  tris  buffer  at  pH  7-5  and  80  (Losada, 
Nozaki,  and  Arnon  [136]). 

this  period  the  appearance  of  the  culture  indicated  a  transitional  formation 
of  elemental  sulphur.  The  results  are  in  agreement  with  the  following 
sequence  of  reactions,  in  which  thiosulphate  is  the  donor  of  electrons  that 
are  activated  by  light  and  used  either  for  the  assimilation  of  carbon  and 


120 

1        1        1         1        I        1 

100 

/ 

80 

/Complete 

60 
40 

y    ^--^NH^CI 

20 

-      /y^          -NajSzOj 

0 
-20 

^-^- — •     "^ 

-—^-^^^ 

-40 

1         1         1         1         1         1 

0        10      20      30      40     50      60 
minutes 

Fig.  19.  Effect  of  No  and  NH4CI  on  photoproduction  of  H2  from  thiosulphate 
by  Chromatiiini  cells.  Experimental  conditions  as  described  for  Fig.  16.  5  /xmoles 
NH4CI  and  No  gas  were  used  as  indicated  (Losada,  Nozaki,  and  Arnon  [136]). 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      377 

nitrogen  into  cellular  substance  or  for  the  photoproduction  of  hydrogen 
gas. 


s,o.r  +  2OH  - 


light 


light 


S  +  SOr  +  2e-  +  H2O 


S  +  80H  -  -^^  SO  =  +  6f  -  +  4H2O 


loHoO 


V  10H++10OH- 


Sum :    So03=  +  5H0O  ^^  zSO^  +  8e  -  +  loH  + 

The  photoproduction  of  hydrogen  gas  approached  the  stoicheiometry 
of  the  first  reaction,  i.e.  for  one  molecule  of  thiosulphate  used,  two  elec- 
trons and  two  protons  were  combined  with  the  aid  of  hydrogenase  and 
evolved  as  Hg. 

'  PHOTOPRODUCTION     OF  Hj     FROM    THIOSULFATE 

— I r        T         1         I 


^moles  thiosulfate  added -~  10 


-o-o-  none 
~~  10  (heated 


cells) 


10       20       30       40       50 

minutes 

Fig.  20.  Photoproduction  of  hydrogen  gas  by  Chromotiimi  cells  as  a  function 
of  added  thiosulphate.  The  control  treatment  contained  cells  that  have  been  heated 
at  loo^  for  lo  min.  Other  experimental  conditions  are  described  for  Fig.  i6  (Losada, 
Nozaki,  and  Arnon  [136]). 

With  succinate  as  the  electron  donor,  the  proposed  sequence  of  reac- 
tions which  results  in  the  photoproduction  of  hydrogen  is  represented  as 
follows : 

Succinate  +  zFe^  +cyt >  fumarate  +  zFe-  +cvt  +  2H  + 


2Fe2+cyt  +  2[Chl] 


->  2Chl  +  2Fe'^  ^cvt 


light 


2Chl— ^-^2[Chl]++2e 

2f'-  +  2H  +  '!I^!Z!!:!!^H2 


Sum :    succinate 


light 


>  fumarate  +  H., 


378  DANIEL    I.    ARNON 

The  results  with  thiosulphate  provided  the  first  experimental  evidence 
for  a  light-dependent  hydrogen  evolution  from  an  inorganic  electron  donor 
by  a  photosynthetic  organism  [136,  cf.  143].  Photoproduction  of  hydrogen 
was  first  observed  in  algae  by  Gaffron  and  Rubin  [140]  and  in  photo- 
synthetic  bacteria  by  Gest  and  Kamen  [138].  In  algae  the  photoproduction 
of  hydrogen  seemed  to  depend  on  internal  electron  donors  of  metabolic 
origin  [140]  whereas  photoevolution  of  hydrogen  by  photosynthetic 
bacteria  appeared  to  depend  on  exogenous  organic  acids  and  CO2  [138, 
141,  142,  143,  139,  144,  145]. 

The  evolved  hydrogen  has  previously  been  ascribed  to  photodecom- 
position  of  water  [140,  145]  or  to  decomposition  of  a-ketoglutaric  acid 
[139].  We  regard  the  photoproduction  of  molecular  hydrogen  from  thio- 
sulphate (or  succinate)  as  evidence  for  a  "non-cyclic"  electron  flow 
mechanism  in  bacterial  photosynthesis  as  depicted  below: 


H, 


11  + 


hydrogenase 
thiosulphate  — -=^  cytochromes  — "- — >  chlorophyll 
sulphate 

light 

PHOTOFIXATION    OF    NITROGEN    GAS 

Nitrogen  fixation  by  photosynthetic  organisms  [149,  141,  139,  150-153] 
may  also  be  viewed  as  resulting  from  a  non-cyclic  electron  flow  in  which 
electrons  pass  from  an  external  electron  donor,  via  cytochromes,  to 
chlorophyll  excited  by  light,  and  thence  to  molecular  nitrogen. 

This  interpretation  was  substantiated  by  using  thiosulphate  and 
succinate  as  electron  donors  for  fixation  of  nitrogen  gas  by  illuminated 
Chromatiiun  cells  [154,  155]-  Figure  21  shows  photofixation  of  nitrogen 
gas  with  thiosulphate  as  the  electron  donor  and  Fig.  22  shows  photo- 
fixation  of  nitrogen  with  succinate  as  the  electron  donor.  In  both  cases, 
Chromatium  cells  fixed  N.,  only  in  light.  Fixation  ceased  when  the  light  was 
turned  off  and  resumed  when  the  light  was  turned  on  again.  The  depend- 
ence of  N2  fixation  on  light  and  an  external  electron  donor,  was  confirmed 
with  the  use  of  ^^N  isotope*  (Table  VIII). 

In  the  case  of  thiosulphate,  photofixation  of  Ng  was  greatly  increased 
by  the  addition  of  oxaloacetate  which  probably  acted  as  an  amino  group 

*  We  are  indebted  to  Dr.  C.  C.  Delwiche  for  the  determinations  of  ^^N. 


PHOTOSYNTHETIC  PHOSPHORYLATION"  AND  THE  ENERGY  CONVERSION  PROCESS       379 


Fig.  21.  Effect  of  thiosulphate  and  oxaloacetate  on  photofixation  of  nitrogen 
gas  by  Chromatiimi  cells.  The  reaction  mixture  included  in  a  final  volume  of  2-9 
ml.,  01  g.  of  washed  cells,  suspended  in  a  modified  nutrient  solution,  pH  72 
(from  which  nitrogen  compounds  were  omitted).  20  /^moles  each  of  thiosulphate 
and  oxaloacetate  were  added  as  indicated,  o  •  i  ml.  of  20",,  KOH  was  placed  in  the 
centre  wells  of  the  Warburg  manometer  flasks.  The  reaction  was  run  at  30  .  Gas 
phase,  nitrogen.  Illumination  50  000  Lux  (Losada,  Xozaki,  Tagawa,  and  Arnon 
[155,  154]).     , 

acceptor.   Succinate  ser\ed  both   as  electron   donor  and  as  source  of  a 
carbon  skeleton  that  is  needed  for  accepting  an  amino  group. 

As  shown  in  Fig.  23,  photofixation  of  X.,  with  thiosulphate  was 
inhibited  by  ammonia  (cf.  [149]).  Howe\  er,  neither  ammonia  nor  nitrogen 
gas  inhibited  COo  fixation  by  illuminated  Chronuitium  cells  when  thio- 


TAHLK  VIII 

Effect  of  Light  and  Electron  Donors  on  Fixation  of  Molecular  Nitrogen 

BY  Chromatiiim  Cells 

(Losada,  Xozaki,  Tagawa,  and  Arnon  [155,   154]) 


Treatment 


Atom  percent  ^^X 
excess 


Dark,  succinate 

Dark,  thiosulphate 

Light 

Light,  succinate 

Light,  thiosulphate 


oxaloacetate 


oxaloacetate 


0-004 

0  -003 
0-288 

1  -049 
1-467 


40  fimoles  each  of  thiosulphate,  oxaloacetate  and  succinate  were  added  as 
indicated.  Other  experimental  conditions  as  in  Fig.  21,  except  that  no  KOH 
was  included,  and  the  gas  atmosphere  was  90*^0  argon  and  10",,  X,,  containing 
30  atom  percent  excess  ^*X.  Reaction  time  was  2  hr. 


38o 


DANIEL    I.    ARNON 


20  40 

minutes 

Fig.  22.  Effect  of  succinate  on  photofixation  of  nitrogen  gas  by  Chromatium 
cells.  Experimental  conditions  as  described  in  Fig.  21,  except  that  thiosulphate  and 
oxaloacetate  were  omitted.  20  /imoles  of  succinate  were  added  as  indicated  (Losada, 
Nozaki,  Tagawa,  and  Arnon  [155,  154]). 


sulphate  was  the  electron  donor  (Table  IX).  These  results  suggest  that 
ammonia  or  Ng  did  not  inhibit  the  flow  of  electrons  that  are  required  for 
CO2  assimilation  (by  way  of  DPNH.,  or  TPNHg).  At  high  light  intensity, 
when  the  electron  flux  is  large  enough  to  cope  with  the  requirements  of 


Fig.  23.  Effect  of  ammonia  on  photofixation  of  nitrogen  gas  with  thiosulphate 
by  Chromatium  cells.  Experimental  conditions  were  the  same  as  described  for  Fig. 
21  except  that  30  //.moles  of  NH4CI  were  added  as  indicated  (Losada,  Nozaki, 
Tagawa,  and  Arnon  [155,  154]).  The  addition  of  ammonia  to,  or  the  omission  of 
thiosulphate  and  oxalacetate  from,  the  complete  system  prevents  nitrogen  fixation. 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      381 

TABLE  IX 

Effect  of  Nitrogen  (Jas  and  Ammonia  on   CO^   Fixation  by   Illuminated 
Chromatium  Cells  with  Thiosulphate  as  Electron  Donor 

(Losada,  Xozaki,  and  Arnon  [136]) 

Total  i^CO,  fixed 
Treatment  counts/min. 

(thousands) 

Argon  524 

Argon  +  thiosulphate  2190 

No  +  thiosulphate  2238 

Argon  +  thiosulphate  +  NH4CI                                             2436 

The  reaction  mixture  contained  in  a  total  volume  of  3  ml. :  50  mg.  of  washed 
cells,  suspended  in  2-3  ml.  of  a  modified  nutrient  solution  from  which  nitrogen 
compounds  were  omitted,  and  the  following  in  micromoles :  tris  buffer,  pH 
7'5>  15°;  thiosulphate,  20;  and  sodium  bicarbonate  labelled  with  ^^C,  i -o.  30 
^tmoles  of  NH4CI  were  added  as  indicated.  Gas  phase,  argon  or  No  as  indicated. 
The  reaction  was  carried  out  at  30  . 

both    X.>    fixation    and    CO^    assimilation,    both    processes    can    occur 
simultaneously. 

CELL-FREE    NITROGEN    FIXATION 

In  preliminary  experiments,  when  cell-free  Chromatium  preparations 
were  supplied  in  the  dark  with  a  mixture  of  hydrogen  and  nitrogen  gas, 

TABLE  X 

No  Fix.^TioN  in  the  Dark  uy   Cell-Free  Extracts  of  Chromatium  Supplied 

WITH  Hydrogen  Gas 

(Losada,  Nozaki,  Tagawa,  and  Arnon  [155,  154]) 

Cjas  uptake,  mm.  pressure  change 


Cofactors  added  q^^  phase  q^^  ^^^^^ 

Ho  50%  Ho  +  50".,  N., 

None  o  —11 

DPN  -  22  -  62 

The  reaction  mixture  contained,  in  a  final  volume  of  3  ml. :  cell-free  extract 
containing  0-27  mg.  bacteriochlorophyll,  and  the  following  in  niicromoles :  tris 
buflFer,  pH  7-5,  150;  MgCla,  5;  and,  where  indicated,  DPN,  0-5  (added  jointly 
with  benzyl  viologen,  0-2).  The  gas  phase  was  in  one  case  hydrogen  gas  and  in 
the  other  a  mixture  of  nitrogen  and  hydrogen  in  equal  volume.  KOH  was  present 
in  the  centre  wells  of  the  manometer  vessels.  The  reaction  was  run  for  45  min. 
at  25°  in  the  dark. 


382  DANIEL    I.    ARNON 

they  absorbed  more  gas  than  in  the  control  treatment  in  which  the  gas 
phase  consisted  solely  of  hydrogen  (Table  X).  This  seems  to  indicate  that 
cell-free  Chromatium  preparations  were  fixing  Ng,  with  the  aid  of  hydrogen 
as  the  electron  donor — an  interpretation  that  w^as  strengthened  by  experi- 
ments with  ^^N  isotope  (Table  XI).  These  findings  support  the  view 
that  the  role  of  light  in  photofixation  of  Ng  is  to  generate  electrons  with  a 
reducing  potential  that  is  at  least  equal  to  that  of  Ho.  When  H.,  was  supplied 
in  the  gas  phase,  light  was  no  longer  necessary  for  the  fixation  of  N2.  It 
seems  that  pyridine  nucleotides  mediate  the  reduction  of  N.2  (Tables  X 
and  XI).  However,  the  experiments  on  N2  fixation  by  cell-free  Chromatium 
preparations  are  at  an  early  stage  and  the  drawing  of  final  conclusions 
would  be  premature. 

TABLE  XI 

^'"No  Fixation  in  the  Dark  by  Cell- Free  Extracts  of  C/inmKitiiini 
(Losada,  Nozaki,  Tagawa,  and  Arnon  [155,  154]) 


No. 

Reductant  added 

i^N  atom 

per 

cent  excess 

Experiment  A 

Experiment  B 

I 
2 
3 

None 

DPNHo 

H, 

0-0255 
0-0480 
0-0362 

0-0274 
0-0430 

The  reaction  mixture  contained  in  a  final  volume  of  3  ml. :  cell-free  extract, 
containing  0-3  mg.  bacteriochlorophyll,  and  the  following  in  micromoles :  tris 
buffer,  pH  7-8,  100;  MgCl.,,  5;  benzyl  viologen,  0-2.  Treatments  i  and  3  received 
0-5  /^tm  DPN,  and  Treatment  2,  i  /^tm  DPNHo.  All  vessels  received  0-5  atmos- 
phere nitrogen  containing  96  atom-",,  excess  ^^N.  Treatment  3  received  in  addition 
0-5  atmosphere  hydrogen  gas.  The  experiment  was  run  at  25"  for  2  hr.  in  the 
dark. 

To  recapitulate,  the  photofixation  of  No  and  the  photoproduction  of 
Ho,  from  electron  donors  such  as  thiosulphate  or  succinate,  are  taken  as 
evidence  for  a  non-cyclic  electron  flow  mechanism,  that  supplements  the 
cyclic  mechanism  for  ATP  production.  Preliminary  experiments  indicate 
that  the  non-cyclic  electron  transport  in  Chromatium  that  results  in 
pyridine  nucleotide  reduction  is  coupled  with  the  formation  of  ATP  [155]. 

A  diagrammatic  representation  of  the  proposed  non-cyclic  electron 
flow  mechanism  in  photosynthetic  bacteria  is  shown  in  Fig.  24.  Three  of 
the  external  electron  acceptors  have  now  been  identified :  pyridine  nucleo- 
tides, nitrogen  gas,  and  protons.  It  seems  likely  that  protons  serve  as 
electron  acceptors  and  hydrogen  gas  is  evolved  when  electrons  activated 
by  light  become  surplus,  i.e.  when  they  are  not  consumed  in  metabolic 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVTIRSION  PROCESS      383 

TABLE  XII 

Hydrogen   Evolution   from   Reduced   Methyl   Violo(;en   by   Cell- Free 
Hydrogenase  FROM  Chrotnotiuni 

(Ogata,  Nozaki,  and  Arnon,  [91]) 

/(moles  Ho  evolved/ 
10  min./mg.  chl. 


Complete  system 
Methyl  viologen  omitted 
Na2S,04  omitted 
Hydrogenase  omitted 


12-8 

none 
none 
none 


Complete  systeni  contained,  in  a  final  volume  of  3-0  ml.,  cell-free  suspension 
(PS)  containing  0-4  mg.  bacteriochlorophyll  and  80  /nmoles  tris  buffer,  pH  7-2. 
o-i  ml.  of  20",,  KOH  was  present  in  the  centre  well  and  16  ^imoles  of  methyl 
viologen  was  added  to  the  sidearm.  Methyl  viologen  was  reduced  by  adding  Na.2S.2O4 
to  the  same  sidearm  while  gassing  with  argon.  The  reaction  was  carried  out  at  30" 
in  the  dark. 

reactions  as  in  the  reduction  of  CO.^  via  pyridine  nucleotides  or  in  the 
photofixation  of  N.,. 

The  hydrogenase  present  in  ChromoUuui  particles  that  catalyzes 
hydrogen  evolution  in  the  light  can  also  catalyze  hydrogen  gas  evolution 


text. 


LIGHT 


Non-cyclic     electron    transport  in  Chromatium 
Fig.   24.   Scheme  for  non-cyclic  electron  flow  in  Chroniatiian.  Details  in  the 


in  the  dark  (Table  XII)  when  electrons  are  supplied  at  a  sufficiently 
reducing  potential,  as  for  example  by  hydrosulphite  (cf.  [132]).  Methyl 
viologen  was  required  as  a  catalyst  in  this  reaction  (compare  [156,  1^7]). 


384 


DANIEL    I,    ARNON 


13.  The  photoreductant  in  plants:  non-cyclic  photophosphorylation 

Photosynthetic  bacteria  can  reduce  pyridine  nucleotide  either  with 
molecular  hydrogen  in  the  dark  or  with  a  less  reduced  electron  donor, 
organic  or  inorganic,  in  the  light.  Green  plants  do  not  ordinarily  contain 
hydrogenase,  hence  they  cannot  use  hydrogen  gas  during  photosynthesis 
for  reducing  pyridine  nucleotide  in  the  dark.  They  use  water  as  the 
electron  donor.  The  reduction  of  pyridine  nucleotides  with  electrons 
donated  by  water  requires  a  considerable  input  of  energy  which  in  photo- 
synthesis is  supplied  by  light. 


2  3 

//M  TPN  added 

Fig.  25.   Stoicheiometry  of  oxygen  evolution  and  ATP  formation  resulting 
from  the  photochemical  reduction  of  TPN  (Arnon,  Whatley,  and  Allen,  [95,  158]). 


As  already  mentioned,  isolated  chloroplasts  were  known  to  reduce 
TPN  in  light  with  an  accompanying  evolution  of  oxygen  [42,  43,  5].  This 
was  regarded  as  a  Hill  reaction  in  which  TPN  served  as  the  hydrogen 
acceptor.  There  was  no  evidence  that  this  photochemical  reduction  of 
TPN  was  in  any  way  linked  with  photosynthetic  phosphorylation. 
Recently,  however,  the  relation  of  photosynthetic  phosphorylation  to  the 
photoreduction  of  TPN  which  at  first  seemed  remote,  was  found  to  be 
direct  [95,  104].  In  the  presence  of  ADP  and  orthophosphate  (P),  the 
photoreduction  of  TPN  and  oxygen  evolution  was  coupled  with  the 
formation  of  ATP  in  accordance  with  equation  (4). 


2TPN  +  2ADP+2P  +  2H0O  ->  2TPNH2  +  O0  +  2ATP 


(4) 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       385 

Under  appropriate  experimental  conditions  [158]  the  evolution  of  one 
mole  of  oxygen  was  accompanied  by  the  reduction  of  two  moles  of  TPN, 
and  the  esterification  of  2  moles  of  orthophosphate  (Fig.  25).  The 
stoicheiometry  of  this  reaction  was  the  same  when  TPN  was  replaced  by 
ferricyanide.  With  either  TPN  [159]  or  ferricyanide  [95,  104]  the  rate  of 
oxygen  evolution  is  greatly  increased  when  it  is  coupled  with  phosphoryla- 
tion. The  conventional  Hill  reaction  could  thus  be  viewed  as  an  uncoupled 
photophosphorylation,  i.e.  a  photochemical  electron  transport  that  is 
proceeding  without  its  normally  associated  phosphorylation  reaction. 

It  was  proposed  elsewhere  [94,  i]  that  the  reduction  of  TPN  by 
chloroplasts  in  Reaction  4  involves  a  non-cyclic  electron  flow  mechanism. 
Reaction  4  may  thus  be  viewed  as  being  analogous  to  the  non-cyclic 
electron  flow  in  bacteria  (Fig.   24)  and  diff"ering  from  it  only  in  those 


/iA  oxygen  formed 


fiM  TPNH2  formed 


3-7 


0-3 


-CI 


+  CI 


•CI 


+  CI 


Fig.  26.  Effect  of  chloride  on  reduction  of  TPX  and  evolution  of  oxygen. 
The  reaction  mixture  contained  in  a  volume  of  3  ml.  chloroplasts  (Pi,)  containing 
o  •  25  mg.  chlorophyll ;  and  the  following,  in  micromoles :  tris  acetate  buffer,  pH 
8-2,  80;  TPN,  4;  and  a  partly  purified  preparation  of  photosynthetic  phosphopyri- 
dine  nucleotide  reductase.  The  plus  chloride  treatment  received  10  /xmoles  KCl. 
Oxygen  evolution  was  measured  manometrically,  and  the  TPNHj  formed  was 
measured  by  its  absorption  at  340  m/n  (Bove,  Bove,  Whatley,  and  Arnon  [103]). 

aspects  that  reflect  the  special  enzymic  composition  of  chloroplasts. 
Unlike  photosynthetic  bacteria,  chloroplasts  contain  neither  X.j-fixing 
enzymes  nor  hydrogenase.  As  a  consequence,  the  electron  acceptor  end 
of  the  non-cyclic  electron  flow  mechanism  in  chloroplasts  can  be  coupled 
neither  to  photofixation  of  nitrogen  nor  to  photoproduction  of  hydrogen 
gas,  but  only  to  COg  reduction  (by  way  of  TPNH.,). 

The  most  characteristic  difference  between  the  non-cyclic  electron 
flow  mechanism  of  chloroplasts  (equation  (4))  and  bacteria  (Fig.  24)  is  in 
the  electron  donor  system.  In  chloroplasts  the  eieciron  donor  is  water 
(i.e.  OH  ~)  whereas  bacteria  cannot  use  water  but  use  inorganic  or  organic 
electron  donors  such  as  thiosulphate  or  succinate  [94,  136]. 

VOL.  U. 2C 


386  DANIEL    I.    ARNON 

This  interpretation  of  the  difference  between  the  non-cycHc  electron 
flow  mechanism  in  chloroplasts  and  in  photosynthetic  bacteria  is  sup- 
ported by  recent  evidence  that  it  is  experimentally  possible  to  replace 
water  as  the  electron  donor  in  non-cyclic  photophosphorylation  by 
chloroplasts. 

As  was  already  mentioned  (Section  7),  photosynthetic  reactions  of 
chloroplasts  in  which  oxygen  is  liberated  require  chloride,  hence  Reaction  4 
could  not  proceed  in  the  absence  of  chloride.  As  shown  in  Fig.  26,  on 
omitting  chloride  from  the  reaction  mixture  (a  step  that  included  purifica- 
tion of  those  reagents  that  contained  chloride  impurities)  TPN  reduction 
and  oxygen  evolution  ceased  and  photophosphorylation  was  abolished 
(Table  XIII). 

TABLE  XIII 

Effect   of   Chloride   on   Non-Cyclic   Photophosphorylation  by   Isolated 

Chloroplasts 

(Bove,  Bove,  Whatley,  and  Arnon  [103]) 

Micromoles  ATP  formed 
Experiment  Electron  acceptor 


chloride  +  chloride 


A 

Ferricyanide 

o- 1 

B 

Ferricvanide 

0-3 

C 

TPN 

0-7 

D 

TPN 

0-7 

3-3 
3-7 
3-6 

4-2 


These  results  indicated  that  chloroplasts  deprived  of  chloride  cannot 
use  water  as  the  electron  donor  in  Reaction  4.  It  was  possible,  however, 
that  they  could  use  other  electron  donors  that  did  not  involve  an  oxidation 
of  water  (i.e.  OH  )  and  a  resultant  oxygen  evolution.  Vernon  and  Zaugg 
[160]  have  found  that  chloroplasts  which  are  incapable  of  photochemical 
oxygen  evolution,  retain  the  capacity  for  photoreduction  of  TPN  with 
ascorbate  (jointly  with  catalytic  amounts  of  2,6-dichlorophenol  indophenol) 
as  the  electron  donor. 

Table  XIV  shows  that  using  an  ascorbate  electron  donor  system, 
chloroplasts  carried  out  a  "bacterial"  type  of  non-cyclic  photophos- 
phorylation in  which  ATP  formation  and  TPN  reduction  were  not 
accompanied  by  an  evolution  of  oxygen.  The  participation  of  water  (OH  ~) 
as  an  electron  donor  was  prevented  here  by  the  omission  of  chloride  and 
also  by  the  addition  of  dichlorophenyl  dimethylurea  as  an  inhibitor  of 
oxygen  evolution  (cf.  [160]). 

These  results  support  the  view  (cf.  [121])  that  the  evolution  of  oxygen 
in  non-cyclic  photophosphorylation  by  chloroplasts  (and  hence  in  photo- 
synthesis of  green  plants)  is  not  fundamental  to  the  key  photosynthetic 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       387 

TABLE  XIV 

Non-Cyclic   Photophosphorylation   by   Chloroplasts  with   Ascorbate  as 

THE  Electron  Donor 

(Whatlev,  Dieterle,  and  Arnon  [161]) 


Effective 

Oxygen 

TPN 

ATP 

No. 

Addenda 

electron 

evolved 

reduced 

formed 

donor 

(/Liatoms) 

(/xmoles) 

(lumoles) 

I. 

None 

Water 

3-0 

3-4 

2-4 

2. 

CMU 

None 

0 

0-5 

0-2 

3. 

CMU, 

ascorbate 

Ascorbate 

0 

3-2 

3-4 

Treatment  i  contained:  washed  chloroplast  fragments  (P1S2),  prepared  in 
the  absence  of  chloride,  containing  o -5  mg.  chlorophyll;  0-05  ml.  purified  spinach 
phosphopvridine  nucleotide  reductase,  and  the  following  in  micromoles:  tris/ 
acetate  buffer,  pH  80,  40;  MgSOj,  5;  K0H3-PO4,  10;  ADP,  10;  TPN,  4;  and 
KCl,  10.  In  Treatment  2  KCl  was  omitted  and  2  x  10^  m  p-chlorophenyl- 
dimethylurea  (CMU)  was  added.  Treatment  3  was  the  same  as  Treatment  2 
except  that  20  /^tmoles  ascorbate  and  0-2  /^mole  2,6-dichlorophenol  indophenol 
were  added  (cf.  [160]).  The  experiment  was  run  for  20  min.  at  15"  (at  a  light 
intensity  of  2000  foot  candles). 

The  omission  of  chloride  (Fig.  26)  and  the  addition  of  CMU,  a  powerful 
inhibitor  of  oxygen  evolution,  prevented  the  use  of  water  as  an  electron  donor  in  the 
chloroplast  system.  Catalytic  amounts  of  dichlorophenol  indophenol  served  as 
an  electron  carrier  [160]  between  ascorbate  and  the  chloroplast  system. 


events,  i.e.  ATP  formation  and  TPN  reduction.  Oxygen  evolution  occurs 
when  water  (OH  "),  on  donating  an  electron  to  the  photosynthetic  particle, 
becomes  oxidized  to  oxygen.  Under  special  experimental  conditions,  when 
ascorbate  displaces  water  as  the  electron  donor,  no  oxidation  of  OH  - 
occurs,  only  the  oxidation  of  ascorbate  [i6o].  This  concept  of  the  non- 
cyclic  photophosphorylation  in  chloroplasts  is  represented  in  Fig.  27. 

The  proposed  mechanism  assigns  to  cytochromes  a  role  in  transporting 
electrons  from  the  electron  donor  system  to  chlorophyll.  Cytochromes  are 
known  to  accept  electrons  from  ascorbate  but  the  suggestion  that  a  photo- 
synthetic  cytochrome  svstem  mediates  the  transfer  of  electrons  from  OH  " 
to  chlorophyll  is  put  forward  only  as  a  working  hypothesis  [i].  This 
hypothesis  implies  that  the  chlorophyll-cytochrome  complex  must 
generate  a  sufficient  oxidizing  potential  to  drive  the  reaction  2H.2O— *0.2  + 
4H+  +  4^^,  which  at  25-  and  at  pH  7,  has  E'^=  +0-815  V.  It  might  be 
argued  that  cytochrome  /',  the  most  oxidizing  cytochrome  now  known  to 
occur  in  chloroplasts,  has  a  redox  potential  lower  than  oxygen  [162],  i.e. 
Eq—  +0-365  V.  However,  it  would  be  premature  to  conclude  that  our 
knowledge  of  redox  potentials  of  cytochromes  in  chloroplasts  is  now 
complete. 


388  DANIEL    I.    ARNON 

The  proposed  reactions  from  OH  "  to  oxygen  evolution  appear  to  be 
thermodynamically  feasible.  The  energy  contribution  of  one  einstein  of  red 
light,  about  43  Kcal.,  is  equivalent  to  a  potential  of  i  -9  V.  per  faraday, 
and  is  sufficiently  large,  after  making  allowances  for  TPN  reduction  and 
ATP  formation,  to  endow  a  chlorophyll-linked  cytochrome  with  a  redox 
potential  more  oxidizing  than  0-815  V.,  as  is  needed  for  oxygen  evolution. 

,'--,  Reductase  ^    ^. , 
;  e-.i  »  PN 


Chi     r  +  'j^mmfmm  Cyt 


^■T^ 


HA 


LIGHT  02'^0H-< — H2O 

Non- cyclic    photophosphorylation  (chloroplasts) 
Fig.  27.   Scheme     for     non-cyclic     photophosphorylation     in     chloroplasts. 
Details  in  the  text.  Chloride  is  required  for  oxygen  evolution. 

It  must  be  emphasized  that,  in  our  present  state  of  knowledge,  the 
proposed  mechanism  for  oxygen  evolution  must  remain  tentative.  The 
possibility  exists  that  the  transfer  of  electrons  from  OH  ^  to  cytochromes 
requires  an  auxiliary  input  of  light  energy  via  a  photosynthetic  pigment.* 

14.  Oxygen-dependent  cyclic  photophosphorylation 

The  mechanisms  of  photosynthetic  phosphorylation  in  chloroplasts 
discussed  thus  far  include  anaerobic  cyclic  photophosphorylation  (Figs.  4 
and  5)  and  non-cyclic  photophosphorylation  (Fig.  27).  Recent  work  by 
Tsujimoto  et  al.  [92]  suggests  the  operation  in  chloroplasts  of  a  third 
mechanism,  an  oxygen-dependent  cyclic  photophosphorylation. 

As  was  already  discussed  in  Section  4,  a  catalytic  role  for  oxygen  was 
envisaged  in  explaining  the  first  experiments  on  photosynthetic  phos- 
phorylation, in  which  the  presence  of  oxygen  was  required  but  no  oxygen 
consumption  was  observed  [13].  Interest  in  the  role  of  oxygen  was 
heightened  when  several  laboratories  reported  that  at  low,  "micro- 
catalytic",  concentrations  of  FMN  or  vitamin  K  (Fig.  3),  photophos- 
phorylation remained  dependent  on  oxygen  [54-56]. 

*  Note  added  in  proof.  Experimental  evidence  for  a  separate  light  reaction 
responsible  for  oxygen  evolution  has  now  been  obtained,  (cf.  M.  Losada,  F.  R. 
Whatley  and  D.  I.  Arnon,  Nature,  Land.  190,  606-610,  1961.) 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      389 

From  the  standpoint  of  cellular  physiology  it  was  interesting  to 
contrast  the  role  of  oxygen  in  ATP  formation  in  photosynthesis  with  that 
in  respiration.  The  participation  of  oxygen  as  the  terminal  electron 
acceptor  in  oxidative  phosphorylation  has  conferred  a  marked  superiority 
on  respiration  over  fermentation,  in  the  efficiency  of  converting  the  free 
energy  of  substrate  into  the  energy  of  the  pyrophosphate  bonds  of  ATP. 
Was  the  efficiency  of  conversion  of  light  energy  into  ATP  also  increased 
by  the  presence  of  oxygen  : 

To  answer  this  question,  photophosphorylation  by  chloroplasts  was 
investigated  in  air  and  in  nitrogen,  at  different  concentrations  of  FAIX  or 
vitamin  K,  and  particularly,  at  a  limiting  light  intensity,  when  the  efficiency 


LIMITING   LIGHT   orx)    HIGH    CHLOROPHYLL 


0003   001       005  01    ,0-3 
//moles  vit,  K3  added 


0003   001      0-05  0-1     0  3      10 
//moles  FMN  added 


Fig.  28.  Effect  of  FMX  and  vitamin  K^  concentration  on  cyclic  photophos- 
phorylation by  spinach  chloroplasts  in  nitrogen  and  air,  at  a  low  light  intensity. 
The  reaction  mixture  included,  in  a  final  volume  of  3  o  ml.,  chloroplast  fragments 
(Cij)  containing  1-5  mg.  chlorophyll;  and  in  micromoles :  tris  buffer,  pH  8-3, 
80;  MgS04,  5;  K.H3-PO4,  15;  ADP,  15;  TPX,  03  (only  in  the  FMN  series). 
FMN  or  vitamin  K^  was  added  as  indicated.  The  reaction  was  run  for  30  min.  at 
an  illumination  of  2  000  Lux  (Tsujimoto,  Hall,  and  Arnon,  [92]). 


of  the  energy  conversion  process  could  be  best  observed  (compare  Sec- 
tion 8).  The  results  are  shown  in  Fig.  2S. 

In  limiting  light,  the  highest  rate  of  photophosphorylation  was  obtained 
in  nitrogen  at  a  concentration  of  approximately  10^  m  of  either  FMN  or 
vitamin  K.  No  photophosphorylation  occurred  in  nitrogen  without  added 
cofactors  but  when  these  were  added  at  an  optimal  concentration,  the 
anaerobic  system  was  about  twice  as  efficient  in  con\erting  light  energy 
into  ATP  as  the  aerobic  system. 

The  experiments  represented  by  Fig.  28  were  carried  out  with  relatively 
high  concentrations  of  chloroplast  material.  Under  these  conditions  the 
aerobic  system  showed  little  increase  in  photophosphorylation  from  adding 
FAIN  or  vitamin  K.  However,  high  concentrations  of  chloroplast  material 
were  found  to  be  necessary  to  insure  the  effective  operation  of  the  anaerobic 
FMN  system.  The  anaerobic  vitamin  K  system  functioned  optimally  at 


390  DANIEL    I.    ARNON 

lower  concentrations  of  chloroplast  material  suggesting  that  it  required 
less  or  fewer  of  the  chloroplast  factor(s)  than  were  required  for  the 
anaerobic  FMN  system.  These  chloroplast  factors  for  the  FMN  system 
appeared  to  be  bound  in  the  grana  fraction  and  were  not  supplied  by  an 
aqueous  extract  of  chloroplasts. 

On  comparing  the  aerobic  and  anaerobic  systems  under  conditions 
when  they  responded  optimally  to  the  addition  of  cofactors,  a  marked 
difference  was  observed,  depending  on  the  presence  or  absence  of  oxygen, 
in  the  effect  of  two  inhibitors,  o-phenanthroline  and  CMU  (p-chloro- 
phenyldimethylurea)  (a  gift  of  Dr.  C.  E.  Hoffman).  The  results  are  shown 
in  Table  XV." 

TABLE  XV 

Effect    of    o-Phenanthroline     (o-P)     and     Dichlorophenyldimethylurea 
(CMU)  on  Cyclic  Photophosphorylation  in  Nitrogen  or  Air 

(Tsujimoto,  Hall,  and  Arnon  [92] 

Percentage  inhibition 

Treatment  , * , 

CMU  o-P 

Nitrogen,  FMN  25  20 

Nitrogen,  vit.  K3  19  27 

Air,  FMN  97  77 

Air,  vit.  K3  85  64 

In  the  nitrogen  series  the  illumination  was  2000  Lux  for  30  min.  and  the 
reaction  mixture  included,  in  a  final  volume  of  3  ml.  chloroplast  fragments  (Ci,) 
containing  i  •  5  mg.  chlorophyll  and  o  ■  3  /umole  of  FMN  or  vit.  Kg.  In  the  air 
series  the  illumination  was  50  000  Lux  for  5  min.  and  the  reaction  mixture  in- 
cluded chloroplast  fragments  (Cj,)  containing  i  mg.  chlorophyll  and  0-003  Mi^iole 
of  FMN  or  vitamin  K3.  The  final  inhibitor  concentrations  were,  3  x  10^^  M 
for  o-phenanthroline  and  2  x  10"  m  for  CMU.  Other  common  components  of 
the  reaction  mixture  were,  in  micromoles:  tris  buffer,  pH  8-3,  80;  K2H^-P04, 
15;  and  MgS04,  5. 

In  agreement  with  findings  of  Wessels  [54],  Jagendorf  and  Avron  [55] 
and  Nakamoto  et  a/.  [56],  o-phenanthroline  and  CMU,  in  the  presence  of 
air,  inhibited  photophosphorylation  in  the  FMN  and  vitamin  K  systems. 
Relatively  little  inhibition  by  these  two  inhibitors  was  observed  in  an 
atmosphere  of  nitrogen.  In  other  experiments,  not  reported  here,  phen- 
azine  methosulphate  was  found  to  differ  from  FMN  and  vitamin  K  in  that 
its  pathway  was  resistant  to  inhibition  by  o-phenanthroline  and  CMU, 
both  in  air  and  in  nitrogen. 

o-Phenanthroline  and  CMU  are  powerful  inhibitors  of  oxygen  evolu- 
tion by  illuminated  chloroplasts  (cf.  [54-56]).  It  seems  likely,  therefore, 
that  as  was  concluded  earlier  by  Wessels  [54]  and  Nakamoto  et  al.  [56], 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       39 1 

oxygen  evolution  is  a  component  step  in  the  "aerobic"  photophosphoryla- 
tion  catalyzed  by  FAIN  or  vitamin  K,  and  that  molecular  oxygen,  when 
present,  acts  as  an  electron  acceptor  in  photosynthetic  phosphorylation. 
This  conclusion  is  supported  by  the  observed  effect  of  chloride  on  cyclic 
photophosphorylation  with  vitamin  K  and  FMN  in  air  and  in  nitrogen 
(Table  XVI).  The  omission  of  chloride  had  scarcely  an  effect  on  photo- 
phosphorylation in  nitrogen,  but  it  se^■erely  inhibited  photophosphoryla- 
tion in  air,  which  depends  on  the  photochemical  evolution  of  oxygen. 

TABLE  XVI 
Effect  of  Chloride  on  Cyclic  Photophosphorylation  in  Nitrogen  or  Air 

(Tsujimoto,  Hall,  and  Arnon  [92]) 

/Ltmoles  P  esterified 
Treatment 


—  chloride  +  chloride 


Nitrogen,  FMN  5-1  5-7 

Nitrogen,  vit.  K3  9-7  9-9 

Air,  FMN  o  •  5  6  ■  i 

Air,  vit.  K3  04  .  5-5 

Experimental  conditions  as  in  Table  XV,  except  that  chloroplasts  were 
prepared  in  0-5  M  sucrose  and  chloride-free  reagents  were  used.  0-2  mg.  chloro- 
phyll was  used  in  the  air  series,  and  2  •  5  mg.  chlorophyll  per  vessel  was  used  in 
the  nitrogen  series.  The  reaction  was  run  for  30  min. 

The  similarity  in  the  effects  of  chloride,  o-phenanthroline,  and  CMU, 
either  in  air  or  in  nitrogen,  on  the  FMN  and  vitamin  K  pathways,  under 
the  modified  experimental  conditions  which  we  now  use,  has  blurred  the 
distinction  between  the  two  pathways  that  was  made  on  the  basis  of  earlier 
inhibitor  experiments  [89].  Apart  perhaps,  from  the  greater  dependence 
of  the  FMN  pathway  on  TPN  [89]  (a  dependence  that  has  not  yet  been 
reinvestigated  under  the  new  experimental  conditions),  what  seems  now 
to  distinguish  the  two  anaerobic  pathways  is  the  greater  requirement,  in 
the  case  of  FMN,  for  a  higher  concentration  of  chloroplast  material. 

The  participation  of  oxygen  in  cyclic  photophosphorylation  may 
increase  the  overall  rate  of  ATP  formation  but  only  when  light  is  abundant 
and  phosphorylation  is  limited  by  a  low  concentration  of  cofactors. 
However,  present  evidence  indicates  (Fig.  28)  that,  in  contrast  to  oxidative 
phosphorylation,  the  intervention  of  molecular  oxygen  in  photosynthetic 
phosphorylation  is  an  energy-wasteful  step  that  lowers  the  efficiency  of  the 
anaerobic  cyclic  photophosphorylation  process  when  light  is  limiting. 

On  the  basis  of  evidence  now  available,  the  participation  of  oxygen  as 
a   catalyst   in   cyclic   photophosphorylation   may   be   represented   by  the 


392  DANIEL    I.    ARNON 

diagram  in  Fig.  29.  Here  the  electron  flow  mechanism  (marked  by  a  heavy 
Hne)  is  composed  of  two  parts.  The  first  part  is  completed  when  electrons 
expelled  from  chlorophyll  are  accepted  by  O2  and,  in  combination  with 
protons,  form  water.  In  the  second  part,  these  electrons  are  replaced  by 
those  donated  by  OH  ",  with  a  concomitant  evolution  of  oxygen,  as  was 
described  for  the  non-cyclic  electron  flow  pathway  for  chloroplasts  (Fig. 
27).  The  proposed  mechanism,  in  which  oxygen  participates,  provides 
for  an  exchange  between  molecular  oxygen  and  the  oxygen  of  water  and  is 
in  agreement  with  the  ^^O  exchange  data  recently  reported  by  Nakamoto 
and  Vennesland  [163]  and  Jagendorf  [164]. 


le 


— ^ 

COfox— COfred. 


I 


O2  ^,"2 


—  H2O 

I 


Chi 


+:'^ 


-    Cytox.^Cyt.ed. 


LIGHT  ~P-^DP [^ 

02-dependent  cyclic  photophosphorylation 

Fig.  29.  Scheme  for  oxygen-dependent  cyclic  photophosphorylation  in  chloro- 
plasts. Details  in  text. 

In  summary  then,  FMN  and  vitamin  K  seem  to  catalyze  two  pathways 
of  cyclic  photophosphorylation,  one  anaerobic  and  one  catalyzed  by 
molecular  oxygen  (cf.  [62]).  The  anaerobic  pathway,  when  investigated  in 
an  atmosphere  of  nitrogen,  requires  appreciable,  although  still  catalytic, 
concentrations  of  cofactors  and,  particularly  in  the  case  of  FMN,  high 
concentrations  of  chloroplast  material  that  evidently  supply  the  additional 
factor(s)  needed  for  the  efficient  conversion  of  light  energy  into  ATP  under 
anaerobic  conditions.  The  ox3^gen-dependent  pathway  for  FMN  or  vitamin 
K  is  catalyzed  by  very  low,  "microcatalytic",  concentrations  of  these 
cofactors  and  is  much  less  dependent  on  additional  chloroplast  material 
than  the  anaerobic  pathway. 

These  findings  are  interpreted  to  mean  that  oxygen,  when  present  in  a 
system  catalyzed  by  either  FMN  or  vitamin  K,  is  able  to  compete  effec- 
tively with  cytochromes  for  the  electrons  of  cyclic  photophosphorylation. 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS       393 

Once  the  electrons  are  accepted  by  oxygen  and  form  water,  the  cycHc 
pathway  can  be  maintained  only  by  a  release  of  electrons  in  the  oxygen- 
forming  reaction  of  non-cyclic  photophosphorylation  in  chloroplasts 
(Section  13).  By  contrast,  phenazine  methosulphate  catalyzes  the  transfer 
of  electrons  to  cytochrome  so  effectively  [90]  that  it  is  able  to  prevent  their 
"escape"  to  oxygen  and  hence  the  phenazine  methosulphate  pathway 
remains  an  "anaerobic"  one  even  when  molecular  oxygen  is  present. 

As  far  as  efficiency  of  conversion  of  light  energy  into  ATP  is  con- 
cerned, it  appears  from  experiments  at  limiting  light  intensities,  that  the 
anaerobic  cyclic  photophosphorylation  with  FAIN  or  vitamin  K  is  more 
efficient  than  with  phenazine  methosulphate  (Figs.  9  and  10).  Also,  the 
anaerobic  FMN  and  vitamin  K  cyclic  pathways  are  more  efficient  than 
their  oxygen-dependent*  counterparts  (Fig.  28).  These  findings  suggest  the 
participation  of  more  than  one  phosphorylation  site  in  the  anaerobic  FMN 
and  vitamin  K  pathways  (compare  Fig.  5  with  Figs.  4  and  29). 

15.  Relation  of  cyclic  to  non-cyclic  photophosphorylation  in 

chloroplasts 

The  ability  of  isolated  chloroplasts  to  carry  out  both  cyclic  (Fig.  5)  and 
non-cyclic  photophosphorylation  (Fig.  27)  raises  the  question  of  the 
mutual  relation  of  these  two  processes.  Specifically,  what  effect  would  the 
addition  of  one  of  the  cefaclors  of  cyclic  photophosphorylation  have  on 
the  reduction  of  TPN  and  evolution  of  oxygen  which  accompany  ATP 
formation  in  non-cyclic  photophosphorylation  ? 

As  shown  in  Figs.  30  and  31,  the  addition  of  either  FMN  or  vitamin  K 
altered  non-cyclic  photophosphorylation  profoundly.  ATP  formation  was 
sharply  increased,  whereas  oxygen  evolution  and  the  accumulation  of 
reduced  TPN  were  abolished.  It  appears,  therefore,  that  cyclic  photo- 
phosphorylation is  a  more  "tightly  coupled"  mechanism  for  converting 
light   energy   into   ATP   than   non-cyclic   photophosphorylation.    In   the 

*  Distinct  from  the  oxygen-dependent  cyclic  photophosphorylation  discussed 
here  is  the  "oxidative  photosynthetic  phosphorylation"  [165]  by  chloroplasts  in 
which  oxygen  consumption  was  induced  by  a  joint  use  of  a  dye  (trichlorophenol 
indophenol),  an  inhibitor  (o-phenanthroline  or  CMU)  and  DPXHo.  The  corres- 
pondence of  the  term  "oxidative  photosynthetic  phosphorylation'  to  oxidative 
phosphorylation  by  mitochondria  appears  to  be  fortuitous.  The  role  of  DPNH., 
in  this  chloroplast  system,  was  not  that  of  a  physiological  electron  donor  but  that 
of  a  non-specific  reducing  agent  for  the  dye,  one  of  several  reducing  agents  that 
were  effective.  That  the  consumption  of  oxygen  was  artificially  induced  and  was 
only  a  feature  of  the  special  system  used,  is  made  clear  by  the  authors'  observations 
that  "there  was  ample  energy  released  by  the  dark  oxidation  of  the  DPNH  to 
form  the  high  energy  phosphate  bonds.  Nevertheless,  the  reaction  gave  no  phos- 
phorylation unless  the  system  was  illuminated,  even  though  the  light  caused  no 
increase  in  the  rate  of  oxygen  consumption"  [165]. 


394  DANIEL    I.    ARNON 

presence  of  the  requisite  cofactors,  cyclic  photophosphorylation  is  capable 
of  diverting  all  the  absorbed  light  energy  for  the  formation  of  ATP,  and 
suppressing  TPN  reduction  and  Og  evolution.  It  is  assumed  that  the 
intact  cell  has  suitable  regulatory  mechanisms  for  keeping  cyclic  and  non- 
cyclic  photophosphorylation  in  balance. 


-o  8 


< 

3. 


minutes 

Fig.   30.  Photophosphorylation  and  oxygen  evolution  by  isolated  chloroplasts 
in  the  presence  and  absence  of  FMN  (Arnon,  Whatley,  and  Allen,  [95,  158]). 

TABLE  XVII 

Effect  of  FMN  and  Vitamin  K3  on  Photophosphorylation  and  Oxygen 
Evolution  Linked  to  TPN  Reduction 


(Arnon,  Whatley,  and  Allen  [158]) 


FMN  or 
vitamin  K3 

FMN 

system 

Vitamin 

K3 

system 

' 

' 

^ 

added 

P  esterified 

O2  evolved 

P  esterified 

O2  evolved 

(jxTnoles) 

(fimoles) 

(/Lcatoms) 

(/Ltmoles) 

(/tatoms) 

none 

5-6 

3-6 

5-6 

3-6 

0-0002 

6-5 

4-2 

6-1 

3-6 

0-0005 

7-4 

3-8 

7-5 

2-9 

o-oci 

7-9 

3-3 

8-0 

2-3 

0003 

8-4 

I  -2 

9-6 

0-9 

o-oi 

Q-O 

0-4 

lO-O 

09 

The  marked  increase  in  phosphorylation  accompanied  by  a  total 
abolition  of  oxygen  evolution  and  TPNHo  accumulation,  shown  in  Figs.  30 
and  31,  occurred  on  adding  o  •  i  /nmoles  of  FMN  or  o  -2  ^moles  of  vitamin 
K  (in  a  final  volume  of  3  ml.).  However,  the  addition  of  even  extremely 
minute  amounts  of  either  FMN  or  vitamin  K  had  a  measurable  effect  on 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS 

TABLE  XVIII 


395 


Effect  of  Phenazine  Methosulphate  (PMS)  on  Phosphorylation  and  Oxygen 
Evolution  Linked  to  TPN  Reduction 

(Arnon,  Whatley,  and  Allen  [158]) 


PMS  added 

P  esterified 

0., 

evolved 

(/nmoles) 

(/Ltmoles) 

(m 

moles) 

none 

4-5 

4-0 

0-003 

6-1 

4-0 

o-oi 

9-0 

3-2 

o-i 

lO-O 

0-8 

Reaction  4.  Table  XVII  shows  that  the  addition  of  as  httle  as  0-0002- 
0-0005  /^rnoles  of  FMN  or  vitamin  K  increased  ATP  formation  without 
appreciably  depressing  oxygen  evolution  (and  the  corresponding  TPNHg 
accumulation).  Similar  eifects  were  observed  on  adding  small  amounts  of 
phenazine  methosulphate  (Table  XVIII). 


-12 


10  ^ 


5: 
3. 


10         20  10 

minutes 

Fig.  31.  Photophosphorylation  and  oxygen  evolution  by  isolated  chloroplasts 
in  the  presence  and  absence  of  vitamin  K3  (Arnon,  Whatley,  and  Allen  [95,  158]). 

Non-cyclic  photophosphorylation  provides  the  three  products  of  the 
light  phase  of  photosynthesis :  O.,,  TPNHo,  and  ATP.  Cyclic  photo- 
phosphorylation supplies  only  ATP  and  the  participation  of  this  reaction 
in  COo  assimilation  would  be  needed  only  if  the  ATP  formed  in  non- 
cyclic  photophosphorylation  were  insufficient  for  COo  assimilation  to  the 
level  of  carbohydrate.  Evidence  that  this  is  indeed  the  case,  and  that  both 


396  DANIEL    I.    ARNON 

cyclic  and  non-cyclic  photophosphorylation  are  required  for  COg  assimila- 
tion, has  recently  been  obtained  by  Trebst  et  al.  [34]. 

Trebst  et  al.  [34]  have  investigated  CO2  assimilation  by  isolated 
chloroplasts  in  a  catalytic  system,  i.e.  one  in  which,  as  in  an  intact  cell, 
TPNHo  and  ATP  were  present  in  catalytic  amounts  and  COg  fixation  was 
therefore  possible  only  in  the  light  while  TPNHo  and  ATP  were  being 
continuously  regenerated  at  the  expense  of  absorbed  light  energy.  CO2 
assimilation  was  then  investigated,  under  three  conditions :  {a)  when  the 


/ 


PHOSPHOGLYCHriA'i'E 


PHEWL/ WATER - 


Fig.  32.  Radioautograph  of  a  chromatogram  showing  products  of  photo- 
synthetic  ^*C02  assimilation  by  illuminated  chloroplasts  in  the  absence  of  added 
FMN  (Trebst,  Losada,  and  Arnon  [34]). 

photochemical  phase  was  limited  to  non-cyclic  photophosphorylation, 
[b)  when  the  photochemical  phase  w^as  limited  to  cyclic  photophosphoryla- 
tion, and  (c)  when  the  photochemical  phase  included  both  [a)  and  {b). 

Figures  32  and  33  show  that  under  conditions  {a)  and  (6)  COo  assimila- 
tion was  limited  almost  entirely  to  the  formation  of  phosphoglycerate.  As 
shown  in  Fig.  34,  the  formation  of  sugar  phosphates,  which  is  taken  as  a 
measure  of  a  reductive  (photosynthetic)  COo  assimilation  in  this  recon- 
stituted chloroplast  system,  was  observed  only  in  case  (c)  when  a  proper 
balance  was  established  between  cyclic  and  non-cyclic  photophosphorylation. 

In  the  experiments  illustrated  by  Figs.  32,  33,  and  34,  the  balance 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS      397 

between  the  non-cyclic  and  cyclic  photophosphorylation  was  maintained 
by  adding  different  amounts  of  one  of  the  catalysts  of  cyclic  photo- 
phosphorylation, FMN,  vitamin  K  or  phenazine  methosulphate  (cf. 
Tables  XVII  and  XVHI).  Concordant  results  were  also  obtained  by 
Trebst  et  al.  [36]  with  inhibitor  experiments.  For  example,  using  the 
uncoupling  effect  of  ammonia  [104,  36]  on  both  cyclic  and  non-cyclic 
photophosphorylation,  it  was  possible  to  suppress  the  formation  of  ATP 
by  illuminated  chloroplasts  without  inhibiting  the  reduction  of  TPN. 


# 


PHOSPHOGLYCERATE 


P«»OL/ WATER - 


Fig.  33.  Radioautograph  of  a  chromatogram  showing  products  of  photo- 
synthetic  ^^COo  assimilation  by  illuminated  chloroplasts  supplied  with  0-15 
(umoles  FMN  (Trebst,  Losada,  and  Arnon  [34]). 


Under  these  conditions,  COg  fixation  was  completely  abolished  except 
when  the  added  "acceptor"  substance  for  CO2  was  ribulose  diphosphate. 
In  that  case  a  single  product,  phosphoglyceric  acid  was  formed  by  the 
carboxylase  reaction  which  does  not  depend  on  added  ATP  (cf.  review 
[166]).  However,  no  sugar  formation  occurred  because  the  phosphoglyceric 
acid  could  not  be  reduced  by  TPXH.,  in  the  absence  of  ATP. 

Parallel  experiments  of  Losada  et  al.  [35]  on  specific  enzyme  systems 
in  chloroplasts  fortified  these  lines  of  evidence  and  supported  the  con- 
clusion, that  in  a  reconstituted  "catalytic"  chloroplast  system  (in  which 


398  DANIliL    I.    ARNON 

CO2  assimilation  can  occur  only  in  the  light),  non-cyclic  photophos- 
phorylation  alone  does  not  provide  sufficient  ATP  for  a  reductive  assimila- 
tion of  CO.^to  the  level  of  carbohydrate.  Additional  ATP  must  be  supplied 
by  cyclic  photophosphorylation. 


^^k  OHYOROXYACETONE 
i^^»  PHOSPHATE 

PHOSFHOGLYCERATE 


fi0^" 


MONOPHOSPHATES 
(GLUCOSE,  FRUCTOSE) 


DIPHOSPHATES 
(FRUCTOSE,  RIBUUOSE) 


.( 


PHCMOL/\«ATlR- 


Fk;.  34.  Radioautograph  of  a  chromatogram  showing  products  of  photo- 
synthetic  ^^COo  assimilation  by  ilkiminatctl  cliloroplasts  supplied  with  o-ooi 
/jmoles  FMN  (Trebst,  Losada,  and  Anion  I34I). 

16.  The  energy  conversion  concept  in  photosynthesis 

The  concept  of  photosynthesis  to  which  we  w-ere  led  in  the  6  years 
since  the  process  was  first  completely  localized  in  isolated  chloroplasts 
[13-15]  differs  from  the  conventional  view  of  photosynthesis  that  it  is 
mainly  a  process  of  CO2  assimilation.  Photosynthesis  appears  to  be  first 
and  foremost  a  process  for  converting  sunlight  into  chemical  energy  and 
this  conversion  is  more  directly  associated  with  phosphorus  than  with 
carbon  assimilation.  In  the  light  of  present  knowledge,  photosynthesis  may 
be  defined  as  the  synthesis  of  cellular  substances  at  the  expense  of  chemical 
energy  formed  by  photochemical  reactions.  This  definition  inclucies,  but 
is  not  limited  to,  CO2  assimilation. 

In  both  bacterial  and  plant  photosynthesis  the  photosynthetic  events 
proper  are  limited  to  the  formation  of  adenosine  triphosphate  and  reduced 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS     399 

pyridine  nucleotide  by  cyclic  and  non-cyclic  photophosphorylation.  This 
transformation  of  light  into  the  common  currency  of  cellular  energy  is 
fundamentally  independent  of  carbon  dioxide  assimilation.  There  is  no 
particular  reason  why  adenosine  triphosphate  or  a  photochemically 
generated  reductant  could  not  be  used  for  driving  endergonic  cellular 
processes  other  than  COo  assimilation. 

The  photoassimilation  of  acetate  by  Chromatitim  is  a  case  of  photo- 
synthesis without  either  oxygen  evolution  or  CO2  reduction  [121].  So  is 
the  light-dependent  conversion  of  glucose  into  starch  [128].  All  these 
light-driven  reactions  are  also  known  to  occur  in  the  dark  in  non-chloro- 
phyllous  cells,  but  in  this  respect  they  resemble  "photosynthetic"  CO., 
assimilation  which  occurs,  by  essentially  the  same  pathway,  in  non-photo- 
synthetic  bacteria  [32,  33].  Other  manifestations  of  the  photosvnthetic 
process,  now  under  active  investigation,  are  the  photofixation  of  nitrogen 
and  the  photoproduction  of  hydrogen  gas.  Usually,  these  reactions  would 
be  considered  as  being  distinct  from  photosynthesis  proper  but  according 
to  our  present  concept  these  examples  represent  photosynthetic  events 
because  they  are  being  dri\en  by  light  energy. 

In  this  view  of  photosynthesis,  CO.,  assimilation,  although  quantita- 
tively the  dominant  form  of  photosynthesis  on  our  planet,  is  funda- 
mentally only  a  special  case  of  the  use  and  storage  of  light  energy.  COg 
assimilation  proper,  in  both  green  plants  and  photosynthetic  bacteria, 
consists  of  exclusively  dark  reactions  that  are  not  peculiar  to  photo- 
synthesis.* The  familiar  accumulation  of  carbon  compounds  as  carbo- 
hydrates during  photosynthesis  in  green  plants  constitutes  storage  of 
trapped  light  energy.  The  first  products  of  photosynthesis  in  green  plants 
[94,  35],  ATP  and  TPNH^,  are  present  in  the  cell  only  in  catalytic  amounts 
and  cannot  be  stored  to  any  appreciable  degree  for  future  use,  whereas 
carbohydrates  or  fats  can. 

The  proposal  that  ATP  formation  is  a  fundamental  event  in  photo- 
synthesis has  been  made  earlier,  notably  in  1944  by  Emerson  et  al.  [167], 
but,  as  was  recently  pointed  out  by  Umbreit,  "the  early  experiments  were 
not  adequate  to  demonstrate  it"  [167].  Without  sufficient  experimental 
evidence,  the  theoretical  proposals  of  Umbreit  and  his  associates  could 
not  be  adequately  defended  against  the  theoretical  objections  levelled 
against  them  (as  for  example  by  Rabinowitch  [75,  p.  229]),  particularly 
since  later,  the  first  experiments  with  ^^P  to  test  the  occurrence  of  light- 
induced   phosphorylation    in    cell-free   systems   led   to    negative    results. 

*  A  similar  conclusion  was  also  reached  by  investigators  of  the  carbon  path  in 
photosynthesis  [166,  i66a].  Calvin  [i66a]  wrote  recently:  "  The  reduction  of  carbon 
dioxide,  we  now  have  every  reason  to  suppose,  occurs  in  a  series  of  reactions 
which  can  take  place  entirely  in  the  dark.  In  fact,  all  the  enzyme  systems  that 
we  now  know  participate  in  the  conversion  of  CO.^  to  carbohydrates  have  been 
found  in  a  wide  variety  of  organisms,  many  of  which  are  not  photosynthetic." 


400  DANIEL   I.  ARNON 

Aronoff  and  Calvin,  who  made  these  experiments  with  spinach  grana, 
reported  that  "there  is  no  direct  connection  between  hght  and  the  gross 
formation  of  organic  phosphorus  compounds"  [i68]. 

17.  Photosynthesis  and  biochemical  evolution 

The  insight  into  the  mechanism  of  photosynthesis,  gained  from  cell- 
free  experiments  with  chloroplasts  and  chromatophores,  permits  us  to 
interpret,  with  somewhat  enhanced  confidence,  certain  aspects  of  bio- 
chemical evolution  which  we  have  already  discussed  elsewhere  [95,  121]. 

The  beginning  of  photosynthesis  may  be  viewed  as  an  emergence  of 
a  porphyrin  that  gave  rise  to  chlorophyll  and  permitted  the  cell  to  use  for 
metabolic  purposes  the  energy  of  sunlight.  This  primitive  photosynthesis 
consisted  only  of  anaerobic  cyclic  photophosphorylation.  No  oxygen  was 
evolved  and  no  photochemically  formed  reductant  was  required  for  the 
photoassimilation  of  say,  acetate,  or  for  the  assimilation  of  COg,  as  long  as 
hydrogen  gas  was  present  in  the  atmosphere.  Oparin  [169]  and  Miller  and 
Urey  [170]  have  summarized  the  evidence  that  in  the  early  periods  of 
evolution  of  life  forms,  the  environment  contained  hydrogen  gas  and 
simple  carbon  compounds  such  as  acetate.  This  primitive  type  of  photo- 
synthesis is  still  seen  today  in  photosynthetic  bacteria.  Chromatium,  for 
example,  is  capable  of  using  molecular  hydrogen  for  reducing,  in  the  dark, 
the  pyridine  nucleotide  that  is  needed  for  CO2  assimilation,  or  of  photo- 
assimilating  acetate  without  the  aid  of  an  external  reductant. 

The  harnessing  of  light  energy  for  the  synthesis  of  ATP  was  an  event 
of  supreme  importance  to  the  cell.  It  provided  the  cell,  in  an  anaerobic 
environment,  with  a  much  more  efficient  mechanism  than  fermentation  for 
the  formation  of  ATP  that  was  needed  for  the  transformation  of  existing 
carbon  compounds,  into  fats,  carbohydrates,  proteins,  etc.  Cyclic  photo- 
phosphorylation gave  the  anaerobic  photosynthetic  cell  a  mechanism  which, 
in  efficiency  of  ATP  formation,  is  comparable  with  the  process  of  oxidative 
phosphorylation  in  aerobic  cells,  that  followed  it  later  in  the  evolutionary 
scale. 

From  the  point  of  view  of  biochemical  evolution,  one  of  the  most 
interesting  findings  in  cell-free  photosynthesis  was  that  higher,  aerobic 
plants  have  retained  to  this  day  the  anaerobic  cyclic  photophosphorylation 
as  a  mechanism  for  making  ATP  while  sharing  with  other  organisms  in  the 
acquisition  of  the  process  of  oxidative  phosphorylation  by  mitochondria. 

As  hydrogen  gas  vanished  from  the  primitive  atmosphere,  the  photo- 
synthetic cell  became  dependent  on  an  enzymic  apparatus  for  generating 
photochemically  a  strong  reductant,  from  such  electron  donors  as  succinate 
or  thiosulphate.  Light  energy  now  served  a  dual  purpose.  It  supplied  ATP 
by  cyclic  photophosphorylation  and  it  provided  electrons  for  reducing 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS     4OI 

pyridine  nucleotides  by  a  non-cyclic  electron  flow  mechanism.  In  organ- 
isms which  contain,  or  can  adaptively  form,  hydrogenase  or  nitrogenase 
(photosynthetic  bacteria  and  algae),  this  phase  of  photosynthesis  can  also 
be  observed  today  as  a  photoproduction  of  molecular  hydrogen  or  photo- 
fixation  of  nitrogen  gas. 

The  mechanism  of  anaerobic  cyclic  photophosphorylation  appears  to 
have  remained  essentially  unchanged  and  constitutes  today  the  common 
denominator  of  all  photosynthetic  cells.  The  difl^erences  between  bacterial 
and  plant  photosynthesis  seem  to  have  arisen  from  evolutionary  trans- 
formations of  the  non-cyclic  electron  flow  mechanism.  When  it  first 
emerged,  the  non-cyclic  electron  flow  mechanism  was  probably  of  the 
bacterial   type.    It   could   accept   electrons   from   several   electron   donor 


1 

r 
TPN_ 

/• 

• 

/ 

DPN_ 

•                        ^ 

^ 

//            No  enzyme 

Ir. K- ^ 

TPN 

10  20 

minutes 


30 


Fig.  35.  Pyridine  nucleotide  reductase  from  Chromatiiim.  Experimental 
conditions  as  in  Table XIX,  except  that  KoH^'-POj  and  ADP  were  omitted.  4  /xmoles 
DPN  or  TPN  were  added  as  indicated.  (Losada,  Nozaki,  Tagawa,  and  Arn(  n 
[155];  Whatley,  Dieterle,  and  Arnon  [161]). 

substances  (thiosulphate,  succinate,  etc.)  but  not  from  water.  Water 
became  an  electron  donor  in  the  non-cyclic  electron  flow  mechanism  only 
with  the  emergence  of  plant  photosynthesis. 

As  was  already  discussed,  the  use  of  water  as  an  electron  donor,  and 
the  resultant  evolution  of  oxygen,  are  not  essential  for  the  key  events  in 
the  non-cyclic  electron  transport,  the  reduction  of  TPX  and  the  coupled 
formation  of  ATP.  When,  under  special  experimental  conditions,  ascorbate 
replaced  water  as  an  electron  donor  [i6o],  chloroplasts  formed  TPNH., 
and  ATP  without  the  oxidation  of  water,  i.e.  without  oxygen  evolution 
(Table  XIV). 

The  basic  similarity  of  the  non-cyclic  electron  flow  mechanisms  in 
bacteria  and  chloroplasts  is  strengthened  by  the  recent  isolation  by 
Losada  et  al.  [155]  of  a  photosynthetic  pyridine  nucleotide  reductase  from 


402  DANIEL   I.  ARNON 

Chromatiiim  and  R.  riihnim.  As  shown  in  Fig.  35  the  bacterial  enzyme 
catalyzed  the  photochemical  reduction  of  pyridine  nucleotides  by  chloro- 
plasts  that  have  been  deprived  of  their  own  pyridine  nucleotide  reductase. 
The  bacterial  PN-reductase  was  similar  to  the  chloroplast  PN-reductase 
in  reducing  TPN  preferentially  to  DPN  (cf.  [86,  148]). 

The  reduction  of  TPN  was  coupled  with  oxygen  evolution  when  the 
bacterial  enzyme  was  added  to  a  chloroplast  preparation  that  by  itself 
could  not  reduce  TPN  and  thereby  evolve  oxygen  (Table  XIX).  These 
findings  again  support  the  conclusion  that  TPN  reduction  and  oxygen 
evolution  are  basically  separate  phenomena.  The  bacterial  PN-reductase 
cannot  bring  about  a  coupling  of  pyridine  nucleotide  reduction  with 
oxygen  evolution  in  a  bacterial  system. 

TABLE   XIX 

Photochemical  Oxygen  Evolution  Catalyzed  by  Pyridine  Nucleotide 
Reductase  from  Chromatium 

(Losada,  Tagawa,  Nozaki,  and  Arnon  [155];  Whatley,  Dieterle,  and  Arnon  [161] 


0.>  evolved 

Minutes 

(juatoms) 

5 

0-75 

10 

I  -60 

20 

2-78 

30 

3-97 

TPN  reduced 

(/Ltmoles) 

0-76 

1-40 

2-39 

3-i8 

The  reaction  mixture  contained  in  a  final  volume  of  3  ml. :  washed  chloroplast 
fragments  containing  0-3  mg.  chlorophyll;  and  the  following  in  micromoles:  tris 
buffer,  pH  7-8,  100;  MgCl,,  5;  ADP,  10;  K.,H3"-P04,  10;  TPN,  6;  and  a  purified 
pyridine  nucleotide  reductase  preparation  from  Chromatium.  The  reaction  was 
run  at  15°  in  the  light. 

Non-cyclic  photophosphorylation  enabled  green  plants  to  form  a  CO2 
reductant  at  the  expense  of  light  energy  with  the  aid  of  an  ubiquitous 
substance,  water,  and  in  this  way  to  invade  and  live  autotrophically  in 
areas  devoid  of  reduced  sulphur  compounds  or  of  other  electron  donors  of 
restricted  distribution.  The  resultant  proliferation  of  plant  growth  was 
responsible  for  releasing  to  the  atmosphere  the  oxygen,  locked  in  the  water 
molecule,  by  the  only  known  important  mechanism  capable  of  accomplish- 
ing this,  photosynthesis  of  green  plants  [169,  170]. 

Once  molecular  oxygen  became  available,  the  way  was  open  for  bio- 
chemical evolution  to  progress  toward  aerobic  metabolism.  The  oxygen- 
independent  cyclic  photophosphorylation  by  chlorophyll-containing 
particles  could  now  be  paralleled  by  an  efficient  biological  utilization  of  the 
energy  of  chemical  substrates  through  the  mechanism  of  oxidative  phos- 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CONVERSION  PROCESS     403 

phorylation  of  mitochondria.  Photosynthesis  of  green  plants  now  provided 
both  the  substrates  and  oxygen  to  make  oxidative  phosphorylation  and 
aerobic  life  on  this  planet  possible. 

An  interesting  aspect  of  the  relation  between  photosynthetic  and 
oxidative  phosphorylation  in  biochemical  evolution  is  the  common 
phylogenetic  relationship  between  proplastids  and  mitochondria,  as  it  was 
recently  reported  by  Miihlethaler  and  Frey-Wyssling  [171].  Their  obser- 
vations on  proplastid  development  in  embryonic  cells  suggest  that  mito- 
chondria followed  rather  than  preceded  chloroplasts  as  functional 
organelles  in  cellular  metabolism.  This  is  in  harmony  with  the  biochemical 
evidence,  since  photosynthetic  phosphorylation  by  chlorophyll-containing 
particles,  being  independent  of  molecular  oxygen,  could  occur  before 
oxidative  phosphorylation  by  mitochondria,  which  requires  molecular 
oxygen  (95). 

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Discussion 

Bergeron  :  The  thiosulphate  and  light-driven  fixation  of  nitrogen  were  not 
cell-free  extract  studies,  is  that  correct  ? 

Arnon:  Yes. 

Bergeron  :  Do  you  know  how  far  the  thiosulphate  reaction  is  from  the 
chromatophores  ? 

Arnon  :  The  thiosulphate  reduces  the  cytochromes  of  the  chromatophores,  so 
it  must  be  close. 

Allfrey  :  Is  there  any  evidence  that  polynucleotides  are  involved  here  ?  There 
is  a  little  RNA  and  perhaps  a  little  DNA  in  the  chloroplast. 

Arnon  :  There  is  nothing  in  our  evidence  to  rule  that  out. 

Allfrey  :  Does  ribonuclease  affect  any  of  these  processes  ? 

Arnon  :  It  has  not  been  tried. 

DiscHE:  What  is  the  relation  between  your  non-cyclic  phosphorylation  and  the 
phenomenon  which  Ochoa  and  Vishniac  describe. 

Arnon  :  The  Vishniac  and  Ochoa  system  involved  a  collaboration  between 
chloroplasts  and  mitochondria.  Chloroplasts  reduced  the  pyridine  nucleotide, 
which  then  had  to  be  given  to  mitochondria  to  carry  out  the  phosphorylation 
reaction.  Thus  the  phosphorylation  reactions  proper  were  those  of  oxidative 
phosphorylation  by  mitochondria.  In  photosynthetic  phosphorylation  no  mito- 
chondria are  involved ;  the  chloroplasts  do  it  themselves.  In  the  Vishniac  and 
Ochoa  phosphorylation  by  mitochondria  oxygen  is  ro/WMwe</;  in  non-cyclic  photo- 
phosphorylation  by  chloroplasts  you  will  observe  that  oxygen  is  produced.  More- 
over, in  oxidative  phosphorylation  by  mitochondria,  DPNH2  is  oxidized  to  DPN, 
in  non-cyclic  photophosphorylation  by  chloroplasts  it  is  just  the  reverse,  TPN  is 
reduced  to  TPNH,. 

DisCHE :  I  would  say  that  this  leads  to  the  question  of  the  source  of  the  hydro- 
gen ;  didn't  Vishniac  and  Ochoa  say  that  hydrogen  came  from  water  ? 

Arnon  :  There  is  no  conflict  between  that  statement  and  the  experimental  facts 
of  non-cyclic  photophosphorylation. 

Dische:  Have  you  any  evidence  that  phosphorylation  and  reduction  take  place 
in  the  same  process  ? 

Arnon:  Yes,  that  is  definite.  It  has  been  confirmed  in  several  other  laboratories. 

Dische:  But  I  think  that  in  the  mitochondria  TPN  is  not  a  good  phosphoryla- 
ting  agent. 


PHOTOSYNTHETIC  PHOSPHORYLATION  AND  THE  ENERGY  CON^^RSION  PROCESS    409 

Arnon:  DPN  is  not  reduced  at  all  by  light  in  chloroplasts.  Chloroplasts  are 
specific  for  TPN ;  DPN  does  not  work. 

LooMis:  I  would  say  that  one  of  your  slides  indicates  that  oxygen  was  being 
released  too,  because  light  affecting  the  chloroplasts  would  release  an  oxygen. 

Arnox  :  Quite  so.  This  is  a  difference  between  the  oxidativ^e  phosphorylation 
by  mitochondria  in  the  Vishniac  and  Ochoa  system  and  non-cyclic  photophos- 
phorylation  by  chloroplasts.  In  their  system  oxygen  had  to  be  supplied,  in  our 
system  oxygen  is  an  excreted  by-product. 

Vernon  :  This  morning  you  said  there  was  some  evidence  for  photophos- 
phor>-lation  accompanying  DPN  reduction.  Could  you  expand  on  this  ? 

Arnon:  Yes,  we  have  evidence,  which  is  not  yet  as  extensive  as  we  wish,  that 
non-cyclic  electron  transfer  in  photosynthetic  bacteria  is  accompanied  by  phos- 
phorylation, as  would  be  expected  from  our  postulation ;  our  basic  view  is  that  we 
get  a  phosphorylation  whenever  cytochrome  is  oxidized.  When  we  supply  electrons 
from  an  exogenous  electron  donor  through  cytochromes  to  chlorophyll  and  thence 
to  DPN,  the  cytochromes  get  oxidized  by  chlorophyll  as  the  electron  transfer 
occurs.  We  now  have  evidence  that  phosphorylation  is  also  coupled  with  these 
reactions  in  photosynthetic  bacteria. 

GoLDACRE :  In  Xitel/a,  chloroplasts  which  are  free  in  the  cytoplasm  can  often 
be  seen  rotating  at  the  rate  of  several  rotations  a  second  even  in  expressed  cytoplasm 
outside  the  cell.  This  conceivably  can  be  the  result  of  a  flow  of  current  tangential 
to  the  surface.  We  have  heard  a  lot  about  the  movement  of  electrons,  and  the 
evolution  of  hydrogen  and  oxygen,  and  potential  differences,  and  I  was  wondering 
if  under  any  conceivable  arrangement  of  the  components  of  chloroplasts  you  could 
get  current  flowing  over  part  of  the  surface  of  the  chloroplast. 

Arnon  :  I  would  not  wish  to  speculate  so  far  afield.  Suffice  it  to  say  that  accord- 
ing to  our  present  view  what  is  really  important  is  that  we  form  chemical  energy 
from  light.  Once  the  cells  form  ATP,  the  cell  can  use  it  for  different  metabolic 
purposes.  With  the  availability  of  ATP  as  a  general  kind  of  cellular  currency  other 
energy-requiring  cellular  phenomena  are  possible  and  would  not  necessarily  have 
to  be  connected  with  specific  electron  transfer  reactions. 

Frenkel  :  Warburg  in  recent  publications,  has  stressed  the  importance  of  COo 
for  the  Hill  chloroplast  reaction.  I  am,  therefore,  interested  in  Dr.  Arnon 's  views 
as  he  indicated  that  CO.,  plays  no  role  in  the  Hill  reaction. 

Arnon:  May  I  just  make  this  comment.  Warburg  has  proposed  that  COo 
reacts  catalytically  in  the  Hill  reaction  and  that  CO^  cancels  out  in  the  overall 
balance  of  the  reaction.  There  is  nothing  in  our  work  which  excludes  this  possi- 
bility. W^hat  we  do  maintain,  however,  is  that  there  is  no  uet  COo  fixation  because 
our  experiments  were  done  without  added  COo  and  in  the  presence  of  KOH. 


The  Mechanism  of  the  Hill  Reaction  and  Its 
Relationship  to  Photophosphorylation* 

BiRGIT    VeNNESLAND 

Department  of  Biochemistry,  University  of  Chicago, 
Chicago,  III.,  U.S.A. 

Introduction 

The  purpose  of  this  paper  is  an  exploration  of  the  interrelationship  of 
some  of  the  chemical  reactions  brought  about  by  the  particulate  structures 
derived  from  the  chloroplasts  of  higher  plants.  Such  green,  chlorophyll- 
bearing  particles  have  been  called  grana.  This  term  is  used  here  to  mean 
the  insoluble  fragments  obtained  when  chloroplasts  are  disrupted  in  water. 

Washed  grana  have  been  shown  to  catalyze  two  rather  different  types 
of  reactions  which  both  involve  a  conversion  of  light  energy  into  chemical 
energy.  One  of  these  is  the  well-known  Hill  reaction  [i,  2];  that  is,  the 
photoreduction  of  an  added  oxidant,  accompanied  by  O2  evolution.  The 
other  is  the  equally  well-known  reaction  of  photophosphorylation,  dis- 
covered bv  Arnon  and  his  associates  [3],  and  by  Frenkel  [4],  working  in 
Lipmann's  laboratory.  In  this  latter  reaction,  orthophosphate  and  ADP  are 
converted  to  ATP  at  the  expense  of  light  energv.  The  question  I  would  like 
to  explore  is  the  nature  of  the  relationship  between  these  two  rather 
different  phenomena.  I  would  also  like  to  bring  up  the  problem  of  the 
relationship  between  the  reactions  catalyzed  by  washed  grana  and  the 
reactions  of  photosynthesis  in  the  intact  leaf.  Let  me  say  at  once  that  I 
intend  to  deal  only  with  selected  aspects  of  these  problems,  and  most 
particularly  with  some  aspects  which  appear  to  have  been  relatively 
neglected.  Let  me  say  also,  in  advance,  that  I  do  not  think  I  have  reached 
any  decisive  conclusion  about  the  nature  of  the  relationship  between 
photophosphorylation  and  the  Hill  reaction.  The  following  account  is  a 
progress  report  to  trace  the  development  of  our  thinking. 

If  one  wishes  to  avoid  controversy,  the  safest  w^ay  of  representing  the 
Hill  reaction  is  in  a  form  which  is  noncommittal  about  mechanism.  Thus, 
equations  i  and  2  show  the  reaction  with  quinone  and  with  ferricyanide 

*  Supported  by  a  grant  from  the  National  Science  Foundation. 


412  BIRGIT   VENNESLAND 

respectively,  and  indicate  only  the  chemical  identity  of  the  initial  reactants 
and  final  products. 

Q  +  H.O ^H.Q  +  iOo  (i) 

2Fe(CN)r+H20  ■ >  2Fe(CN)^-+2H+  +  iOo  (2) 

The  possible  mechanism  of  the  Hill  reaction  can  be  represented 
schematically  in  many  ways  [5].  A  formulation  which  has  been  used 
quite  frequently  is  depicted  in  Scheme  i,  p.  413.  At  the  top  is  a  diagram 
often  employed  to  represent  the  oxidation-reduction  reactions  underlying 
the  Hill  reaction.  Photons,  in  the  presence  of  chlorophyll,  supply  the 
energy  for  a  dismutation  reaction  which  results  in  the  formation  of 
a  reductant  XH,  and  an  oxidant,  YOH,  from  X  and  Y  and  water.  The 
reductant  is  available  to  reduce  an  added  oxidant,  the  Hill  reagent; 
and  the  oxidant  somehow  excretes  its  oxygen  in  the  form  of  O^.  The 
photon-requiring  step,  shown  in  equation  i,  is  sometimes  referred  to  as 
the  "splitting  of  water".  X  and  Y  are  generally  regarded  as  "built-in" 

hv 

Chi 

/  x,y\ 

/      \ 

XH  YOH  — ^  O, 

X+Y+HOH >XH+YOH  (i) 


XH  +  Hill  reagent >  X  +  reduced  Hill  reagent  (2) 

YOH >i02  +  YH  (3a) 

YH  +  Hill  reagent >  Y  + reduced  Hill  reagent  (3a') 

2YOH >J02  +  2Y  +  H,0  (3b) 

Scheme  i 

components  of  the  chloroplast.  Since  water  is  required  to  balance  the 
equation  for  the  Hill  reaction,  it  is  convenient  to  bring  it  in  at  an  early 
stage.  We  should  note,  however,  that  it  is  impossible  to  denote  how  the 
elements  of  water  participate  in  the  reaction  unless  we  know  the  structural 
formulae  of  all  the  reaction  components.  Equations  2  and  3  in  Scheme  i 
show  various  steps  in  the  process  of  reconverting  XH  and  YOH  to  X  and  Y. 
The  regeneration  of  X  is  pictured  as  an  oxidation  of  XH  by  the  Hill  oxidant 
in  equation  2.  Two  possible  ways  of  regenerating  Y  are  shown.  One 
alternative  involves  the  formation  of  a  second  reductant,  YH  (equation  3a), 
which  reduces  another  molecule  of  oxidant  (equation  3a').  The  other 
alternative  shown  is  a  dismutation  between  two  molecules  of  YOH 
(equation  3b). 


HILL    REACTION    AND    ITS    RELATIONSHIP    TO    PHOTOPHOSPHORYLATION       413 

As  a  reminder  of  my  ignorance  of  the  intermediates  of  the  Hill  reaction, 
I  have  liked  to  draw  it  occasionally  as  shown  in  Scheme  2.  Here  the  initial 
reaction  is  formulated  as  an  oxygen  transfer  from  OR  to  X  (equation  i)  and 
the  regeneration  steps  are  shown  in  equations  2  and  3. 

hv 

Chi 

/      \ 

/OR,  X\ 

/  \ 

R  OX >  10, 

OR  +  X >R  +  OX  (i) 


(H,Oj 

R  +  Hill  reagent >  OR  +  reduced  Hill  reagent  (2) 

OX >X  +  iO.,  (3) 

SchetJie  2 

One  might  just  as  well  formulate  the  first  reaction  as  an  electron  trans- 
fer, but  the  resulting  pictures  tend  to  look  more  complicated.  What  should 
be  emphasized  is  that  Schemes  i  and  2  are  intended  to  represent  more  or 
less  the  same  process,  and  that  we  should  guard  against  the  danger  of 
reading  more  information  into  such  schemes  than  is  actually  justified  by 
experimental  evidence. 

Photophosphorylation  with  a  catalytic  amount  of  cofactor 

Let  us  proceed  to  the  nature  of  the  relationship  of  the  process  of 
photophosphorylation  to  the  reactions  of  Scheme  2. 

The  net  process  of  "cyclic"  photophosphorylation  is  depicted  in 
equation  3. 

ADP=^-  +  Pf- >ATP3+OH-  (3) 

'      cofactor  ^•^' 

This  was  the  first  type  of  photophosphorylation  recognized  [6-8].  It  is 
characterized  by  the  fact  that  only  a  catalytic  amount  of  cofactor  is  added 
to  the  chloroplast  preparation — the  cofactor  being  anv  of  a  large  number  of 
oxidation-reduction  compounds  which  must  be  added  (in  addition  to 
Mg++),  to  elicit  the  photophosphorylation  process.  It  w^as  apparent  from 
the  first  work  of  Frenkel  that  the  occurrence  of  a  Hill  reaction  is  certainly 
not  necessary  for  the  occurrence  of  photophosphorylation.  Frenkel  worked 
with  bacterial  chromatophores  which  have  never  been  shown  to  cause  a 
photoevolution  of  Oo,  but  which  give  an  excellent  photophosphorylation 
reaction  [4,  9]. 

There  is  abundant  evidence,  however,  that  chromatophores  catalyze 


414  BIRGIT   VENNESLAND 

oxidation-reduction  dismutations,  and  that  these  are  associated  with  the 
photophosphorylation  process  [10,  11].  The  apparent  association  of  photo- 
phosphorylation  with  electron  transport  or  flow  suggested  an  obvious 
analogy  to  mitochondrial  oxidative  phosphorylation.  The  simplest  way  of 
picturing  this  analogy  is  to  insert  the  ATP-generating  step  into  an  oxida- 
tion-reduction back  reaction  between  the  oxidant  and  reductant  generated 
in  the  light,  as  shown  in  Scheme  3. 


OX >  +0.. 


R+OX  +  Pi  +  ADP >OR  +  X+ATP 

Scheme  3 

This  is  the  interpretation  which  was  adopted  initially  and  which  has  been 
generally  accepted  [8,  11-14].  ^^  ^^  consistent  with  the  fact  that  the  O2 
evolving  step  is  not  a  required  part  of  the  photophosphorylation  reaction. 
It  explains  why  chromatophores  and  green  grana  have  this  step  in  common, 
that  is — because  they  both  cause  an  appropriate  oxidation-reduction 
dismutation — and  it  explains  why  the  overall  Hill  reaction  may  be  inhibited 
without  necessarily  inhibiting  photophosphorylation. 

With  a  mechanism  such  as  that  depicted  in  Scheme  3,  the  function  of 
the  cofactor  is  to  serve  as  a  bridge  between  the  reductant,  R,  and  the 
oxidant,  OX,  which  are  regarded  as  built-in  chloroplast  components.  The 
need  for  such  a  bridge  highlights  the  separation  of  R  and  OX  in  the 
chloroplast.  Whatever  the  cause  of  this  separation,  whether  it  be  geometry 
or  the  absence  of  an  adequate  catalyst,  R  and  OX  must  not  back-react  freely 
or  the  system  would  not  be  able  to  store  any  chemical  energy.  The  cofactor 
is  pictured  as  closing  a  gap  and  thus  permitting  electron  flow  along  an 
electron  transport  chain  from  R  to  OX.  The  generation  of  high-energy 
phosphate  bonds  is  considered  to  occur  in  association  with  the  electron 
flow,  in  analogy  to  mitochondrial  oxidative  phosphorylation. 

Though  it  is  a  reasonable  picture,  Scheme  3  cannot  be  regarded  as  a 
statement  of  known  fact.  We  do  not  actually  know  the  site  of  high-energy 
phosphate  bond  generation.  All  that  can  be  said  with  reasonable  certainty 
is  that  reaction  [3]  occurs  and  that  the  cofactor  is  alternately  reduced  and 
oxidized  while  this  reaction  takes  place.  It  has  been  suggested  more 
recently  [15-17]  that  the  generation  of  high-energy  phosphate  bonds  may 


HILL    REACTION    AND    ITS    RELATIONSHIP    TO    PHOTOPHOSPHORYLATION       415 

occur  in  association  with  the  photon-requiring  oxidation-reduction 
reaction  (i.e.  equation  i  in  Scheme  2).  This  is  a  possibihty  that  has  to  be 
considered.  The  mechanism  in  Scheme  3  has  the  advantage  of  providing 
an  analogy  to  mitochondrial  oxidative  phosphorylation.  The  grana  contain 
cytochrome  b  [16,  18]  and  the  quinone  derivative  Q255  [19,  20],  and  these 
components  are  very  similar  to  some  of  the  mitochondrial  constituents 
thought  to  be  rather  intimately  involved  in  the  oxidative  phosphorylation 
process  [21-25].  In  mitochondrial  oxidative  phosphorylation,  the  phos- 
phate "  pick-up  "  occurs  during  a  dark  reaction  with  a  negative  free-energy 
change.  This  would  presumably  also  be  the  case  in  Scheme  3.  If  the 
phosphorylation  were  associated  with  a  reaction  occurring  against  a 
thermochemical  gradient  and  consequently  requiring  light  energy,  one 
might  expect  it  to  be  quite  different  in  nature. 


Stoicheiometric  photophosphorylation 

If  the  mechanism  shown  in  Scheme  3  is  accepted  for  the  purpose  of 
the  argument,  a  question  may  be  posed  with  the  aid  of  the  diagram  in 
Scheme  4.  Here  a  catalytic  amount  of  cofactor  is  visualized  as  shuttling  in 
the  manner  indicated  by  the  equations.  Does  the  phosphorylation  occur 
during  reaction  A  or  B  ?  This  question  requires  an  admission  that  there 
may  actually  be  several  sites  for  photophosphorylation.  Let  us  assume  at 
this  stage  that  there  is  only  one  site. 


h 

V 

Chi 

/   \ 

/ 

\ 

A 

B 

R OX 


cofactor 

A.  Oxidized  cofactor +R >  reduced  cofactor  +  OR 

B.  Reduced  cofactor  +  OX >  oxidized  cofactor  +  X 

Scheme  4 

There  is  convincing  evidence  that  if  the  phosphorylation  occurs  as 
depicted  in  Scheme  3  and  at  only  one  site,  then  this  site  must  be  A  and 
not  B.  It  has  been  shown  that  photophosphorylation  can  be  coupled  to  the 
net  photoreduction  of  Hill  reagents  such  as  TPN,  ferricyanide,  and 
naphthoquinone  sulphate  [8,  26-31].  Arnon  has  termed  the  process 
"stoicheiometric"  photophosphorylation,  to  distinguish  it  from  "cyclic" 
photophosphorylation.  The  stoicheiometry  of  the  photophosphorylation 


41 6  BIRGIT   VENNESLAND 

coupled  with  the  reduction  of  quinone,  ferricyanide  and  TPN  is  shown  in 
Equation  4,  5,  and  6,  respectively.  These  equations  have  been  balanced 

Q  +  ADP2-  +  Pf-  +  H+ >HoQ  +  ATP3  +1O0  (4) 

2Fe(CN)3-  +  ADP2-  +  Pf-  >  2Fe(CN)^-  +  ATP3-  +  H+  +  i02        (5) 

TPN  +  +  ADP2  +  Pf- >  TPNH  +  ATP^  +  iQ,  (6) 

completely  with  respect  to  hydrogen  ions.  Note  that  water  is  not  required 
to  satisfy  the  stoicheiometry  (since  the  oxygen  may  be  regarded  as  coming 
from  the  phosphate),  but  that  hydrogen  ions  are  consumed  during  the 
reaction  with  quinone  and  formed  during  the  reaction  with  ferricyanide, 
whereas  there  is  no  net  formation  or  consumption  of  acid  during  the 
reaction  with  TPN.* 

Equations  4-6  show  that  the  P/ze  ratio  for  all  three  reactions  is  one. 
The  photoreduction  of  TPN  requires  addition  of  a  soluble  protein  to  the 
grana  [32-34],  and  so  may  be  assumed  to  be  somewhat  indirect,  but 
reactions  4  and  5  do  not  require  such  a  soluble  activator.  This  and  other 
evidence  has  shown  that  TPN  is  not  a  natural  mediator  in  the  reactions 
with  most  other  Hill  reagents  [30].  The  photoreduction  of  TPN  is  dis- 
cussed in  the  paper  by  Dr.  Davenport,  and  so  will  not  be  further  mentioned 
here.  It  is  generally  agreed  that  the  Hill  reactions  per  se  can  readily  be 
dissociated  or  uncoupled  from  the  generation  of  high-energy  phosphate 
bonds.  Ageing  of  almost  any  preparation  of  chloroplasts  or  grana  usually 
(but  not  always)  results  in  a  loss  of  their  capacity  for  catalyzing  photo- 
phosphorylation  before  the  loss  of  their  capacity  for  catalyzing  the  various 
Hill  reactions.  Here  again  there  is  an  obvious  analogy  to  the  behaviour  of 
mitochondria. 

The  role  of  oxygen  in  cyclic  photophosphorylation 

After  the  occurrence  of  stoicheiometric  photophosphorylation  had  been 
demonstrated,  a  re-examination  of  the  process  of  cyclic  photophos- 
phorylation has  led  to  the  conclusion  that  it  generally  occurs  in  a  manner 
somewhat  more  complicated  than  that  depicted  in  Scheme  3.  If  the  chloro- 
plasts have  an  unimpaired  oxygen-evolving  system,  cyclic  phosphorylation 

*  The  equations  are  written  for  one  ionization  state  of  each  of  the  three  forms 
of  phosphate  (Pf~,  ADP^",  and  ATP^~).  Since  there  will  be  two  ionization  states 
of  phosphate  present  at  the  pH's  generally  employed  for  carrying  out  these  reac- 
tions, a  complete  description  of  the  reaction  would  be  more  complicated  than  that 
indicated.  The  overall  change  in  acid-base  balance  indicated  by  the  equation  is, 
however,  largely  correct.  The  equations  have  been  balanced  in  detail  to  show  what 
is  meant  by  the  statement  that  the  chemical  nature  of  X  and  Y  must  be  known 
before  we  can  say  how  the  elements  of  water  participate  in  their  oxidation-reduc- 
tion reactions. 


HILL    REACTION    .\ND    ITS    RELATIONSHIP    TO    PHOTOPHOSPHORYLATION      417 

with  a  catalytic  amount  of  cofactor  does  not  involve  reoxidation  of  the 
reduced  cofactor  bv  OX,  but  reoxidation  by  molecular  oxygen,  as  shown  in 
Scheme  5   [31,  33,   35].   Such  systems  cause  a  rapid  isotopic  exchange 

//.' 

Chi 


;/-  P  \ 

R OX >iO. 

Hill  oxidant 

reduced,  and 

reoxidized 

byO^ 
Scheme  5 

between  ^^Oo  and  water,  and  the  rate  of  exchange  is  as  fast  or  faster  than 
the  rate  of  phosphorylation  [36].  The  reoxidation  of  reduced  cofactor  is 
partly  enzymic  and  partly  non-enzymic.  In  the  latter  case  H2O2  is  formed 
[31].  The  proportion  of  non-enzymic  auto-oxidation  increases  with 
increasing  O.,  tension  as  evidenced  by  increased  hydrogen  peroxide 
production,  without  necessarily  changing  the  yield  of  ATP  [35].  This  may 
be  taken  as  additional  evidence  that  the  phosphorylation  does  not  occur 
during  the  oxidation  of  reduced  cofactor.  Warburg  et  al.  [31]  have 
described  these  phenomena  for  naphthoquinone  sulphate  as  a  cofactor, 
and  we  have  studied  them  with  riboflavin  monophosphate  [37]  and  with 
menadione  [35,  38].  In  all  these  cases  there  is  photophosphorylation 
associated  with  a  Alehler  reaction  [39]. 

An  exception  to  the  behaviour  of  the  cofactors  just  described  is 
provided  by  the  N-methylphenazonium  salts  and  the  related  substance 
pyocyanine  [11,  12,  40  44].  With  these  compounds  as  cofactors  for  photo- 
phosphorylation, the  predominant,  though  not  the  only,  mode  of  cycling 
appears  to  be  genuinely  anaerobic;  that  is,  OX  is  diverted  to  oxidize  the 
reduced  cofactor.  Such  phosphorylation  is  characterized  by  a  low  sensitivity 
to  inhibitors  of  the  oxygen-evolving  process  such  as  orthophenanthroline, 
chlorophenyldimethylurea  and  related  compounds,  or  high  concentrations 
of  tris  buffer  [37,  41,  45,  46].  In  the  presence  of  these  inhibitors,  oxygen 
becomes  strongly  inhibitory,  as  would  be  expected  from  the  fact  that  the 
reduced  cofactors  are  auto-oxidizable,  and  that  if  oxygen  excretion  is  not 
possible  the  cofactor  system  must  be  poised  so  as  to  discharge  equivalent 
amounts  of  R  and  OX.  The  oxygen  destroys  this  poising.  In  many  respects, 
chloroplasts  with  inhibited  oxygen  evolution  show  a  behaviour  toward 
cofactors  reminiscent  of  that  of  bacterial  chromatophores.  The  occurrence 
of  the  anaerobic  cycling  confirms  the  conclusion  drawn  from  the  data  with 
chromatophores,  that  O,  evolution  is  not  a  necessary  accompaniment  for 
phosphor}dation, 

VOL.  n. 2E 


41 8  BIRGIT   VENNESLAND 

Photoreduction  of  ferricyanide  and  of  trichlorophenol  indophenol 

Some  of  the  most  interesting  results  with  coupled  stoicheiometric 
photophosphorylation  have  been  obtained  in  a  study  of  the  reduction  of 
ferricyanide  in  the  presence  of  intact  chloroplasts  by  Jagendorf,  et  al.  at 
the  McCoUum  Pratt  Institute  [26-29].  These  investigations  started  with 
the  original  observation  of  Arnon  [8],  that  the  rate  of  photoreduction  of 
ferricyanide  could  be  increased  by  addition  of  ADP  and  inorganic  ortho- 
phosphate.  The  Baltimore  group  showed  that  with  fresh  chloroplasts  this 
increase  w'as  a  striking  phenomenon.  The  rate  of  photoreduction  of  ferri- 
cyanide was  about  200  /^.moles  per  mg.  chlorophyll  per  hour,  in  the 
absence  of  the  phosphorylation  system,  and  about  800  /xmoles  per  mg. 
chlorophyll  per  hour  in  the  presence  of  added  orthophosphate  and  ADP. 
If  the  chloroplasts  were  washed  in  slightly  acid  isotonic  medium,  they 
were  "  uncoupled  ",  in  the  sense  that  they  caused  ferricyanide  reduction  at 
a  maximum  rate  in  the  absence  of  the  phosphorylating  system.  Un- 
coupling could  also  be  achieved  by  addition  of  ammonia,  or  of  a  small 
amount  of  the  dye,  trichlorophenol  indophenol.  Separate  examination  of 
the  behaviour  of  the  dye  showed  that  it  was  an  excellent  Hill  reagent  and 
that  its  rate  of  photoreduction  was  always  at  least  as  fast  or  faster  than  the 
rate  of  photoreduction  of  ferricyanide  under  any  conditions.  The  photo- 
reduction of  the  dye  was  not  associated  with  coupled  phosphorylation, 
however,  and  not  subject  to  stimulation  by  added  ADP  and  orthophosphate 
to  any  significant  extent. 

All  the  phenomena  described  up  to  this  point  can  be  accommodated 
within  a  simple  mechanistic  picture  involving  only  one  phosphorylation 
site,  designated  as  A  in  Scheme  4.  The  system  behaves  as  though  the  dye 
can  by-pass  the  phosphorylation  site,  reacting  more  or  less  directly  with  R. 
Such  a  picture  is  supported  by  the  results  of  Witt  et  al.  [47],  who  con- 
cluded that  the  transfer  of  electrons  to  indophenol  dyes  is  a  simpler  and 
more  rapid  process  than  is  the  transfer  to  ferricyanide.  Their  evidence  was 
based  on  a  study  of  the  kinetics  of  disappearance  of  a  light-induced 
absorption  increase  at  515  m^u  in  isolated  chloroplasts. 

"Oxidative"  photophosphorylation 

The  behaviour  of  trichlorophenol  indophenol  with  chloroplasts  exhibits 
an  idiosyncrasy  which  cannot  be  so  easily  explained  by  the  assumption  of 
one  photophosphorvlation  site  at  A  in  Scheme  4,  unless  one  makes  some 
fundamental  additions  to  or  changes  in  the  Scheme.  Although  there  is  no 
photophosphorylation  associated  with  the  reduction  of  oxidized  dye,  one 
can  demonstrate  a  synthesis  of  ATP  from  orthophosphate  and  ADP  when 
the  reduced  dye  is  oxidized  by  molecular  oxygen  in  the  presence  of 


HILL    REACTION    AND    ITS    RELATIONSHIP    TO    PHOTOPHOSPHORYLATION      419 

illuminated  chloroplasts.  This  was  discovered  by  Dr.  David  Krogmann 
who  has  made  an  extensive  study  of  the  phenomenon  [48-51].  Most  of  his 
work  has  been  done  with  trichlorophenol  indophenol,  but  the  behaviour 
in  which  we  are  interested  is  manifested  by  phenol  indophenol  itself,  and 
by  many  of  its  derivatives  [51]. 

The  unique  behaviour  of  trichlorophenol  indophenol  may  be  sum- 
marized as  follows.  A  catalytic  amount  of  dye  supports  a  moderate  rate  of 
photophosphorylation  (50-100  ^umoles  per  mg.  chlorophyll  per  hour)  but 
only  in  the  presence  of  oxygen.  Under  these  circumstances  the  dye  causes 
an  oxvgen  exchange  between  molecular  oxygen  and  water  at  a  rate  com- 
mensurate with  the  best  photophosphorylation  rates  which  can  be  induced 
by  dye.  The  dye  is  rapidlv  photoreduced  and  less  rapidly  photo-oxidized, 
and  the  phosphorylation  accompanies  the  latter  process.  The  reaction  is 
not  inhibited  by  cvanide.  In  order  to  obtain  maximum  photophosphoryla- 
tion rates,  the  dye  must  be  kept  in  the  reduced  state.  A  catalytic  amount 
of  dve  is  therefore  emploved,  with  an  excess  of  reducing  agent,  such  as 
ascorbate,  glutathione  or  reduced  diphosphopyridine  nucleotide.  With  the 
latter  reductant,  which  does  not  itself  cause  much  H.^Oo  generation,  the 
best  P  jze  ratio  for  oxidative  photophosphorylation  has  been  shown  to  be 
about  two,  and  the  photo-oxidation  of  dye  by  O2  has  been  shown  not  to 
involve  H^O.,  production.  The  process  of  oxidative  phosphorylation  occurs 
with  washed  chloroplast  fragments  and  is  relatively  insensitive  to  reagents 
which  inhibit  the  Hill  reaction  (e.g.  orthophenanthroline,  chlorophenyl 
dimethyl  urea,  concentrated  tris  buffer,  etc.). 

Scheme  6  represents  an  attempt  to  show  how  all  the  above  facts  can  be 
accommodated  to  the  same  photophosphorylation  site  as  that  localized  at 

hv 
Chi 


R< — >     dye     < — >  OX >  hO.^ 


O., 


p,~p 


Hill  Reaction : 

.A.     R  +  oxidized  dye >  OR  +  reduced  dye 

B.  OX >  lO.  +  X 

"  Photo-oxidative  "  photophosphorylation  : 

C.  R  +  iOo  +  2Pi  +  2ADP >OR  +  2ATP 

D.  OX  +  reduced  dye >  X  +  oxidized  dye 

Scheme  6 


420  BIRGIT   VENNESLAND 

A  in  Scheme  3.  Scheme  6  is  drawn  to  indicate  that  the  dye  reacts  rather 
directly  with  OX  as  well  as  with  R. 

In  addition  to  the  first  oxidation-reduction  dismutation  to  form  R  and  OX, 
the  Hill  reaction  with  oxidized  dye  would  include  reactions  A  and  B. 
To  explain  photo-oxidative  photophosphorylation  we  assume  also  that 
molecular  oxygen  can  substitute  for  the  other  Hill  reagents  to  reoxidize  R 
by  way  of  a  phosphorylating  electron  transport  chain.  The  regeneration  of 
OR  and  of  X  should  be  understood  to  proceed  according  to  reactions  C 
and  D  when  a  sufficient  amount  of  reduced  dye  is  present.  The  electron 
transport  chain  may  contain  plastoquinone  and  cytochrome  b.  The 
cyanide-insensitive  auto-oxidizability  of  cytochrome  b  would  be  compatible 
with  a  position  at  this  point. 

It  should  be  understood  in  connection  with  Scheme  6,  that  though  the 
occurrence  of  reaction  A  excludes  the  occurrence  of  reaction  C  for  a  given 
molecule  of  R,  and  B  similarly  excludes  D,  nevertheless,  all  the  reactions 
could  be  occurring  simultaneously  in  a  given  chloroplast  suspension,  with 
the  relative  rates  determined  by  the  concentrations  of  reduced  and  oxidized 
dye  and  by  the  oxygen  tension.  "Oxidative"  photophosphorylation  is 
rather  slow  relative  to  other  types  of  photophosphorylation,  although  the 
Hill  reaction  with  the  dye  is  quite  rapid.  Thus  reactions  A  and  B  represent 
the  preferred  reaction  sequence. 

There  is  one  very  serious  difficulty,  however,  with  the  otherwise  rather 
plausible  picture  in  Scheme  6.  If  reactions  C  and  B  can  both  occur,  then 
why  should  one  need  a  cofactor  at  all  to  elicit  photophosphorylation  or 
oxygen  exchange  ?  If  the  system  is  really  constituted  as  shown  in  Scheme  6, 
it  should  form  ATP  in  the  light  while  it  evolves  and  reconsumes  Og, 
whether  a  cofactor  is  added  or  not.  It  is  established,  however,  that  grana 
unsupplemented  by  cofactor  do  not  cause  photophosphorylation  or 
oxygen  exchange  at  an  appreciable  rate. 

In  order  to  get  around  this  difficulty  one  must  either  postulate  different 
or  additional  phosphorylation  sites,  or  one  must  establish  a  necessity  for 
added  cofactor  in  the  oxygen  evolving  step  whereby  O2  and  X  are  formed 
from  OX.  Reactions  3a  and  3a'  of  Scheme  i  show  in  principle  how  the 
oxygen  evolving  step  might  be  dependent  on  cofactor.  Here  the  evolution 
of  oxygen  involves  the  formation  of  a  second  reductant,  which  is  reoxidized 
by  a  Hill  reagent. 


The  COo  requirement  of  the  Hill  reaction 

A  rather  specific  mechanism  for  the  Hill  reaction  recently  proposed  by 
Warburg  [31]  is  depicted  in  Scheme  7. 


HILL   REACTION   AND    ITS   RELATIONSHIP   TO    PHOTOPHOSPHORYLATION      42 1 

Quinone  +  COo  +  HjO  +  H3PO4  =  Hydroquinone  +  H2PO3-O-COOOH 
HoPOg-O-COOOH  +  H.O  =  H3P04  +  0,  +  HCOOH 
Quinone  +  HCOOH  =  Hydroquinone  +  CO2 

Net  change :  zQuinone  +  2H2O  =  aHydroquinone  +  O2 

Scheme  7 

Warburg's  proposal  is  based  in  part  on  the  demonstration  that  CO2  is  an 
essential  requirement  for  Hill  reactions,  whether  catalyzed  by  grana  or  by 
preparations  oi  Ch/ore/la  cells  [31,  52,  53,  54].  In  Warburg's  mechanism, 
the  reduction  of  quinone  is  pictured  as  occurring  in  two  separate  steps. 
First  one  mole  of  quinone  is  reduced  with  the  simultaneous  formation  of  a 
phosphorylated  peroxide  of  carbonate.  This  peroxide  is  then  converted  to 
Oo  and  "nascent"  formate,  and  the  latter  substance  reduces  a  second 
molecule  of  quinone.  Scheme  8  shows  how  the  major  flaw  in  Scheme  6  is 
corrected  by  the  insertion  of  the  elements  of  Warburg's  reaction  sequence. 
What  was  needed  was  a  requirement  for  a  cofactor  in  the  oxygen  evolving 
step.  This  need  is  provided  by  the  requirement  of  an  oxidant  for  (CO)  (i.e. 
carbon  at  the  oxidation-reduction  stage  of  formate).  The  continued 
operation  of  the  catalytic  mechanism  requires  that  [CO]  must  somehow  be 
reoxidized,  and  we  assume  that  this  oxidation  cannot  be  eftected  by  O,, 
even  though  R  can  be  reoxidized  by  O.,,  with  accompanying  phosphoryla- 
tion. It  should  be  noted  that  the  mechanism  shown  in  Scheme  8  does  not 


Reduced 
Hill  reagent  Hill  reagent 


/  \  CO2  < (CO) 

/       Hill  reagent      ^                               V                               /^  <-'hl 
/          or                              \                                \.^             ^/        ^"' 
R O,  OX (CO;,) ^O, 

2Pi         2  ~  P  ~  P  ? 

Sc/wnw  8 

provide  for  any  retention  of  reduced  carbon,  in  keeping  with  the  fact  that 
the  grana  cause  no  net  fixation  of  COo.  The  grana  are  presumably  deficient 
in  the  means  of  causing  removal  of  [CO]  in  a  normal  manner.  It  should  also 
be  noted  that  Scheme  8  accounts  nicely  for  the  observed  facts  that  the  P/2^ 
ratio  is  about  one  for  photophosphorylation  coupled  to  net  reduction  of  a 
Hill  reagent,  whereas  the  P/2f  ratio  is  about  two  for  phosphorylation 
coupled  to  photo-oxidation  of  reduced  dye.  The  reduced  dye  is  assumed 


422  BIRGIT    VENNESLAND 

to  be  oxidized  by  OX,  with  an  accompanying  reoxidation  of  R  by  Og,  over 
an  electron  transport  chain  which  gives  a  coupled  phosphorylation  of  2 
moles  of  ATP  per  atom  of  O  reduced.  When  the  Hill  reagent  is  reduced, 
it  replaces  O2  as  an  oxidant  for  R,  but  for  each  mole  of  Hill  reagent  reduced 
by  R,  an  equal  amount  is  reduced  by  [CO].  If  no  net  ATP  synthesis  occurs 
in  the  latter  reaction,  the  average  F  jze  ratio  must  be  one. 

Because  of  the  importance  of  the  demonstration  that  CO,  is  a  required 
component  of  the  Hill  reaction,  it  seemed  desirable  to  verify  Warburg's 
conclusion,  particularly  for  the  indophenol  dyes  which  appear  to  react 
more  immediately  with  the  oxidation-reduction  components  of  the  grana 
than  does  a  Hill  reagent  such  as  ferricyanide.  In  these  experiments  (which 
have  been  done  together  with  Dr.  Babette  Stern  [35,  55]),  we  wished  to 
measure  both  the  rate  of  oxygen  evolution  and  the  rate  of  reduction  of  Hill 
reagent.  Several  /xmoles  of  Hill  reagent  are  required  in  order  to  obtain 
reasonably  accurate  rate  measurements  of  O.,  evolution  by  the  manometric 
procedures  employed  in  our  laboratory.  Because  of  its  intense  pigmenta- 
tion, trichlorophenol  indophenol  could  not  be  employed  in  these  amounts. 
The  reduced  dye  is  rapidly  oxidized  by  ferricyanide,  however,  in  a  non- 
enzymic  reaction.  We  therefore  used  ferricyanide  with  a  catalytic  amount 
of  trichlorophenol  indophenol.  The  procedure  involved  a  determination  of 
the  relative  rates  of  photoreduction  of  the  ferricyanide  in  the  presence  and 
absence  of  CO,,.  The  CO.,  was  removed  by  the  use  of  KOH  in  the  centre 
well  of  the  Warburg  vessel. 

If  measurements  were  made  with  fresh  grana  after  the  usual  equilibra- 
tion period  in  the  dark  of  about  15  min.,  little  or  no  difference  was  noted  in 
reaction  rates.  It  is  thus  easy  to  understand  why  the  CO.,  effect  on  the  Hill 
reaction  has  often  been  overlooked.  If  the  dark  equilibration  was  extended 
over  a  period  of  several  hours,  however,  a  marked  effect  of  CO.,  developed. 
The  COo  appears  to  be  tenaciously  held  by  the  preparation,  and  its  removal 
by  KOH  in  the  centre  well  required  a  prolonged  preincubation  in  the  dark. 
The  longer  this  preincubation,  the  greater  the  CO.,  effect,  as  measured  by 
the  ratio  of  the  photoreduction  rate  in  the  presence  of  CO2  to  the  photo- 
reduction  rate  in  its  absence. 

The  above  procedures  were  worked  out  before  the  latest  papers  of 
Warburg  and  Krippahl  [53,  54]  were  available  to  us.  It  is  of  interest  that 
the  details  of  procedures  we  have  employed  are  rather  different  from  those 
used  in  the  Dahlem  laboratory,  but  that  the  conclusions  are  in  agreement. 
We  used  dye  with  ferricyanide,  saturating  light  with  a  small  amount  of 
grana,  and  a  long  preincubation  in  the  dark.  Warburg  and  Krippahl 
employed  ferricyanide  without  dye,  excess  grana,  limiting  light,  and  a 
preincubation  period  of  i  hr.  in  the  light.  They  employed  a  new  mano- 
metric procedure  to  show  how  the  rate  of  photoreduction  of  ferricyanide 
varies  with  CO.,  tension,  and  they  also  demonstrated  that  the  effect  of  CO2 


HILL    REACTION    AND    ITS    RELATIONSHIP    TO    PHOTOPHOSPHORYLATION      423 

TABLE  I 
Effect  of  CO^  and  of  Trichlorophenol  Indophenol  (TCP)  on 

THE    PhOTOREDUCTION    OF    FeRRICYANIDE 

Ferricyanide  reduced 
(;LimoIes/mg.  chlorophyll/hr.) 

No  CO2     i-sf'oCO. 

No  TCP  73  93 

003  /imole  TCP  107  240 

006  /tmole  TCP  107  230 

o- 10 /imole  TCP  88  204 

Reaction  mixtures  contain  100  /nmoles  of  pyrophosphate  buffer  of  pH  6-8,  and 
spinach  grana  containing  02  mg.  chlorophyll  in  a  total  volume  of  3-0  ml.  After 
preincubation  in  the  dark  for  2  hrs.,  20  /xmoles  of  ferricyanide  were  tipped  in  from 
the  side  arm  and  the  lights  were  turned  on.  T  =  20  ,  4000  ft. -candles  white  light. 
Gas  phase,  either  No  (with  KOH  in  centre  well),  or  i    s",,  COo  in  No. 

tension  on  the  Hill  reaction  rate  with  grana  was  the  same  as  the  effect  of 
COo  tension  on  the  rate  of  photosynthesis  in  the  intact  leaf.  Twelve  years 
ago,  Boyle  [56]  reported  that  COo  was  required  for  the  photoreduction  of 
quinone.  His  findings  have  received  little  attention,  presumably  because 
of  his  failure  to  define  appropriate  experimental  conditions  for  duplicating 
his  results. 

The  experiment  summarized  in  Table  I  was  one  of  a  series  carried  out 
to  determine  the  optimal  concentration  of  dye  for  demonstration  of  the 

TABLE  II 
Effect  of  CO.,  and  pH  on  the  Hh.l  Reaction 


Initial  pH 


COo  Ferricyanide  reduced 

(i-5"o)  (/xmoles/mg.  chlorophyll/hr.) 


6-8  -  72 

6-7  +  152 

67  +  173 

6-7  -  48 


6-6 


45 


The  reaction  mixtures  contained  100  /tmoles  of  sodium  pyrophosphate  buffer, 
initially  of  pH  6-8,  40  /xmoles  of  KCl,  0-07  /xmole  of  trichlorophenol  indophenol, 
and  kohlrabi  grana  containing  0-2  mg.  chlorophyll,  with  water  to  make  a  final 
volume  of  3  -o  ml.  HCl  was  added  to  adjust  the  pH  in  the  absence  of  CO.,.  The 
dark  preincubation  was  for  2-5  hr.  20  /itmoles  of  fi^rricyanide  were  tipped  in  from 
the  side  arm  at  the  onset  of  illumination  with  white  light,  4000  ft. -candles. 
Illumination  was  for  20  min.  T  —  20". 


424  BIRGIT   VENNESLAND 

CO2  eflFect.  The  results  show  that  the  rate  of  photoreduction  of  ferricyanide 
alone,  though  stimulated  by  COo,  is  still  relatively  slow  even  in  the  presence 
of  COg.  When  dye  is  present  together  with  CO2,  however,  the  rate  of 
photoreduction  of  ferricyanide  is  considerably  faster  than  the  rate  observed 
when  either  dye  or  CO2  is  absent.  Since  it  seems  unlikely  that  CO2  should 
be  required  for  the  reduction  of  ferricyanide  by  reduced  dye,  the  data 
suggest  that  it  is  the  photoreduction  of  the  dye  itself  which  is  COg- 
dependent.  It  also  seems  unlikely  that  the  CO2  effect  is  a  pH  effect,  since 
the  addition  of  a  small  amount  of  dye  has  no  appreciable  effect  on  the  pH. 
Table  II  shows  an  experiment  to  verify  this  conclusion.  The  small  decrease 
in  pH  brought  about  by  added  COg  was  duplicated  by  addition  of  HCl, 
with  no  stimulatory  effect  on  the  Hill  reaction. 

Finally,  and  most  importantly,  the  CO2  effect  could  be  shown  to  be 
freely  reversible  [55].  A  preparation  of  grana  which  has  lost  activity  by 
prolonged  incubation  in  the  presence  of  KOH  is  rapidly  reactivated  if  COg 
is  added  back  a  few  minutes  prior  to  the  photoreduction  assay.  A  repre- 
sentative experiment  illustrating  the  reactivation  is  shown  in  Table  III. 

We  regard  the  above  experiments  as  a  partial  confirmation  of  Warburg's 
results.  The  requirement  of  COo  for  the  Hill  reaction  is  another  discovery 

TABLE  III 
Reversibility  of  the  CO.,  Effect  on  the  Hill  Reaction 

Reaction  rate  in  /xmoles  per 
mg.  chlorophyll  per  hour 

rrocedure  Ferricyanide      Pressure  increase 

reduced  calculated  as  O2 

(4  X   ytimoles  O2) 

I  '5%  CO.,  in  No  present  in  dark  and  light  108  (144) 

N2,  and  no  CO2  in  dark  and  light  66  62 
No  CO2  in  dark,  1-5%  COo  in  N2  added  15 

min.  before  assay  in  light  loi  (142) 

The  reaction  mixtures  contained  100  /imoles  of  sodium  pyrophosphate  buffer 
of  pH  6-8,  40  /tmoles  of  KCl,  0-07  ;umole  of  trichlorophenol  indophenol,  and 
spinach  grana  containing  0-2  mg.  chlorophyll.  Samples  were  preincubated  in  the 
dark  for  2  •  5  hr.  Assay  in  light  was  for  40  min.  Other  conditions  are  those  given  for 
Table  I.  Figures  in  brackets  include  CO2  given  off  from  the  bicarbonate  of  the 
medium  as  the  result  of  acid  formation  attending  ferricyanide  reduction. 

of  major  significance,  which  should  be  listed  with  the  many  notable 
achievements  of  the  Dahlem  laboratory.  Although  our  experimental 
techniques  are  not  as  elegant  as  those  employed  by  Warburg  and  Krippahl, 
we  feel  our  results  have  additional  reinforcing  value  because  they  were 


HILL   REACTION   AND   ITS   RELATIONSHIP   TO   PHOTOPHOSPHORYLATION      425 

obtained  in  a  somewhat  different  way,  and  they  demonstrate  clearly  the 
need  for  CO2  in  the  photoreduction  of  trichlorophenol  indophenol,  a 
different  Hill  reagent  from  the  quinone  and  ferricyanide  employed  by 
Warburg.  The  demonstrated  need  for  COo  in  all  Hill  reactions  examined 
cannot  easily  be  explained  in  terms  of  other,  previously  described  CO.,- 
fixing  reactions,  nor  can  it  easily  be  explained  away  as  an  artifact  or  side 
effect, 

I  have  already  indicated  the  advantages  gained  from  adding  Warburg's 
mechanism  for  the  Hill  reaction  to  the  schemes  we  have  been  using  to 
represent  the  process  of  photophosphorylation.  The  diagram  shown  in 
Scheme  8  represents  our  present  working  hypothesis  regarding  the  manner 
in  which  the  Hill  reaction  operates  in  grana,  and  the  locus  of  the  ATP- 
generating  phosphorylation  site  in  relation  to  the  other  reactions.  The 
diagram  is  subject  to  amplification  and  modification.  We  may  speculate 
that  X  might  include  cytochrome  /.  This  would  be  in  keeping  with 
Kamen's  first  postulated  chemical  step  after  photon  absorption  [57].  (See 
also  the  paper  by  Kamen  in  the  present  volume.)  One  might,  in  fact, 
borrow  Kamen's  first  step  and  insert  this  initial  step  for  the  oxidation- 
reduction  between  cytochrome  and  chlorophyll  directly  into  Scheme  8, 
equating  R  with  reduced  chlorophyll,  and  OX  with  oxidized  cytochrome. 
Two  such  reactions  would  be  required,  however,  for  the  two  electron 
change  shown.  Scheme  8  has  been  drawn  to  show  a  second  photon- 
requiring  step  at  the  stage  where  the  peroxide  of  carbonate  splits  out 
oxygen.  Warburg  has  not  stated  explicitly  [31]  which  of  the  reactions  in 
Scheme  7  require  photons,  but  it  seems  reasonable  to  conclude  that  the 
second  step  in  Scheme  7  would  require  light.  As  Scheme  8  is  drawn,  the 
dismutation  of  OR  and  X  to  R  and  OX  would  also  require  light.  Two 
different  photon-requiring  reactions  would  be  in  line  with  the  phenomena 
of  the  so-called  "second"  Emerson  effect  [57-60],  which  is  possibly 
related  to  the  activation  by  blue  light  described  by  Warburg  [61].  The 
latter  phenomenon  appears  to  be  a  catalytic  effect,  however,  whereas  the 
Emerson  effect  does  not.  In  this  connection  one  can  speculate  about  the 
possibility  that  the  COo  "sub-cycle"  in  Scheme  8  might  to  some  extent 
operate  independently  of  the  generation  of  R  and  OX.  This  does  not 
appear  to  be  impossible. 

The  relationship  of  phosphate  to  the  COo  "sub-cycle"  requires  some 
special  comment.  Warburg  has  stated  that  in  addition  to  CO.,,  the  Hill 
reaction  also  requires  a  cataKtic  amount  of  phosphate  [31].  This  is 
apparently  the  basis  on  which  he  brings  orthophosphate  into  the  system  to 
participate  in  the  formation  of  the  precursor  of  O2.  In  our  experiments  on 
the  CO2  stimulation  of  the  Hill  reaction  we  found  that  added  orthophos- 
phate had  little  effect  on  the  photoreduction  reaction  rate,  but  none  of  our 
grana  preparations  was  completely  free  of  traces  of  orthophosphate,  so 


426  BIRGIT    VENNESLAND 

these  findings  do  not  necessarily  conflict  with  Warburg's  statement.  It  is 
significant,  however,  that  the  prolonged  incubation  at  20°  which  we 
employ  to  remove  CO2  results  in  a  loss  of  the  ability  of  the  grana  to  give  net 
ATP  synthesis  in  any  photophosphorylation  system.  Furthermore,  the 
ferricyanide-dye  system  employed  does  not  support  ATP  synthesis,  even 
with  fresh  grana.  Thus,  the  operation  of  the  CO.,  sub-cycle  shown  in 
Scheme  8  does  not  appear  to  require  externally  added  ATP.  It  is  of  course 
possible  that  high  energy  phosphate  could  be  transferred  more  directly, 
without  going  through  the  adenylate  system.  It  seems  just  as  likely  that 
the  COo  sub-cycle  is  self-sustaining  with  regard  to  high-energy  bonds. 
The  oxidation  of  formate  by  the  Hill  reagent  involves  a  sufficient  release  of 
free  energy  to  provide  for  the  synthesis  of  a  high-energy  phosphate  bond, 
so  that  no  external  sources  would  be  required.  Scheme  8  was  primarily 
designed  to  show  how  Warburg's  mechanism  for  the  Hill  reaction  can  be 
supplemented  to  account  for  all  the  major  phenomenology  of  photophos- 
phorylation, with  only  one  postulated  phosphorylation  site  similar  in  its 
chemical  components  and  properties  to  the  site  of  mitochondrial  phos- 
phorylation. As  should  have  been  apparent  in  the  development  of  the 
argument,  the  data  do  not  compel  one  particular  choice  among  a  variety 
of  possibilities,  so  that  Scheme  8  should  be  regarded  as  a  flexible  working 
hypothesis  only.  The  reactions  diagrammed  in  this  scheme  are  all  presumed 
to  be  catalyzed  by  washed  grana.  To  explain  the  overall  process  of  photo- 
synthesis, additional  reactions  are  clearly  required.  It  should  be  noted  that 
the  "  formate  "  of  the  diagram  may  be  used  in  part  as  a  source  of  reducing 
power,  in  which  event  it  will  be  reoxidized  to  CO2.  Though  some  of  the 
"formate"  will  probably  also  be  retained  as  fixed  carbon,  the  occurrence 
of  this  extra  COa-fixing  mechanism  does  not  in  any  way  deny  a  functional 
role  to  the  soluble  enzymes  of  the  chloroplast  which  catalyze  other  COg- 
fixing  reactions.  It  is  reasonably  self-evident,  however,  that  if  the  grana 
photoreduce  COo  directly  in  the  manner  indicated  in  Scheme  7,  then  this 
reaction  must  be  regarded  as  the  most  important  "  COo-fixing"  reaction  in 
nature.  It  is  conceptually  incorrect  to  think  of  the  grana  primarily  as 
generators  of  ATP  and  reducing  power  in  the  form  of  reduced  TPN.  The 
CO2  reduction  precedes  the  reduction  of  TPN  instead  of  following  it  [54]. 

The  question  of  a  natural  cofactor 

One  final  precaution  must  be  kept  in  mind.  The  Hill  reagents  and 
phosphorylation  cofactors  used  in  our  studies  with  grana  are  largely 
artificial.  This  is  true  even  for  the  FMN-  and  menadione-stimulated 
photophosphorylation  systems.  Although  FMN  is  certainly  present  in 
chloroplasts,  the  quantities  are  insufficient  to  elicit  any  reasonably  rapid 
rate  of  photophosphorylation.  Menadione  does  not  occur  in  nature,  but 


HILL    REACTION   AND    ITS    RELATIONSHIP   TO    PHOTOPHOSPHORYLATION      427 

the  chloroplasts  contain  a  large  amount  of  the  chemically  related  lipid  Q255 
or  plastoquinone  [19,  20].  This  is  bound  in  the  grana  structure,  and  Bishop 
has  shown  that  it  is  essential  for  the  Hill  reaction  [19].  We  have  postulated 
an  associated  role  in  the  generation  of  high-energy  phosphate.  But  it  does 
not  play  a  role  equivalent  to  the  cofactor  which  must  be  added  to  elicit  the 
photophosphorylation.  Examination  of  the  soluble  constituents  of  leaves 
has  shown,  however,  that  the  leaf  does  contain  a  substance,  or  substances, 
which  can  function  as  excellent  cofactors  for  photophosphorylation.  Mv 
associates,  Drs.  David  Krogmann  and  Mary  Stiller,  are  currently  engaged 
in  a  study  of  this  "naturally  occurring"  cofactor.  They  find  that  though 
this  material  is  present  almost  exclusively  in  the  supernatant  when  chloro- 
plasts are  centrifuged  out  of  an  aqueous  medium,  there  is  an  appreciable 
amount  of  it  retained  by  chloroplasts  isolated  in  non-aqueous  medium 
[62,  63].  Such  chloroplasts  do  not  carry  out  photophosphorylation.  The 
cofactor  can  be  extracted  from  them  and  added  back  to  an  equivalent 
amount  of  chlorophyll  in  the  form  of  active  chloroplasts.  Calculated  on 
this  basis,  there  is  sufficient  cofactor  in  the  chloroplasts  isolated  in  non- 
aqueous medium  to  elicit  photophosphorylation  at  at  least  one-quarter  of 
the  maximum  rate  achieved  with  FMN  or  menadione.  With  larger  amounts 
of  natural  cofactor,  the  maximum  photophosphorylation  rate  is  as  high  as 
the  maximum  achieved  with  FMN  or  menadione.  The  photophosphoryla- 
tion with  the  natural  cofactor  is  oxygen-dependent.  In  some  respects  its 
behaviour  suggests  that  it  is  an  orthohydroquinone  derivative.  Such 
substances  are  widelv  distributed  in  leaves.  Among  them  are  the  flavonoids 
quercetin  and  catechin,  and  related  compounds,  and  the  ortho-dihy- 
droxycinnamic  acid  derivative,  cafi^eic  acid,  with  the  related  depside, 
chlorogenic  acid.  All  of  these  substances,  when  tested,  proved  to  be  good 
cofactors  for  photophosphorylation.  It  is  probably  in  these  groups  of 
compounds  that  we  will  find  a  substance  or  substances  which  might  serve 
in  the  leaf  to  elicit  ATP  formation.  Before  we  understand  completely  how 
the  energy  of  the  photons  is  transmitted  chemically  to  the  energy  con- 
suming steps  in  metabolism,  we  may  have  to  learn  a  good  deal  more  about 
the  nature  of  the  interaction  of  the  "natural  cofactor  or  cofactors"  with 
the  oxidation-reduction  components  of  the  grana.  In  this  surmise,  as  in 
others,  we  are  following  the  lead  of  Warburg,  who  hinted  many  years  ago 
at  a  functional  role  in  photosynthesis  for  a  naturally  occurring  ortho- 
hydroquinone [64]. 

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712  (1959)- 

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2783  (1959)- 

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HILL    REACTION   AND    ITS   RELATIONSHIP   TO    PHOTOPHOSPHORYLATION      429 

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(1949)- 


Discussion 

Jagexdorf:  It  seems  to  me  that  there  is  a  chance  that  Krogmann's  data  on  the 
oxygen-requiring  phosphorylation  with  a  dye  could  still  be  explained  if  high 
concentrations  of  the  dye  were  really  an  uncoupler  and  the  function  of  the  ascorbate 
is  to  maintain  the  dye  in  the  reduced  fomi  so  that  you  have  a  little  bit  of  the 
oxidized  dye  to  be  an  acceptor  in  the  usual  scheme  making  ATP.  I  think  you  may 
be  able  either  to  rule  this  out  or  prove  it  one  way  or  the  other,  by  experiments  with 
the  indophenol  dye  and  ferricyanide.  Were  you  able  to  see  stoicheiometric  phos- 
phorylation during  the  reduction  of  a  mixture  of  a  catalytic  amount  of  the  dye  and 
a  lot  of  ferricyanide  ? 

Vennesland  :  We  have  seen  no  ATP  synthesis  with  the  dye-ferricyanide 
combination.  I  should  mention  that  the  grana  which  we  were  using  had  been 
incubated  for  several  hours  at  20  in  dilute  suspensions  and  would  probably  not 
make  ATP  with  any  cofactor.  When  they  are  fresh  they  can  make  ATP  with 
ferricyanide.  I  do  agree  with  you  that  there  are  alternative  ways  of  explaining  the 
phenomena.  I  think  that  we  must  still  be  ver\-  flexible  in  our  views.  The  mechanism 
presented  is  the  best  working  hypothesis  at  the  present  time. 

Smith  :  Is  the  intermediate  compound  between  COo  and  formate  phos- 
phorylated  or  is  it  a  peroxyformate  ? 

Vennesl.^nd  :  The  intermediate  which  Warburg  postulates  is  a  phosphate  of  a 
peroxide  of  carbonate. 

Smith  :  The  question  arises  as  to  whether  there  is  a  high-energy  bond  in  this 
intermediate  ? 


430  BIRGIT   VENNESLAND 

Vennesland  :  As  Warburg  writes  it  I  should  say  there  is  a  high-energy  bond 
and  you  would  presumably  get  an  energy  boost  for  the  liberation  of  oxygen  from 
this  high-energy  bond. 

Lynen  :  We  have  recently  been  interested  in  the  interaction  of  COo  with  biotin 
and  I  should  like  to  ask  whether  you  have  tried  the  effect  of  avidin  on  this  process  ? 

Vennesland  :  No,  we  haven't  tried  it. 

Dische:  In  your  experiments  the  rate  of  the  Hill  reaction  declined  with  time. 
Is  it  not  possible  that  the  effect  of  CO^  is  simply  to  slow  down  the  decline  of  the 
reaction  ? 

Vennesland  :  We  could  reactivate  the  inhibited  reaction  with  CO.,. 

Lowenstein  :  If  you  want  to  leave  phosphate  out  of  the  scheme  entirely,  then 
it  is  easier  to  envisage  oxygen  being  split  out  of  performic  acid  than  out  of  car- 
bonic acid. 

Vennesland:  I  don't  think  the  evidence  that  I  have  available  makes  it  possible 
to  choose  between  two  such  alternatives. 

Lowenstein:   It  is  hard  to  visualize  oxygen  being  split  out  of  carbonate  as 
oxygen  (O2). 

Vennesland  :  I  think  Warburg  envisages  the  reaction  as  occurring  on  the 
chlorophyll,  with  the  COo  bound  in  some  way.  The  bound  CO^  could  have  quite 
different  chemical  properties  from  free  COo. 


Electron  Transport  and  Phosphorylation  in 
Light-Induced  Phosphorylation* 

Herrick  Baltscheffsky 

The  Wenner-Gren  Institute  for  Experimental  Biology, 
University  of  Stockholm,  Szveden 

For  some  time  we  have  been  studying  light-induced  phosphorylation 
(photosynthetic  phosphorylation,  photophosphorylation)  in  photosynthetic 
bacteria  and  also,  to  some  extent,  in  green  plants.  In  these  studies  inhibitors 
of  oxidative  phosphorvlation  in  animal  mitochondria  have  been  employed, 
in  an  attempt  to  obtain  information  about  electron  transport  and  phos- 
phorvlation reactions  in  light-induced  phosphorylation,  both  per  se  and  as 
compared  with  the  svstem  for  oxidative  phosphorylation. 

Washed  chromatophores  of  Rhodospirillum  rubrum  were  used  in  the  case 
of  bacteria  and  washed  spinach  chloroplasts  in  the  case  of  green  plants.  In 
these  two  systems  light-induced  phosphorylation  was  discovered  about  6 
years  ago,  first  by  Arnon  et  al.  [i]  in  plants  and,  somewhat  later,  by 
Frenkel  [2]  in  bacteria.  In  fact,  most  of  the  present  knowledge  about  the 
light-induced  formation  of  adenosine  triphosphate  (ATP)|  stems  from 
studies  with  these  materials. 

Some  results  from  our  earlier  investigations,  which  were  made  at  high 
light-intensities  and  under  aerobic  conditions  and  which  have  been  described 
in  detail  recentlv  [37],  ^vill  be  summarized  in  the  first  two  figures. 

Figure  i  shows  our  proposed  scheme  for  the  electron  transport  in  light- 
induced  phosphorylation  of  R.  rubrum  [3].  The  sites  of  action  of  certain 
inhibitors  and  of  the  stimulatory  agent  phenazine  methosulphate  (PAIS) 
are  also  indicated.  As  is  seen  from  the  Figure,  it  is  possible  in  this  bacterial 
system  to  choose  between  two  pathways  for  the  electron  transport.  In  what 
may  be  called  "the  physiological  pathway  "  it  is  assumed  that  the  electrons 
are  transferred  from  the   photochemical   reductant   to  flavoprotein  and 

*  This  work  has  partly  been  carried  out  in  collaboration  with  Mrs.  M.  Balt- 
scheffsky (mainly  in  experiments  with  bacteria)  and  Miss  B.  Arwidsson  (mainly 
in  experiments  with  plants). 

t  Abbreviations :  ATP,  adenosine  triphosphate ;  DPNH,  reduced  diphospho- 
pyridine  nucleotide;  FAD,  flavinadenine  dinucleotide ;  FMN,  flavin  mono- 
nucleotide; PMS,  phenazine  methosulphate;  HOQNO,  2-/?-heptyl-4-hydroxy- 
quinoline-N-oxide ;  m,  moles  per  litre;  LIP,  light-induced  phosphorylation. 


432  HERRICK   BALTSCHEFFSKY 

further  to  a  compound  X,  which  represents  the  site  of  action  for  HOQNO 
[8]  and  antimycin  A  [9],  and  then  to  the  photochemical  oxidant.  This  is  a 
minimum  scheme,  based  on  our  own  results,  and  there  probably  exist 
more  electron  carriers  in  the  chain.  For  example,  the  participation  of 
cytochromes  was  indicated  in  earlier  work  by  Smith  and  M.  Baltscheffsky 
[10].  In  what  may  be  called  "the  PMS-pathway "  added  PMS,  serving  as 
a  link  between  the  flavoprotein  and  the  oxidant,  gives  a  new,  "artificial" 

Light 

. ' 1 

reductant »-  flavoprotein >-  X >■  oxidant 

atebnn    \  HOQNO 

antimycin  A 


PMS 
Fig.  I.  Electron  transport  in  LIP  chromatophores  of  R.  riibrum. 

electron  transport  chain.  When  this  pathway  is  used,  the  transport  of 
electrons  from  the  reductant  to  the  oxidant  along  "the  physiological 
pathway"  can  be  ehminated  by  inhibition  at  X  with  HOQNO  or 
antimycin  A  [9,  11]. 

Light 


[ 


reductant 


1 


FMN,  FAD 
menadione 
PMS,  pyocyanine 


Fig.  2.  Electron  transport  in  LIP  in  spinach  chloroplasts. 


In  green  plants,  the  ATP-formation  which  is  linked  to  cyclic  electron 
transport  is  almost  totally  dependent  upon  the  addition  of  an  electron 
carrier.  Our  tentative  view  about  the  electron  transport  in  isolated  spinach 
chloroplasts  is  given  in  Fig.  2.  A  great  similarity  is  seen  between  this 
scheme  and  that  proposed  earlier  by  Jagendorf  [12].  The  main  difference 
is  that  flavoprotein  has  been  included  as  an  obligatory  member  in  the 
physiological  electron  transport  chain.  The  experimental  background  for 
this  scheme  has  been  presented  earlier  [5]. 

In  recent  experiments  aiming  at  an  estimation  of  the  efficiency  of  light- 
induced  phosphorylation  in  vitro  we  have  used  three  different  approaches 


LIGHT-INDUCED    PHOSPHORYLATION  433 

to  this  problem,  namely,  inhibitor  studies,  measurements  of  the  quantum 
requirement  of  light-induced  ATP-formation,  and  work  with  pre-aged 
preparations.  It  may  be  pointed  out,  that  all  these  experiments,  except 
for  some  inhibitor  studies,  have  been  done  with  bacterial  chromatophores, 
where  the  above-mentioned  possibility  of  working  with  two  different 
pathways  for  the  electron  transport  has  been  utilized. 

Recently,  McMurray  and  Begg  [13]  reported  that  an  antibiotic  named 
valinomycin  completely  uncoupled  the  oxidative  phosphorylation  in 
animal  mitochondria.  The  effect  of  this  agent  upon  light-induced  phos- 
phorylation in  bacteria  and  in  plants  was  tested  [7,  14]. 


^    60 


^     40 


20 


0  I  2  3  6 

/yM  valinomycin 

Fig.  3.  Effect  of  valinomycin  on  LIP  in  R.  rubrnvi.  The  experimental  details 
were  as  described  in  ref.  [3].  The  final  concentrations  of  the  various  agents  were, 
where  added  :  3  3  x  io~-'  m  ATP,  and  3  -3  x  io~*  M  PMS.  =  the  series  without 
PMS;  •  =  the  series  with  PMS.  Without  PMS,  100%  activity  =  34- 5  «b  ortho- 
phosphate  esterified  in  20  min.  With  PMS,  ioo"o  activity  =  40- 5%  orthophos- 
phate  esterified  in  6  min.  In  all  samples  the  "OD^oo  "  was  0-28. 


In  chromatophores  oi  R.  nibrmii  (Fig.  3)  less  than  micromolar  concen- 
trations of  the  antibiotic  gave  a  partial  inhibition  when  "the  physiological 
pathway"  for  electron  transport  was  used.  Titration  to  much  higher  con- 
centrations of  inhibitor  showed  a  levelling  off  at  about  50"o  inhibition.  On 
the  other  hand,  phosphorylation  in  "  the  PMS-pathway"  was  not  significantly 
inhibited.  Such  an  absence  of  effect  with  low  concentrations  of  inhibitor 
was  encountered  also  in  the  experiments  with  spinach  chloroplasts,  as  is 
demonstrated  in  Table  I.  Irrespective  of  whether  menadione  (vitamin  K3), 
FININ,  FAD,  or  PMS  was  used  as  added  electron  carrier,  no  marked 
inhibition  was  seen  wdth  the  low  concentrations  of  valinomycin. 

VOL    n. 2F 


434  HERRICK    BALTSCHEFFSKY 

The  results  with  vahnomycin  (Fig.  3)  are  given  the  following  tentative 
explanation  (Fig.  4).  In  R.  riibriifri,  "the  physiological  pathway"  is  con- 
nected with  tzvo  phosphorylation  sites,  one  of  which  is  sensitive  to  vahno- 
mycin; thus  we  have  the  50",,  inhibition.  The  valinomycin-sensitive  site  is 
by-passed  when  "the  PMS-pathway "  is  used;  thus  we  have  no  inhibition. 
In  other  words,  the  P/2e^  ratio  is  assumed  to  be  2  in  "the  physiological 
pathway"  and  i  in  "the  PMS-pathway".  According  to  this,  the  rate  of 
electron  transport  in  the  latter  as  compared  to  that  in  the  former  is, 
obviously,  twice  as  high  as  would  be  assumed  from  a  direct  comparison  of 
the  rates  of  phosphate  esterification.  The  valinomycin-insensitive  phos- 
phorylation site,  which  would  be  common  for  both  pathways,  may  well  be, 
in  some  way,  closely  linked  to  the  primary  photochemical  reaction,  which 
has  been  implied  in  Fig.  4  by  connecting  this  phosphorylation  with  the 

TABLE  I 

Effect  of  Valinomycin  on  LIP  in  Spinach  Chloroplasts 

The  experimental  details  were  as  described  in  ref.  [5].  The  final  concentrations  of 
the  various  agents  were,  where  added:  33  x  10  *  m  ATP,  io~*  m  menadione, 
I  •  3  X  10^*  M  FMN,  io~*  M  FAD,  and  2  x  iq-^  m  PMS.  In  the  menadione,  FMN, 
FAD  and  PMS-series,  respectively,  the  values  for  100%  activity  were  58,  28,  57 
and  53  ?b  orthophosphate  esterified  and  the  chlorophyll  content  in  each  sample 
o*  12,  0-07,  o- 12,  013  mg.  respectively.  The  time  for  the  experiments  was  6  min. 


Valinomycin 

Per 

cent  of  I 

nitial 

activity 

(/xM) 

menadione 

FMN 

FAD 

PMS 

— 

100 

100 

100 

100 

o- 1 

98 

113 

108 

95 

I 

98 

84 

96 

96 

3 

90 

62 

75 

114 

6 

78 

40 

33 

98 

photochemical  oxidant.  Consequently,  there  may  be  two  kinds  of  electron 
transport-linked  phosphorylation  in  the  light-induced  formation  of  ATP 
in  bacterial  chromatophores,  one  being  similar  to  and  the  other  different 
from  electron  transport  phosphorylation  in  respiring  mitochondria,  as  is 
visualized  from  the  data  obtained  with  valinomycin. 

In  spinach  chloroplasts  (Fig.  4)  the  absence  of  inhibition  by  low 
concentrations  ofvalinomycin  is  tentatively  taken  to  indicate  the  functioning 
of  only  one  phosphorylation  site,  corresponding  roughly  to  the  valinomycin- 
insensitive  one  in  bacteria.  The  possibility  cannot  be  excluded,  that  ATP- 
formation  at  a  site  of  phosphorylation,  similar  to  the  valinomycin-sensitive 
one  in  bacteria,  has  been  uncoupled  in  the  spinach  chloroplasts  during  the 
preparation. 

By  measuring  the  quantum  requirement  for  the  formation  of  ATP  we 


LIGHT-INDUCED    PHOSPHORYLATION 


435 


have  obtained  further  indications  that  there  may  be  two  sites  of  phos- 
phorylation in  the  bacteria,  and  that  one  is  by-passed  in  "the  PAIS-path- 
way".  In  these  experiments,  which  were  made  at  the  Johnson  Research 
Foundation*  in  Philadelphia,  my  wife  and  I  were  fortunate  to  have  the 
collaboration  of  Dr.  John  Olson  of  Brandeis  University.  Experimental 
details  of  this  work  are  published  elsewhere  [15].  As  is  seen  in  Table  II, 
the  quantum  requirement  for  the  formation  of  one  molecule  of  ATP  was 
about  (although  not  quite)  twice  as  high  in  "the  PMS-pathway "  as  in 

R.  rubrum  chromatophores 
Light 


reductant- 


-»-flavoprotein- 


.PMS, 

HOQNO 
SN  5949 

antimycin  A 

I 

-»-X oxidant 


Vahnomycin 

uncouples     |  does  not 


© 


uncouple  1 


0 


spinach  chloroplasts 
Light 


FMN, FAD 

menadione  f \ 

PMS,  pyocyaninej  I 


reductant ►-  flavoprotein- 


.9- 


oxidant  — ►-  O2 


Valinomycin 

does  not  |^ 
,-'-,        uncouple  ~^ — 
uncoupled  [    'P   ',  {  ~P 


Fig.  4.  Light-induced  phosphorylation. 


"the  physiological  pathway".  This  is  to  be  expected  if  there  are  twice  as 
many  phosphorylation  sites  in  the  latter  pathway  as  in  the  former. 

Some  of  the  preparations  used  in  the  studies  on  the  quantum  require- 
ment for  ATP-formation  were  highly  active,  giving  values  of  600-700 
/imoles  of  orthophosphate  esterified  per  hour  per  mg.  chlorophyll  (in  a 
volume  of  3  ml.).  This  was  obtained  in  the  absence  of  any  added  electron 
carriers.  The  bacterial  extracts  were  prepared  in  the  manner  reported 

*  The  generous  support  of  Dr.  Britton  Chance,  head  of  the  Johnson  Research 
Foundation,  is  gratefully  acknowledged. 


436  HERRICK   BALTSCHEFFSKY 

TABLE  II 

Quantum  Requirement  for  LIP  in  Chromatophores  of  R.  rubrum 

The  wavelength  of  the  light  was  862  m/<..  In  experiments  I  and  II  sand  had 
been  used  for  the  grinding,  in  experiment  III  alumina  had  been  used.  The  values 
were  obtained  from  the  initial  slopes  of  the  curves  for  esterification  of  orthophos- 
phate  versus  light-intensity. 


Experiment 

Quanta  absorbed 

per 

molecule  ATP  formed 

No. 

No  addition 

PMS  +  HOQNO 

I 
II 

III 

6±i 

8±i 

IO±2 

8±i 
>  11 

[3],  except  that  the  intact  cells  were  disrupted  by  grinding  with  sand  instead 
of  alumina.  The  method  was  developed  last  year  in  this  laboratory  [16]. 
Pre-ageing  the  chromatophores  at  55-57°  influenced  light-induced 
phosphorylation  as  is  shown  in  Fig.  5.  It  is  the  third  type  of  experiment 
suggesting  that  phosphorylation  may  occur  at  two  sites  in  "the  physio- 
logical pathway"  for  the  electron  transport  and  at  only  one  site  in  "the 
PMS-pathway  ".  As  is  evident  from  the  figure,  pre-ageing  decreases  the 
phosphorylation  much  more  in  the  "physiological"  system  than  in  the 


PMS+  HOQNO 


No  additions 


Pre-aging  (min) 

Fig.   5.  LIP  in  R.  rubrum  after  pre-ageing.  The  experiment  was  performed 
immediately  after  the  pre-ageing,  which  was  made  at  55-57  • 

system  where  PMS  and  HOQNO  have  been  added.  Thus  the  phos- 
phorylation at  the  single  site  of  ATP-formation  in  "the  PMS-pathway" 
appears  to  be  more  stable  than  that  at  the  valinomycin-sensitive  site  which 
according    to    our    hypothesis    exists    in    "the    physiological    pathway" 


LIGHT-INDUCED    PHOSPHORYLATION  437 

together  with  the  above-mentioned,  more  stable  site.  If  the  curve  in  Fig.  5 
for  "the  physiological  pathway"  represents  the  decrease  of  phosphoryla- 
tion at  two  sites,  one  of  them  being  the  same  as  that  in  the  curve  indicating 
a  high  stability  of  the  phosphorylation  in  the  system  containing  PMS  and 
HOQXO,  the  curve  for  the  stability  of  the  other  phosphorylation  is  given 
by  the  broken  line  in  Fig.  5. 

TABLE  III 

Requirement  for  a  Reducing  Agent  in  Aerobic  LIP  in 
Chromatophores  of  R.  riibrum 

The  experimental  details  were  as  described  in  ref.  [3].  Twice  w-ashed  "chro- 
matophores" were  used.  The  final  concentrations  of  the  various  agents  were, 
where  added:  33  x  io~*  m  ATP,  3-3  x  io~*  M  succinate,  33  x  io~^  m  DPNH, 
10"^  M  ascorbate,  3  3  x  10^  m  PMS,  and  2  x  lo^"  m  HOQNO.  The  time  for  the 
experiment  was  20  min.  in  the  absence  and  6  min.  in  the  presence  of  PMS.  The 
6-min.  values  have  been  recalculated  to  20-min.  values,  assuming  earlier  shown 
linearity. 

.....  Per  cent  orthophosphate 

Additions  .r-    , 

estenned 

—  0-2 

Succinate  9 

DPNH  9 

Ascorbate  10 

PMS  +  HOQNO  20 

Succinate  +  PMS  +  HOQNO  22 


It  was  found  by  Frenkel  and  by  Geller  and  Lipmann  in  the  early  days 
of  research  on  light-induced  phosphorylation  [17]  that  washed  chromato- 
phores of  R.  ruhrum  under  aerobic  conditions  needed  catalytic  amounts  of 
hydrogen  donor,  for  example  DPXH  or  succinate,  in  order  to  produce 
ATP  in  the  light.  Ascorbate  may  be  used  instead  of  these  agents  [18]  as 
is  shown  in  Table  III.  High  concentrations  of  ascorbate  are  needed  to  give 
maximum  effect  (Fig.  6).  "The  PMS-pathway ",  however,  functions 
without  any  addition  of  hydrogen  donor  under  our  standard  conditions 
(Table  III).  The  reason  for  this  can  be  ascribed  to  light-induced  reduction 
of  PMS,  which  then  provides  the  necessary  reducing  equivalents  to  the 
phosphorylation  system.  This  explanation  has  recently  been  given  by 
Geller  and  Lipmann  [19],  who  have  demonstrated  that  an  added  hydrogen 
donor  is  indeed  needed  for  phosphorylation  when  the  light-induced 
reduction  of  PMS  is  inhibited  by  avoiding  light  of  wavelengths  where 
this  compound  absorbs.  Their  conclusion  was  tested  with  our  system  and 
confirmed. 


438  HERRICK    BALTSCHEFFSKY 

The  earlier  general  attempts  to  explain  the  requirement  for  hydrogen 
donor  [17,  19]  may  be  substituted  with  a  more  definite  hypothesis  on  the 
basis  of  the  effect  of  ascorbate  combined  with  recent  results  reported  by 
Chance  and  Nishimura  [20].  Ascorbate  has  been  used  to  reduce  directly 
mitochondrial  cytochrome  c  in  experiments  designed  to  determine  the  P/O 
ratio  in  the  span  cytochrome  c  to  oxygen  [21,  22].  From  the  known  redox- 
potential  of  cytochrome  c^  [23]  it  may  be  assumed  that  ascorbate  in  a 
similar  manner  reduces  this  electron  carrier,  which  has  been  reported  to 
participate  in  the  electron  transport  of  light-induced  phosphorylation  in 
R.  tiibrum  [10].  In  w^hat  appears  to  be  a  primary  photochemical  reaction 
cytochrome  c^  of  Chromatium  becomes  rapidly  oxidized  in  the  light,  even 


12  16 

mM  ascorbate 

Fig.  6.  Effect  of  ascorbate  on  LIP  in  R.  rubnim.  Ascorbate  was  the  only 
reducing  agent  added  (cf.  ref.  [3]).  The  bacteria  had  been  disrupted  by  grinding 
with  sand. 


at  8o°K.  [20].  The  logical  assumption  has  been  made  that  chlorophyll  is 
reduced  in  this  reaction  [20].  If  the  electron  transport  is  initiated  by  light- 
induced  electron  transfer  from  cytochrome  r.,  to  chlorophyll  it  is  clear  that 
some  cytochrome  c.^  must  he  present  in  the  reduced  form  in  order  for  the  system 
to  operate.  Aerobically,  this  criterion  may  not  be  fulfilled,  and  a  hydrogen 
donor  which  causes  either  enzymic  or  chemical  reduction  of  an  appro- 
priate amount  of  cytochrome  f.,  has  to  be  present.  According  to  our 
hypothesis  the  function  of  any  used  hydrogen  donor  in  aerobic  light- 
induced  phosphorylation  of  R.  rubrum  is  to  reduce  an  adequate  portion  of 
cytochrome  c.y  (Fig.  7). 

Table  IV  shows  some  difl^erences  between  menadione  and  PMS  as 
electron  carriers  in  bacterial  light-induced  phosphorylation  [7].  Quanti- 
tatively, PMS  is  a  much  more  potent  stimulating  agent  than  menadione, 


LIGHT-INDUCED    PHOSPHORYLATION 


439 


which  gives  an  appreciable  degree  of  stimulation  only  in  pre-aged  prepara- 
tions [6].  However,  also  with  PMS,  in  preparations  with  activities  of  about 
600  /xmoles  of  orthophosphate  esteriiied  per  hour  per  mg.  chlorophyll,  or 


chlorophyll 
(reduced) 

DPNH 
Succinate 


cylochrofne  c, 
(oxidized) 


Qscorbate 


'  Fig.  7. 

higher,  the  degree  of  stimulation  has  been  relatively  low,  as  low  as  50% 
or  less.  The  two  agents  must  stimulate  phosphate  esterification  bv  bridging 
different  parts  of  the  electron  transport  chain,  as  antimycin  A  exerts  a 

TABLE  IV 

Menadione  and  PMS  as  Electron  Carriers  in  LIP  in 
Chromatophores  of  R.  rubrum 

The  experimental  details  were  as  described  in  ref.  [3].  The  final  concentrations 
of  the  various  agents  were,  where  added:  3'3  x  lo"^*  m  ATP,  10"^^  M  menadione, 
and  3  3  X  io~*  M  PMS.  The  time  for  the  experiment  was  20  min.  in  the  absence 
and  6  min.  in  the  presence  of  PMS. 


A 

/Ltmoles  orthophosphate  esterified//;/"  ODgoo" 

Antimycin 

I                                                            II* 

No  addition   Menadione    PMS    No  addition   Menadione 

PMS 

o-i 

25                    28                  91              7                     13 
1-5                   i-i              76              0-4                  0-3 

74 
66 

*  In  experiment  II  the  chromatophores  had  been  partly  destroyed  by  heating 
10  min.  in  a  water-bath  of  55-57    immediately  before  the  actual  experiment. 


Strong  inhibition  only  on  the  stimulation  due  to  addition  of  menadione. 
This  is  seen  especially  clearly  in  the  experiment  with  the  pre-aged  sample. 
A  comparison  between  the  values  obtained  with  the  fresh  and  the  pre-aged 
material  further  show^s  that  the  stability  of  the  three  systems  increases  in 
the  order:  system  with  no  added  electron  carrier,  system  with  added 
menadione,  and  system  with  added  PMS.  This  sequence  has  been  shown 


440  HERRICK    BALTSCHEFFSKY 

in  more  detail  in  other  experiments  [24].  As  another  example  of  the 
similarities  between  respiratory  electron  transport  in  animal  mitochondria 
and  light-induced  electron  transport  in  bacterial  chromatophores  may  be 
mentioned,  that  also  in  mitochondria  the  stimulation  of  electron  transport 
obtained  with  menadione  is  sensitive  to  antimycin  A,  as  was  shown  by 
Conover  and  Ernster  [25],  whereas  that  obtained  with  PMS  is  insensitive, 
as  was  shown  by  Kimura  and  Singer  [26]. 

Earlier  it  has  been  strongly  emphasized  that  electron  transport  in 
oxidative  phosphorylation  of  animal  mitochondria  and  in  light-induced 
phosphorylation  of  plant  chloroplasts  and,  especially,  bacterial  chromato- 
phores shows  several  similarities  [6].  On  the  basis  of  data  given  here  this 
view  is  further  strengthened.  For  two  reasons  we  consider  the  chromato- 
phores of  R.  ruhrum  as  being  most  suitable  for  studies  of  the  kind  presented. 
The  first  is  that  high  rates  of  light-induced  phosphorylation  are  obtained 
without  the  addition  of  any  electron  carrier.  This  means  that  one  does  not, 
at  least  that  way,  introduce  any  artificial  "by-pass"  around  a  smaller  or 
greater  part  of  the  physiological  electron  transport  system.  The  second 
reason  is  that  one  may  select  one  of  two  separate  cyclic  pathways  for  the 
electrons  to  follow,  a  fact  which  has  opened  up  new^  possibilities  to  gain 
more  knowledge  about  the  reactions  involved  in  bacterial  light-induced 
phosphorylation. 

It  has  been  generally  assumed,  that  more  than  one  molecule  of  ATP  may 
be  formed  when  two  electrons  are  transported  through  the  electron 
transport  chain  in  light-induced  phosphorylation,  as  has  long  been  known 
to  be  the  case  in  oxidative  phosphorylation.  The  three  diflFerent  kinds  of 
evidence  given  above  provide,  when  taken  together,  appreciable  support 
for  our  opinion  that  two  different  sites  of  ATP-formation  exist  in  "  physio- 
logical "  light-induced  phosphorylation  of  chromatophores  from  R.  ruhrum, 
i.e.  for  a  F/ze^  ratio  of  2. 

References 

1.  Arnon,  D.  I.,  Allen,  M.  B.,  and  Whatley,  F.  R.,  Nature,  Loud.  174,  394  (1954). 

2.  Frenkel,  A.  W.,y.  Arner.  cheni.  Soc.  76,  5568  (1954). 

3.  Baltscheffsky,  H.,  Biochim.  biophys.  Acta  40,  i  (i960). 

4.  Baltscheffsky,  H.,  and  Baltscheffsky,  M.,  Acta  cheni.  scaud.  14,  257  (i960). 

5.  Baltscheffsky,  H.,  Acta  chem.  scaud.  14,  264  (i960). 

6.  Baltscheffsky,  H.,  Sveusk  kem.  Tidskr.  72,  310  (i960). 

7.  Baltscheffsky,  H.,  Baltscheffsky,  M.,  and  Arwidsson,  B.,  Acta  chem.  scaud.  14, 
1844  (i960). 

8.  Smith,  L.,  and  Baltscheffsky,  M.,  Fed.  Proc.  15,  357  (1956). 

9.  Geller,  D.  M.,  Abstracts  of  the  Vlltli  luteruatioual  Cougress  for  Microbiology 
{Stockholm),  p.  73  (1958). 

10.  Smith,  L.,  and  Baltscheffsky,  yi.,y.  biol.  Che?u.  234,  1575  (1959). 

11.  Baltscheffsky,  H.,  and  Baltscheffsky,  M.,  Acta  chem.  scaud.  12,  1333  (1958). 

12.  Jagendorf,  A.  T.,  Brookhaveu  Symp.  Biol.  11,  236  (1959). 

13.  McMurray,  W.  C,  and  Begg,  R.  W.,  Arch.  Biochem.  Biophys.  84,  546  (1959). 


LIGHT-INDUCED    PHOSPHORYLATION  441 

14.  Baltscheffsky,  H.  (to  be  published). 

15.  Baltscheffsky,  H.,  Baltscheffsky,  M.,  and  Olson,  J.  AI.,  Biochim.  biophys.  Acta 
50,  380  (1961). 

16.  Baltscheffsky,  M.,  Acta  chefn.  scand.  15,  215  (1961). 

17.  Frenkel,  A.  \\'.,J.  biol.  Cherti.  222,  823  (1956). 

18.  Baltscheffsky,  H.,  and  Anvidsson,  B.  (to  be  published). 

19.  Geller,  D.  M.,  and  Lipmann,  ¥.,J.  biol.  Chem.  235,  2478  (i960). 

20.  Chance,  B.,  and  Nishimura,  M.,  Proc.  tiat.  Acad.  Sci.,  Wash.  46,  19  (i960). 

21.  Maley,  G.  F.,  and  Lardy,  H.  A.,^  biol.  Chem.  210,  903  (1954). 

22.  Lehninger,  A.  L.,  Hassan,  M.,  and  Sudduth,  H.  D.,^.  biol.  Chem.  210,  911 

(1954)- 

23.  Vernon,  L.  P.,  and  Kamen,  M.  D.,y.  biol.  Chem.  211,  643  (1954). 

24.  Baltscheffsky,  M.,  and  Baltscheffsky,  H.  (to  be  published). 

25.  Conover,  T.  E.,  and  Ernster,  L.,  Biochem.  biophys.  Res.  Comm.  2,  26  (i960). 

26.  Kimura,  T.,  and  Singer,  T.  P.,  Nature,  Lond.  184,  791  (1959). 


Discussion 

Vernon  :  I  shovild  like  to  discuss  briefly  the  effect  of  ascorbate  on  photo- 
phosphorylation.  There  is  an  alternative  explanation  which  also  agrees  with  the 
earlier  experiments  of  Geller  in  which  he  observed  an  inhibition  of  photophos- 
phorylation  with  high  concentrations  of  succinate  or  with  completely  reduced 
phenazine  methosulphate.  The  experiments  of  Kamen  and  Newton  with  Chro- 
matium  also  indicate  that  if  conditions  are  excessively  reducing,  photophos- 
phorylation  is  inhibited.  In  our  laboratory  we  have  found  that  addition  of  ascorbate 
or  succinate  under  anaerobic  conditions  produces  an  inhibition  of  photophos- 
phorylation  with  chromatophores  of  R.  rubriim.  This  implies  that  in  the  presence 
of  air,  succinate  and  ascorbate  partly  reduce  and  thus  poise  the  electron  transfer 
agents  at  an  appropriate  level,  and  if  an  excess  of  ascorbate  or  succinate  is  added 
the  medium  becomes  over-reducing  and  photophosphorylation  is  inhibited.  I 
think  the  ascorbate  data  can  be  better  interpreted  from  the  point  of  view  of  poising 
of  the  electron  transport  agents. 

Baltscheffsky  :  Well,  the  difference  between  your  point  of  view  and  our  point 
of  view  is  not  one  of  principle  but  one  of  specificity ;  you  say  that  electron  transport 
agents  need  to  be  poised  and  we  go  one  step  further  and  say  that  cytochrome  c^  is 
the  electron  carrier  which  needs  to  be  poised,  that  is,  adequately  reduced  in  order 
for  the  system  to  function  under  aerobic  conditions.  Not  only  ascorbate  but  also 
succinate  and  reduced  diphosphopyridine  nucleotide  act,  we  feel,  by  poising 
cytochrome  c.,. 

Jagendorf  :  I  think  it  is  very  interesting  that  both  you  and  Dr.  Arnon  find  a 
higher  quantum  efficiency  with  low  light  intensities  using  a  system  other  than 
phenazine  methosulphate.  However,  I  worry  about  coming  to  the  conclusion  that 
this  necessarily  means  there  is  one  site  for  phosphorylation  when  phenazine 
methosulphate  is  used  and  two  sites  in  the  other  system.  I  think  there  are  still 
some  alternative  interpretations  open ;  for  instance,  maybe  with  phenazine  metho- 
sulphate you  waste  some  quanta  because  there  are  two  electron  transport  paths 
operating,  one  not  phosphorylating  at  all  and  the  other  one  involving  the  same  two 
phosphorylation  sites  as  with  FMN,  etc.  The  point  is  that  a  direct  measurement 


442  HERRICK   BALTSCHEFFSKY 

of  quantum  efficiency  is  not  quite  the  same  thing  as  a  direct  measurement  of  the 
number  of  phosphorylation  sites. 

Baltscheffsky  :  I  certainly  agree  that  the  quantum  requirement  experiments 
only  imply  a  ratio  of  two  to  one  and  that  they  do  not  give  final  proof  for  it.  Re- 
garding the  possibility  that  phenazine  methosulphate  may  act  at  two  points,  as 
Dr.  Jagendorf  suggested ;  as  long  as  no  experiments  support  such  an  idea  it  seems 
logical  to  continue  to  assume  that  phenazine  methosulphate  provides  only  one 
"by-pass"  around  "the  physiological  pathway".  The  valinomycin  data  which 
were  presented  appear  to  be  inconsistent  with  a  view  that  the  same  two  phos- 
phorylation sites  could  be  involved  in  the  presence  of  phenazine  methosulphate  as 
in  its  absence.  Taken  together  with  the  two  other  types  of  evidence  given,  it  seems 
to  us  that  the  quantum  requirement  experiments  motivate  the  hypothesis  that  you 
have  two  phosphorylation  sites  in  "the  physiological  pathway"  and  only  one  site 
in  "the  phenazine  methosulphate  pathway". 

Williams:  I  just  wanted  to  ask  about  valinomycin  inhibition:  you  wrote  it 
on  one  of  your  schemes  with  the  inhibited  sites  towards  the  reductant  but  did  you 
have  any  evidence  on  this  point  ? 

Baltscheffsky:  No. 

Williams:  You  just  had  to  put  them  somewhere  ? 

Baltscheffsky:  Yes,  we  had  the  phenazine  methosulphate  results  and  we  had 
to  put  in  the  valinomycin-inhibited  step  somewhere  where  phenazine  metho- 
sulphate ' '  by-passes ' '  the ' ' physiological ' '  electron  transport  chain  in  order  to  explain 
the  fact  that  we  did  not  have  an  inhibition  when  this  agent  was  added.  The  essential 
point  is  really  this :  we  have  to  do  here  with  cyclic  electron  transport  both  in  the 
absence  and  in  the  presence  of  phenazine  methosulphate.  What  our  data  indicate  is 
that  one  phosphorylation  site  is  in  the  part  of  the  "physiological  pathway  ",  which 
is  shared  by  "  the  phenazine  methosulphate  pathway  "  and  the  other  site  in  the  part 
which  is  "by-passed"  by  phenazine  methosulphate. 

Arnon:  I  would  like  to  associate  myself  with  Dr.  Baltscheffsky 's  interpretation 
of  the  phenazine  methosulphate  data  as  suggesting  that  there  is  probably  more 
than  one  phosphorylating  site  and  that  by  using  phenazine  methosulphate  we  are 
by-passing  one  of  them.  It  seems  to  me  that  this  is  strongly  supported  by  the 
chemical  evidence  for  the  way  phenazine  methosulphate  acts  and  I  rely  here  on 
the  data  of  Dr.  Massey.  I  am  very  impressed  by  how  rapidly  phenazine  metho- 
sulphate reduces  cytochrome  in  his  experiments.  It  is  a  very  effective  electron 
carrier  to  cytochrome  and  this,  taken  together  with  the  evidence  from  photo- 
phosphorylation  experiments  gives  me  some  confidence  that  this  may  be  the 
correct  interpretation,  namely,  that  in  the  presence  of  phenazine  methosulphate 
electrons  go  directly  to  cytochrome  and  by-pass  any  phosphorylation  sites  prior 
to  the  cytochromes. 


Reduction  of  Dinitrophenol  by  Chloroplasts 

J.  S.  C.  Wessels 

Philips  Research  Laboratories, 

N.  V.  Philips'  G/oeilampenfahrieken, 

Eindhoven^  Netherlands 

Isolated  chloroplasts  are  capable  of  synthesizing  ATP  from  inorganic 
phosphate  and  ADP  in  light,  provided  they  are  supplemented  with 
catalytic  amounts  of  an  electron  carrier  such  as  FMN,  vitamin  K,  or 
phenazine  methosulphate.  This  ATP  formation  was  named  cyclic  photo- 
phosphorylation  by  Arnon  to  distinguish  it  from  the  phosphorylation 
associated  with  the  reduction  of  substrate  amounts  of  TPN  or  K3  Fe(CN)6. 

In  contrast  with  the  oxidative  phosphorylation  of  mitochondria,  which 
is  completely  uncoupled  by  concentrations  of  DNP  (2,4-dinitrophenol)  as 
low  as  io~^-io^*  molar,  cyclic  photophosphorylation  of  chloroplasts 
proved  to  be  rather  insensitive  to  DNP.  Inhibition  was  observed  only  at 
concentrations  at  which  the  Hill  reaction  was  also  blocked,  that  is  at  about 
io~^  molar. 

In  the  course  of  investigations  on  the  effect  of  DNP  on  cyclic  photo- 
phosphorylation it  was  found  that  DNP  is  even  capable  of  catalyzing  the 
generation  of  ATP  by  illuminated  chloroplasts.  As  shown  in  Table  I,  the 
optimal  concentration  of  DNP  under  anaerobic  conditions  is  about  o-6 
/xmole  per  3  ml.  of  reaction  mixture,  which  is  of  the  same  order  of  magni- 
tude as  the  optimal  concentrations  of  vitamin  K3  or  FMN. 

It  has  been  shown  earlier  that  under  anaerobic  conditions  vitamin  K3 
and  FMN  are  involved  in  separate  pathways  for  cyclic  photophosphoryla- 
tion. The  FMN-pathway  proved  to  be  more  sensitive  to  the  poisons 
NHoOH,  NaN3,  KCN,  and  o-phenanthroline  than  the  vitamin  K3- 
pathway.  Cyclic  photophosphorylation  catalyzed  by  DNP  is  similar  to 
phosphorylation  in  the  presence  of  vitamin  K3  as  regards  the  insensitivity 
to  KCN  and  NaNg  and  the  inhibition  by  dicoumarol,  /)-chloromercuri- 
benzoate,  and  CMU  (3-(4-chlorophenyl)-i,i-dimethylurea).  NH.,OH  and 
o-phenanthroline,  however,  are  more  inhibitory  to  cyclic  photophos- 
phorylation  catalyzed  by   DNP  than   to   phosphorvlation   catalyzed   by 


444  J-    S.    C.    WESSELS 

TABLE  I 

DiNITROPHENOL   AS   A    CATALYST    OF    CYCLIC    PhOTOPHOSPHORYLATION 

.  jj.  .         ,         ,     s  umoles  ATP  generated  during 

Additions  (umoles)  ^  .      ^,,       .       .  * 

30-min.  illumination 

0-5  vitamin  K3  14 '4 

0-5  FMN  7-7 

3  DNP  2-5 

1-5  DNP  7-6 

0-6DNP  107 

03 DNP  79 

015  DNP  2-9 

No  addition  o-6 

o  •  6  DNP ;  dark  o  •  5 

0-6DNP  8-6 

0-6DNP  +  30KCN  7-7 

o-6DNP  +  3NaN3  69 

06  DNP  +  3  NH.,OH  09 

06  DNP  +  0012  CMU  2-9 

06  DNP  +  o-3  dicoumarol  3-5 

0-6  DNP  +  o-3  o-phenanthroline  1-7 

0-6  DNP  +  o  •  3 /)-chloromercuribenzoate  2-5 

In  addition,  the  reaction  mixture  included  40  /xmoles  Na  and  K  phosphate 
buffer,  pH  7  5;  10  /xmoles  MgCL;  125  /imoles  glucose;  i  ^umole  ADP;  25  K.M. 
units  of  hexokinase ;  i  ml.  of  a  suspension  of  chloroplasts  in  o- 1  M  tris  (hydroxy- 
methyl)  aminomethane  buffer  pH  7-5,  containing  0-5  mg.  chlorophyll;  and  de- 
ionized  water  to  give  a  final  volume  of  3  -o  ml.  The  reaction  was  carried  out  under 
anaerobic  conditions  in  Warburg  manometer  vessels  as  described  previously  [i]. 

A  number  of  phenols  have  been  tested  for  their  catalytic  activity  in  the 
process  of  cyclic  photophosphorylation  and  the  results  are  given  in  Table  II. 
It  seems  that  the  presence  of  a  nitro  group  is  necessary  though  not  sufficient 
for  the  activity  of  the  phenol  derivatives. 

In  order  to  act  as  an  intermediate  electron  carrier  across  some  gap  in 
the  electron  transport  chain  of  isolated  chloroplasts,  DNP  should  be 
transformed  by  chloroplasts  into  some  reversible  oxidation-reduction 
system.  Actually  it  was  found  that  illuminated  chloroplasts  are  capable  of 
reducing  DNP  quantitatively  to  2-amino-4-nitrophenol,  and  that  the  latter 
compound  can  serve  as  a  cofactor  for  cyclic  photophosphorylation. 

The  reduction  of  DNP  is  dependent  on  light  and  on  anaerobic  condi- 
tions. When  the  mixture  is  kept  in  the  dark  or  when  the  chloroplasts  are 
illuminated  in  air,  DNP  can  be  recovered  nearly  quantitatively  even  after 
several  hours.  This  is  also  the  case  when  boiled  chloroplasts  are  illuminated 
in  the  presence  of  DNP.  The  transformation  of  DNP  into  amino-nitro- 
phenol  proceeds  approximately  twice  as  fast  if  phosphorylating  reagents 


REDUCTION    OF   DINITROPHENOL   BY    CHLOROPLASTS 

TABLE  II 

Catalytic  Activity  of  Phenols  ix  Cyclic  Photofhosphorylation 


445 


Active 


Inactive 


2,4-dinitrophenol 

2,5-dinitrophenoI 

2,6-dinitrophenoI 

2,4-dinitro-5-acetylaminophenol 

2-nitro-4,6-dimethylphenol 

2-nitro-4,5-dimeth\iphenol 

2-nitroresorcinoI 

o-nitrophenol 

w-nitrophenol 

/)-nitrophenol  (slightly  active) 

2-amino-4-nitrophenol 


^-nitrophenylphosphate 

picric  acid 

2,4-dinitro-6-methylphenol 

2,4-dichlorophenol 

pentachlorophenol 

o-cresol 

p-cresol 

o-methoxyphenol 

o-aminophenol 

A«-arninophenol 

^-aminophenol 

^-acetylaminophenol 

o-phenolsLilphonic  acid 

hydroquinone 

catechol 


(phosphate,  ADP,  MgCl^,  gkicose  and  hexokinase)  are  present.  The 
reaction  is  strongly  inhibited  by  lo^^  m  o-phenanthroHne,  4  x  io~''  m  CMU, 
and  lO"^  M  NH.,OH,  which  are  known  to  inhibit  the  Hill  reaction,  and  is 
accompanied  by  oxygen  production  (nearly  i  ■  5  jumole  Oo/jumole  of  DNP). 
The  inhibition  of  the  photoreduction  of  DNP  by  low  concentrations  of 
NH2OH  and  o-phenanthroline  may  explain  the  finding  that  cyclic  photo- 
phosphorylation  catalyzed  by  DXP  is  more  sensitive  to  these  poisons  than 
is  photophosphorylation  in  the  presence  of  ^•itamin  K3,  which  apparently 
is  independent  of  the  formation  of  molecular  oxygen. 

The  photoreduction  of  DXP  is  strongly  stimulated  by  KCN,  but  even 
under  these  conditions  the  rate  is  not  higher  than  about  4 /xmoles  DNP 
reduced/mg.  chlorophyll/hour.  DNP  reduction  thus  proceeds  at  a  much 
lower  rate  than  the  reduction  of  usual  Hill  oxidants,  such  as  indophenol 
dyes  or  ferricyanide.  As  3  x  io~^  molar /)-chloromercuribenzoate  shows  no 
inhibitory  effect,  it  seems  unlikely  that  photoreduction  of  TPN,  which  is 
very  sensitive  to  this  poison,  is  involved  in  the  photoreduction  of  DNP. 
It  is  known,  on  the  other  hand,  that  the  Hill  reaction  is  resistant  to  p- 
chloromercuribenzoate. 

From  these  experiments  we  may  conclude  that  the  ability  of  DNP  to 
catalyze  ATP  synthesis  by  ilhmiinated  chloroplasts  is  due  to  photo- 
reduction of  this  compound. 

Photoreduction  of  DNP  does  not  occur  in  the  presence  of  phosphorylat- 
ing  reagents  and  vitamin  K3  or  FMN.  This  indicates  that  the  conversion 
of  DNP  into  aminonitrophenol  cannot  account  for  the   insensitivity   of 


446  J.    S.    C.    WESSELS 

cyclic  photophosphorylation  to  DNP.  As  regards  the  effect  of  DNP  there 
seems  to  exist  an  obvious  difference  between  the  mechanisms  of  photo- 
synthetic  and  respiratory  generation  of  ATP. 

With  the  exception  of  w-nitrophenol,  all  nitrophenols  which  have  been 
found  to  be  capable  of  catalyzing  cyclic  photophosphorylation  could  be 
converted  into  reversible  oxidation-reduction  systems  by  reduction  of  the 
nitro  group  to  the  amino  group.  It  was  shown,  however,  that  w-nitrophenol 
is  not  reduced  to  w-aminophenol,  but  to  an  intermediate  reduction 
product  which  may  be  identical  with  ;;/-hydroxylaminophenol  or  m- 
nitrosophenol  or,  more  probably,  with  a  conversion  product  of  these 
compounds.  In  this  connection  it  is  of  interest  to  note  that/)-nitrosophenol 
was  also  found  to  be  active  as  a  catalyst  of  cyclic  photophosphorylation. 
At  the  moment  an  effort  is  being  made  to  elucidate  the  structure  of  the 
photoreduction  product  of  w-nitrophenol. 

Chloroplasts  are  also  capable  of  reducing  DNP  in  the  dark  under 
anaerobic  conditions,  but  then  the  presence  of  FMN  and  of  an  excess  of 
TPNH  is  required.  TPNH  cannot  be  replaced  by  DPNH  or  ascorbate, 
nor  FMN  by  vitamin  K3. 

Reduction  of  DNP  has  not  been  observed  under  aerobic  conditions, 
either  in  the  presence  or  in  the  absence  of  KCN.  As  FMN  catalyzes  the 
oxidation  of  TPNH  by  chloroplast  preparations  under  aerobic  conditions, 
it  seems  probable  that  the  dark  reduction  of  DNP  is  due  to  the  presence 
of  some  enzyme  which  can  transfer  electrons  from  TPNH  to  FMN.  This 
enzyme  may  be  TPNH  diaphorase  or  TPNH — cytochrome  c  reductase, 
both  of  which  have  been  shown  to  be  present  in  chloroplasts  by  Avron 
and  Jagendorf  [2],  and  Marre  et  al.  [3],  respectively.  In  accordance  with 
this  view  it  was  demonstrated  that  chemically  reduced  FMN  is  capable 
of  reducing  DNP.  When  a  solution  of  FMN  is  illuminated  anaerobically 
by  white  light  in  the  presence  of  TPNH  or  EDTA,  the  flavin  is  reduced 
reversibly,  as  has  been  shown  recently  by  Vernon  [4].  Subsequent  addition 
of  DNP  in  the  dark  resulted  in  reduction  of  DNP  to  aminonitrophenol. 
TPNH  was  found  to  be  incapable  of  reducing  DNP. 

As  yet  no  indication  has  been  found  that  FMN  has  some  function  in  the 
photoreduction  of  DNP  by  chloroplasts.  The  formation  of  aminonitro- 
phenol in  light  is  affected  neither  by  the  addition  of  FMN,  nor  by  the 
addition  of  the  flavin  antagonists  atebrin  and  chlorpromazine. 


References 

1.  Wessels,  J.  S.  C,  Biochim.  biophys.  Acta  29,  113  (1958). 

2.  Avron,  M.,  and  Jagendorf,  A.  T.,  Arch.  Biochem.  Biophys.  65,  475  (1956). 

3.  Marre,  E.,  and  Servettaz,  O.,  Arch.  Biocheiti.  Biophys.  75,  309  (1958). 

4.  Vernon,  L.  P.,  Biochim.  biophys.  Acta  36,  177  (1959). 


REDUCTION    OF    DINITROPHENOL    BY    CHLOROPLASTS  447 

Discussion 

Vennesland  :  I  would  like  to  comment  on  the  fact  that  you  find  that  hydro- 
quinone  and  catechol  are  not  co-factors  for  cyclic  phosphorylation.  We  have  been 
working  with  a  variety  of  diphenolic  compounds  and  we  find  that  if  we  put  in  a 
high  enough  concentration  of  hydroquinone  or  catechol  or  any  compound  which 
has  this  type  of  structure,  they  will  support  photophosphorylation ;  we  have  to  put 
in  what  appear  to  be  substrate  amounts  but,  nevertheless,  we  think  that  the  reaction 
is  cyclic.  That  is,  the  hydroquinone  and  the  catechol  are  oxidized  mainly  non- 
enzymically — this  is  why  you  need  such  high  concentrations — and  then  the 
quinones  are  reduced  with  accompanying  photophosphorylation.  Have  you  looked 
to  see  what  happens  if  you  go  to  high  concentrations  of  the  substances  you  tested  ? 
Let  me  add  that  the  reason  that  we  are  particularly  interested  in  this  is  that  some 
of  the  flavonoids,  which  are  di-o-hydroxy  compounds,  appear  to  be  good  cyclic 
cofactors.  These  substances  occur  in  leaves,  but  they  have  been  neglected  as 
possible  natural  cofactors  for  photophosphorylation.  Perhaps  if  we  are  looking 
for  substances  that  might  function  in  this  way  physiologically,  the  flavonoids 
should  be  considered. 

Wessels  :  The  compounds  which  were  found  to  be  capable  of  catalyzing  cyclic 
photophosphorylation  showed  an  optimal  activity  at  a  concentration  of  about 
10  ~*  M.  For  this  reason  all  phenols  which  we  have  examined  were  added  in  con- 
centrations ranging  from  10  ~^  to  10^  M.  We  have  not  added  substrate  amounts 
of  any  of  the  substances  compiled  in  the  Table. 


The  Relationship  between  "Methaemoglobin 

Reducing  Factor"  and  " Photosynthetic  Pyridine 

Nucleotide  Reductase" 

H.  E.  Davenport 

University  of  Bristol,  Research  Station, 
Long  Ashton,  Bristol,  England 

In  his  earliest  experiments  with  isolated  chloroplasts  Hill  [i]  found  that 
extracts  of  acetone-dried  leaf  contain  material  capable  of  accepting  hydro- 
gen from  illuminated  chloroplasts  with  the  concomitant  evolution  of 
oxygen.  Although  the  pathway  of  hydrogen  transport  in  this  reaction  has 
not  been  determined,  investigations  on  the  leaf  extracts  led  to  the  first 
demonstration  of  the  need  for  a  naturally  occurring  catalyst  of  reduction 
in  the  Hill  reaction.  Davenport  ct  al.  [2]  showed  that  neither  methaemo- 
globin nor  metmyoglobin  was  reduced  directly  by  illuminated  chloroplasts, 
but  reduction  of  either  was  initiated  by  the  addition  to  the  system  of  an 
extract  of  acetone-dried  leaf.  The  methaemoglobin  reducing  factor  (MRF) 
was  found  to  be  associated  with  a  protein  fraction  in  the  extracts. 

More  recently  [3]  the  active  protein  has  been  obtained  in  a  state  where 
it  is  homogeneous  both  electrophoretically  and  in  the  ultracentrifuge. 
Purification  was  achieved  by  electrophoresis  on  paper  after  a  preliminary 
fractionation  with  ammonium  sulphate.  The  product  is  a  protein  of  small 
molecular  weight  {c.  19  000),  reddish-brown  in  colour,  and  it  is  active  in 
catalyzing  the  photochemical  reduction  of  a  number  of  haem-protein 
compounds  including  cytochromes  b-^  and  c.  The  specificity  of  the  catalyst 
towards  illuminated  chloroplasts  as  a  source  of  reducing  power,  its  high 
catalytic  activity  and  its  localization  in  the  chlorophvll-containing  cells  of 
higher  plants  and  algae  [4]  suggested  that  it  plays  a  part  in  the  transport  of 
hydrogen  in  photosynthesis.  Howe\'er,  the  pattern  of  specificity  towards 
haem-compounds  of  comparatively  oxidizing  potential  did  not  appear  to 
be  relevant  to  the  energetic  requirements  of  carbon  dioxide  reduction. 

]\Iore  significant  in  this  connexion  was  the  isolation  and  partial  purifica- 
tion by  San  Pietro  and  Lang  [5]  of  a  protein  factor  active  in  catalyzing  the 
photochemical  reduction  of  pyridine  nucleotides.  This  "photochemical 
pyridine  nucleotide  reductase"  (PPNR)  was  prepared  by  acetone  frac- 
tionation, isolation  of  the  active  material  as  a  protamine  sulphate  complex 
with  subsequent  recovery  of  the  protein  from  this  complex. 

VOL.  n. — 2  G 


45° 


H.    E.    DAVENPORT 


In  some  preliminary  attempts  to  purify  further  the  protein  obtained 
from  pea  leaves  by  the  method  of  San  Pietro  and  Lang,  using  salt  precipi- 
tation and  electrophoresis  as  additional  preparative  steps,  it  emerged  that 
activity  towards  triphosphopyridine  nucleotide  (TNP)  and  metmyoglobin 
were  associated  in  the  same  protein  fractions  at  all  levels  of  purification  [6]. 
It  appeared  therefore  that  MRF  is  a  highly  purified  form  of  PPNR. 

Methaemoglobin  reducing  factor  as  a  catalyst  of  TPN  reduction 

The  activity  of  MRF  towards  TPN  at  two  stages  of  purification  of  the 
protein  from  pea  leaves  is  shown  in  Fig.  i.  The  final  electrophoretic  step 


200 


01  02  0-3 

mg   protein  added 


0  4 


Fig.  I.  Catalysis  of  TPN  reduction  by  "methaemoglobin  reducing  factor". 
Pea  leaf  protein :  :•  before  electrophoresis ;  •  after  electrophoresis.  Reaction 
mixtures  contained  (in  3  ml.)  added  protein  as  indicated,  spinach  chloroplasts 
containing  0-03  mg.  chlorophyll,  and  the  following  (in  jumoles) :  TPN,  0-4; 
ADP,  0-5;  MgCl.,,  20;  Na.HPOi,  10;  tris  HCl  buffer,  pH  7-7,  150;  NaCl,  40. 
Leaf  protein  was  omitted  from  the  blank  cell. 

gave  a  nine-fold  increase  in  specific  activity.  With  the  purified  protein  the 
particulate  chloroplast  system  was  here  saturated  by  the  addition  of  about 
100  jug.  to  give  a  rate  of  reduction  of  TPN  of  184  /nmoles/mg.  chlorophyll/ 
hr.  On  the  basis  of  a  molecular  weight  of  19  000  this  would  correspond  to 
the  addition  of  5  m/xmoles  of  the  leaf  protein. 


Comparison  of  metmyoglobin  and  TPN  as  hydrogen  acceptors 

The  relative  activity  of  a  PPNR  preparation  from  pea  leaves  in  cata- 
lyzing the  reduction  of  metmyoglobin  and  TPN  is  shown  in  Fig.  2.  The 


RELATIONSHIP   BETWEEN       REDUCING   FACTOR       AND       REDUCTASE 


451 


protein  used  here  had  been  further  purified  by  electrophoresis  on  paper. 
With  either  hydrogen  acceptor  saturation  of  the  chloroplast  system 
occurred  at  about  the  same  concentration  of  added  protein  and  at  this 
saturation  level  TPN  was  i  •  3  times  as  effective  as  metmyoglobin  in  terms 
of  hydrogen  equivalents  transferred.  This  ratio,  in  different  experiments, 
was  found  to  varv  from  1-2  to  1-7  but  the  variation  could  not  be  related 
to  the  method  used  in  preparing  the  leaf  protein. 


300 


200 


0  1  0  2 

mg  protein  added 


03 


Fig.  2.  Comparison  of  the  activity  of  pea  leaf  protein  (PPNR  further  purified 
by  electrophoresis)  in  catalyzing  the  reduction  of  TPN  and  metmyoglobin. 
Reaction  mixture  contained  (in  3  ml.)  leaf  protein  as  indicated,  spinach  chloro- 
plasts  (chlorophyll  0-115  irig-),  and  (in  //moles)  phosphate  buffer  pH  77,  90; 
NaCl,  40;  and  the  following:  TPN,  04;  ADP,  05;  MgCl.,,  15;  •  metmyo- 
globin, 0-26.  Leaf  protein  was  omitted  from  the  blank  cells. 


Stimulation  of  TPN  reduction  by  photophosphorylation 

The  reactions  shown  in  Figs  i  and  2  where  TPN  was  the  hydrogen 
acceptor,  were  carried  out  in  the  presence  of  adenosine  diphosphate  (ADP), 
orthophosphate  and  magnesium  chloride.  The  presence  of  this  phosphate 
acceptor  system  was  found  to  be  essential  for  maximum  reduction  rates 
provided  that  the  chloroplasts  were  at,  or  near,  saturation  with  respect  to 
added  leaf  protein.  At  saturation  the  additional  phosphate-accepting 
ingredients  stimulated  the  reduction  rate  2-5-fold.  It  was  confirmed  that 
inorganic  phosphate  was  incorporated  as  ATP  in  the  molecular  ratio 
I  TPNHo  I  ATP  [7].  This  resuh  is  at  variance  with  the  report  of  Jagen- 
dorf  [8]  that  no  such  stimulation  of  the  rate  of  reduction  of  TPN  during 
phosphorylation  had  been  detected  in  three  laboratories  in  the  United 


452 


H.    E.    DAVENPORT 


States.  From  the  data  shown  in  Fig.  3  it  would  appear  Hkely  that  these 
failures  can  be  attributed  to  the  use  of  rate-limiting  amounts  of  the 
catalytic  protein.  For  maximum  stimulation  all  the  ingredients  of  the 
phosphate-accepting  system  were  found  to  be  required  and  adenosine 
5'-phosphate  did  not  replace  ADP  unless  it  was  supplemented  by  catalytic 
amounts  of  either  ADP  or  ATP. 

In  contrast  with  these  observations  the  rate  of  reduction  of  metmyo- 
globin  was  found  to  be  unaffected  by  the  presence  of  the  phosphate 
accepting  ingredients  and  no  evidence  for  photophosphorylation  has  been 


01  0-2 

mg  protein  added 

Fig.  3.  Effect  of  phosphate  acceptor  system  on  TPN  reduction  catalyzed  by 
pea  leaf  protein  (PPNR  preparation  further  purified  by  electrophoresis).  Reaction 
mixtvires  contained  (in  3  ml.)  leaf  protein  as  indicated ;  spinach  chloroplasts 
(chlorophyll  o-oi6  mg.),  and  (in  /xmoles)  NaCl,  40;  TPN,  0-4;  tris  HCl  buffer, 
pH  70,  150.  Reaction  mixtures  for  points  •  contained  in  addition:  ADP,  0-5; 
Na2HP04,  15;  MgClo,  20.  Leaf  protein  was  omitted  from  the  blank  cells. 


obtained  with  this  hydrogen  acceptor.  Evidence  that  TPN  is  able  to 
compete  with  metmyoglobin  for  hydrogen  produced  in  the  photochemical 
reaction  was  obtained  by  measuring  metmyoglobin  reduction  in  the 
presence  and  absence  of  substrate  amounts  of  TPN.  The  presence  of  TPN 
was  found  to  inhibit  metmyoglobin  reduction  and  this  inhibition  was 
further  enhanced  when  the  phosphate  accepting  system  was  also  present. 
At  the  present  state  of  this  work  it  appears  that  the  haem-protein  and 
TPN  reducing  activities  are  common  properties  of  a  homogeneous  protein. 
The  absence  of  detectable  activity  towards  diphosphopyridine  nucleotide 
emphasizes  a  very  unusual  pattern  of  specificity  towards  hydrogen 
acceptors. 


RELATIONSHIP    BETWEKN       REDUCINC    FACTOR       AND       REDUCTASE  453 

References 

1.  Hill,  R.,  Proc.  roy.  Soc.  B.  127,  192  (1939). 

2.  Davenport,  H.  E.,  Hill,  R.,  and  Whatley,  F.  R.,  Pruc.  roy.  S(jc.  B.  139,  346 
(1952). 

3.  Davenport,  H.  E.,  and  Hill,  R.,  Bwclu'm.'J.  74,  493  (i960). 

4.  Hill,  R.,  Northcote,  D.  H.,  and  Davenport,  H.  E.,  Nature,  Loml.   172,  948 

(1953)- 

5.  San  Pietro,  A.,  and  Lang,  H.  AI.,;7.  biol.  Chem.  231,  21 1  (1958). 

6.  Davenport,  H.  E.,  Biochem.J.  77,  471  (i960). 

7.  Arnon,  D.  I.,  Whatley,  F.  R.,  and  Allen,  M.  R.,  Biuclum.  hiuphys.  Acta  32,  47 

(1959)- 

8.  Jagendorf,  A.  T.,  Fed.  Proc.  18,  974  (1959). 


Discussion 

Jagendorf:  I  should  like  to  mention  here  that  essentially  parallel  researches 
are  going  on  at  Baltimore,  by  Dr.  San  Pietro  and  associates.  If  he  were  here,  he 
could  have  been  given  almost  the  identical  paper  from  his  own  data.  The  identities 
extend  to  the  requirement  for  higher  levels  of  the  reductase  enzyme,  a  three-fold 
stimulation  of  the  rate  of  TPN  reduction  when  the  complete  phosphorylating 
system  is  added,  and  a  response  to  uncouplers. 

Arnon:  I  would  like  to  make  one  brief  comment  and  to  ask  one  question.  I 
think  it  is  very  gratifying  that  Dr.  Davenport  and,  from  what  we  have  just  heard, 
also  Dr.  San  Pietro,  find  that  phosphorylation  increases  the  rate  of  TPN  reduction, 
because  this  brings  into  agreement  the  facts  of  non-cyclic  photophosphorylation 
with  TPN  with  the  earlier  observations  of  the  ferricyanide  system.  Aly  question  is 
whether  you  have  tested  any  connection  between  the  pyridine  nucleotide  reductase 
and  the  photosynthetic  cytochromes  ? 

D.'WENPORT:  Well  as  you  know  the  problem  of  looking  at  cytochromes  in  the 
presence  of  chlorophyll  is  one  which,  as  far  as  I  know,  has  not  been  solved  unless 
Dr.  Chance  can  tell  us  how  it  can  be  done,  so  I  cannot  say. 

Chance  :  Here,  I  cannot  solve  the  problem  of  photosynthetic  cytochromes  but 
I  can  mention  some  very  preliminary  experiments  with  Dr.  San  Pietro  who  was 
good  enough  to  work  with  us  on  PPNR,  and  to  investigate  whether  bleaching 
actually  occurs  in  the  presence  of  the  chloroplasts.  We  did  find  the  pigment  in 
PPNR  to  be  bleached.  We  found  a  difference  spectrum  on  illumination  of  roughly 
23  ^moles  PPNR  and  roughly  25  /(moles  are  bleached  in  the  absence  of  TPN. 
If  TPN  is  present,  bleaching  is  much  less.  The  rate  at  which  the  absorbancy 
change  occurred  was  in  rough  agreement  with  the  rate  at  which  TPN  was  reduced ; 
so  it  is  not  unreasonable  to  believe  that  this  absorbancy  change  has  something  to 
do  with  the  activities.  But  it  is  obvious  that  the  spectrum  doesn't  identify  the 
compound  involved. 


ATP  Formation  by  Spinach  Chloroplasts*t 

Andre  T.  Jagendorf  and  Joseph  S.  Kahn:|: 

Biology  Depaytment  and  McCoUnm- Pratt  Institute, 

The  Johns  Hopkins  University, 

Baltimore,  Md.,  U.S.A. 

The  mechanism  for  conserving  oxidation-reduction  energy  as  ATP  is 
as  much  of  a  challenge  in  chloroplasts  as  it  is  in  mitochondria.  It  is  also  a 
matter  of  comparative  biochemical  interest  to  see  how  closely  these  two 
mechanisms  resemble  one  another. 

Our  efforts  in  this  area  started  when  it  became  possible  to  study  the 
coupling  between  electron  tiow  and  phosphorylation,  thanks  to  the 
discovery  by  Arnon  and  colleagues  [i]  that  ATP  is  formed  during  ferri- 
cyanide  reduction  in  a  Hill  reaction.  The  rate  of  the  Hill  reaction  is 
stimulated  by  simultaneous  phosphorylation,  up  to  3  • :;  times  under  the 
conditions  of  our  experiments  [2].  We  observed  that  arsenate  was  an 
uncoupler — increasing  the  rate  of  electron  flow,  while  inhibiting  phos- 
phorylation— but  only  in  the  presence  of  ADP  [3].  We  have  speculated 
elsewhere  [3,  4,  5]  that  this  means  either  that  a  stable  high-energy  arsenate 
intermediate  is  formed  (analogous  to  the  theoretical  high-energv  phosphate 
intermediate  in  ATP  formation);  or  that  ADP  is  bound  in  a  high-energy 
complex  first,  and  phosphate  addition  is  the  last  step  in  ATP  formation. 
Since  a  stable  high-energy  arsenate  intermediate  seemed  unlikely,  we 
suggested  the  alternative  of  a  high-energy  adenylate  as  the  first  step  in 
ATP  formation. 

The  evidence  for  this  sequence,  the  reverse  of  that  postulated  for 
mitochondria,  was  indirect.  We  have  since  been  able  to  devise  a  more 
direct  experiment  which  indicates  instead  that  a  high-energv  phosphate 
intermediate  is  formed  first,  and  ADP  addition  is  the  last  step.  Our  later 
conclusions  are  in  accord  with  the  conclusions  to  be  drawn  from  recent 
oxygen- 1 8  studies  by  Avron  and  Sharon  [6]  and  by  Schultz  and  Boyer  [7]. 

The  procedure  for  the  more  direct  experiment  [S]  consists  of  illuminat- 
ing chloroplasts  in  the  presence  of  radioactive  phosphate  but  without 
ADP.  A  presumed  high-energy  phosphate  intermediate  (X  ^  ^-P)  has  a 

*  Contribution  No.  329  from  the  McCollum-Pratt  Institute, 
t  Supported  in  part  by  Research  Grant  RG3923  from  the  National  Institutes 
of  Health. 

X  Present  address  :  Dept.  of  Botany,  North  Carolina  State  College,  Raleigh,  N.C. 


456  ANDRE   T.   JAGENDORF   AND   JOSEPH    S.    KAHN 

chance  to  accumulate  under  these  circumstances.  The  Hght  is  then  turned 
off,  stopping  electron  flow  almost  instantaneously.  A  very  brief  time  later 
ADP  is  added,  allowed  to  incubate  for  15  sec.  in  the  dark,  and  then  the 
reaction  mixture  is  killed.  We  find  that  some  [^-P]-ATP  has  formed  in  the 
dark,  which  is  therefore  a  measure  of  the  amount  of  X  ~  ^^P  carried  in  the 
chloroplasts  just  after  irradiation.  The  amount  comes  to  i  mju,mole  per 
1000  m/Ltmoles  of  chlorophyll  in  a  remarkably  reproducible  fashion 
(±20%). 

Although  the  experiment  was  simple  in  concept,  scrupulous  attention 
had  to  be  paid  to  the  controls.  Figure  i  shows  that  the  chloroplasts  were 
pre-incubated  for  8  minutes  in  the  light  with  a  large  amount  of  cold 
phosphate.  This  served  to  convert  any  internal  ADP  to  unlabelled  ATP. 

8min  light 
unlabelled   P, 

i 

Dark,  add  ^^P| 

^  \ 

15  sec  light  Dark  control 

/  \  /  \ 

Dark,  ADP         3%  TCA        Dark,  ADP         3%  TCA 
15  sec  I  15  sec  I 

\  \  \  \ 

37oTCA  ADP  3%  TCA  ADP 

I  n  m  IV 

Fig.  I.  Protocol  for  "pre-loading"  experiment,  designed  to  show  the  existence 
of  a  high-energy  phosphate  intermediate. 


If  this  were  not  done,  internal  ADP  would  combine  with  the  added  ^^p  to 
form  large  amounts  of  p-P]-ATP  during  the  second  illumination. 

Direct  controls,  as  shown  in  Fig.  i,  included  adding  ^-P  without  a 
second  illumination  (treatments  III  and  IV),  or  killing  the  reaction  prior 
to  addition  of  ADP  in  the  dark  (II  and  IV). 

Table  I  shows  the  results  of  one  experiment.  Treatment  I,  representing 
illumination  with  ^-P  followed  by  addition  of  ADP  in  the  dark,  formed 
three  times  as  much  p-P]-ATP  as  any  of  the  others.  In  other  experiments 
this  ratio  has  varied  from  1-5  to  6  (but  the  increment  in  p-P]-ATP  due 
to  illumination  remained  the  same  in  all  experiments).  Thanks  to  the 
several  controls  we  are  sure  that  [^-P]-ATP  was  not  formed  in  a  dark 
reaction,  or  in  the  light  prior  to  the  addition  of  ADP. 

By  contrast  with  the  successful  results  of  this  experiment  no  [^-P]-ATP 
was  ever  found  as  a  result  of  pre-illuminating  with  ADP  and  adding  ^^P 
in  the  dark  afterwards.  We  therefore  tend  to  conclude  that  a  high-energy 
ADP  complex  is  probably  not  an  intermediate  here. 


ATP    FORMATION    BY    SPINACH    CHLOROPLASTS  457 

TABLE  I 

Formation  ok  [''-PJ-ATP  after  Illumination  with  ''-P 


Treatment 

['-P]-ATP          Light-Dark 

No. 

Illumination 

Dark  addition 

(m/nmoles//imole  chlorophyll) 

I 

II 
III 
IV 

Light 
Light 
Dark 
Dark 

ADP 
TCA 
ADP 
TCA 

1-72                       1-04 
o-6o                      0-14 
0-68 
0-46 

All  flasks  received  8  min.  pre-illumination  with  cold  phosphate.  Numbers  refer 
to  protocol  shown  in  Fig.  i.  The  illumination  indicated  is  the  second  one,  of  15  sec. 
duration,  in  the  presence  of  ''-P.  Reaction  mixture  contained  0013  M  tris  pH  80, 
0-0033  ^^  ^IgClo,  0-033  M  NaCl,  0-00003  ^i  phenazine  methosulphate,  0-00033  ^^ 
phosphate,  and  chloroplasts  containing  from  i  to  5  /mioles  of  chlorophyll;  total 
volume  12  ml. 


A  trivial  possibility  in  this  sort  of  experiment  would  be  the  formation, 
in  the  light,  of  first  a  very  small  amount  of  labelled  ATP,  and  then  a  larger 
amount  of  a  compound  on  a  side  pathway  (such  as  carbamyl  phosphate, 
or  other  high-energy  phosphate  compound).  This  secondary  product 
might  be  the  storage  site  for  high-energy  phosphate,  and  pass  it  on  to  the 
large  amounts  of  ADP  added  after  illumination.  This  sequence  would  be 
represented  by  equations  13  : 

X  -  -^^P  +  ADP  -^  [=^-P]-ATP  +  X  (i) 

PPJ-ATP  +  Y  ^  ADP  +  Y  -  3->p  (2) 

Y  -  3-P  +  ADP  ^  Y  +  [3-^P]-ATP  (3) 

If  this  mechanism  were  operating  the  specific  acti\ity  of  phosphate  in 
the  secondary  product  (Y  ~  ^-P),  and  therefore  the  total  amount  of 
p-P]-ATP  formed  in  Reaction  3  v>ould  be  sensitive  to  variations  in  the 
amount  of  unlabelled  ATP,  or  in  specific  activity  of  ATP  present  before 
adding  ADP  in  the  dark.  This  is  not  the  case,  however. 

The  internal  content  of  free  ATP  in  chloroplasts  is  of  the  order  of 
200  400  m/Ltmoles/mg.  chlorophyll.  It  can  be  removed  almost  completely 
by  breaking  the  plastids  open  in  water.  Whether  the  internal  (unlabelled) 
ATP  is  present  or  absent,  i  m/unole  of  [-^-PJ-ATP  is  formed  mg.  chloro- 
phyll, owing  to  pre-illumination  followed  by  ADP  in  the  dark. 

A  second  indication  of  the  absence  of  a  simple  dark  equilibrium 
between  our  stored  intermediate  and  ATP  lies  in  the  constant  amount  of 
p-P]ATP  formed  due  to  light,  over  a  ten-fold  variation  in  specific  activity 
of  internal  ATP  due  to  dark  reactions.  Whether  the  dark  controls  have 


458  ANDRE   T,   JAGENDORF   AND   JOSEPH   S.    KAHN 

o-i6or  2-0  m/xmoles  of  p-P]-ATP,  the  increase  due  to  pre-illumination 
followed  by  ADP  in  the  dark  is  i  m/xmole//xmole  of  chlorophyll. 

The  high-energy  phosphate  intermediate  suggested  by  these  experi- 
ments is  rather  unstable.  Our  measurements  show  it  has  a  half-life  of 
about  4  min.  in  the  reaction  mixture  at  5°. 

Isolating  and  identifying  an  unstable  intermediate  may  be  a  formidable 
job.  We  have  not  yet  attempted  to  do  so.  Instead,  we  have  discovered, 
solubilized  and  partially  purified  an  enzyme  which  might  either  be,  or 
function  close  to,  our  theoretical  X  ~  P. 

The  enzyme  is  one  which  causes  an  exchange  of  the  third  phosphate 
from  ATP  to  ADP.  It  would  appear  to  be  analogous  to  the  ADP-ATP 


70 

1          J 

Equ 

llbnum 

value                                         1 

.-• 

60 

- 

^^,^ ^^^ 

50 

- 

/* 

40 

/ 

30 

/■ 

20 

/ 

10 

- 

1 

1       _i 1 1 1 

6        8        10 
Time  (mm) 


14       16 


Fig.  2.  Time  course  of  ADP-ATP  exchange  reaction.  Reaction  mixture  was 
o-i  ml.  total  volume,  containing  [i*C]-ADP  at  5  x  lo"^  m,  ATP  at  i  x  10^=^  M, 
MgCU  at  3  X  lo"^  M,  tris  at  2  x  10"^  M,  and  solubilized  enzyme,  the  whole  brought 
to  pH  8-0.  Reactions  were  run  at  30"  for  the  length  of  time  shown. 


exchanging  enzyme  isolated  by  Wadkins  and  Lehninger  [9].  Activity  is 
measured  by  incubating  labelled  [^^CJ-ADP,  unlabelled  ATP,  the  enzyme 
and  Mg  for  5  to  10  min.  The  reaction  products  are  separated  chromato- 
graphically  and  counted  separately.  Figure  2  shows  that  the  label  in  ATP 
comes  to  equilibrium  with  that  in  ADP,  in  proportion  to  their  relative 
concentrations.  The  high  value  in  ATP  at  zero  time  is  due  to  a  20% 
contamination  of  p^CJ-ADP  (Schwartz  Biochem.  Co.)  with  [i^CJ-ATP. 

Purification  of  the  enzyme  (to  be  reported  elsewhere)  involved  extrac- 
tion from  chloroplasts  by  blending  with  water,  followed  by  two  acetone 
fractionations  and  several  cycles  of  freezing  and  thawing.  This  results  in  a 
solution  which  has  80"  „  of  its  protein  under  one  peak  in  electrophoresis, 
with  two  minor  components.  Upon  analysis  of  various  fractions  in  a 
preparative    electrophoresis    apparatus    (designed    and    constructed    by 


ATP    FORMATION    BY   SPINACH    CHLOROPLASTS  459 

Mr.  L.  Choules  and  Dr.  R.  B.  Ballentine  at  Johns  Hopkins  [lo])  the  activity 
appears  to  be  associated  with  the  major  component. 

The  enzvme  characteristics  known  so  far  are  compatible  with  a  role 
in  phosphorylation.  The  pH  optimum  is  a  broad  one,  around  7  to  8.  Mg 
ions  are  needed,  with  50",,  activity  at  about  5  x  io~^  m.  Mg  is  replaceable 
by  Mn,  Fe  or  Co  ions.  Although  Ca  has  some  low  activity  by  itself,  it  is 
inhibitory  when  combined  with  Mg.  Zn,  Hg,  and  Cu  are  inhibitors. 
Ammonium  ions  are  weak  inhibitors  also,  with  50",,  inhibition  occurring 
at  I  X  io~^  M.  Dinitrophenol  has  \irtually  no  effect. 

Efforts  to  show  an  irrele\ant  acti\ity  for  this  enzyme  have  so  far  failed. 
There  is  no  ATP--'^-P|  exchange,  no  myokinase  activity,  no  pyrophosphatase 
or  RNAase,  no  phosphatase  for  glucose-6-phosphate,  fructose-6-phos- 
phate,  fructose- 1, 6-diphosphate,  phosphoglyceric  acid  or  phospho- 
glvcerol.  We  can  rule  out  glutamine  synthetase,  protein  phosphokinase 
(either  with  phosvitin  or  casein  as  substrate)  and  polynucleotide  phos- 
phorvlase.  Xo  transfer  of  high-energy  phosphate  to  ADP  occurs  from 
carbamvl  phosphate,  phosphoenolpyruvate,  acetylphosphate,  phospho- 
serine  or  phosphocreatine.  Other  dinucleotides  serve  only  weakly  as 
phosphate  acceptors  from  ATP:  GDP  at  9",,  of  the  rate  of  ADP,  CDP  5"o, 
UDP  19",,,  and  I  DP  14",,.  No  products  are  formed  other  than  ADP  or 
ATP  in  the  usual  reaction,  and  no  other  compounds  (aside  from  Alg  ions) 
need  to  be  added  even  to  the  highly  dialyzed  enzyme. 

In  short,  there  is  no  sign  of  any  reaction  except  for  the  exchange  of  a 
high-energy  phosphate  from  ATP  to  ADP.  There  is  a  very  real  possibility 
that  the  reaction  proceeds  through  a  high-energy  phosphate  on  the  enzyme  : 

ATP  +  E  ^  E  -  P  +  ADP  (4) 

If  this  is  the  case,  the  E  ^  P  might  be  related  to  or  a  part  of  the  X  ^  P 
shown  in  the  pre-loading  experiments.  Our  future  efforts  will  be  devoted 
to  testing  these  aspects  of  the  problem. 

References 

1.  .Arnon,  D.  I.,  Whatley,  F.  R.,  and  Allen,  M.  B.,  Science  127,  1026-1034  (1958). 

2.  .Avron,  M.,  Krogmann,  D.  \\.,  and  Jagendorf,  A.  T.,  Bioc/iitn.  hiuphys.  Acta 
30,  144-153  (1958)- 

3.  Avron,  M.,  and  Jagendorf,  -A.  T.,^'.  biol.  Chetn.  234,  967-972  (1959). 

4.  Jagendorf,  A.  T.,  Brookharoi  Syvip.  Biol.  II,  236-258  (1958). 

5.  Jagendorf,  A.  T.,  Fed.  Proc.  18,  974-984  (1059). 

6.  Avron,  M.,  and  Sharon,  X.,  Biochevt.  biophys.  Res.  Cnmm.  2,  336-339  (i960). 

7.  Schultz,  A.  R.,  and  Boyer,  P.  D.,  Plant  Physiol.  35,  Sitppl.,  xxi  (i960). 

8.  Kahn,  J.  S.,  and  Jagendorf,  A.  T.,  BiocJiem.  biophys.  Res.  Conini.  2,  259-263 
(i960). 

9.  Wadkins,  C.  L.,  and  Lehninger,  A.  L.,  "7.  hiol.  Clieni.  233,   1589-1597  (1958). 
10.    Choviles,  G.  L.,  and  Ballentine,  R.  B.,  Analyt.  Bioclieni.  2,  59  (1961). 


460  ANDRE   T.    JAGENDORF   AND   JOSEPH    S.    KAHN 

Discussion 

Lehninger  :  I  guess  I  do  not  need  to  say  how  delighted  I  am  to  hear  about  the 
occurrence  of  the  ATP-ADP  exchange  in  photosynthetic  phosphorylation.  This 
finding  of  course  establishes  continuity  between  oxidative  and  photosynthetic 
phosphorylation.  The  inhibition  by  ammonium  ion  is  very  interesting  and  I 
wonder  if  you  have  any  evidence  for  a  requirement  of  or  an  inhibitory  effect  of 
either  potassium  or  sodium. 

Jagendorf  :  No,  we  haven't  tested  that.  We  are  suspicious  of  the  significance 
of  ammonium  inhibition  because  it  occurs  with  the  highly  purified  enzyme  without 
any  chloroplasts  present.  The  dinitrophenol  sensitivity  of  your  ATP-exchanging 
enzyme  requires  both  the  presence  of  the  particles  and  re-coupling  back  into  them, 
which  is  much  more  suggestive. 

Lehninger:  Well,  of  course,  DNP  and  ammonium  ion  do  not  necessarily 
uncouple  at  the  same  site. 

Baltscheff-Sky  :  I  have  two  questions.  First,  was  the  light  TCA  control  always 
higher  than  the  dark  TCA  control  ?  Second,  did  you  use  arsenate  instead  of 
phosphate  and  did  you  in  that  case  obtain  an  intermediate ;  if  so,  was  it  more  stable 
or  less  stable  than  the  phosphate  intermediate  ? 

Jagendorf:  The  light  TCA  control  will  always  be  a  little  bit  higher  than  the 
dark  TCA  control  because  in  the  light  when  '^"P  is  present,  all  one  needs  is  a  trace 
of  residual  ADP,  or  a  little  break-down  of  the  large  amount  of  ATP  present  to 
ADP,  and  ^-P-ATP  will  be  formed.  By  being  careful  in  eliminating  ADP  from  the 
system  and  especially  by  using  short  times  to  prevent  ATP  break-down,  we  can 
keep  this  control  down  to  a  minimum.  Also,  if  there  were  a  slow  exchange  reaction 
you  would  see  it  in  the  control  where  TCA  is  added  after  the  light  but  before 
ADP;  I  think  that  the  fact  that  we  have  a  15-sec.  exposure  rather  than  several 
minute  exposure  helps  a  lot  in  that  respect. 

Arsenate  does  inhibit  somewhat  if  you  put  it  in  with  the  phosphate  but  whether 
it  forms  an  intermediate  or  not  I  don't  know.  Even  if  arsenate  did  form  an  inter- 
mediate we  would  not  be  able  to  trap  it  in  a  stable  compound  analogous  to  trapping 
the  high  energy  phosphate  intermediate  as  ATP. 

Vennesland:  Would  you  care  to  comment,  Dr.  Jagendorf,  on  the  mechanism 
of  the  arsenate  efTect  that  you  observed  previously  ?  I  am  referring  to  the  ATP- 
dependent  stimulation  of  ferricyanide  reduction  by  arsenate.  This  appeared  to  be 
a  real  and  rather  striking  phenomenon,  and  I  wonder  how  you  would  explain  it  ? 

Jagendorf  :  Well  yes,  it  is  a  real  phenomenon ;  at  that  time  we  had  only  thought 
of  two  possibilities :  (a)  that  there  was  a  stable  high  energy  arsenate  intermediate, 
or  {b)  that  ADP  comes  first.  Now  we  find  that  ADP  doesn't  come  first  but  another 
possibility  has  occurred  to  us  since ;  perhaps  the  chloroplasts  have  membranes,  not 
the  external  membrane  but  ones  surrounding  the  actual  site  of  phosphorylation. 
If  penetration  of  arsenate  or  phosphate  were  to  require  the  presence  of  ADP  that 
would  explain  the  ADP  requirement  for  arsenate  uncoupling.  However,  when  we 
broke  chloroplasts  up  into  particles,  uncoupling  by  arsenate  still  required  ADP. 
This  experiment  seems  to  argue  against  the  concept  of  arsenate  entry,  which 
leaves  us  now  with  the  feeling  that  there  may  very  well  be  a  stable  high-energy 
arsenate  intermediate  bound  to  the  enzymes. 


ATP   FORMATION   BY   SPINACH   CHLOROPLASTS  46 1 

Smith  :  There  is  a  very  interesting  effect  in  photosynthesis  which  is  caused  by 
irradiation  of  the  plant  with  two  wavelengths  at  the  same  time  which  is  called  the 
Emerson  enhancement.  When  these  two  wavelengths  are  used  together  they  give 
more  photosynthesis  than  when  used  separately.  It  has  been  found  that  when 
these  two  wavelengths  are  given  alternatively  the  enhancement  occurs  as  well  as 
given  simultaneously  and  the  effect  will  last  as  long  as  15  sec.  between  irradiation 
with  the  two  wavelengths  of  light.  In  other  words,  times  can  go  as  high  as  15  sec. 
between  the  radiation  with  the  long  wavelengths  and  that  which  is  absorbed  by 
chlorophyll  b  or  some  form  of  chlorophyll  a,  and  my  colleagues  have  been  wonder- 
ing whether  it  is  phosphorylation  or  something  of  this  nature ;  or  could  one  have 
an  intermediate  of  the  form  that  you  have  suggested  ?  Have  you  done  any  action 
spectrum  on  this  at  all  to  see  whether  it  is  formed  by  chlorophyll  a  or  chlorophyll  h  ? 

Jagendorf:  We  previously  obtained  an  action  spectrum,  and,  as  you  probably 
know,  Hoch  and  Kok,  at  Glenn  L.  Martin  Co.,  Baltimore,  have  run  some  action 
spectra  for  phosphorylation  recently.  The  picture  is  still  a  little  bit  confused,  I 
think,  but  briefly  it  looks  to  me  as  if  the  requirement  for  the  accessory  pigment 
occurs  only  when  oxygen  evolution  occurs.  Now  phosphorylation  can  be  supported 
either  by  an  electron  transport  cycle,  or  by  the  series  of  reactions  leading  to  oxygen 
evolution.  In  the  experiments  of  Hoch  and  Kok  there  seems  not  to  have  been  any 
oxygen  evolution,  and,  correspondingly,  no  accessory  pigment  illumination  was 
needed  for  phosphorylation.  In  our  experiments  I  think  now  that  there  probably 
was  some  oxygen  evolution,  and  we  did  need  accessory  pigment  illumination  for 
phosphorylation.  I  want  to  emphasize  that  the  existing  data  are  not  complete  and 
1  don't  want  to  sound  too  positive.  But  I  think  quite  clearly  Kok  does  find  some 
phosphorylation  going  when  only  chlorophyll  a  is  being  illuminated,  which  would 
rule  out  our  present  intermediate  as  the  one  where  the  second  pigment  participates. 

Packer  :  I  would  like  to  comment  on  the  possibility  of  the  existence  of  an 
arsenate  energy-rich  intermediate  or  sort  of  factoral  approach  in  our  studies  of 
the  swelling  and  shrinking  phenomenon  in  mitochondria.  We  titrated  phosphate 
to  get  a  certain  swelling  level  and  we  soon  noticed  a  change  in  the  level  of  the 
intermediates,  so  the  interesting  thing  was  that  we  could  do  the  same  type  of 
experiment  with  arsenate  and  it  titrated  to  exactly  the  same  swelling  level,  although 
the  arsenate  requirement  is  slightly  different  from  phosphate  and  we  presume 
from  this  that  it  miyht  indicate  that  an  energy-rich  arsenate  intermediate  could 
exist. 


INTACT  CELLULAR  STRUCTURE  AND 
FUNCTION 


Chairman's  Introduction :  Remarks  on  Control  of 
Structure  and  Differentiation  in  Cells  and  Cell 

Systems 

J.    RUXXSTROM 

The  Weuuer-Gyeii  Institute  fur  Experimental  Biology, 
University  of  Stockholm,  Szceden 

I  have  the  honour  of  opening  the  section  on  "  Intact  Cellular  Structure 
and  Function". 

It  must  be  my  first  duty  to  discuss  the  significance  of  the  title  of  our 
section.  It  could  refer  to  researches  in  which  only  intact  cells  were  used  as 
material.  I  do  not  think  such  a  study  would  be  very  rewarding.  Xo  biologist 
would  refrain  from  carrying  out  experiments.  The  title  may  rather  refer  to 
studies  which  aim  at  understanding  the  structure  and  function  at  the  level 
of  the  intact  cell.  With  such  a  definition  of  our  task  the  essential  difii'erence 
between  this  section  and  the  preceding  ones  tends  to  decrease  or  disappear. 
I  suppose  that  everybody  who  studies,  for  example,  control  mechanisms  in 
isolated  mitochondria  or  microsomes  has  the  hope  that  these  mechanisms 
apply  also  to  the  living  cell.  Let  us  call  such  systems  models.  The  more 
complete  these  are  with  respect  to  cellular  components  the  more  they  may 
bear  upon  the  conditions  in  the  intact  cell.  On  the  other  hand  we  must  be 
aware  that  when  we  go  from  the  study  of  cell  components  to  that  of  the 
whole  cell  we  have  to  count  with  new  interactions  that  may  seem  to 
complicate  the  situation  to  a  large  extent. 

A  concentric  approach  by  difi'erent  experimental  methods  seems  to  be 
the  strategy  to  be  adopted.  Neither  of  the  methods  may  be  able  to  give  a 
satisfactory  answer  but  an  increasing  insight  may  be  gained  from  a  com- 
bination of  the  different  approaches.  Sometimes  one  may  also,  on  the  level 
of  the  intact  cell,  distinguish  the  alternation  between  complexity  and 
simplicity  to  which  Dr.  Kendrew  drew  attention  when  he,  on  the  first  day 
of  this  Symposium,  dealt  with  the  structure  of  the  protein  molecule. 

For  a  long  time  many  workers  have  been  interested  in  the  physical 
state,  or  let  us  call  it  the  consistency  of  the  cytoplasm.  Before  the  com- 
plicated electron  micrographs  that  Dr.  Porter  has  shown  us,  this  problem 
seems  at  first  sight  rather  meaningless.  It  is  also  generally  agreed  that  we 
cannot  give  an  overall  estimate  of  the  consistency  of  the  cytoplasm  on  the 

vol..   II. — 2H 


466  J.    RUNNSTROM 

basis  of,  for  example,  centrifuge  experiments.  The  consistency  may  vary 
in  different  regions  in  the  cytoplasm  as  Allen  and  Allen  and  Roslansky 
have  confirmed  in  an  elegant  way  by  studies  on  amoeba  [i,  2].  What  we 
study  by  centrifugation  experiments  or  by  other  methods  may  seem  in  the 
first  place  to  be  the  consistency  of  the  ground  cytoplasm  or  matrix  in  which 
the  other  components  are  embedded.  The  centrifugation  and  other 
methods  may  thus  be  able  to  give  us  certain  information  about  the  changes 
which  occur  in  the  matrix.  These  changes  may  indeed  be  impressive.  A 
stratification  of  the  inclusions  occurs  very  readily  when  an  unfertilized  egg 
of  the  sea  urchin  Arbacia  is  submitted  to  an  acceleration  of  4000-5000  x  g 
for  some  minutes,  whereas  15  min.  after  fertilization  practically  no 
stratification  occurs  under  the  same  conditions.  These  variations  in 
consistency  have  been  particularly  studied  by  Heilbrunn  and  co-workers 
[6,  7,  8].  The  ground  cytoplasm  is  evidently  a  very  complicated  system. 
It  contains  a  number  of  elements  among  them  certainly  fibrous  proteins 
that  may  be  mainly  responsible  for  the  changes  in  consistency.  These  changes 
are  often  characterized  as  gelations  or  solations,  which  expressions  indicate 
variations  in  the  intermolecular  binding  forces.  We  look  forward  to  the  time 
when  electron  microscopy  will  be  able  to  demonstrate  such  changes  in  the 
cytoplasmic  matrix.  As  gelations  and  solations  may  be  localized  in  definite 
regions  of  the  cell  they  may  play  a  role  in  the  cell  machinery,  particularly 
in  the  division  of  the  cell  and  in  difi^erentiation. 

Contraction  may  be  regarded  as  gelation  in  an  accidentally  or  per- 
manently oriented  fibrillar  system.  Its  role  in  amoeboid  movements  will 
be  analvzed  in  Dr.  Allen's  paper,  whereas  Dr.  Gustafson  will  demonstrate 
the  great  role  that  apparently  random  cell  movements  play  in  bringing 
about  strictly  regulated  morphogenetic  processes. 

In  Fig.  I  I  have  roughly  outlined  a  curve  from  a  paper  in  press  [23]. 
The  unfertilized  eggs  of  the  sea  urchin  Paracentrotus  lividus  were  exposed 
for  15  min.  to  varying  concentrations  of  crystalline  trypsin.  After  the 
treatment  the  eggs  were  thoroughly  washed  with  pure  sea  water.  There- 
after the  eggs  were  fertilized.  When  trypsin  concentrations  of  io~^-io^*  % 
were  used  the  pretreatment  caused  blockage  of  segmentation  in  maximally 
80-90%  of  the  eggs.  The  blockage  was  evidently  due  to  a  gelation  of  the 
cytoplasm.  This  was  also  confirmed  by  centrifugation.  Even  before 
fertilization  a  decreased  stratification  was  observed  in  those  eggs  that  had 
been  pretreated  by  trypsin  concentrations  in  the  range  of  maximum  effect 
on  the  cleavage.  The  gelation  caused  by  lower  concentrations  of  trypsin  is 
reversed  by  higher  concentrations  of  the  enzyme.  ATP  in  concentrations 
of  5  X  10^^  M  enhances  the  gelation  effect  if  it  is  added  after  a  pretreatment 
of  the  eggs  with  a  trypsin  concentration  that  is  not  sufficient  to  bring  about 
the  maximum  gelation  of  the  cytoplasm,  cf.  first  arrow  from  the  left  in 
Fig.  I.  Added  ATP  may  in  such  cases  bring  the  eggs  to  the  maximum 


chairman's  introduction  467 

degree  of  gelation.  If  ATP  is  added  following  exposure  of  eggs  to  higher 
concentrations  of  trypsin  it  may  enhance  the  reversal  of  the  gelation. 

In  Ca^~-free  solutions  of  trypsin  this  latter  has  a  lower  gelating  effect. 
Exposure  of  the  eggs  to  glutathione  after  pretreatment  with  trypsin 
enhances  the  reversal  of  gelation.  From  a  number  of  such  experiments  it 
was  evident  that  the  gelation  is  not  directly  caused  by  the  trypsin  treatment 
but  this  latter  activates  an  enzyme  of  the  egg  cell  which  has  the  gelating 
effect.  If  the  dose  (time  x  concentration)  of  trypsin  is  increased  other 
enzymes  are  activated  which  cause  a  reversal  of  the  gelation.  Besides  the 
enzymes  of  cathepsin  B  type  there  are  at  least  three  other  proteolytic 
enzymes  present  in  the  sea  urchin  egg  with  their  optimum  activity  around 
the  neutral  point,  as  was  demonstrated  by  my  colleague  Dr.  G.  Lundblad 


IxlO 


IxlO' 
Percent  cr/st.  trypsin 


2x10 


Fig.  I.  Simplified  curve  from  Runnstrom  [22],  showing  the  effect  of  pre- 
treatment of  unfertilized  eggs  with  low  concentrations  of  crystalline  trypsin.  The 
eggs  were  fertilized  and  the  number  of  uncleaved  eggs  is  plotted  as  a  function  of 
the  trypsin  concentration  (duration  of  pretreatment:  15  min.).  The  block  of 
cleavage  indicates  gelation  processes  in  the  cytoplasm.  If  after  the  trypsin  exposure 
the  eggs  were  transferred  to  5  x  10^  M  ATP  an  enhancement  or  a  removal  of 
gelation  was  observed  according  to  the  level  of  concentration  of  trypsin. 


[16].  He  designates  them  as  Ei,  E2  and  E3,  of  which  the  first  and  last  are 
SH-enzymes.  It  is  not  excluded  that  the  gelating  action  may  be  assigned 
to  one  of  these  enzymes  and  the  reversal  of  gelation  to  two  other  enzymes. 
These  results  seem  to  indicate  the  possibility  that  changes  in  consistency 
of  the  cytoplasm  may  be  controlled  by  enzymes  of  proteolytic  character. 

If  homogenates  of  unfertilized  eggs  were  subjected  to  a  treatment  with 
ribonuclease  a  considerable  activation  of  the  proteolytic  enzymes  occurs. 
The  interpretation  was  that  these  enzymes  are  attached  to  the  ribonucleo- 
protein  granules  of  the  microsome  system  w^here  they  probably  have  been 
synthesized. 

In  conjunction  with  my  colleagues  Hagstrom  and  Low  [26]  I  demon- 
strated that  the  same  holds  true  for  a  factor  which  gelates  the  jelly  coat 
surrounding  the  egg.  This  coat  consists  of  a  complex  of  polysaccharides 


468  J.    RUNNSTROM 

and  protein,  cf.  [27].  We  call  the  active  agent  "jelly  precipitating  factor" 
which  may  be  identical  with  the  antifertilizin  of  F.  R.  Lillie  [14].  Later  on 
Hagstrom  found  that  direct  treatment  of  the  surface  of  intact  unfertilized 
eggs  with  ribonuclease  also  causes  the  precipitation  of  the  jelly  coat. 
Ribonucleoprotein  granules  are  thus  probably  present  also  in  the  surface 
of  the  eggs  as  was  earlier  suggested  by  the  work  of  Lansing  and 
Rosenthal  [12]. 

In  phase  contrast  it  is  easy  to  see  dark  spots  in  the  surface  layer  of  the 
egg,  the  so-called  hyaline  layer.  The  spots  represent  evidently  groups  of 
ribonucleoprotein  granules.  Upon  brief  treatment  with  ribonuclease  in  sea 
water  they  vanish.  The  hyaline  layer  is  rich  in  acid  mucopolysaccharides. 
The  structureless  layer  is  perforated  by  numerous  villi.  These  villi  are  the 
carriers  of  the  ribonucleoprotein  granules  which  are  attached  to  vesicles 
or  tubules.  According  to  my  view  the  microsome  system  extends  into  the 
tips  of  the  villi.  Electron  micrographs  put  at  my  disposal  by  my  colleague 
B.  Afzelius  are  in  keeping  with  this  view.* 

Under  certain  conditions  the  villi  may  be  strongly  enlarged  and  in  such 
giant  villi  both  groups  of  dark  granules  and  of  lipoprotein  tubules  w^ere 
observed.  In  certain  cases  the  tubules  could  be  followed  as  continuous 
structures  deeply  into  the  endoplasm  where  they  were  seen  to  be  connected 
with  the  astrospheres  in  the  architecture  of  which  tubular  lipoprotein 
structures  seem  to  play  a  role.  One  may  assume  that  the  microsome  system 
of  the  villi  control  the  state  of  the  cell  surface. 

At  the  segmentation  the  hyaline  layer  concentrates  to  the  equator  of  the 
egg  along  with  the  villi  and  their  content  of  microsomal  elements.  A  release 
of  a  gelating  agent  occurs  equatorially  at  the  onset  of  segmentationf  [24]. 

Cell  differentiation  is  also  on  the  programme  for  the  discussion  today. 
Dr.  W.  F.  Loomis  has  discovered  how  a  relatively  simple  factor,  carbon 
dioxide,  may  induce  the  formation  of  genital  cells  in  the  fresh  water 
cnidarian.  Hydra. 

If  I  may  be  allowed  to  persist  in  talking  about  our  own  results,  I  shall 
go  back  to  the  experiments  on  the  gelating  action  of  trypsin  on  the  sea 
urchin  eggs.  Relatively  few  of  the  eggs  develop  that  were  exposed  to 
io~^-io^^  ^'1,  trypsin.  If  we  examine  the  larvae  obtained  after  about  24  hr., 
cf.  Fig.  2,  we  can  distinguish  three  types :  (a)  rather  normal  larvae  with 

*  It  may  be  referred  to  Fig.  22  in  [27].  Vesicular  and  tubular  structures  are 
seen  in  the  villi,  cf.  also  Mercer  and  Wolpert  [19].  In  the  electron  micrograph  [27] 
150  A  granules  could  readily  be  seen  to  surround  at  least  some  of  the  vesicles.  The 
reproduction  does  not  give  justice  to  the  original  in  this  respect.  What  is  seen  in 
phase  contrast  as  dark  spots  corresponds  certainly  to  groups  of  granules  surround- 
ing a  vesicle. 

f  This  is  also  the  stage  in  which  the  groups  of  ribonucleoprotein  granules  are 
best  observed  in  phase  contrast. 


chairman's    INTRODUCTION'  469 

endomesoderm  formed,  (b)  larvae  with  a  rather  normal  ectoderm  but  the 
endomesoderm  dissolved  into  large  evidently  pathological  cells,  (c)  larvae 
which  are  animalized,  i.e.  the  whole  larva  consists  only  of  ectoderm, 
whereas  endomesoderm  has  not  or  only  incompletely  developed.  Let  us 
now  consider  a  certain  group  of  cells  in  the  endomesoderm  of  the  normal 
larva,  viz.  that  marked  bv  a  square  in  Fig.  2(a).  From  our  experiments  we 
must  infer  that  these  cells  have  the  potentialitv  both  for  endomesodermal 
and  for  ectodermal  differentiation.  In  the  diagram,  Fig.  3(^/)'  the  larger 
horizontal  \ector  (^  eg)  indicates  the  pathway  of  syntheses  which  have  a 
specifically  endomesodermic  trend.  The  opposite  horizontal  smaller 
vector  (An)  represents  a  pathway  for  syntheses  which  have  an  animal  or 
ectodermic  trend.  Moreover  there  are  other  vectors  that  represent  trends 


(b) 


(c) 


¥lG.  2.  ia),  {h),  (r),  three  possible  alternatives  of  differentiation  in  eygs  that  had 
been  pretreated  in  the  unfertilized  stage  with  6-5  x  lo"^',,  trypsin.  In  (a)  develop- 
ment is  normal,  a  certain  region  marked  by  a  square  becomes  normal  endoderm. 
In  (b)  the  ectoderm  forms  a  continuous  layer  that  presents  a  lower  degree  of 
differentiation  than  (a).  The  endomesoderm  is  dissociated  in  rounded  cells.  The 
region  corresponding  to  the  marked  region  in  (a)  consists  of  dissociated  cells  showing 
tendency  for  cytolysis.  In  (c)  ectodermization  or  animalization  has  occurred.  The 
marked  cells  again  constitute  an  epithelium  but  this  has  ectodermic  character. 
Some  few  mesenchyme  cells  appeared. 


that  are  common  to  cells  on  all  le\els  in  the  larva.  The  \egetal  pathwav 
dominates  in  this  region  but  the  animal  pathwav  is  not  suppressed 
altogether.  It  contributes  to  the  character  of  the  cells  on  this  level.  In  this 
wav  one  explains  the  fine  gradation  in  the  properties  and  beha\"iour  of  the 
cells  which  Dr.  Gustafson  has  demonstrated  and  certainly  will  refer  to 
later  today.  In  larvae  of  the  tvpe  of  Fig.  2{a)  no  injurv  was  observed, 
whereas  in  a  larva  of  the  type  Fig.  2{b)  the  threshold  of  injury  evidently  was 
low.  In  the  larval  region  marked  by  the  square  protein  synthesis  gradually 
became  blocked,  with  ensuing  dissociation  of  the  cells  gradually  followed 
by  cytolysis.  The  break  up  of  the  synthetic  pathways  is  also  indicated 
diagrammaticallv  in  Fig.  T,{b).  The  larvae  of  type  Fig.  2(r)  demonstrate 
however,  that  the  vegetal  pathway  evidently  has  a  lower  threshold  than 
the  animal  one.  The  vegetal  pathway  mav  now  be  eliminated  (jr  reduced. 
In  this  way  animalization  results,  cf.  also  diagram,  I-'ig.  t,{c).  W'q  find  that 


470  J.    RUNNSTROM 

the  marked  region  now  consists  of  cells  that  are  typical  for  a  certain  region 
of  the  ectoderm.  The  cause  of  the  injury  was  in  the  cases  Fig.  2(6)  and 
2{c)  the  pretreatment  with  a  low  concentration  of  trypsin.  This  has 
induced  the  activation  of  certain  proteolytic  enzymes  probably  of  proteo- 
lytic character  in  the  egg.  If  the  activation  is  limited  or  reversed  normal 
development  occurs  (Fig.  2(0)).  If  this  does  not  occur  the  activation 
continues  and  the  proteolytic  activity  is  such  that  the  synthetic  pathways 
may  be  interrupted.  It  was  concluded  previously  that  the  vegetal  pathway 

An    X -^.- >-  Veg  (q) 

D  '■ 

An   >^ • -*■  Veg  (b) 

An  X • *■  Veg  (c) 

Fk;.  3.  Diagram  to  illustrate  the  condition  with  respect  to  the  vegetal  and 
animal  vectors  or  pathways  at  the  level  of  the  square  in  the  larvae  represented  in 
Fig.  2.  In  (o)  the  vegetal  pathways  of  synthesis  dominate  (Veg),  but  certain  path- 
ways of  animal  synthetic  processes  are  also  open.  The  four  shorter  lines  represent 
pathways  that  are  common  to  all  levels  in  the  embryo.  In  (b)  both  the  vegetal  and 
the  animal  pathways  are  interrupted.  In  (c)  the  vegetal  pathways  have  been 
reduced  with  a  compensatory  increase  in  the  animal  pathways  which  bring  about 
an  animalization  of  the  marked  region. 

is  more  easily  blocked  than  the  animal  one  which  is  the  cause  of  animaliza- 
tion shown  in  Fig.  2(r).  The  animal  or  animalized  cells  are  in  general  more 
resistant  to  injury  than  the  vegetal  ones.  The  vegetal  pathway  gives  the 
impression  of  being  more  unstable.* 

*  The  recent  results  of  Leone  [13]  are  well  in  keeping  with  the  views  presented 
above.  He  carried  out  a  set  of  experiments  on  the  effects  of  ribonuclease  on 
embryos  of  the  sea  urchin  Arbacia  lixiila.  He  found  that  the  enzyme  tends  to 
inhibit  development  in  general  and  especially  the  differentiation  of  endoderm, 
without  animalizing  the  embryos.  His  results  may  be  explained  on  the  basis  of  our 
diagram  Fig.  3  {b).  On  a  vegetal  level  both  the  animal  and  vegetal  pathways  are 
interrupted  by  the  treatment  with  the  ribonuclease  because  the  formation  of 
ribonucleic  acid  is  the  prerequisite  for  protein  synthesis  along  both  pathways.  As 
a  consequence  also  the  prerequisite  for  animalization  of  the  vegetal  region  is 
lacking.  The  somewhat  greater  resistance  of  the  animal  region  of  the  embryo 
found  by  Leone  corresponds  to  the  result  illustrated  by  our  Fig.  2(6).  The  latter 
case  may  correspond  to  a  stronger  activation  of  hydrolytic  enzymes  in  the  vegetal 
as  compared  with  the  animal  region.  On  the  other  hand  structural  differences  may 
play  a  role  in  making  the  sensitive  sites  of  the  macromolecules  in  question  more  or 
less  accessible  to  the  attack  of  the  enzymes.  The  vegetal  part  of  the  embryo  is  also 
more  sensitive  to  disturbances  in  electrolvte  composition  of  the  medium,  as  to  lack 
of  K+or  SOr,  cf.  [21]. 


CHAIRMAN  S    INTRODUCTION  47 1 

The  vectors  of  Fig.  3  may  primarily  represent  pathways  of  protein 
synthesis.  As  experiments  with  labelled  precursors  show,  incorporation  of 
for  example  ['^C]-leucine  into  proteins  runs  parallel  with  the  incorporation 
of  [^^C]-adenine  into  ribonucleic  acid,  cf.  Markman  [18].  It  is  evident  that 
the  ribonucleic  acid  plays  the  same  fundamental  role  in  protein  synthesis 
in  the  sea  urchin  material  as  elsewhere.  As  far  as  it  is  possible  to  resolve 
the  sites  of  incorporation,  that  of  the  ribonucleic  acid  precursor  (e.g.  [^^C]- 
adenine)  occurs  primarilv  in  the  nuclei.  It  seems  thus  probable  that  the 
synthesis  of  ribonucleic  acid  on  which  the  protein  svnthesis  is  dependent 
occurs  in  the  nucleus.  I  trust  that  the  question  about  the  site  of  ribonucleic 
acid  formation  will  be  more  deeply  discussed  in  the  paper  by  Dr.  Prescott 
who  refers  to  another  material,  viz.  amoebae. 

ft 

An    Veg 


AP>^ 


•-VP 


Fig.  4.  Diagram  of  the  vegetal  and  animal  vectors  over  the  whole  embryo, 
VP  vegetal  pole,  AP  animal  pole.  Vectors  representing  the  opposite  synthetic 
pathways  on  the  same  embryonic  level  form  the  same  angle  with  the  vegetal- 
animal  axis  VP-AP. 


In  this  context  anabolic  processes  other  than  ribonucleic  acid  and 
protein  synthesis  must  be  disregarded.  It  may  only  be  noticed  in  passing 
that  my  colleague  Immers  found  also  indications  of  a  parallelism  between 
incorporation  of  amino  acids  into  proteins  and  an  incorporation  of  ["^'S]- 
sulphate  into  mucopolysaccharides. 

Figure  4  gives  a  diagram  of  the  vegetal  and  the  animal  \ectors  of 
ribonucleic  acid  and  protein  synthesis  over  the  whole  embryo  from  the 
vegetal  (VP)  to  the  animal  pole  (AP).  The  levels  in  the  embryo  are  given 
by  the  angles  that  the  vectors  form  with  the  baseline  (AP-VP).  There  is 
no  need  to  emphasize  how  grossly  simplified  this  picture  is.  Nevertheless 
it  may  be  of  some  use.  Different  kinds  of  ribonucleic  acids  must  be  formed 
in  the  nuclei  where  they  presumably  receive  their  "  information"  from  the 
deoxyribonucleic  acid,  cf.  discussion  in  [3]. 

As  indicated  in  Fig.  t,{(i),  certain  pathways  of  synthesis  may  be  inde- 
pendent of  the  level  in  the  animal  vegetal  system.  In  Fig.  4  emphasis  is 


472  J.    RUNNSTROM 

laid,  however,  on  the  pathways  that  are  responsible  for  the  animal  and 
vegetal  trends  of  differentiation.  The  scheme  will  indicate  that  the  dif- 
ferences between  the  different  embryonic  levels  are  primarily  of  a  quanti- 
tative rather  than  of  a  qualitative  nature.  One  of  our  simplifications  is 
certainly  to  refer  to  animal  or  vegetal  pathways  instead  of  to  families  of 
animal  and  vegetal  pathways.  It  is  to  assume  that  each  single  pathway 
corresponds  to  the  formation  of  one  specific  ribonucleic  acid.  The  intensity 
of  its  formation  is,  however,  regulated  in  the  system.  According  to  our 
view  the  primary  control  is  exerted  by  an  animal  and  a  vegetal  cytoplasmic 
centre.  Each  of  these  produce  certain  agents  which  spread  in  the  direction 
of  the  opposite  pole,  cf.  [21,  25].  This  view  is  well  supported  by  a  great 
number  of  experiments,  involving  operative  separations  and  transplanta- 
tions, or  transformations  obtained  by  chemical  means  as  those  described 
above,  cf.  [9,  15,  21]. 

The  main  point  of  attack  of  the  controlling  agents  is  probably  the 
nucleus  and  particularly  the  synthesis  of  ribonucleic  acid  within  the 
nucleus  [18,  25]. 

So  far  chemical  changes  of  the  ribonucleic  acids  have  not  been  directly 
demonstrated  during  the  development  of  the  sea  urchin  embryo,  cf.  [5]. 
It  is  of  some  interest  that  by  certain  cytochemical  tests  differences  between 
the  nuclear  ribonucleic  acids  of  the  animal  and  those  of  the  vegetal  em- 
bryonic region  could  be  demonstrated.  The  tendency  for  "unmasking"  of 
the  phosphate  groups  of  the  ribonucleic  acid  seems  to  be  greater  in  the 
former  than  in  the  latter,  cf.  [10,  17]  and  a  recent  review  [25].  These 
differences  become  obvious  only  in  the  stage  in  which  the  primary  mesen- 
chyme begins  to  immigrate.  The  gastrulation  initiates  more  direct  inter- 
actions between  the  germ  layers.  This  holds  not  only  for  the  material  so 
far  considered — the  sea  urchin  embryo — but  also  for  amphibia  and 
vertebrates  in  general.  I  have,  however,  to  refrain  from  details. 

In  the  progress  of  differentiation  mechanisms  arise  that  stabilize  the 
attained  differentiations.  These  mechanisms  may  act  by  repressions  to 
some  extent  analogous  to  those  found  in  bacterial  systems,  cf.  [11,  20].  As 
well  known,  one  has  here  been  able  to  distinguish  two  kinds  of  genes,  the 
structural  gene  and  the  regulating  gene,  the  latter  operating  bv  production 
of  repressor.  Just  as  "structural"  genes  underlying  for  example  the 
animal  and  vegetal  pathways  are  activated  in  the  course  of  early  embryonic 
development,  regulating  genes  may  also  gradually  be  activated.  The 
repressors  produced  may  act  at  the  cytoplasmic  or  the  nuclear  level.*  The 
latter  kind  of  repression  may  possibly  be  realized  in  the  work  of  Briggs  and 
King,  cf.  [4],  dealing  with  transplantations  of  nuclei  from  embryonic 
nuclei  of  frog  into  enucleated  egg  cells.  Such  nuclei  are  able  to  promote 

*  This  may  be  the  mechanism  of  "canalization"  in  the  sense  of  Waddington 
[29]. 


CHAIRMAN  S    INTRODUCTION  473 

development  of  the  egg  but  in  the  late  gastrula  or  in  the  neurula  stage 
restrictions  occur  that  have  been  demonstrated  particularly  in  endodermic 
nuclei.  With  respect  to  stabilization  a  variety  of  different  conditions  must 
be  expected  according  to  the  organism  under  consideration.  In  vegetalized 
sea  urchin  larvae  islets  of  ectoderm  may,  under  certain  conditions,  arise 
within  the  vegetal  endoderm  demonstrating  a  late  revival  of  apparentlv 
extinct  pathwavs  [22].  Certain  forms  of  regeneration  in  adult  organisms 
may  mean  a  similar  revival.* 

Xo  doubt  the  views  established  in  microbiological  research  would  allow 
a  unitary  conception  of  the  early  labile  embryonic  determination  and  of 
the  later  stabilization  which  still  leaves  the  door  open  for  revival  of  sup- 
pressed pathways.  To  get  the  firm  basis  for  generalizations  of  this  kind  an 
intensified  interaction  between  morphogenetic,  genetic  and  biochemical 
studies  will  be  necessary. 

My  rapid  account  on  differentiation  started  from  a  rather  special  case. 
Nevertheless  it  may  have  demonstrated  some  of  the  principles  and  prob- 
lems involved  in  this  function  that  results  in  a  gradual  unfolding  of  a 
multitude  of  structures.  The  function  is  based  on  the  structure  of  the 
genetic  system  but  without  the  interactions  that  arise  during  the  develop- 
ment the  normal  pattern  of  differentiation  would  not  be  realized. 


References 

1.  Allen,  R.  D.,jf.  biophys.  hi(jchem.  Cytnl.  8,  379  (i960). 

2.  Allen,  R.  D.,  and  Roslansky,  ].,jf.  hinpliv!;.  biochem.  Cytol.  6,  437  (1959). 

3.  Brachet,  J.,  and  Chantrenne,  H.,  Cold  Spr.  Horb.  Svmp.  quant.  Biol.  21,  329 
(1956). 

4.  Briggs,  R.,  and  King,  T.  J.,  /;/  "The  Cell",  \'()1.  I,  ed.  J.  Brachet  and  A.  E. 
Mirsky.  Academic  Press,  New  York,  537  (1959). 

5.  Elson,  D.,  Gustafson,  T.,  and  Chargaff,  E.,y.  biol.  Cheni.  209,  21X5  (1954). 

6.  Heilbrunn,  1..  X.^'J.  exp.  Zool.  30,  211  (1920). 

7.  Heilbrunn,  L.  y.,jf.  e\p.  Znol.  34,  417  (1921). 

8.  Heilbrunn,  L.  V.,  "The  Dynamics  of  Living  Pr(>t()plasm  ".  Academic  Press, 
New  York  (1956). 

9.  Horstadius,  S.,  Biol.  Rev.  14,  132  (1939). 

10.  Immers,  J.,  Exp.  Cell  Res.  10,  546  (1956). 

11.  Jacob,  F.,  and  Monod,  J.,  C.  R.  Acad.  Sci.,  Paris  249,  12N2  fi95Q)- 

12.  Lansing,  A.  L,  and  Rosenthal,  T.  B.,jf.  cell,  cnnip.  Physial.  40,  337  (1952). 

13.  Leone,  W,  Acta  embryo,  et  morph.  exp.  3,  146  (i960). 

14.  Lillie,  F.  R.,  "Problems  of  Fertilization".  Chicago  L'niversity  Press,  Chicago 

(1919)- 

15.  Lindahl,  P.  E.,  XaturTcissoiscliafteii  29,  673  (1941). 

16.  Lundblad,  G.,  Ark.  Kemi  7,  127  (1954). 

17.  Alarkman,  B.,  Exp.  Cell  Res.  12,  424  (1957). 

*  I  am  indebted  to  Dr.  F.  Jacob,  Paris,  for  stimulating  discussions  concerning 
the  subject  of  this  paragraph. 


474  J-    RUNNSTROM 

i8.  Markman,  B.,  Exp.  Cell  Res.  23,  118  (1961). 

19.  Mercer,  E.  H.,  and  Wolpert,  L.,  Exp.  Cell  Res.  14,  629  (1958). 

20.  Pardee,  A.  B.,  Jacob,  F.,  and  Monod,  ].,jf.  mol.  Biol.  I,  165  (1959). 

21.  Runnstrom,  J.,  "Verh.  Deutsch.  Zool.  Ges.  in  Tubingen",  32  (1954). 

22.  Runnstrom,  J.,  Arkiv.  Zool.  10,  523  (1957). 

23.  Runnstrom,  J.,  Exp.  Cell  Res.  22,  576  (1961). 

24.  Runnstrom,  J.,  Exp.  Cell  Res.  23,  145  (1961). 

25.  Runnstrom,  J.,  Pathologie  et  Biologie  (in  press)  (1961). 

26.  Runnstrom,  J.,  Hagstrom,  B.,  and  Low,  H.,  Exp.  Cell  Res.  8,  235  (1955). 

27.  Runnstrom,  J.,  Hagstrom,  B.  E.,  and  Perlmann,  P.,  in  "The  Cell",  Vol.  I,  ed. 
J.  Brachet  and  A.  E.  Mirsky.  Academic  Press,  New  York,  327  (1959). 

28.  Runnstrom,  J.,  and  Kriszat,  G.,  Ark.  Zool.  13,  95  (i960). 

29.  Waddington,  C.  H.,  "Principles  of  Embryology".  Allen  and  Unwin,  London 
(1954)- 


The  Central  Problems  of  the  Biochemistry  of 
Cell  Division 

Daniel  Mazia 

Department  of  Zoology,  L  nirersitv  of  Ca/ifornia, 
Berkeley,' Calif.,  U.S.A. 

The  frame  of  reference  for  any  consideration  of  cell  division  is  the 
whole  cell.  Entering  division,  it  acquires  poles  and  an  equator.  When  we 
contemplate  the  reproduction  and  distribution  of  the  genetic  equipment 
in  the  mitotic  cvcle,  we  can  no  longer  confine  ourselves  to  questions 
concerning  the  molecular  character  of  genetic  information,  but  now  must 
consider  how  it  is  packaged  into  chromosomes.  The  problems  of  the 
chromosome  involve  us  in  behaviour  and  mo\ement,  and  not  merely  the 
control  of  biosynthesis.  The  movements  are  rapid  and  orderlv ;  the 
distances  travelled  are  very  long  by  molecular  standards.  The  cell  as  a 
whole  divides  itself  in  a  way  that  is  consistent  both  in  timing  and  in 
geometry.  At  the  end  of  the  cycle,  we  have  two  full-fledged  cells,  each  with 
the  capacity  for  living  its  own  pri\ate  life,  where  previouslv  we  had  one. 
The  whole  operation  of  cell  division  is  not  only  a  large-scale  operation, 
being  played  on  a  cellular  stage  in  micron  dimensions,  but  is  also  a  purpose- 
ful one  in  an  intelligible  and  unpanglossian  sense. 

Having  to  deal  with  these  problems  of  large-scale  structure,  of  large- 
scale  polarity,  with  complex  but  sensibly  co-ordinated  movements,  and 
with  precise  timing,  the  student  of  cell  division  does  not  have  to  be 
reminded  of  the  need  to  correlate  Biological  Structure  and  Function,  the 
theme  of  this  Symposium.  The  correlations  are  built  into  his  every  problem. 

I  hope  that  this  introduction,  which  has  been  intended  as  a  descriptive 
characterization  of  the  problem  of  cell  division,  is  not  interpreted  as  an 
apology  for  its  difficulty  or  complexity.  Of  course,  cell  division  is  complex 
in  a  sense  in  which  a  single  biosynthetic  step,  for  example,  is  not,  but  it  is 
an  analyzable  complexity.  We  can  analyze  it  into  unit  processes  which  are 
not  quite  so  formidable  in  themselves.  Such  an  analysis  has  recently  been 
discussed  by  me  [i].  The  difficulties  are  real,  but  we  may  take  the  opti- 
mistic view  that  they  arise  chiefly  from  our  lack  of  a  biochemistry  of 
structure  and  of  multimolecular  phenomena.  This  could  hardly  have  been 
asked  of  the  infancy  or  adolescence  of  biochemical  science,  but  we  mav 
now  expect  it  from  its  maturity. 


476  DANIEL    MAZIA 

It  is  demonstrable,  in  a  sufficiently  lengthy  treatise  on  cell  division  (cf. 
[2]),  that  the  problems  of  cell  division  are  linked  in  some  way  to  almost  all 
of  the  biochemical  problems  of  the  cell,  yet  certain  events  may  be  regarded 
as  being  specifically  related  to  division.  Some  of  these  have  been  partly 
amenable  to  biochemical  attack  or  speculation,  and  these  will  be  outlined. 

I.  DNA  synthesis 

So  far  as  we  know,  the  life  of  an  individual  cell  does  not  depend  on 
continued  synthesis  of  DNA.  With  some  exceptions  that  should  not  be 
ignored  (cf.  [3]),  the  doubling  of  DNA  may  be  regarded  as  a  preparation 
for  division.  One  of  the  truly  important  discoveries  of  modern  cyto- 
chemistry has  been  the  demonstration  that  DNA  synthesis — in  cells  that 
divide  by  mitosis,  which  includes  all  plant  and  animal  cells-  takes  place 
between  divisions,  in  anticipation  of  division,  and  not  during  the  mitotic 
period.  In  differentiated  multicellular  organisms,  certain  categories  of  cells 
do  not  divide,  and  these  generally  do  not  synthesize  DNA,  but  retain  the 
DNA  received  from  the  division  at  which  they  arose.  Since  many  such 
cells  can  be  made  to  synthesize  DNA  and  to  divide  under  carcinogenic 
influences  or  merely  by  removing  them  from  the  organism,  their  failure  to 
synthesize  DNA  can  be  viewed  as  an  inhibition  imposed  by  their  environ- 
ment. If  DNA  synthesis  takes  place  at  all,  it  generally  goes  all  the  way  to  a 
doubling  of  the  original  amount.  In  a  larger  view,  the  "regulation"  or 
"  control "  in  the  sense  of  a  modulation  of  time  or  rate  or  ultimate  amount 
synthesized  is  not  a  serious  problem  for  the  present.  The  questions  of 
"control"  are:  (i)  How  can  DNA  synthesis  be  totally  suppressed  by  a 
great  variety  of  organismal  factors ;  hormones,  immunity  factors,  etc. ;  and 
(2)  why  does  it  stop  when  the  original  dose  of  DNA  has  just  doubled  ? 
The  first  question,  one  predicts,  will  be  solved  as  a  straightforward  though 
profound  biochemical  problem,  involving  such  variables  as  the  induction 
of  the  polymerizing  enzyme  or  of  enzymes  providing  the  nucleotide  pre- 
cursors or  in  terms  of  direct  inhibition  of  the  enzymes  assuming  that  they 
are  always  present.  There  are,  however,  structural  factors  of  major 
importance.  These  express  themselves  in  the  fact  that  DNA  synthesis,  in 
cells  of  higher  organisms  can  take  place  only  during  the  phase  of  the  cell 
cycle  from  telophase  to  the  next  prophase,  when  the  chromosomes  are  so 
thoroughly  extended  or  uncoiled  that  they  are  not  resolvable  with  the 
microscope.  It  cannot  take  place  during  the  mitotic  period  when  the 
chromosomes  are  coiled  into  the  compact  packages  by  which  we  recognize 
them.  The  condensation  of  the  chromosomes  for  mitosis  is  intelligible  in 
terms  of  the  requirements  for  moving  them  about.  The  fact  that  this 
condition  is  incompatible  with  DNA  synthesis  explains  the  discontinuity 
of  such  synthesis  in  the  life-history  of  cells  of  higher  organisms.  The  recent 


THE    CENTRAL    PROBLEMS    OF    THE    BIOCHEMISTRY    OF    CELL    DIVISION         477 

evidence  that  DNA  synthesis  may  be  continuous  during  the  hfe  cycle  of 
bacteria  creates  no  paradox ;  it  may  merely  be  telling  us  that  the  bacterial 
genetic  apparatus  is  not  required  to  go  through  a  mitotic  cycle. 

I  know  of  no  reasonable  speculation  to  account  for  the  fact  that  DNA 
in  cells  of  higher  organisms  only  doubles,  after  which  synthesis  ceases  until 
the  cell  has  divided.  If  such  a  limitation  is  not  inherent  in  the  enzyme 
system  it  may  be  referable  to  chromosomal  organization,  which  will  be 
considered  next. 

2.  Reproduction  of  the  chromosomes 

Even  if  one  regards  the  chromosome  genetically  as  a  package  of  DNA, 
the  problems  of  cell  division  draw  our  attention  to  packing  as  well  as  to  its 
contents,  and  even  to  the  handles  by  which  it  is  carried.  No  one  doubts 
that  the  chromosome  is  not  only  large,  but  also  chemically  complex.  It 
contains  at  least  as  much  protein  as  DNA,  and  lipids  and  RNA  have  also 
been  included  in  estimates  of  its  composition. 

Some  fundamental  questions  concerning  the  chemical  structure  of 
chromosomes  have  been  under  study  for  20  years  or  more  without  being 
resolved.  One  such  question,  rephrased  in  contemporary  form,  is  whether 
a  single  chromosome  can  be  viewed  as  an  enormously  extended  DNA 
molecule.  Such  a  state  of  affairs  would  greatly  simplify  the  theoretical 
structure  of  genetics,  for  it  would  abolish  an  otherwise  necessary  distinction 
between  coarse  and  fine-structural  genetic  phenomena,  and  would  tend 
to  validate  the  phage  or  the  bacterial  chromosome  as  a  general  genetic 
model.  Experimentally,  the  question  takes  this  form:  is  the  chromosome  a 
DNA  continuum  to  which  discrete  protein  units  are  attached  (cf.  [4]), 
a  protein  continuum  to  which  discrete  DNA  units  are  attached  (cf.  [5,  6]), 
an  assembly  of  nucleoprotein  macromolecules  which  are  linked  to  each 
other  by  bonds  weaker  than  covalent  bonds  (cf.  [7]),  or  is  it  composed  of 
alternating  segments  of  DNA  and  protein  ?  Unfortunately,  there  is 
plausible  evidence  for  all  of  these  views.  Quite  apart  from  the  elegancies 
of  genetic  theory,  we  need  a  decisi\e  answer  to  this  simple  question 
before  we  can  make  a  pointed  attack  on  chromosome  heliaviour  in  cell 
division. 

A  second  question  concerning  the  complexity  of  the  chromosomes 
concerns  their  fundamental  multiplicity.  To  what  extent  are  they  composed 
of  bundles  of  genetically  identical  units,  representing  redundancy  of  genetic 
information. 

Thus  far,  each  method  of  attack  on  the  question  leads  to  a  different 
answer.  The  geneticists  prefer  that  each  chromosome  be  a  single  element, 
for  if  it  is  a  bundle  of  identical  elements  the  interpretation  of  mutation 
becomes  difficult  in  a  number  of  ways.  The  cytologists  and  the  students 
of  chromosome  breakage  have  preferred  a  two-imit  chromosome  and  can 


478  DANIEL    MAZIA 

adduce  convincing  visual  evidence  for  it  (e.g.  [(S]).  The  electron  micro- 
scopists  present  us  with  evidence  of  a  still  higher  level  of  multiplicity, 
though  not  with  innnense  numbers  (e.g.  [9,  10]). 

There  is  still  a  third  level  at  which  the  complexity  of  the  chromosome 
must  be  considered;  this  involves  elements  which  are  not  "genie"  in  the 
usual  sense,  but  may  be  consistent  functional  parts  of  a  given  chromosome. 
One  is  the  nucleolus  (and  nucleolar  substance).  In  manv  cells,  there  are 
compact  nucleolar  bodies,  associated  with  definite  regions  of  given  chromo- 
somes. In  addition,  it  has  recently  been  shown  that  a  nucleolar  substance 
is  associated  A\ith  other  regions  of  the  chromosomes  [i  i].  Functionally,  the 
nucleolar  equipment  may  be  regarded  as  that  part  of  the  chromosome  which 
operates  at  the  RNA  stage  of  the  DNA-RNA-protein  relationship.  (A 
more  specific  statement  would  be  difficult  to  make,  and  need  not  concern 
us  here.)  A  second  functional  eonipoiient  of  the  chromosome,  and  the 
one  that  is  ot  crucial  importance  for  cell  di\ision,  is  the  kinetochore  or 
centromere.  This  is  a  distinct  body,  associated  with  each  chromosome, 
which  is  absolutely  essential  for  its  movements  in  ceM  di\  ision.  N'isually  it 
appears  as  the  point  at  which  the  chromosome  is  engaged  by  the  mitotic 
apparatus;  more  crudely  put,  it  is  the  "motor"  of  the  chromosome.  It  is 
not  only  essential  but,  if  lost,  is  irreplaceable.  In  short,  it  meets  one  of  the 
fundamental  recpiirements  of  a  reproducing  element.  We  know  nothing 
about  its  chemistry  nor  about  any  of  its  mechanisms,  but  its  beha\  iour  is 
as  exact  and  reproducible  as  that  of  any  element  of  the  cell. 

It  has  been  necessary  to  summarize  the  evidence  tor  a  chemical  com- 
plexity of  the  chromosome  in  order  to  raise  an  important  biochemical 
question  of  cell  di\  ision  ;  when  can  we  say  that  the  whole  chromosome  has 
reproduced  and  how  does  it  reproduce  }  We  are  asking  a  question  with 
which  Molecular  Biology  is  bound  to  be  confronted :  how  does  a  complex 
and^ — on  a  molecular  scale     "three  dimensional"  body  reproduce  ? 

I  ha\e  recently  suggested  [i]  that  the  reproduction  of  the  whole  chromo- 
some is  carried  out  by  a  "generati\e  "  method.  Starting  with  a  complete 
chromosome,  its  DNA  first  reproduces  by  a  genuine  replication  mechanism. 
This  is  the  exent  ot  concepfion  of  a  new  chromosome.  We  now  have  a 
complete  chromosome  plus  an  additional  allotment  of  DNA.  The 
"daughter"  DNA  now  serves  as  the  seed  or  centre  for  the  development  of 
a  complete  daughter  chromosome.  This  takes  time,  and  will  not  be 
completed,  according  to  our  fragmentarv  e\idence,  until  the  time  of  the 
next  following  di\  ision. 

This  picture  ot  a  generati\e  reproduction  of  the  chromosome  is  more 
easily  understood  from  a  diagram  than  from  a  \erbal  description  (Fig.  i). 
It  gi\es  us  a  sinifile  reason  why  the  chromosome  is  fundamentally  a  duplex 
structure.  In  any  system  reproducing  bv  a  generative  scheme,  where  a 
period  ot  dexelopment  is  required  between  conception  and  parturition,  we 


THE   CENTRAL    PROBLEMS    OF   TIIK    BIOCHEMISTRY    OF   CELL    DIVISION         479 

expect  to  find  two  generations  in  existence  at  the  same  time.  The  double 
cliromosome  is  not  always  strictly  double;  during  the  period  of  develop- 
ment it  contains  two  sets  of  DNA  but  may  consist  of  a  complete  parent 
chromosomal  unit  and  an  incomplete  daughter  chromosomal  unit. 

This  generative  scheme  of  chromosome   reproduction   seems   rather 
comjilicated,  but  no  niori'  so  than  the  better  known  case  of  the  reproduction 


END    DIVISION 


END  INTERPHASE 


END  /NAETA PHASE 


Fui.  I.  Diayrani  ol  a  possihlc  ^tncrativc-  plan  of  iipioiluction  ot  the-  whole- 
chromosome.  Chromosome  is  represented  as  genetically  duplex  at  all  times. 
DN.A  (symbolized  as  double  helix)  replicates  durinji;  interphase  and  is  simultaneous 
joined  to  histone,  which  may  be  synthesized  at  the  same  time  or  earlier.  Other 
chromosomal  proteins  (P)  are  synthesized  or  incorporated  later.  In  this  version, 
the  complete  reproduction  of  a  chromosome  strand,  including  the  reproduction 
and  splittinji;  of  the  kinetochores  (K)  is  not  completed  until  anaphase.  If  this  is  so, 
the  division  will  sind  one  "  old  "'  ami  one  "  new  "  straiul  to  each  pole.  I  f  the  rtpro- 
duction  is  completed  before  anaphase,  the  four  units  may  split  at  random.  'The 
reproduction  of  the  nucleolus  takes  place  during  division;  the  old  nucleolus  breaks 
down  at  prophase  and  two  niw  iiuekoli  appear  at  anaphase. 


of  bacterial  \iriKses.  There,  tlie  parent  "soma"  seems  to  be  rejected 
entirely,  the  DNA  reproduces  to  conceive  many  new  units,  and  the 
complete  imits  are  later  de\ eloped  aroimd  the  DNA  "seeds".  To  imagine 
that  the  whole  bacteriophage  reproduces  in  a  single  steji  would  now  seem 
absiu'd. 

These  questions  concerning  the  reproduction  oi  the  whole  chromosome 


480  DANIEL   MAZIA 

may  or  may  not  be  important  for  our  understanding  of  its  genetic  functions, 
but  they  may  be  the  heart  of  many  problems  of  cell  division :  the  way  in 
which  the  DNA  synthesized  in  one  generation  is  distributed  among 
descendants,  the  relation  between  the  timing  of  genetic  reproduction  and 
the  timing  of  cell  division,  the  realization  of  mutational  events,  and  all  of 
the  problems  of  the  realization  of  two  fully  operational  chromosome  sets 
from  one.  Some  of  the  experimental  problems  are  out  of  reach  at  present, 
but  others  are  simple  enough.  For  example,  there  is  preliminary  evidence 
[12]  that  some  of  the  chromosomal  proteins  are  in  fact  made  at  a  different 
time  than  the  time  of  DNA  synthesis.  On  the  other  hand,  the  histones 
seem  to  be  incorporated  into  chromosomes  in  parallel  with  DNA 
synthesis  [13]. 

The  reproduction  of  the  nucleolar  equipment  is  rather  unusual  in  that 
the  original  material  is  given  up  by  the  chromosome  in  prophase  and  two 
nucleoli,  containing  at  least  some  new  RNA  [14],  appear  at  late  anaphase. 
There  is  no  substantial  chemical  evidence  concerning  the  reproduction  of 
the  kinetochores,  although  there  are  many  interesting  cytological  inferences. 

3.  Chromosomes  in  the  mitotic  cycle 

The  structural  behaviour  of  the  chromosomes  in  the  mitotic  cycle  is 
one  of  the  dramatic  events  of  the  life-history  of  the  cell.  As  everyone 
knows,  the  chromosome  substance,  which  is  so  highly  extended  and 
attenuated  between  divisions  that  individual  chromosomes  cannot  be 
discerned,  undergoes  during  prophase  a  "condensation"  which  most 
cytologists  attribute  to  superimposition  of  several  orders  of  helix  formation. 
The  sense  of  this  event  is  clear  enough  in  terms  of  the  mechanics  of 
mitosis;  the  genetic  material  can  be  transported  in  compact  packages. 

A  little  can  be  said  about  the  physiological  import  of  this  "condensa- 
tion". As  has  already  been  mentioned,  the  condensed  state  of  the  chromo- 
some seems  to  be  incompatible  with  its  ability  to  synthesize  DNA ;  the 
evidence  for  this  is  quite  good.  A  case  can  be  made  for  the  proposition  that 
the  genetic  function  of  the  chromosomes,  the  control  of  synthesis,  is 
interrupted  during  the  period  when  they  are  condensed  [i]  but  we  need 
not  discuss  this  now.  Since  the  prophase  coiling  of  the  chromosomes  is 
the  most  convenient  signal  that  a  given  cell  is  committed  to  division,  its 
initiation  and  mechanism  represent  a  major  problem,  and  one  that  has 
excited  considerable  and  stimulating  speculation  [15,  16].  Many  of  the 
cytological  hypotheses  postulate  various  changes  in  structural  composition, 
such  as  the  packing  of  the  primary  genetic  threads  into  a  "  matrix",  but  I 
am  afraid  that  most  of  what  has  been  said  represents  ingenious  inference. 
We  simply  do  not  possess  any  solid  facts  that  bear  directly  on  chromosome 
coiling. 


THE    CENTRAL   PROBLEMS   OF   THE   BIOCHEMISTRY   OF    CELL   DIVISION        48 1 

On  the  other  hand,  cvtochemical  research  has  yielded  some  sohd  facts 
on  changes  of  chromosome  composition  during  the  mitotic  cycle :  (i)  There 
is  an  "  RNA  cycle".  Chromosomes  acquire  RNA  during  prophase,  carry 
it  through  the  period  of  their  mitotic  movements,  and  give  it  up  at  the 
end  of  the  mitotic  period  [17].  This  is  not  to  say  that  interphase  chromo- 
somes do  not  contain  some  RXA,  but  the  fact  that  they  acquire  more  of 
it  and  distribute  it  bv  the  mitotic  mechanism  raises  some  interesting 
speculative  possibilities  [i<S].  (2)  There  is  a  cycle  of  changes  in  staining 
properties  which  can  be  interpreted  as  the  incorporation  of  phospholipids 
in  chromosomes  at  prophase  and  its  release  from  the  chromosomes  at  the 
end  of  the  mitotic  period  [19].  (3)  The  chromosomes  lose  their  nucleolar 
substance,  including  an  unknown  component  identified  by  its  reactions 
with  silver,  during  prophase  and  reacquire  it  at  telophase  [11].  Various 
inferences  can  be  made  about  these  changes,  but  none  of  them  is  very 
compelling  as  yet.  All  we  can  say,  and  it  is  not  trivial,  is  that  the 
mitotic  cvcle  does  involve  some  important  changes  in  chromosome 
chemistry. 

!  4.  The  mitotic  apparatus :  general 

Once  the  chromosomes  have  reproduced,  the  problem  of  mitosis  is  to 
separate  the  daughters  and  to  collect  them  into  two  equivalent  nuclei.  The 
astoundinglv  precise  events  can  be  described  in  a  formal  way:  (i)  There 
are  two  poles ;  (2)  Each  of  a  pair  of  sister  chromosomes  may  be  engaged  by 
(or  "attracted"  to)  one  pole  and  the  two  may  not  be  engaged  by  the  same 
pole.  The  realities  are,  fortunately,  embodied  in  a  definite  structure,  which 
we  call  a  mitotic  apparatus  [20]. 

The  general  structural  features  of  the  mitotic  apparatus  are  these : 
(i)  It    is    a    "solid"    coherent    body,    having   properties    sufficiently 
different  from  the  rest  of  the  cvtoplasm  to  permit  its  isolation,  which  will 
be  described  below. 

(2)  It  is  looselv  describable  as  a  rather  unstable  gel  in  which  there  are 
oriented  regions  or  structures  which  are  observed  as  fibres,  both  at  the 
submicroscopic  and  microscopic  levels  [21].  In  contrast  with  the  rest  of 
the  cytoplasm,  it  does  not  contain  mitochondria  or  other  larger  particles, 
but  it  does  contain  smaller  particles  similar  to  those  seen  elsewhere  in  the 
cell  [22]. 

(3)  The  "  fibres  ",  observed  either  in  fixed  material  or  in  living  material 
with  the  aid  of  polarization  optics,  appear  to  include  specific  connections 
between  chromosomes  and  poles.  Visually,  it  would  appear  that  chromo- 
somes are  engaged  to  the  poles  bv  these  chromosomal  fibres  and  are 
"pulled"  to  the  poles  by  them.  At  least,  the  fibres  do  predict  the  paths  the 
chromosomes  will  follow. 

VOL.  II. 2  I 


482  DANIEL   MAZIA 

(4)  In  animal  cells  at  least,  and  conceivably  in  all  cells  dividing  by 
mitosis,  the  poles  are  not  an  abstraction,  but  are  represented  physically  by 
particles  called  "  centrioles  ".  The  polarization  of  mitosis  by  these  remark- 
able particles  depends  on  their  power  of  self-reproduction  and  on  the  fact 
that  they  move  apart,  following  their  reproduction,  in  a  definite  way  that 
is  superficially  describable  as  a  "repulsion".  The  movement  is  almost 
certainly  not  an  actual  repulsion,  and  does  not  follow  an  inverse-square 
relationship  [14].  While  the  reproduction  and  movement  of  the  centrioles 
is  a  major  problem  in  the  analysis  of  mitosis,  I  shall  say  no  more  about  it 
here,  but  will  refer  to  a  recent  publication  of  ours  [23].  There  is  a  lot  to 
say  about  centrioles,  but  not  as  chemistry. 

In  the  above  summary,  I  have  not  discussed  the  progress  that  has  been 
made  in  the  electron-microscope  study  of  the  mitotic  apparatus.  This  has 
been  reassuring  (cf.  [24])  to  the  extent  that  it  has  confirmed  the  "existence  " 
of  the  reasonable  structure  that  had  been  inferred  from  accumulated 
cytological  knowledge,  but  does  not  necessarily  make  life  simpler  for  the 
chemist  with  his  instinctive  homogenizer. 

5.  Isolation  of  the  mitotic  apparatus :  the  stability  problem 

The  mitotic  apparatus  is  a  large  body,  clearly  seen  in  living  cells  of 
many  kinds.  Sometimes  it  would  seem  to  occupy  a  very  large  fraction  of 
the  cell's  volume.  By  using  eggs  of  marine  animals,  which  may  be  obtained 
in  mass  quantities  and  which  divide  synchronously  following  fertilization 
in  the  laboratory,  we  may  obtain  sufficient  material  for  the  isolation  of  the 
mitotic  apparatus  for  chemical  study.  The  difficulty  of  achieving  such  an 
isolation  arises  from  the  fact  that  the  apparatus  is  so  unstable ;  if  we  break 
open  the  cell  in  any  of  the  media  that  are  so  satisfactory  for  other  sub- 
cellular structures,  the  mitotic  apparatus  simply  falls  apart. 

It  now  seems  to  me  that  the  instability  of  the  mitotic  apparatus  is 
perhaps  the  most  interesting  of  all  the  problems  of  its  structure,  and 
perhaps  holds  the  key  to  many  other  problems  of  cell  structure  which  we 
have  been  compelled  to  ignore.  I  shall  return  to  this  point  in  a  later  section. 
Experience  other  than  attempts  to  isolate  the  mitotic  apparatus  attests  to 
its  instability.  It  seems  to  disappear  from  the  living  dividing  cell,  sometimes 
reversibly,  under  many  chemical  treatments,  under  high  pressures,  at 
extremes  of  temperature,  etc.  It  loses  its  characteristic  orientation  very 
easily  in  vivo.  Its  structure  and  orientation  may  well  be  the  expression  of 
an  equilibrium  between  dissociated,  and  oriented-associated  molecules,  an 
equilibrium  that  is  sensitive  to  many  variables.  Such  an  equilibrium  has 
been  discussed  by  Inoue  [21].  If  this  is  a  proper  approach  to  the  stability 
of  the  mitotic  apparatus — and  I  now  think  it  is — we  could  hope  to  isolate 
it  in  one  of  two  ways.  The  easiest,  and  the  one  which  first  succeeded  in  the 


THE   CENTRAL    PROBLEMS    OE   THE   BIOCHEMISTRY   OF   CELL   DIVISION        483 

hands  of  Dr.  Katsuma  Dan  and  myself  [20],  is  to  stabilize  the  structure 
artificially,  risking  the  distortion  of  some  of  its  chemical  properties  but  at 
least  obtaining  it  as  a  pure  isolate  for  brute  analysis.  In  effect,  our  earlier 
methods — and  the  ones  on  which  a  good  deal  of  our  present  information 
depends — rested  on  the  stabilization  of  the  mitotic  apparatus  by  immersing 
dividing  cells,  usuallv  sea  urchin  eggs,  in  30",,  ethanol  at  —  10  .  F'ollowing 
this  stabilization,  we  could  free  and  clean  the  mitotic  apparatus  by  dispers- 
ing the  rest  of  the  cell,  which  did  not  appear  to  be  stabilized,  with  various 
detergents  and  other  dispersing  agents.  For  the  second  step,  we  in  our 
laboratorv  have  most  often  used  digitonin,  although  ATP  and  urea 
(unpublished  experiments  with  Dr.  Rollin  Hotchkiss)  were  also  effective. 
When  I  refer  to  results  obtained  by  these  methods,  I  shall  generally 
speak  of  the  "  alcohol-digitonin  method  ". 

A  second  and  more  demanding  approach  to  a  more  natural  isolation  is 
to  attempt  to  mimic,  in  the  isolation  medium,  the  conditions  in  the  cell 
which  provide  for  the  stability  of  the  mitotic  apparatus.  This  could  well 
be  hopeless  according  to  the  above-mentioned  hypothesis  of  a  dynamic 
stabilitv.  If  the  cell  must  be  continuously  active  in  some  way  to  sustain  the 
structure,  of  the  mitotic  apparatus,  then  this  activity  could  be  mimicked 
only  if  it  were  expressed  in  some  simple  terminal  product  or  condition. 
The  approach  to  such  a  method  depended  on  the  hypothesis  (discussed  by 
Mazia  [25])  that  the  molecular  interactions  responsible  for  the  structure  of 
the  mitotic  apparatus  involved  sulphur  bonds  and  possibly  S — S  bonds. 
On  the  basis  of  experience  that  will  not  be  discussed  here,  it  was  imagined 
that  an  intermolecular  ( — SH)-(S- -S)  equilibrium  might  be  "poised"  in 
one  direction  or  another  by  an  appropriate  SH  or  S — S  reagent.  To  poise 
it  in  the  direction  of  S — S,  we  chose  dithiodiglycol  (OHCH.,CH.,S — 
SCHoCHoOH).  Whether  or  not  the  reasoning  was  correct — and  one  must 
admit  that  it  was  somewhat  woollv — this  line  of  attack  finally  was  success- 
ful. The  mitotic  apparatus  could  be  isolated  in  a  medium  consisting  of 
isotonic  (i  m)  dextrose  or  sucrose,  lo^^  m  versene,  and  0-15  M  dithiodi- 
glvcol  at  pH  6 -0-6 -3.  An  important  point  is  that  it  is  unstable  if  the 
dithiodiglvcol  is  omitted  and  becomes  unstable  even  after  isolation  if 
this  substance  is  removed.  In  our  more  recent  work,  the  sucrose  medium 
has  proved  to  be  preferable  to  dextrose  for  the  purpose  of  eliminating  other 
cvtoplasmic  particles ;  otherwise,  I  am  not  sure  that  it  makes  much 
difference  which  sugar  is  used. 

Thus  this  method,  which  I  will  refer  to  as  the  DTDG  method,  is 
comparable  to  those  used  for  other  kinds  of  particles  with  the  exception  of 
the  requirement  for  dithiodiglycol.  It  must  be  said  that  we  have  not  yet 
compared  dithiodiglvcol  with  related  compounds.  The  essential  steps  of 
the  isolation  are  these:  (i)  Sea  urchin  eggs  are  inseminated  and  immedi- 
atelv  transferred  to  a  medium  of  Ca-free  sea  water,  versene  (o-oi  m),  and 


484  DANIEL   MAZIA 

mercaptoethylgluconamide  (o-ooi-o-oi  m).  The  purpose  of  the  mer- 
captoethylghiconamide,  to  which  we  were  introduced  by  Dr.  David 
Doherty  of  Oak  Ridge,  is  to  block  the  hardening  of  the  surface  layers  and 
fertilization  membrane  by  disulphide  formation.  The  principle  depends 
on  discoveries  made  at  the  Wenner-Grens  Institute.  (2)  The  fertilization 
membranes  are  removed  mechanically  by  passing  the  eggs  through  a  fine 


Fig.  2.  Mitotic  apparatus  isolated  directly  from  sea  urchin  eggs  by  a  new 
method,  ciescribed  in  the  text.  After  isolation,  the  preparation  was  exposed  to 
5  X  10^*  M  CaCia.  which  stabilizes  it  and  sharpens  the  appearance  of  the  fibres. 

silk  filter.  (3)  The  eggs  are  washed  in  Ca-free  sea  water.  This  is  very 
important,  as  traces  of  Ca  interfere  with  the  isolation.  (4)  As  they  approach 
metaphase,  the  eggs  are  washed  in  a  mixture  of  9  parts  isotonic  dextrose 
to  I  part  sea  water  to  lower  the  ionic  strength  of  the  medium.  (5)  At  the 
desired  stage  of  mitosis  they  are  suspended  in  the  sucrose-versene-DTDG 
medium.  Gentle  shaking  by  hand  suffices  to  break  the  eggs  and  free  the 


THE    CENTRAL    PROBLEMS    OF    THE    BIOCHEMISTRY    OF    CELL    DIVISION         485 

mitotic  apparatus,  suspended  in  a  smooth  homogenate  of  cytoplasm. 
(6)  The  mitotic  apparatus  may  readily  be  purified  and  washed  by  very 
low  speed  (200  500  g)  centrifugation.  Fortunately,  most  of  the  smaller 
particles  remain  suspended  in  the  dense  sucrose  medium. 

The  following  properties  of  the  mitotic  apparatus  as  isolated  bv  the 
DTDG  method  are  pertinent  and  interesting: 

(i)  They  are  extremely  sensitive  to  Ca  and  J\Ig,  becoming  irreversibly 
stabilized.  A  Ca"^  concentration  of  5  x  10"^  m  suffices  to  stabilize  them.  In 
this  form  they  are  beautiful  to  behold  (Fig.  2)  because  the  fibres  become 
highly  condensed,  but  we  cannot  dissolve  them  for  further  chemical  work. 

(2)  They  are  osmotically  sensitive,  swelling  and  shrinking  as  the 
sucrose  concentration  is  varied.  They  disperse  at  low  sucrose  concentrations. 

(3)  They  may  be  dissolved  in  a  number  of  ways  if  they  have  not  "  seen  " 
Ca++  or  Mg+^.  For  enzvme  studies  we  dissolve  them  in  isotonic  (0-53  m) 
KCl  at  pH  8.  This  has  the  advantage  that  the  osmotically  sensitive  yolk 
particles  which  are  a  major  contaminant  are  not  lysed  and  can  be  separated 
by  centrifugation,  along  with  other  particles,  probably  ribosomes,  that  are 
known  to  be  embedded  in  the  structure  [22]. 

It  would  be  brash  to  suggest  that  this  isolation  represents  achievement 
of  the  goal  of  obtaining  a  fully  natural  mitotic  apparatus.  A  more  limited 
objective  was  to  obtain  the  mitotic  apparatus  in  a  form  suitable  for  studies 
of  enzyme  activity,  especially  of  enzymes  concerned  with  ATP.  The 
methods  employing  alcohol  and  detergents  did  not  preserve  such  activitv ; 
while  the  DTDG  method  does.  A  second  objecti\e,  and  indeed  a  long-term 
ideal  of  these  studies,  was  to  obtain  the  isolated  mitotic  apparatus  as  an 
effective  "model"  in  the  Weber  [26]  sense  of  the  term.  So  far,  this  has 
failed.  We  have  found  no  conditions  imder  which  the  isolated  mitotic 
apparatus  will  mo\e  chromosomes. 

6.  Survey  of  the  chemistry  of  the  isolated  mitotic  apparatus 

The  alcohol-digitonin  method  provided  clean  preparations  of  mitotic 
apparatus  which  retained  the  essential  and  expected  morphological 
features,  and  which  could  be  regarded  as  suitable  for  the  study  of  some  of 
the  major  structural  macromolecules.  Much  of  the  information  that  has 
been  obtained  has  already  been  reviewed,  and  I  shall  only  list  the  findings. 

I.  The  mitotic  apparatus  isolated  by  the  old  method  consists  largelv 
of  protein.  Conjugation  of  RNA  to  the  protein  has  been  studied  in  some 
detail,  but  comparable  studies  have  not  been  made  on  conjugation  of  lipid 
or  carbohydrates.  There  seemed  to  be  little  point  in  analvzing  for  lipids 
after  isolation  with  digitonin,  and  we  have  tended  to  formulate  the  struc- 
tural problems  pretty  much  in  terms  of  protein  chemistrv.  In  view  of 
electron   microscopic   evidence   describing  the   filaments   of  the   mitotic 


486  DANIEL   MAZIA 

apparatus  as  tubular,  and  suggesting  the  presence  of  structures  reminiscent 
of  endoplasmic  reticulum  in  the  apparatus  in  some  cases,  we  are  now 
reconsidering  the  possible  significance  of  lipoproteins  in  the  mitotic 
apparatus.  The  newer  method  of  isolation  has  made  this  practicable. 

2.  The  RNA  content  is  relatively  high.  The  last  studies  [27]  give  a 
figure  of  about  (yi[j.  When  the  major  proteins  components  are  put  into 
solution,  and  partly  purified,  they  behave  as  ribonucleoproteins.  At  one 
time,  it  was  thought  that  adenylic  nucleotides  predominated  [8]  but  this 
was  erroneous.  Better  analyses  recovered  from  the  mitotic  apparatus 
as  RNA  having  about  the  same  nucleotide  composition  as  the  averaged 
RNA  of  the  whole  cell  (sea  urchin  egg)  [27].  The  present  hypo- 
thesis is  that  the  structure  proteins  are  in  fact  ribonucleoproteins.  If 
so,  we  are  confronted  with  the  question  of  function  of  the  RNA ;  there  is 
no  reason  to  think  that  mitotic  structure  or  action  involves  protein  synthesis. 

My  current  speculations  about  the  role  of  RNA  in  the  structure  of  the 
mitotic  apparatus  take  the  following  form.  If  RNA  carries  information 
regarding  amino  acid  sequences  in  proteins,  perhaps  this  information  may 
be  used  for  the  "recognition"  of  proteins  as  well  as  for  their  synthesis. 
Could  such  a  recognition  function  be  involved  in  the  assembly  of  a 
structure  by  association  of  like  molecules  ?  Of  course,  the  RNAs  need  not 
recognize  proteins ;  they  might  recognize  each  other.  If  the  speculation  is 
unsupported,  it  does  call  attention  to  a  question  that  is  easily  overlooked : 
how  does  genetic  information  operate  in  governing  the  structure  of  the 
cell  in  its  larger  sense,  as  well  as  the  structure  of  its  component  molecules  ? 

3.  By  electrophoretic  and  ultracentrifugal  criteria  [27]  and  by  immuno- 
logical criteria  [28]  the  major  protein  composition  of  the  mitotic  apparatus 
appears  to  be  simple,  probably  deceptively  so.  Two  or  at  most  three 
components  can  be  detected,  and  one  of  them  predominates.  It  seems  to 
be  a  protein  whose  molecular  weight  is  r.  315  000  [27]. 

4.  Amino  acid  analysis  [29],  data  cited  by  Mazia  [30],  shows  striking 
similarities  between  the  major  protein  of  the  mitotic  apparatus  and  actin 
from  vertebrate  muscles.  This  might  be  a  matter  of  chance,  but  might 
also  point  to  some  common  properties  of  structure  proteins  involved  in 
biological  movement. 

7.  Origin  of  the  mitotic  apparatus 

It  has  already  been  mentioned  that  the  mitotic  apparatus  occupies  a 
considerable  part  of  the  volume  of  a  dividing  cell.  Analyses  of  sea  urchin 
eggs  and  mitotic  apparatus  isolated  from  these  eggs  shows  that  the  mitotic 
apparatus  represents  an  investment  of  at  least  io°o  of  all  the  protein  in 
the  cell.  If  the  compositional  studies  cited  above  are  not  entirely  deceptive, 
this  is  mostly  protein  of  one  or  a  few  kinds.  The  mitotic  apparatus  is  not 


THE    CENTRAL    PROBLEMS   OF   THE   BIOCHEMISTRY   OF   CELL    DIVISION         487 

seen  in  cells  except  when  they  are  dividing.  The  question  is  whether  this 
amount  of  structuie  protein  is  made  as  the  mitotic  apparatus  is  formed  or  is 
made  earlier  and  assembled  at  the  time  of  division.  For  the  sea  urchin  egg, 
the  answer  seems  to  be  that  it  is  preformed.  This  was  shown  by  H.  A. 
Went  [28]  by  immunological  means.  He  has  demonstrated  that  the  egg 
before  division  contains  all  of  the  antigens  that  can  be  recovered  from  the 
isolated  mitotic  apparatus. 

While  such  a  finding,  if  general,  would  suggest  that  the  actual  formation 
of  the  apparatus  is  a  problem  of  assemblv  and  not  of  svnthesis,  it  does  not 
follow  that  the  synthesis  of  structural  protein  for  the  mitotic  machinery  is 
not  one  of  the  important  problems  of  the  biochemistry  of  cell  di\ision.  The 
egg  is  a  special  case,  a  cell  which  is  provided  with  enough  proteins,  includ- 
ing enzvmes,  for  a  long  period  of  de\elopment  and  it  undergoes  little  or 
no  net  growth.  In  a  growing  population,  each  cell  would  have  to  provide 
the  protein  for  the  mitotic  apparatus  of  the  next  division,  or  at  least  half 
of  it  if  it  "inherited"  half  from  the  previous  di\ision.  But  it  would  be 
important  for  our  thinking  about  the  control  of  cell  division  if  the  protein 
of  the  mitotic  machinerv  had  to  be  svnthesized  in  anticipation  of  a  future 
division.  As  Swann  [31]  has  pointed  out,  the  di\'ersion  of  proteins  and 
protein  svnthesis  into  or  from  the  formation  of  the  mitotic  apparatus  may 
be  an  interesting  factor  in  differentiation  and  the  control  of  division. 

8.  Thiol  chemistry  and  cell  division 

The  alchemists  never  succeeded  in  transmuting  sulphur  into  gold,  but 
the  biochemists  may  yet  do  so.  It  is  an  extraordinary  fact  that  theories  in 
which  thiols  plaved  a  central  part  have  been  prominent  in  discussions  of 
the  biochemistry  of  cell  division  ever  since  there  were  such  discussions. 
One  need  only  cite  Louis  Rapkine,  whose  work  on  a  glutathione  cycle 
during  mitosis  [^2]  was  the  stimulus  to  much  contemporary  work  (dis- 
cussed bv  Mazia  [2^],  Stern  [t,^])-  Among  others  who  early  felt  that  thiol 
biochemistrv  somehow  lav  at  the  heart  of  the  cell  division  problem  was 
Hammett  [34],  and  there  were  others.  In  recent  years,  the  study  of  what 
we  in  our  laboratory  call  the  "Thiology"  of  cell  division  -confessing  to 
an  ingredient  of  faith  as  well  as  of  good  works  in  this  line  of  study — has 
de\eloped  in  a  number  of  ways:  in  studies  on  metabolic  regulations 
associated  with  thiols,  in  studies  of  the  relation  between  soluble  and 
protein  SH  in  the  dividing  cell  [30,  35,  36,  37],  in  studies  on  the  inter- 
ference with  cell  division  bv  SH  compounds  [25,  3S],  in  the  demonstration 
of  the  participation  of  interesting  sulphur-containing  nucleotidepolypetide 
complexes  in  cell  division  in  algae  [39]  and  in  observations  on  a  specific 
role  of  sulphur-containing  amino  acids  in  the  synchronization  of  division 
in  algae  [40]. 


488  daniel  mazia 

(a)  bonding  of  the  mitotic  APPAR.\TUS 

So  far  as  the  mitotic  apparatus  is  concerned,  the  present  picture  is 
confusing,  although  not  in  an  unconstructive  way.  The  original  design  of 
an  isolation  method  by  Dan  and  myself  was  based  on  an  hypothesis  that 
S — S  bonds  were  involved  in  the  polymerization  of  macromolecules  into 
a  coherent  mitotic  apparatus.  We  proceeded  first  in  a  seemingly  strange 
way,  artificially  stabilizing  the  mitotic  apparatus  by  deliberately  making 
more  S — S  bonds  by  oxidation  with  peroxide,  but  it  did  work.  When  a 
method  was  developed  which  avoided  the  use  of  such  an  oxidizing  agent, 
the  isolated  mitotic  apparatus  seemed  to  be  an  S — S  bonded  structure, 
soluble  only  by  methods  which  reduce  such  bonds  [30].  Then  this  turned 
out  to  be  a  partial  oxidation  artifact,  for  it  was  discovered  by  Dr.  Zimmer- 
man in  our  laboratory  that  the  freshly  isolated  apparatus  which  had  been 
given  no  chance  to  oxidize  could  be  dissolved  by  salyrgan  and/)-chloromer- 
curibenzoate.  At  the  same  time,  Kawamura  and  Dan  [36]  showed  by 
cytochemical  means  that  the  mitotic  apparatus  during  the  stages  at  which 
it  was  forming  was  strikingly  rich  in  protein-SH,  which  became  less 
prominent  during  the  terminal  stages  of  mitosis.  It  would  be  satisfactory, 
and  would  meet  all  the  facts,  to  suppose  the  following:  (i)  That  the 
apparatus  contains  SH  groups  in  closely  apposed  pairs,  easily  oxidized  but 
not  necessarily  existing  as  S — S  links  in  the  living  condition,  and  (2)  That 
a  linkage  of  unknown  character  between  these  vicinal  SH  groups  holds 
the  molecules  together  in  the  structure  of  the  mitotic  apparatus.  Such  a 
link  might  be  split  by  agents  such  as  salyrgan  and  PCMB.  There  is  in- 
direct evidence  for  the  occurrence  of  such  thiol,  non-S — S,  linkages  in 
other  situations  (reviewed  by  Jensen  [41])  but  the  nature  of  the  bond  is 
unknown.  I  take  it  that  most  chemists  are  not  happy  with  the  idea  of 
hydrogen  bonding  through  SH,  although  it  has  been  defended.  In  general, 
the  idea  that  intermolecular  hydrogen  bonding  is  prevalent  in  the  mitotic 
apparatus  has  been  an  appealing  one  (e.g.  Gross  [42])  and  it  has  seemed  to 
some  that  an  hypothesis  of  extensive  S — S  bonding  was  not  consistent 
with  the  instability  of  the  mitotic  apparatus.  Thus,  the  situation  was  that 
the  hypothesis  of  an  S — S  bonded  system  led  to  certain  positive  results 
but  tests  of  the  hypothesis  always  favoured  the  implication  of  thiol  groups 
in  some  other  way.  It  was  a  looser  hypothesis  concerning  sulphur  bonding 
that  led  to  the  development  of  the  DTDG  method  of  isolating  the  mitotic 
apparatus  in  a  more  native  condition.  The  speculations  involved  will  be 
discussed  in  a  later  section. 

(b)  the  thiol  cycle 

It  should  have  been  stressed  earlier  that  the  instability  of  the  mitotic 
apparatus  is  not  merely  an  inconvenience  for  its  isolation ;  it  is  a  funda- 


THE   CENTRAL    PROBLEMS    OF   THE   BIOCHEMISTRY   OF    CELL   DIVISION        489 

mental  property  of  mitosis  in  the  living  cell.  The  mitotic  apparatus  cannot 
be  observed  as  an  organized  structure  in  the  cell  when  it  is  not  dividing, 
though  certain  parts  of  it  concerned  with  the  centrioles  may  be  present. 
It  can  be  said  to  appear  when  it  is  "needed"  and  to  disappear  when  its 
work  is  done.  One  way  of  saying  this  is  that  the  intracellular  conditions 
permit  its  stability  during  the  period  of  division  and  no  longer  do  so  when 
division  is  completed.  This  loose  statement  leads  to  a  very  speciiic  ques- 


Pseudocentrotus    depressus 


30        40        50        60        ^0        80 
Time     after    fertilization 


120    130 


Fig.  3.  Fluctuation  of  a  TCA-soluble  protein  or  polypeptide  during  the 
division  cycle  in  a  sea  urchin  egg  {Pseitdocoitrotus  depressus),  and  non-fluctuation 
of  glutathione.  Upper  curve;  total  — SH  soluble  in  25 "o  trichloroacetic  acid. 
Lines  B,  D,  E,  F,  G.  Soluble  — SH  after  extraction  of  eggs  with  saturated 
ammonium  sulphate  (B,  D),  after  precipitation  of  protein  from  TCA  extract  (E), 
and  after  dialysis  of  TC.\  extract.  C  shows  oxidized  glutathione  (from  Sakai  and 
Dan  [35]). 

tion;  is  the  intracellular  environment  ditTerent  during  division  and 
between  divisions  .'  It  was  already  demonstrated  by  Rapkine  in  193 1  [32] 
that  the  period  of  division  was  characterized  by  a  remarkable  fluctuation 
in  the  soluble  SH  content  of  the  cell;  the  "cycle"  involved  a  striking 
decrease  in  soluble  SH  during  the  early  phases,  up  to  about  metaphase, 
and  an  increase  during  the  later  phases.  Rapkine  identified  the  TCA- 
soluble  SH  component  as  glutathione.  The  situation  was  confused  when 
attempts  to  confirm  the  glutathione  cycle  as  such  failed  (e.g.  [43]),  but  a 
brilliant  study  by  Sakai  and  Dan  [35]  resolved  the  problem.  The  cycle 


490  DANIEL   MAZIA 

does  exist  but  it  is  not  a  fluctuation  of  glutathione.  Rather,  it  involves  a 
protein  or  polypeptide  that  is  soluble  in  TCA  but  is  precipitable  by  other 
protein  precipitants  (Fig.  3). 

Thus  it  can  be  confirmed  that  there  is  a  major  fluctuation,  during 
division,  of  an  SH-carrying  molecule  which  may  very  well  be  viewed  as 
"environmental"  and  not  part  of  the  structure  of  the  mitotic  apparatus. 
Other  and  equally  interesting  fluctuations  of  the  intracellular  medium  may 
well  be  found,  but  this  is  the  one  we  now  know  and  can  speculate  about. 
One  obvious  speculation  is  that  the  fluctuation  is  a  controlling  factor  in  the 
mechanochemical  operations  of  the  mitotic  apparatus,  in  the  same  sense 
that  other  mechanochemical  systems,  non-biological  systems  or  "models" 
of  biological  derivation,  can  be  driven  by  appropriate  changes  in  their 
surroundings. 

A  second  speculation,  and  one  more  relevant  to  the  problems  we  have 
been  discussing,  is  that  the  Sakai-Dan  cycle,  the  successor  to  the  gluta- 
thione cycle,  may  account  for  the  assembly  and  stability  of  the  mitotic 
apparatus,  during  the  division  period,  and  its  instability  at  the  end  of  the 
division  period.  Some  years  ago  I  proposed  a  mechanism  of  how  this  could 
take  place  by  the  reduction  of  intramolecular  disulphide,  followed  by 
reoxidation  to  form  intermolecular  disulphide  links  [30,  30a].  For  reasons 
given  above,  I  would  no  longer  stress  the  importance  of  conventional  S — S 
bonds  as  such,  but  the  principle  may  yet  hold  up  in  a  more  refined 
version  involving  other  intermolecular  associations  through  thiol  groups. 
I  will  return  to  this  point  below. 

9.  The  mitotic  apparatus  and  ATP :  the 
energetics  of  cell  division 

Since  cell  division  involves  the  movement  of  the  chromosomes  as  well 
as  the  formation  of  a  rather  elaborate  structure,  we  can  certainly  assume 
that  it  has  its  price  in  energy.  Attempts  to  assess  this  price  as  an  excess 
oxygen  consumption  have  led  to  the  conclusion  that  it  is  probably  not  very 
great,  but  in  any  case  the  payment  does  not  seem  to  be  made  during  the 
visible  phases  of  division  but  beforehand.  An  increased  oxygen  consump- 
tion during  division  itself  is  not  observed;  indeed,  Zeuthens'  extensive 
experiments  (summarized  by  Zeuthen  [44])  show  a  slight  decline  in 
respiration  during  the  division  period.  Similarly,  inhibitors  of  respiration, 
glycoloysis,  or  oxidative  phosphorylation  do  not  block  division  once 
mitosis  has  begun,  but  can  prevent  it  if  imposed  before  a  "point  of  no 
return"  just  before  the  active  phases  of  division.  These  findings  have  led 
to  the  valuable  hypothesis  of  an  "energy  reservoir"  (Swann  [31]  and 
earlier).  In  some  kinds  of  cells,  such  an  energy  reservoir  has  not  yet  been 
detected  as  a  pool  of  a  known  high-energy  compound.  In  one  case,  the 


THE   CENTRAL    PROBLEMS    OF   THE   BIOCHEMISTRY    OF    CELL    DIVISION         491 

Tetrahymena  cell  which  divides  by  a  mechanism  different  from  ordinary 
mitosis,  Plesner  [45]  has  demonstrated  a  build-up  of  nucleoside  triphos- 
phates in  anticipation  of  division.  As  I  have  pointed  out  elsewhere  [i],  the 
principle  of  an  energy  reservoir  for  division  need  not  necessarily  be  inter- 
preted in  terms  of  a  tangible  pool  of  high-energy  compounds,  and  in  fact 
there  are  some  difficulties  with  this  simple  view.  Another  possibility  is  that 
the  mitotic  apparatus  itself  is  the  energy  reservoir  in  the  sense  that  it  is 
assembled  in  an  activated  form,  and  is  driven  through  its  manoeuvres  by 
environmental  changes  such  as  the  SH  cycle  discussed  above. 

Another  and  simple  way  of  attacking  the  energetics  of  cell  division  has 
sound  precedents.  This  is  the  examination  of  its  reactions  with  ATP  or 
other  conventional  energy  sources.  No  event  in  the  historv  of  the  bio- 
chemistry of  muscle  contraction  was  more  portentous  than  the  discovery 
by  Engelhardt  and  Ljubimova  that  the  proteins  involved  in  contraction 
included  an  ATPase  acti^"ity,  even  though  the  outcome  was  not  as  simple 
as  might  have  been  hoped.  It  is  natural  to  ask  the  same  question  of  the 
mitotic  apparatus,  and  this  became  possible  when  the  DTDG  method 
became  available.  Xo  ATPase  actixity  could  be  obtained  with  the  mitotic 
apparatus  isolated  by  the  alcohol  digitonin  method.  The  studies  with  the 
new  method  were  begun  by  Dr.  R.  M.  Iverson  and  completed  bv  Dr.  R.  R. 
Chaffee.  Using  straightforward  methods  of  assay  analogous  to  those  used 
for  muscle  and  mitochondrial  ATPases,  the  following  information  has  been 
obtained,  and  will  be  published  in  full  elsewhere,  (i)  \\'hen  the  mitotic 
apparatus  is  isolated  and  purified  by  the  DTDG  method,  dissohed  in 
isotonic  KCl,  the  supernatant  following  high-speed  centrifugation  shows 
a  substantial  ATPase  activity.  The  sediment,  representing  the  particles 
associated  with  and  embedded  in  the  apparatus,  also  shows  an  activity 
attributable  to  yolk,  etc.,  but  it  has  different  properties  with  respect  to 
metal  activation,  etc.  It  is  assumed,  for  the  present,  that  the  activity  of  the 
supernatant  is  that  of  the  "fibrous"  component  of  the  apparatus,  which  is 
dissolved  in  the  isotonic  KCl.  (2)  The  pH  optimum  is  about  8-4.  (3)  The 
activity  is  highly  dependent  on  divalent  ions,  and  Alg^  "  is  three  times  as 
effective  as  Ca"^.  Manganese  is  slightly  less  efl'ectixe  than  magnesium. 
(4)  The  enzyme  does  not  split  ADP  or  glycerophosphate.  (:;)  The  enzyme 
is  highly  specific  for  ATP.  It  does  not  split  UTP,  CTP,^or  GTP.  The 
splitting  of  ITP  proceeds  at  a  rate  half  that  of  ATP  or  less.  I  do  not  know 
whether  there  is  a  precedent  for  this  degree  of  ATPase  specificity. 

So  far,  no  other  enzyme  of  the  mitotic  apparatus  has  been  studied. 
Whether  the  disco\  ery  of  a  rather  specific  ATPase  is  important  for  our 
picture  of  cell  division  obviously  depends  on  our  point  of  view.  It  does 
seem  to  link  the  mitotic  apparatus  to  muscle  and  certain  other  motile 
structures  such  as  flagella,  by  analogy  at  least,  ^^>  are  bound  to  suspect 
that  the   energetics   of  mitosis   are   conventional   enough   to   in\"ol\-e  the 


492  DANIEL    MAZIA 

splitting  of  ATP.  On  the  other  side,  such  a  reaction  can  be  only  an  exiguous 
part  of  a  complex  biochemical  picture,  and  it  is  not  obvious  where  to 
turn  next. 

10.  A  speculation  on  the  structure  of  the  mitotic  apparatus 
and  on  cellular  structure 

The  instability  of  the  mitotic  apparatus  is  remarkable,  the  more  so 
when  we  consider  that  its  job  is  to  move  massive  chromosomes  over  long 
distances.  As  further  evidence  of  chemical  instability,  I  may  cite  the 
experience  of  those  who  have  attempted  to  fix  it  for  electron  microscopy 
with  osmium  tetroxide  or  other  conventional  fixatives.  While  beautiful 
results  have  been  obtained  in  some  cases,  there  are  others  in  which  fixation 
is  capricious  and  still  others  where  it  seems  to  be  impossible  to  preserve 
fine  structure.  It  is  suggested  that  the  mitotic  apparatus,  especially  in 
larger  cells,  will  not  always  "  stand  still  "  long  enough  following  damage  to 
the  cell  to  be  properly  fixed  before  disintegrating. 

As  we  have  seen,  the  idea  that  the  mitotic  apparatus  was  bonded 
through  protein-sulphur  has  had  a  certain  predictive  success,  but  the  theory 
that  it  was  a  simply  vulcanized  system,  bonded  through  conventional  and 
stable  S — S  links,  has  not  been  substantiated.  In  the  development  of  the 
dithiodiglycol  procedure  to  stabilize  the  apparatus  for  isolation,  w^e  turned 
to  a  more  dynamic  conception  of  sulphur  bonds.  This  was  founded  on  the 
growing  body  of  evidence  (reviewed  by  Jensen  [41])  that  (SH)-(S — S) 
interchange  existed  and  was  perhaps  a  common  phenomenon.  The  mitotic 
apparatus  was  viewed  as  a  massive  aggregate  in  which  there  were  numerous 
pairs  of  S  atoms  located  close  to  each  other,  and  in  which  S-to-S  linkages 
were  opening  and  closing  all  the  time.  As  a  statistical  disulphide  structure, 
its  stability  would  depend  on  the  probability  of  the  existence  of  a  sufficient 
number  of  S-to-S  linkages  at  a  given  time,  and  it  was  imagined  that  this 
probability  could  be  "poised"  at  a  given  level  by  introducing,  in  the  total 
system,  and  S — S  compound  such  as  dithiodiglycol.  This  would  influence 
the  level  of  S-to-S  linkage  in  the  protein,  acting  not  quite  as  a  conventional 
stoicheiometric  oxidant  but  as  a  kind  of  "buff^er"  determining  the  trend 
of  electron  flow  to  and  from  the  protein-SH.  Such  a  view  may  be  out- 
rageously naive,  but  it  was  in  fact  the  basis  for  the  isolation  of  the  mitotic 
apparatus  with  dithiodiglycol. 

This  is  not  the  place  to  review  the  body  of  evidence  concerning 
disulphide  interchanges.  They  can  take  place  in  systems  containing  SH 
and  S- — ^S  [46,  41]  and  can  take  place  between  two  S — S  compounds  under 
the  action  of  ionizing  radiation  [47].  The  point  is  that  a  structural  system 
based  on  S-to-S  interactions  between  proteins  can  be  viewed  as  a  dynamic, 
sensitive,  and  unstable  one  given  the  right  conditions.  One  imagines  that 


THE   CENTRAL    PROBLEMS   OF   THE   BIOCHEMISTRY   OF   CELL    DIVISION         493 

in  these  interactions  the  mitotic  apparatus  conditions  are  governed  by 
the  fluctuating  of  thioldisulphide  systems  such  as  the  protein  of  the 
Sakai-Dan  cycle,  and  that  the  success  of  the  dithiodiglycol  method 
depends  on  a  mimicking,  and  no  more,  of  such  a  svstem  in  the  hving 
celL 

Perhaps  we  mav  speculate  a  step  further.  If  the  mitotic  apparatus  is 
such  a  dynamic  thiol-disulphide  svstem,  is  it  not  possible  that  the  protein- 
to-protein  interaction  includes  not  only  S  S  bonds  and  SH  groups  in  a 
state  of  dynamic  interchange,  but  also  sites  where  pairs  of  SH  groups  are 
only  half-oxidized  ?  It  is  likely,  from  the  Michaelis  principle  of  two-step 
oxidation  that  such  intermediates  have  at  least  a  transitory  existence.  Is  it 
conceivable,  in  a  structure  composed  of  so  many  interacting  molecules, 
that  there  is  an  appreciable  number  of  such  sites  at  anv  given  time,  and 
that  they  are  a  factor  in  the  stability  of  the  mitotic  apparatus  in  vivo.  This 
speculation  would  view  the  apparatus  as  having  some  of  the  properties  of 
a  gigantic  free  radical,  and  this  is  something  that  we  hope  to  test.  For  the 
time  being,  one  speculates  in  this  way  because  everyday  experience  with 
the  apparatus  shows :  that  it  is  high  in  protein  SH,  at  least  after  fixation  [36], 
that  it  behaves  in  isolation  procedures  as  a  structure  that  depends  on 
sulphur-to-sulphur  links,  and  yet  it  certainly  does  not  fit  our  image  of  a 
stable  S — S  bonded  structure.  This  proposal  cannot  be  defended  in  any 
rigorous  way,  but  I  wished  to  mention  it  here  because  it  is  in  fact  the 
predictive  basis  for  our  current  work  on  the  mitotic  apparatus. 

These  problems  of  stability  are  not  confined  to  the  mitotic  apparatus, 
but  to  other  structures  of  the  cell  whose  existence  is  inferred  for  good 
reasons  but  which  do  not  assert  themselves  either  after  biochemical 
isolations  or  common  electron-microscopic  fixation.  Examples  are  the  gel 
states  of  the  cytoplasm  studied  by  the  late  L.  V.  Heilbrunn  and  his  school 
and  the  structure  involved  in  intracellular  streaming,  discussed  in  this 
symposium  by  Robert  Allen.  It  will  not  be  surprising  if  the  structure  of  the 
cell  does,  after  all,  include  a  level  of  intermolecular  organization  so 
dynamic  and  so  sensitive  that  it  has  escaped  our  rather  violent  direct 
attacks  so  far.  Such  ideas  of  a  "protoplasmic"  organization,  popular  in  an 
earlier  era  of  cell  biology  but  often  rejected  as  being  bevond  experimental 
consideration,  may  yet  become  accessible  to  test. 

II.  Concluding  comments 

The  first  step  in  building  a  bridge  is  to  span  a  chasm  with  a  simple 
cable,  and  this  is  what  is  called  for  if  Molecular  Biology  is  to  come  to  grips 
with  the  uncomfortably  complex  problems  of  the  whole  cell  such  as  cell 
division.  An  individual  experimenter  can  ignore  such  problems  of  the 
higher  levels  of  cellular  organization  for  the  benefits  of  working  with  clean 


494  DANIEL   MAZIA 

and  simple  systems,  but  biology  as  a  whole  cannot  afford  to  do  so.  In  this 
essay,  I  have  tried  to  point  out  some  links  between  the  formidable  prob- 
lems of  cell  division  and  the  existing  trends  of  Molecular  Biology  and 
Biochemistry. 


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THE   CENTIL\L   PROBLEMS   OF   THE   BIOCHEMISTRY   OP   CELL   DIVISION        495 

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Discussion 

Ch.^rgaff  :  I  am  wondering  about  the  composition  of  the  mitotic  preparations 
which  you  have  made  in  the  presence  of  lead  thioglycol,  in  this  case  without  the 
use  of  detergents,  you  presumably  do  not  move  very  much  of  the  mass.  Do  you 
find  lipids  in  the  spindle  preparations  r 

MazL'\  :  I  feel  certain  that  we  do  recover  lipoproteins,  but  chiefly  because  of 
our  diflriculties  in  sedimenting  the  proteins  of  the  dissolved  mitotic  apparatus  by 
procedures  that  gave  good  patterns  with  the  product  of  the  older  method. 
Obviously,  your  question  cannot  be  answered  until  we  have  used  chemical  and 
ultracentrifugal  methods  that  are  appropriate  for  studies  of  lipoproteins. 

Ch.arg.\ff:  Dr.  Murray  and  I  studied  the  eftect  of  colchicine  and  its  reversal 
by  inositol  on  the  metaphase  arrest.  We  got  the  impression  that  the  mitotic 
apparatus  consisted  in  part  of  lipoprotein  which  after  application  of  detergents 
you  wouldn't  expect  to  find  any  more.  The  second  question  I  have  concerns  the 
state  of  the  sulphur.  Do  you  have  any  cysteine  determinations  on  your  prepara- 
tions ?  What  form  would  the  sulphhydrul  exist  in  ? 

M.AZLA :  Yes,  we  have  found  one  cysteine  per  18  000  units  of  molecular  weight, 
weight.  This  isn't  very  much,  but  neither  is  the  mitotic  apparatus  very  stable. 

Mitchell  :  Could  you  tell  us  more  about  the  occurrence  of  the  special  proteins 
of  the  mitotic  apparatus  during  the  resting  phases  of  the  cell  ?  I  imagine  that  what 
you  said  in  the  earlier  part  of  your  talk — that  these  proteins  are  probably  always 
present,  or  at  least  are  present  in  the  unfertilized  egg — means  that  we  do  not  have 
to  postulate  the  synthesis  of  a  special  supernumerary  apparatus  for  division. 

M.AZi.^ :  In  the  immunological  studies  it  was  found  that  the  antigens  charac- 
teristic of  the  mitotic  apparatus  were  present  at  all  stages  of  early  development 


496  DANIEL   MAZIA 

but  could  not  be  detected  in  those  adult  tissues— gut,  lantern  muscle,  and  mature 
testis — which  we  were  able  to  study.  The  optimistic  view  of  these  results  is  that 
the  presence  of  the  proteins  of  the  mitotic  apparatus  is  an  anticipation  of  division, 
that  they  will  be  present  in  cells  that  will  divide  in  the  future  but  not  in  cells  that 
will  no  longer  divide.  Thvis,  they  are  already  present  in  the  ovary,  presumably  in 
ripening  oocytes,  but  are  no  longer  present  in  the  mature  testis,  in  which  all  of 
the  maturation  divisions  are  over  for  a  long  time  to  come. 

A  problem  which  has  not  yet  been  resolved  satisfactorily  is  whether  the  proteins 
of  the  mitotic  apparatus  are  related  to  those  of  cilia  and  flagella.  The  common 
denominator,  of  covirse,  is  the  homology  of  the  centrioles  around  which  the 
mitotic  apparatus  is  organized,  and  the  basal  particles  of  the  cilia  and  flagella. 

Peters  ;  I  find  this  enormously  interesting  and  it  does  seem  that  this  conception 
that  Dr.  Mazia  has  given  us  clears  up  one  of  the  main  difficulties  in  thinking  about 
the  cytomosaic  (cytoskeleton),  but  of  course  we  have  still  in  front  of  us  the  awful 
question  as  how  can  this  become  integrated  from  the  cell  surface  ? 

Mazia  :  I  would  like  to  have  developed  a  further  speculation  concerning  the 
paradox  of  the  existence  of  so  much  RNA  in  the  mitotic  apparatus.  We  still  have 
no  reason  at  all  to  think  that  this  structure  is  concerned  with  protein  synthesis, 
and  we  have  reason  to  think  that  a  substantial  amount  of  RNA  is  associated 
directly  with  the  protein  (or  lipoprotein)  making  up  the  "  fibrous  "  structure.  If  the 
RNA  plays  a  specific  or  information-carrying  role,  and  if  this  is  not  concerned 
with  protein  synthesis,  I  wonder  whether  we  could  visualize  it  as  a  "recognition 
RNA".  If  molecules  are  to  associate  with  each  other  to  form  an  orderly  structure, 
we  do  have  a  problem,  long  recognized,  of  specific  interaction  or  "recognition". 
One  general  view  of  genetic  action  is  that  it  dictates  specificity  at  the  level  of  cell 
structure  as  well  as  at  the  level  of  the  structure  of  enzymes.  Could  not  the  genes 
dictate  structure,  communicating  information  as  conjugated  RNA,  to  the  molecules 
of  which  structures  are  built  ? 

Chargaff  :  Have  you  examined  the  reactivity  of  the  isolated  fibrous  protein 
with  anti-bodies  prepared  from  proteins  of  the  muscle  cells  ? 

Mazia  :  We  have  done  the  experiments  you  suggest,  comparing  the  mitotic 
apparatus  of  sea  urchin  eggs  with  extracts  of  the  lantern  muscles  of  the  same 
species  of  sea  urchin.  The  results  were  negative,  but  could  possibly  be  explained 
away  by  problems  of  diflfusion  of  the  muscle  proteins  in  the  agar  gels  used  for  the 
precipitin  tests.  However,  Holtzer  and  colleagues  have  obtained  negative  results 
when  they  attempted  to  stain  the  mitotic  apparatus  of  chick  cells  in  culture  with 
fluorescent  antibodies  against  chick  muscle  proteins. 


Studies  on  the  Cellular  Basis  of  Morphogenesis 
in  the  Sea  Urchin 

T.    GUSTAFSON 

The  Wenner-Gren  Institute  for  Experimental  Biology, 
University  of  Stockholm,  SzvedeJi 

The  task  of  de\  elopmental  physiology  is  not  only  to  elucidate  how  the 
cells  in  an  embryo  become  biochemically  different  from  each  other,  e.g. 
how  some  cells  get  the  capability  to  produce  and  to  accumulate  heart 
myosin,  cerebrosides,  glucose  6-phosphatase,  rhodopsin,  etc.  We  also  want 
to  understand  how  the  cells  become  arranged  into  various  well-organized 
complicated  organ  structures  such  as  hearts,  brains,  kidneys,  eyes,  etc., 
which  in  turn  are  integrated  to  form  whole  organisms. 

As  a  point  of  departure  in  our  attempt  at  an  analysis  we  may  state  that 
morphogenesis  of  the  organs  and  the  organism  as  a  whole  reflects  the 
molecular  events  going  on  in  its  cells.  We  are  permitted  to  make  this 
statement  for  many  reasons,  e.g.  treatment  of  an  embryo  with  agents 
which  interfere  with  the  physical  and  chemical  events  in  the  cells,  also 
brings  about  characteristic  alterations  in  the  anatomical  development  of 
the  embryo.  As  an  example  I  may  mention  that  o-iodosobenzoic  acid, 
which  is  capable  of  oxidizing  certain  SH  groups,  suppresses  the  develop- 
ment of  the  entomesodermal  elements  in  the  sea  urchin  larva,  which  there- 
fore only  develop  into  an  ectodermal  vesicle.  A  cytosine  analogue,  2-thio-5- 
methyl  cytosine,  has  the  same  effect.  Furthermore,  it  is  generally  accepted 
that  genes  exert  their  action  by  determining  the  kind  of  enzymes  a  cell  can 
form,  and  we  begin  to  look  upon  the  hereditary  morphological  deficiencies 
as  the  result  of  disturbances  of  cellular  metabolism. 

The  problem  we  are  confronted  with  is  to  define  how  the  molecular 
events  are  translated  into  organ  structures  distributed  according  to  a 
characteristic  and  reproducible  pattern.  We  may  approach  this  problem 
from  many  different  directions.  One  way  is  to  describe  the  metabolic 
pattern  of  cells  in  different  presumptive  organ  regions  in  the  embryo.  But, 
even  a  detailed  biochemical  dissection  of  an  embryo  may — I  fear — fail  to 
answer  our  fundamental  questions  about  morphogenesis,  e.g.  how  a  certain 
part  of  the  blastula  wall  invaginates  to  form  an  archenteron  and  how  the 
archenteron  becomes  subdivided  into  coelomic  sacs  and  other  derivatives. 

VOL.  n. — 2K 


498  T.    GUSTAFSON 

One  reason  for  this  apparent  difficulty  to  bridge  the  gap  between  the 
molecular  and  the  organ  level  is  probably  differences  in  language,  con- 
cepts, and  slogans  used  by  the  workers  in  these  two  fields.  Therefore,  in 
order  to  find  a  common  point  where  anatomy  and  molecular  biology  can 
meet,  it  seems  logical  to  try  to  reduce  the  complex  processes  at  the  organ 
level  into  morphological  activities  of  individual  cells.  Could  we,  for 
instance,  show  that  the  moulding  of  a  certain  organ  shape  depends  upon 
the  formation  of  pseudopods,  upon  changes  in  the  adhesive  properties  of 
the  cells,  etc.,  then  we  could  begin  to  discuss  the  molecular  background  for 
these  cellular  phenomena. 

The  translation  of  the  phenomena  at  the  organ  level  to  a  cellular 
language  is  greatly  facilitated  by  the  use  of  time-lapse  cinematography. 
The  sea-urchin  larva  is  a  suitable  object  for  such  a  study,  as  it  is  transparent 
enough  to  allows  observations  of  the  morphological  activities  of  all  its 
individual  cells.  Furthermore,  its  anatomical  organization  is  not  too 
complicated,  and  finally,  much  is  known  about  the  biochemical  differentia- 
tion of  its  cells. 

The  application  of  time-lapse  cinematography  to  the  developing  sea- 
urchin  larva  raises  some  technical  problems,  as  the  larvae  swim  around. 
We  have,  however,  been  able  to  overcome  this  difficulty  by  catching  the 
larvae  in  the  meshes  of  a  nylon  net  to  which  crystals  of  calcium  carbonate 
have  been  attached.  The  crystals  make  small  indentations  in  the  larvae  and 
keep  them  in  a  constant  position  without  interfering  with  their  normal 
development  [i].  I  will  try  to  give  some  examples  of  the  results  we  have 
obtained  with  this  simple  technique  used  in  combination  with  conventional 
time-lapse  filming.* 

*  The  photographs  in  this  paper  are  all  reproduced  from  our  i6-min.  reversal 
films  of  developing  larvae  of  Psammechiuiis  milioris. 


Fig.  I.  The  formation  of  the  primary  mesenchyme  and  the  archenteron  in  a 
larva  of  the  sea  urchin  Psamniecliimis  miliaris.  a-d  show  the  release  of  the  primary 
mesenchyme  cells  which  is  brought  about  by  a  pvilsatory  activity  and  the  resulting 
change  in  shape  of  the  cells  in  combination  with  a  decrease  in  their  adhesion  for 
each  other  and  for  the  hyaline  membrane ;  in  d  one  of  the  cells  has  begun  to  form 
a  pseudopodium  with  which  it  migrates  along  the  blastula  wall  (to  the  right).  In  e 
the  primary  mesenchyme  cells  have  settled  down  to  form  a  characteristic  ring-like 
structure.  /  shows  the  end  of  the  primary  invagination  of  the  archenteron,  which 
is  brought  about  by  pulsatory  activity  and  the  resulting  change  in  shape  of  the 
cells  at  the  archenteron  tip  ;  it  also  shows  the  first  sign  of  the  formation  of  a  pseudo- 
podium (protuberance  to  the  left  on  the  archenteron  tip),  g  and  h  show  diflferent 
stages  in  the  secondary  phase  of  invagination  of  the  archenteron,  which  is  brought 
about  by  the  contractile  pseudopodia  formed  from  the  archenteron  tip.  The  time 
covirse  of  invagination  is  shown  in  Fig.  3.  Time  interval  between  a  and  li  6  hr.  48 
min.  Picture  series  from  a  single  i6-mm.  time-lapse  film.  Magnification  430  x  . 


CELLULAR    BASIS    OF    MORPHOGEXESL^    LX    THE    SEA    LRflllX 


499 


500  T.    GUSTAFSON 

It  is  for  practical  reasons  suitable  to  denote  the  lowest  part  of  the 
blastula  (the  region  at  the  vegetal  pole)  as  zone  i,  the  next  as  zone  2,  etc. 
These  regions  correspond  to  the  presumptive ; 

1.  primary  mesenchyme 

2.  secondary  mesenchyme 

3.  coelomic  sacs 

4.  oesophagus 

5.  stomach 

6.  proctodoeum 

7.  ectoderm 

The  films  indicate  that  the  morphogenetic  activity  in  these  zones  can  be 
partly  reduced  to  similar  types  of  cellular  activities.  One  of  these  is  a 
pulsatory   activity   of  the    cell   surfaces    bordering   the    blastocoel.    The 


Fig.  2.  Further  example  of  the  primary  invagination  {a  and  h)  and  of  secondary 
invagination  (c). 

pulsations  involve  a  centripetal  translocation  of  cytoplasm,  which  causes 
the  cells  to  become  thicker  at  their  centripetal  than  at  their  centrifugal 
ends.  The  pulsatory  activity  is  often  a  forerunner  of  an  emission  of  thin 
pseudopods  (filopods)  from  the  inner  cell  surfaces.  These  pseudopods 
either  collapse  or  attach  to  the  ectoderm  and  contract.  A  third  change 
involves  a  decrease  or  increase  in  adhesion  between  the  cells  in  the 
blastula  or  gastrula  wall.  These  activities  may  be  closely  related  to  each 
other.  It  looks,  at  least,  that  a  vigorous  pulsation  can  be  modified  into  a 
"shooting"  out  of  pseudopods. 

I  will  try  to  give  some  examples  how  these  three  changes  in  cellular 
activity  co-operate  in  the  morphogenesis  of  the  sea-urchin  larva : 

A.  The  pulsatory  activity  of  the  cells  and  their  resulting  change  in 
shape  may  bring  about  their  release  into  the  blastula  cavity.  This,  how- 
ever, only  occurs  if  their  adhesion  to  each  other  and  to  the  hyaline  mem- 
brane, which  covers  the  blastula  surface,  is  low.  The  entrance  of  the 


CELLULAR    BASIS    OF    MORPHOGENESIS    IN    THE   SEA   URCHIN  5OI 

primary  mesenchyme  into  the  blastula  cavity  is  an  example  of  this  activity, 
cf.  Fig.  I  and  [i,  6].  If  the  celkilar  adhesiveness  remains  unchanged,  on 
the  other  hand,  the  pulsatory  activity  and  their  change  in  shape  bring 
about  an  invagination  of  the  body  wall.  The  first  phase  of  invagination  of 
the  archenteron  and  the  onset  of  the  evagination  of  the  coelomic  sacs  from 
the  archenteron  tip  are  examples  of  such  a  process,  cf.  Figs,  i,  2  and  [3,  6]. 
B.  The  pseudopod  activity  brings  about  a  strong  extension  of  the 
invaginated  or  evaginated  regions  of  the  body  wall.  The  second  phase  of 
invagination,  cf.  Figs,  i,  2  and  [i,  3],  and  the  extension  of  the  coelomic  sac 
rudiments  [3]  are  brought  about  by  such  a  mechanism.  The  bending  of 


8  hours 


Fi<i.  3.  Diagram  of  the  course  of  invagination  of  the  archenteron  in  a  larva  of 
Psdnimechiiius  miliaris  (the  larva  in  Fig.  i).  Inner  height  of  the  archenteron  in 
microns  is  plotted  against  relative  age  in  hours,  the  first  stage  arbitrarily  called 
o  hr.  The  pseudopodal  activity  is  symbolized  by  the  horizontal  lines  below  the 
curve  to  the  right,  each  line  representing  one  visible  pseudopodium ;  dashed  line : 
pseudopodium  intermittently  visible ;  wavy  line  indicates  direct  contact  between 
the  secondary  mesenchyme  and  the  ectoderm.  The  schematic  drawing  to  the  left 
gives  the  appearance  of  the  larva  in  three  diflFerent  stages  corresponding  to  the 
marks  below  the  abscissa. 


the  archenteron  tip  towards  the  presumptive  stomodaeum  region  of  the 
ectoderm  (Fig.  4  and  [2])  is  another  result  of  pseudopod  contractions.  The 
contractions  of  the  pseudopods  will  finally  pull  the  pseudopodia-forming 
cells  out  from  the  archenteron  tip  and  a  so-called  secondary  mesenchyme 
is  thus  formed.  The  pseudopod  activity  finally  brings  about  a  rapid 
migration  of  the  liberated  primary  as  well  as  secondary  mesenchyme  cells. 

C.  The  role  of  a  decrease  in  adhesion  between  the  cells  in  the  wall  in 
the  larva  has  already  been  exemplified.  A  strong  increase  in  adhesion 
between  the  cells  may  cause  them  to  increase  their  contact  surfaces  so  that 
they  become  more  or  less  cylindrical.  This  appears  to  be  the  basis  for  the 
formation  of  the  ciliary  bands  and  the  ciliary  plate  in  the  animal  pole. 

The  activities  mentioned  appear  to  be  released  according  to  a  simple 


502  T.    GUSTAFSON 

time-space  pattern.   As   an   example   we   may  study  the   release   of  the 
pulsatory  activity.  This  activity 

starts  in  zone  i  and  brings  about  a  release  of  the  primary  mesen- 
chyme cells  into  the  blastocoel; 

continues  in  zone  2  where  it  brings  about  the  primary  invagination 
of  the  archenteron  rudiment ; 

continues  in  zone  3  where  it  brings  about  an  early  evagination  of 
the  coelomic  sacs ; 

continues  in  zone  4  where  it  may  contribute  to  the  morphogenesis 
(a  rounding  up)  of  the  oesophagus; 

continues  (occasionally)  in  zone  4. 


Fig.  4.  The  bending  of  the  archenteron  towards  the  presumptne  stumodaeum. 
a  shows  how  contact  has  been  formed  between  the  archenteron  tip  and  the  ventral 
side  by  means  of  pseudopods.  The  tension  in  the  pseudopods  has  caused  the 
formation  of  cones  of  attachment  in  the  ectoderm  and  pulled  out  cells  from  the 
archenteron  tip,  the  whole  of  which  will  gradually  (through  the  pull  of  several 
pseudopods  of  this  kind)  be  forced  to  make  contact  with  the  ventral  side.  In  b  the 
contact  has  been  established.  The  archenteron  tip  is  sometimes  met  by  the 
invaginating  stomodaeum  rudiment,  an  invagination  which  recalls  the  primary 
invagination  of  the  archenteron. 

The  pulsatory  activity  in  the  other  zones  generally  fails,  with  some 
exceptions.  It  may  thus  occur  (preliminary  studies)  in  the  ectoderm  where 
it  mav  contribute  to  the  invagination  of  the  presumptive  stomodaeum  [2]. 
It  also  occurs  in  the  regions  between  the  extending  arms  and  thus  con- 
tributes to  their  elongation  [4]. 

The  difference  in  the  morphogenetic  activity  of  the  consecutive  zones 
is  not  onlv  a  time  difference.  There  also  appears  to  be  a  decrease  in  the 
capability  of  the  cells  to  pulsate  and  to  emit  pseudopods  and  an  increase 


CELLULAR   BASIS    OF   MORPHOGENESIS    IN   THE    SEA    URCHIN  503 

in  their  mutual  adhesion  and  their  adhesion  to  the  hyahne  membrane. 
These  differences  might  be  summarized  as  differences  in  the  "strength  of 
their  mesenchymal  properties"  of  the  cells  in  the  different  zones  (a 
provisional  and  perhaps  vague  and  misleading  concept).  This  intensity 

is  highest  in  zone  i :  The  cells  pulsate  and  lose  their  adhesion  for 
each  other  and  for  the  hyaline  membrane  before  the  pseudopodal 
activity  starts. 

It  is  weaker  in  zone  2 :  The  cells  pulsate  but  their  adhesive  proper- 
ties onlv  gradually  decrease  and  the  cells  are  only  released  as  a 
result  of  a  strong  pull  of  their  pseudopods. 

It  is  weaker  in  zone  3  :  The  coelom  cells  show  some  pulsatory 
activitv  and  form  pseudopods  but  they  never  pull  themselves  out 
but  remain  connected  to  each  other  to  form  cell  sheets,  the  walls  of 
the  coelomic  sacs. 

It  is  weaker  in  zone  4 :  The  oesophagus  cells  pulsate  somewhat,  but 
never  emit  pseudopods.  There  are,  however,  contractile  elements 
within  the  wall  of  the  oesophagus,  bringing  about  its  periodic 
contraction.  One  can  imagine  that  these  elements  are,  in  a  sense, 
equivalent  to  contractile  pseudopods.  The  main  difference  may  be 
that  thev  never  shoot  out  from  the  wall. 

It  is  still  weaker  in  zone  5 :  The  stomach  cells  may  occasionally 
pulsate  but  no  contractile  elements  are  present  and  the  cells  remain 
connected  to  a  sheet,  the  wall  of  the  stomach. 

This  graded  change  in  properties  appears  to  continue  within  the 
ectoderm.  The  cells  of  this  germ  layer  do  not  pulsate  (with  the  exceptions 
mentioned  earlier)  but  the  adhesion  between  the  cells  is  different  in 
different  regions.  The  cells  in  the  thin  epithelial  sheets  can  be  assumed  to 
have  a  comparativelv  low  adhesion  for  each  other.  In  some  zones,  however, 
the  mutual  adhesion  increases,  and  the  cells  therefore  increase  their  mutual 
contact  surfaces  and  become  cylindrical  or  more  or  less  hexagonal.  This 
occurs  in  the  most  animal  region,  the  animal  plate,  and  in  the  ciliary  band 
which  extends  from  it.  (This  rearrangement  of  the  cells  is  no  doubt 
responsible  for  the  ventral  flattening  of  the  gastrula.) 

If  this  interpretation  is  correct,  there  is  thus  a  more  or  less  continuous 
spectrum  of  morphogenetic  properties  along  the  animal-vegetal  axis  of 
the  larva.  The  closelv  packed  ciliary  cells  in  the  animal  plate  and  the 
ciliarv  bands  represent  one  end  of  this  spectrum,  the  primary  mesenchyme 
cells  derived  from  the  zone  at  the  vegetal  pole  represents  another  extreme 
case.  It  is  tempting  to  relate  this  spectrum  in  cellular  activities  to  the 
animal-vegetal  gradients,  so  familiar  to  the  embryologists,  cf.  [7]. 

The  film   and  this   review  suggest  that  the  ectoderm  has   a  rather 


504  T.    GUSTAFSON 

restricted  capability  for  strong  deformations.  It  may  change  its  shape  some- 
what (e.g.  form  a  ciHary  plate  and  ciliary  bands,  undergo  a  dorso-ventral 
flattening  and  form  arm  buds)  as  a  result  of  a  change  in  adhesion  between 
some  of  its  cells.  When  it  deforms  strongly,  the  forces  required  are  pro- 
vided by  the  mesenchymal  and  mesodermal  elements :  the  extension  of  the 
arms,  Fig.  5,  and  the  scheitel  (the  dorsal  extension  of  the  ectoderm),  Fig.  6, 
are  thus  dependent  upon  the  clusters  of  skeleton  forming  mesenchyme 
cells  which  collect  at  the  tips  of  the  skeleton  spicules  and  push  the  ecto- 
derm forwards.  As  a  further  example,  the  dilatation  of  the  mouth  appears 


a 


mmh 


Fig.  5.  Anal  arms  (seen  from  the  anal  side)  in  larvae  of  Psamnucliiinis  miliaris 
in  early  pluteus  stages,  a,  arm  with  the  typical  cluster  of  mesenchyme  attached 
to  and  forming  the  growing  skeleton.  The  extension  of  the  arm  rudiment  is 
brought  about  by  the  pressure  of  the  mesenchyme  cluster.  The  region  between  the 
two  arms  (right  arm  outside  the  picture)  shows  a  pulsatory  activity  which  brings 
about  an  invagination,  b,  the  pulsatory  activity  between  the  anal  arms  is  supple- 
mented by  the  emission  of  contractile  pseudopodia  which,  for  example,  attach  to 
the  skeleton  and  exert  a  tension  which  partly  causes  a  release  of  the  pseudopod- 
forming  ectoderm  cells. 


to  depend  upon  the  contractility  of  the  oesophagus.  (The  expansion  of  the 
stomach  rudiment  into  a  thin-walled  vesicle  is  also  greatly  dependent  upon 
a  contractility  outside  the  rudiment  itself,  i.e.  is  brought  about  by  the 
hydrostatic  pressure  generated  by  the  contractions  of  the  oesophagus.) 
The  ectoderm  may,  however,  acquire  a  pulsatory  and  pseudopodal  activity 
in  certain  regions,  i.e.  in  the  regions  on  the  ventral  side  which  early  have 
been  in  close  contact  with  the  ventral  clusters  of  primary  mesenchyme.  I 
refer  to  the  invaginating  regions  between  the  arm  rudiments.  The  in- 
vaginations are  brought  about  by  pulsatory  and  pseudopodal  activity. 
Fig.  5,  and  thus  are  reminiscent  of  the  invagination  of  the  archenteron 
rudiment.   It  may  be  permitted  to  suggest  that  the  ectoderm  in  these 


CELLULAR   BASIS   OF   MORPHOGENESLS    IN    THE    SEA    URCHIN  505 

regions  has  acquired  certain  mesenchymal  properties  as  a  result  of  its  close 
contact  with  the  primary  mesenchyme.  One  might  denote  this  as  a  kind 
of  induction. 

The  ectoderm  is,  however,  not  only  a  toy  with  which  the  mesenchyme 
plays — it  is  to  a  great  extent  the  ectoderm  which  guides  the  mesenchymal 
pseudopodia  and  thus  the  morphogenesis  of  the  entomesoderm.  One  may 
thus  say  that  the  ectoderm  serves  as  a  kind  of  template  for  the  entomeso- 
derm, but  of  course  not  a  template  in  the  biochemical  sense. 

How  does  the  ectoderm  guide  the  mesenchymal  pseudopods  ?  The 
films  indicate  that  the  ectoderm  in  some  regions  has  a  high  "stickiness" 
for  the  pseudopods,  i.e.  permits  the  pseudopods  to  attach  strongly.  The 


Fig.  6.  A  developing  scheitel  (dorsal  extension  of  the  ectoderm)  in  a  young 
pluteus  stage.  The  extension  of  the  ectoderm  is  brought  about  by  the  plug  of 
mesenchyme  attached  to  the  growing  skeleton  rod  in  the  same  way  as  in  the 
extending  arms,  cf.  Fig.  4. 

Stickiness  is  lower  in  other  regions.  There  is,  in  other  words,  a  charac- 
teristic pattern  of  stickiness  at  the  inner  surface  of  the  ectoderm.  But  how 
do  the  mesenchymal  pseudopods  find  the  areas  of  high  stickiness  ? 

The  films  show  that  the  pseudopods  of  the  primary  mesenchyme  are 
very  long  and  numerous  and  that  they  appear  to  explore  the  whole  inner 
surface  of  the  ectoderm,  cf.  Fig.  7.  During  this  apparently  random  ex- 
ploration they  come  in  contact  with  regions  of  low  stickiness  as  well  as 
regions  where  the  stickiness  is  high.  In  the  latter  case  they  attach  to  the 
ectoderm  and  contract  and  thereby  carry  the  cell  body  in  the  direction  of 
the  sticky  region.  In  the  latter  case  the  pseudopods  may  either  collapse — 
to  be  succeeded  by  new  pseudopods — or  continue  their  random  exploration 
until  they  reach  a  region  where  the  stickiness  of  the  ectoderm  is  high.  The 
primary  mesenchyme  as  a  whole  will  therefore  gradually  arrange  itself 
into  a  pattern  which  corresponds  to  the  high  points  of  stickiness  of  the 


5o6  T.    GUSTAFSON 

ectoderm — the  "template"  [5].  The  attachment  of  the  cells  to  the  ecto- 
derm do  not  appear  to  be  permanent,  however.  Preliminary  observations 
indicate  that  the  contacts  have  a  rather  restricted  life  time,  and  the  mesen- 
chyme cells  may  thus  change  their  distribution  as  a  response  to  a  further 
elaboration  of  the  pattern  of  stickiness  of  the  ectoderm.  I  may  finally 
mention  that  the  adhesion  between  the  primary  mesenchyme  and  the 
ectoderm  may  be  determined  by  the  same  factor  that  determines  the 
adhesion  between  the  cells  within  the  ectoderm :  The  primary  mesenchyme 
cells  thus  seem  to  accumulate  in  those  regions  where  the  ectoderm  cells 


Fig.  7.  Primary  mesenchyme  cells  with  exploring  thin  pseudopods  (filopods). 
Drawing  from  a  time-lapse  film  of  a  young  gastrula.  The  mesenchyme  cells  have 
already  arranged  themselves  into  a  ring  (part  of  which  is  seen  in  the  Figure)  but 
still  explore  the  ectoderm  in  regions  far  outside  the  ring  level. 


will  come  close  together  (become  more  or  less  cylindrical),  e.g.  to  form 
ciliated  bands.  The  phenomenon  of  random  exploration  is  also  applicable 
to  the  pseudopods  formed  by  the  secondary  mesenchyme  and  the  coelomic 
sacs. 

As  a  general  conclusion  of  this  brief  review  we  may  state  that  the 
morphogenesis  of  the  sea  urchin  larva,  in  spite  of  its  relative  complexity, 
is  not  completely  obscure  but  appears  to  be  resolvable  into  a  restricted 
number  of  morphological  cellular  activities,  which  appear  to  be  released 
according  to  a  simple  time-space  pattern,  i.e.  as  a  wave  proceeding  from 
the  lower  (vegetal)  pole  towards  the  higher  (animal)  pole.  The  activities  in 
the  different  zones  bear  a  certain  relationship  to  each  other,   i.e.  the 


CELLULAR    BASIS    OF    MORPHOGENESIS    IX    THE    SEA    URCHIN  507 

ditference  can  partly  be  reduced  to  a  quantitative  change  of  some  basal 
activities.  It  may  furthermore  be  permitted  to  state  that  pseudopodal 
elements  play  an  important  role  in  morphogenesis.  Such  elements  are  no 
doubt  excellent  morphogenetic  tools  as  they  not  only  provide  a  force  which 
gives  rise  to  translocations  and  deformations,  they  also  find  the  suitable 
direction  for  the  forces  by  random  exploration — and  thereby  contribute 
to  a  proper  integration  of  the  organ  rudiments  to  form  an  organism  fit  for 
survi\al  [2]. 

It  is  easv  to  make  a  long  list  of  problems  for  future  research.  One  of  the 
most  important  problems  is  how  the  time  sequence  for  the  release  of  the 
processes  concerned  is  determined,  how  the  borders  between  the  indi\"idual 
organ  rudiments  is  determined  and  why  they  are  so  sharp,  and  why  the 
future  development  of  the  cells  in  the  different  regions  diverge.  The  only 
point  I  will  make  in  this  connection  is  that  I  think  that  the  control  of  the 
time-sequence  may  be  a  strategic  point  where  the  analysis  should  start.  A 
properlv  controlled  time-sequence  mav  serve  as  a  good  basis  for  a  feed-back 
control  of  development  of  less  ad\anced  rudiments  bv  older  ones. 

Finally,  may  I  add  a  personal  comment :  I  think  that  the  gap  between 
the  organ  level  and  the  molecular  events  can  be  bridged  if  we  trv  to  under- 
stand the  biochemical  basis  for  pseudopodal  formation,  pulsatory  activity 
and  changes  in  adhesion  between  the  cells  and  similar  phenomena. 
Willmer  [S]  has  indicated  one  wav  in  which  such  relationships  between 
cellular  morphology  and  the  biochemical  level  can  be  studied.  I  refer  to 
his  work  with  the  amoeba  Xotg/eria  i^nihcri  which  he  is  able  to  transform 
from  an  amoeboidic  cell  into  a  flagellate  one,  and  vice  versa,  merely  by  a 
change  in  its  chemical  milieu.  And  Runnstrom,  cf.  [7],  has  long  ago 
focused  our  attention  on  the  metabolic  gradients  in  the  egg  which  no 
doubt  appear  to  be  paralleled  by  gradients  in  morphological  behaviour  of 
the  cells.  And  therefore,  as  a  final  personal  confession  to  the  participants 
in  this  symposium,  where  much  is  said  about  oxidati^■e  phosphorylation: 
the  day  may  come  when  I,  or  at  least  mv  grandchildren,  begin  to  look 
upon  the  discussion  between  "phosphorylatiAe  fans"  as  something  more 
than  a  bullfight. 

References 

1.  Gustafson,  T.,  and  Kinnandtr,  H.,  Exp.  Cell  Res.  II,  36  (1956). 

2.  Gustafson,  T.,  and  Kinnander,  H.,  E.xp.  Cell  Res.  21,  361  (1960). 

3.  Gustafson,  T.,  and  Wolpert,  L.,  Exp.  Cell  Res.  22,  437  (ig6i). 

4.  Gustafson,  T.,  and  Wolpert,  L.,  Exp.  Cell  Res.  22,  509  (1961). 

5.  Gustafson,  T.,  and  Wolpert,  L.,  Exp.  Cell  Res.  (in  press). 

6.  Kinnander,  H.,  and  Gustafson,  T.,  Exp.  Cell  Res.  19,  278  (i960). 

7.  Runnstrom,  J.,  I'erluuidl.  Deittsch.  Zool.  Ges.  in  Tiibingen,  p.  32  (1954). 

8.  Willmer,    ¥..    X.,    "Cytology   antl    Evolution"".    Academic    Press,    Xew    York, 
London  ( i960). 


5o8  T.    GUSTAFSON 

Discussion 

Allen  :  This  is  a  remarkable  demonstration  of  the  importance  of  cell  movement 
in  embryonic  development.  I  was  particularly  interested  to  observe  that  there  are 
two  kinds  of  amoeboid  movement  represented  which  were  ordinarily  separated  by 
those  of  us  who  are  interested  in  cellular  motility.  I  see  formation  of  both  lobopodia 
and  filopodia ;  the  principal  difference  between  these  two  kinds  of  pseudopodia  is 
that  the  pattern  of  streaming  in  the  lobopodium  is  that  of  a  fountain,  whereas  that 
in  a  filopodium  is  two  directional  streaming.  So  far  we  do  not  know  whether  these 
two  kinds  of  movement  have  similar  mechanisms.  I  wonder  if  you  have  looked 
carefully  at  the  filopodia  to  find  if  there  is  in  fact  streaming  in  two  directions  ? 

Gustafson:  No,  I  haven't,  but  during  my  last  sojourn  at  Kristineberg's 
Zoological  Station  we  filmed  larvae  for  days  at  one-second  intervals  just  to  investi- 
gate the  dynamics  of  the  pseudopods. 

Holter:  I  was  very  much  interested  in  your  evidence  for  areas  of  stickiness 
that  seem  to  play  a  determining  role  in  morphogenesis.  Isn't  there  any  possibility 
to  determine  chemically  by  means  of  surface  reactions,  what  would  be  the  reason 
for  this  surface  stickiness  ? 

Gutsafson:  I  have  not  tried,  but  I  am  very  interested  to  do  so. 

RuNNSTROM :  I  can  tell  you  that  in  our  experiments  very  low  trypsin  concen- 
trations (treatment  with  lo  ~^-io^  ",,  trypsin  for  15  min.)  induce  stickiness  of  the 
sea  urchin  egg.  This  may  indicate  a  possible  role  for  proteolytic  enzymes. 

Gustafson  :  In  this  connection  I  may  mention  that  if  one  treats  the  eggs  with 
very  weak  detergent  solution  one  completely  changes  the  pattern  of  development 
of  the  ectoderm :  the  mesenchyme  ring  and  the  main  ciliated  band  form  at  wrong 
places  and  so  on.  This  may  give  some  indication  of  what  lies  behind  the  stickiness 
in  the  cells. 

Porter  :  Your  observations  suggest  that  there  might  be  some  guiding  frame- 
work in  the  blastocoele  for  the  mesenchymal  cells ;  is  there  fibrous  material  there  ? 

Gustafson  :  One  can  often  see  a  lot  of  particles  in  the  blastocoele  which  swim 
around  with  great  speed.  This  suggests  that  there  are  no  rigid  structures  in  the 
blastocoele  at  this  stage  of  development.  Occasionally,  however,  one  can  see 
particles  lined  up  and  vibrating  together  in  a  way  which  suggests  the  presence  of 
some  submicroscopic  or  at  least  thin  and  transparent  fibres. 

Runnstrom  :  As  shown  by  my  colleague  J.  Immers,  there  are  sulphated 
polysaccharides  present  in  the  blastocoele  of  the  sea  urchin  embryo.  In  a  late 
blastula  stage  these  polysaccharides  become  linked  to  proteins.  The  migrating 
cells  which  Ciustafson  has  studied  are  in  fact  surrounded  by  a  coat  of  a  protein-acid 
polysaccharide  complex,  a  fact  that  probably  is  of  importance  for  understanding  the 
behaviour  of  the  migrating  cell.  If  the  coat  is  imperfectly  formed  the  migration  of 
the  cells  is  disturbed  or  prevented  (the  latter  occurs  following  pronounced 
animalization  of  the  larvae). 


Cell  Differentiation :  A  Problem  in  Selective  Gene 
Activation  Through  Self-Produced  Micro-Environ- 
mental Differences  of  Carbon  Dioxide  Tension 

\V.  F.  LooMis 

The  Loomis  Laboratory, 
Greenwich,  Conn.,  U.S.A. 


It  is  an  interesting  fact  that  the  subject  matter  of  most  of  this  sym- 
posium, i.e.  DNA,  RNA,  ribosomes,  mitochondria,  etc.,  concerns  the 
Uving  cell  as  it  was  present  on  this  earth  a  billion  years  ago,  before 
Darwinian  evolution  even  started.  In  those  dark  ages,  before  there  were 
metazoa  of  any  kind,  the  primary  inventions  of  protein  and  nucleic  acid 


Fig.  I.  Relative  sizes  of  a  sulphur-bottomed  whale- 
and  the  African  elephant  "Jumbo"  (from  Lull). 


-the  largest  living  animal- 


synthesis  were  combined  to  etfect  the  miracle  of  replication.  Once  this  had 
been  achieved,  a  second  series  of  inventions  could  begin,  inventions  by 
which  replication  could  lead  to  differentiation  and  larger  and  larger  multi- 
cellular organisms  arise.  InterceWuhr  chemistry  in  other  words  is  needed 
to  explain  how  thirty  quadrillion  cells  of  about  a  hundred  different  types 
co-operate  to  make  a  sulphur-bottomed  whale  (Fig.  i).  Embryologically, 
of  course,  this  vast  number  of  cells  is  derived  by  clonal  growth  from  a 
single  fertilized  ovum. 

Basically,  the  problem  is  one  of  selective  gene  activation.  Since  the 
nucleus  of  the  fertilized  egg  contains  all  the  genetic  information  needed  to 
make  each  of  the  final  differentiated  cells  present  in  the  adult  body,  it  is 
clear  that  only  part  of  this  information  is  used  in  any  one  cell.  Take,  for 
example,  the  insulin-secreting  cells  of  the  Islets  of  Langerhans.  Sanger 


5IO  W.    F.    LOOMIS 

and  his  colleagues  have  shown  that  insulin  has  the  structure  given  in 
Fig.  2.  A  glance  at  this  structure  shows  that  the  "one  gene,  one  enzyme" 
theory  must  include  the  incredible  fact  that  "  one  gene  "  can  contain  the 
10'"  or  so  "bits"  of  information  needed  to  synthesize  such  a  protein 
from  an  amino  acid  pool.  The  striking  fact  is  that  this  insulin-synthesiz- 
ing gene  is  present  but  unused  in  all  the  other  cells  within  the  body. 
What  is  it  then  that  selects  which  genes  are  activated  where  ?  What  are 
the  activating  agents  ?  Whence  do  they  come,  and  how  do  they  reflect 
the  embryo-as-a-whole  with  all  its  nearly  magical  powers  of  self- 
regulation  ? 

Even  single-celled  animals  are  capable  of  demonstrating  selective  gene 
activation,  for  Sonneborn  [i]  has  shown  that  paramecia  possess  eight 
different  sets  of  flagellal-protein-synthesizing  genes,  but  the  expression  of 
one  set  automatically  inhibits  the  expression  of  the  remaining  seven.  It  is 
as  if  a  Paramecium  were  a  player  piano  with  eight  different  tunes  stored 
on  rolls  within  the  piano  stool.  The  selection  of  any  one  tune  for  conversion 
from  genotype  to  phenotype  automatically  prevents  the  expression  of  the 
other  seven  rolls. 

Most  of  the  gene-activating  agents  we  know  today  come  under  the  cate- 
gory of  maturation  hormones,  chemicals  that  activate  long-dormant 
genes  during  metamorphosis  or  adolescence.  This  paper  will  not  consider 
the  various  steroid,  amino  acid  and  protein  hormones  that  fall  into  this 
category,  for  clearly  they  are  not  responsible  for  the  beginnings  of  develop- 
ment when  the  complex  glands  responsible  for  their  manufacture  are  not 
yet  present.  Simple  animals  such  as  hydra  contain  no  endocrine  glands 
and  indeed  no  circulatory  system,  yet  they  demonstrate  cellular  dif- 
ferentiation and  produce  seven  different  types  of  adult  cells  from  their 
original  zygote.  Clearly  there  is  a  chemical  progression  to  development,  a 
series  of  causes  where  early  effects  produce  later  results  almost  auto- 
matically as  envisioned  long  ago  by  Aristotle  in  his  famous  passage  from 
De  Generatione  Aniwalium: 

"It  is  possible,  then,  that  A  should  move  B,  and  B  move  C:  that  in 
fact  the  case  should  be  the  same  as  with  automatic  machines  shown  as 
curiosities.  For  the  parts  of  such  machines  while  at  rest  have  a  sort  of 
potentiality  of  motion  in  them,  and  when  any  external  force  puts  the  first 
of  them  in  motion,  immediately  the  next  is  moved  in  actuality." 

What  then  is  "A"  in  Aristotle's  list,  the  agent  that  operates  even  in  the 
blastula  and  gastrula  ?  How  can  the  ecto-,  endo-,  and  mesoderm  differ  so 
much  from  each  other  at  such  an  early  date  when  they  were  all  descended 
from  the  zygote  just  a  few  cell  generations  before  ?  My  purpose  in  this 
lecture  is  to  propose  the  hypothesis  that  Artistotle's  "A"  is  in  fact  carbon 
dioxide,  and  that  carbon  dioxide  tension — pCOo — is  the  first  self-produced 
regulator  in  embryological  development. 


CELL   DIFFERENTIATION 


511 


/ 


o- 


U—rr. X- 


U 


X     2. 
Z— O 

X      % 

z~< 


512  W.    F.    LOOMIS 

It  is  a  curious  fact  that  many  people  react  negatively  to  the  mere 
mention  of  carbon  dioxide  tension,  saying,  "Oh,  we  know  all  about  COg. 
There  is  nothing  new  in  that."  Most  of  them,  of  course,  do  not  know 
all  about  CO2,  or  even  about  the  crucial  diiferences  between  free  and 
combined  COg.  What  they  do  remember  is  the  headache  they  experienced 
studying  this  subject  in  their  graduate  student  days.  Such  at  least  was  my 
experience,  and  it  was  only  when  hard  experience  in  the  laboratory  forced 
me  to  the  conclusion  that  pCOg  was  the  active  variable  in  my  experimental 
system  that  I  finally  sat  down  and  attempted  to  master  the  subject  both 
theoretically  and  experimentally. 

This  was  about  4  years  ago.  Before  that,  I  had  found  that  hydra 
differentiated  sexually  into  mature  males  and  females  when  they  were 
grown  in  crowded  cultures,  but  did  not  do  so  when  grown  in  isolation. 
Clearly,  the  question  was,  "What  is  in  the  water  of  crowded  cultures  that 
makes  them  differentiate  along  this  new  pathway,  activating  these'  pre- 
viously dormant  genes  ?"  Attempts  to  take  crowded  water  and  use  it  to 
turn  a  single  hydra  sexual  were  unsuccessful  until  it  was  realized  that 
simple  aeration  could  remove  the  active  ingredient.  Here  then  was  a  clear- 
cut  system  with  which  to  study  some  of  the  chemical  variables  that  control 
cellular  differentiation. 

Our  first  finding  was  that  "crowded  water"  contained  less  dissolved 
oxygen  than  did  water  in  which  only  single  hydra  had  been  grown.  This 
suggested  that  lowered  oxygen  tension  was  the  operative  variable.  Further 
experiments  showed  that  this  was  not  the  case :  lowered  oxygen  tension 
accompanied  sexual  differentiation  in  hydra  but  did  not  cause  it.  Appar- 
ently some  gas  accumulated  in  the  water  of  crowded  cultures  that  induced 
hydra  to  differentiate  along  the  sexual  pathway  rather  than  along  the 
asexual.  What  was  this  differentiation-controlling  gas  ?  Analysis  by  infra- 
red spectrophotometry,  mass  spectrography  and  gas-liquid  partition 
chromatography  showed  that  water  from  crowded  cultures  of  hydra 
contained  increased  amounts  of  gaseous  CO.,  but  no  detectable  amounts 
of  any  gases  other  than  those  known  to  be  in  normal  air.  Since  earlier 
experiments  had  shown  that  no  amount  of  bound  CO2,  such  as  bicar- 
bonates  and  carbonates,  could  induce  sexual  differentiation  in  hydra,  it 
seemed  necessary  to  conclude  that  gaseous  CO.,  dissolved  in  water  was  the 
mysterious  variable  involved.  Secondary  variables  such  as  ammonia  might 
also  be  operating  in  the  system,  but  the  ability  of  free  COo  to  affect  cellular 
differentiation  seemed  inescapable.  This  conclusion  was  strengthened  by 
finding  [2]  that  uncrowded  hydra  could  be  turned  sexual  by  growing  them 
in  fresh  culture  water  that  had  been  artificially  enriched  with  CO2  gas 
(Table  I).  This  experiment  has  now  been  repeated  in  our  laboratory  seven 
times  and,  so  far,  has  always  reproduced  the  published  results.  I  need 
hardly  say  that  this  is  a  hard  rock  of  fact  in  a  field  of  variable  and  con- 


CELL   DIFFERENTIATION 

TABLE  I 

Control  of  Sexual  Differentiation  in  Hydra  bv  fCO., 


513 


Vessel 

I 

2 

3 

4 

5 

6 

7 

8 

Culture  water  shaken  w 

•ith 

100%  0,  (ml.) 

15 

14 

10 

5 

Culture    water   shaken 

wi 

th 

10%  CO.,  and  90",, 

0. 

(ml.) 

— 

0 

I 

5 

10 

Initial  pCOj 

o- 

0% 

o- 

6° 

b 

2- 

CO' 
"  ,0 

5- 

6% 

Day 

Percentage  of  sexual  forms 

I 

0 

0 

0 

0 

0 

0 

0 

0 

2 

0 

0 

0 

0 

0 

0 

0 

0 

3 

0 

0 

0 

0 

0 

0 

0 

0 

4 

0 

0 

0 

0 

0 

0 

0 

0 

5 

0 

0 

0 

0 

0 

0 

0 

0 

6 

0 

0 

0 

0 

0 

0 

0 

0 

7 

0 

0 

0 

0 

0 

0 

0 

0 

8 

0 

0 

0 

0 

10 

0 

0 

0 

9 

0 

0 

10 

30 

70 

70 

70 

60 

10 

0 

0 

60 

50 

100 

100 

100 

100 

1 1 

0 

0 

70 

60 

100 

100 

100 

100 

12 

0 

0 

100 

60 

ICG 

100 

100 

TOO 

13 

0 

0 

100 

70 

100 

TOO 

100 

100 

flicting  results  that  is  most  comforting  to  an  experimenter.  Before  free 
CO.,  was  recognized,  thirteen  separate  variables  had  had  to  be  controlled 
for  the  2  or  3  weeks  needed  for  such  experiments.  Thus,  after  4  years  of 
work,  I  was  forced  to  investigate  carbon  dioxide  tension  or  pCO.^.  How 
rewarding  this  has  been  will  appear  in  the  account  below,  for  it  not  only 
appears  that  pC02  is  the  chief  variable  responsible  for  sexual  differentiation 
in  hydra,  but  that  this  factor  plays  a  large  and  varied  role  in  many  other 
biological  phenomena  connected  with  growth  and  differentiation  [3]. 

Let  us  start  from  the  beginning.  Almost  all  animal  cells  respire. 
Crowding  respiring  cells  together  therefore  induces  partial  anaerobiosis  in 
the  centre  of  a  cell  aggregate  that  consists  of  (i)  lowered  oxygen  tension, 
and  (2)  increased  PCO2.  Two  inverse  gradients  exist  therefore  in  such  a 
cell  mass,  the  pCO.,  gradient  resembling  the  temperature  of  the  sun  in  that 
it  is  highest  in  the  centre  and  lowest  on  the  outside  surface.  Both  of  these 
gradients  may  operate  at  times  to  create  disparity  within  a  previously 
homogeneous  group  of  cells.  Between  the  two,  pCO._,  would  seem  to  be  the 
more  likely  candidate  for  Aristotle's  "A"  because  it  enters  into  such  a  wide 
variety  of  cellular  reactions  as  the  synthesis  of  purines  and  pyrimidines, 

VOL.  11. — 21. 


514  W.    F.    LOOMIS 

the  maintenance  of  oxaloacetate  levels  as  well  as  playing  a  vital  role  in 
setting  the  pH  of  the  interior  of  the  cell.  In  contrast  to  this  high  reactivity, 
molecular  oxygen  combines  almost  solely  with  cytochrome  oxidase  and 
that  in  a  manner  that  is  independent  of  the  level  of  oxygen  tension  except 
at  the  lowest  levels  [4].  As  a  possible  regulator  of  cellular  differentiation, 
therefore,  CO2  is  a  more  likely  candidate  than  oxygen ;  in  addition,  it  has 
the  cybernetic  advantage  of  being  actively  produced  rather  than  used  up 
with  the  resulting  difference  that  pCOg  increases  from  nearly  zero  at  the 
surface  to  its  highest  point  at  the  centre,  while  oxygen  tension  levels  do 
the  reverse  and  hence  are  far  more  at  the  mercy  of  the  over-all  environ- 
ment. 

Before  continuing,  certain  facts  concerning  CO2  must  be  reviewed,  for 
they  are  vital  to  any  understanding  of  the  subject.  Thus,  it  is  well  known 
that  pH,  or  concentration  of  the  hydrogen  ion,  is  something  quite  different 
from  the  total  amount  of  acid  present  in  a  buffered  solution.  In  the  same 
way,  pCOa,  or  the  partial  pressure  of  dissolved  CO.,  gas,  is  something 
quite  different  from  the  total  amount  of  CO2  that  may  be  in  a  solution  in 
such  hydrated  forms  as  bicarbonate  and  carbonate.  Only  free  gaseous  CO2 
is  given  off  by  a  solution  on  simple  aeration ;  both  free  and  bound  CO2  are 
given  off  following  the  addition  of  acid. 

Everyone  knows  the  characteristic  taste  of  dissolved  free  CO2  gas,  for 
the  taste  buds  of  the  tongue  are  uniquely  sensitive  to  this  variable  as  found 
in  beer,  champagne  and  similar  carbonated  beverages.  A  vivid  demonstra- 
tion of  this  fact  can  be  arranged  in  the  laboratory  by  simply  filling  a  large 
syringe  with  gas  from  a  tank  of  COo  and  then  shaking  it  with  a  small 
amount  of  water  that  has  subsequently  been  drawn  into  the  syringe.  Since 
this  water  sample  has  been  equilibrated  with  100%  of  an  atmosphere  of 
COo  (760  mm.  Hg),  it  has  a  PCO2  of  100%  atm.  The  basic  fact  is  that 
pressures  of  any  dissolved  gas  equalize  whenever  gas  and  water  phases  are 
shaken  together;  if  the  gas  phase  has  a  partial  pressure  of  pCOg  of  100% 
atm.,  then  the  water  phase  within  the  syringe  has  an  equal  PCO2.  It  should 
be  noted  that  syringes  are  extremely  useful  because  they  provide  the 
operator  with  an  adjustable  volume  that  is  always  automatically  main- 
tained at  a  pressure  of  i  atm.  In  contrast  to  this  simplicity,  the  concentra- 
tioti  of  free  COo  dissolved  within  the  water  phase  changes  with  both  the 
temperature  and  the  ionic  strength  of  the  solution,  for  Henry's  Law  states 
that  [COo]  =  cc  pCOo  and  both  temperature  and  ionic  strength  affect  the 
solubility  coefficient  a. 

If  the  water  in  such  a  syringe  is  now  expressed  into  a  small  beaker  and 
placed  beside  a  similar  beaker  of  plain  water,  the  demonstrator  may 
remark,  "Oh,  I  have  forgotten  which  is  which.  I  wonder  which  has  the 
PCO2  of  100%  atm.  and  which  has  a  PCO2  equal  to  that  of  air  ? "  (0-03% 
atm.).  At  this  point  the  onlooker  may  be  challenged  to  figure  out  some 


CELL   DIFFERENTIATION 


515 


way  of  finding  out  which  beaker  is  which,  using  only  his  five  unaided 
senses.  Since  no  bubbles  of  any  kind  are  visible  in  either  vessel,  no  visual 
difference  may  be  detected.  Eventually  the  onlooker  hesitatingly  takes  a 
sip  from  each  of  the  beakers,  whereupon  a  look  of  certainty  crosses  his 
face  as  he  remarks,  "This  is  the  beaker  that  has  the  high  pCOo.  It  is 
unmistakable." 

Further  experiments  can  be  conducted.  For  example,  does  water  made 
equally  acid  (pH  3-7)  with  HCl  taste  the  same  ?  (No.)  If  a  pCOa  of  100"  „ 
atm.  is  unmistakable,  can  the  human  tongue  detect  a  pCOg  of  50^0  atm.. 


Fig.  3.  General  view  of  pilot-plant  ior  Chlorella  production  designed  and  built 
by  Arthur  D.  Little,  Inc.,  Cambridge,  Mass.  Note  plastic  cover  to  photosyn- 
thesizing  trough  and  large  vertical  tower  for  treating  culture  medium  with  CO.,  gas. 


or  of  25",,  atm.  ?  Does  bicarbonate  with  an  equal  total  concentration  of 
dissolved  CO.^  taste  the  same  r  The  answers  are  quickly  and  vividly 
obtained,  for  onlv  gaseous  CO.,  dissolved  in  water  affects  the  taste  buds 
of  the  tongue  in  the  manner  that  is  specific  to  beer  and  other  carbonated 
waters. 

The  respiratory  centre  of  the  brain  also  contains  cells  that  are  sensitive 
to  pCO.,  as  a  variable  different  from  pH.  It  is  these  cells  that  regulate  our 
breathing  in  such  a  way  that  the  percentage  of  CO.,  in  the  base  of  our  lungs 
is  held  at  S\V,'o  CO.,,  a  physiological  mechanism  that  guarantees  that  our 


5l6  W.    F.    LOOM  IS 

arterial  blood  shall  have  a  pCOo  of  5-3",,  atm.  at  all  times.  Many  students 
believe  that  breathing  is  regulated  by  CO^  as  an  indirect  means  of  con- 
trolling the  oxvgen  in  the  blood.  In  fact,  the  body  is  immune  to  changes  in 
oxygen  tension  within  wide  limits  as  may  be  demonstrated  by  the  lack  of 
any  bodily  reaction  to  breathing  pure  oxygen  when  at  sea-level. 

Living  cells  then  react  to  free  or  gaseous  CO2  in  a  manner  that  is 
different  from  their  reaction  to  the  bicarbonate  ion.  This  difference  be- 
tween pCO.,  and  total  CO.,  is  fundamental  to  any  understanding  of 
biological  crowding  effects  and  stems  from  the  little-known  fact  that  CO2 


Gas  exchange 
tower   X 


Control 
Compressor  house 


Algal  suspension 
Culture  medium 
COg-air 
Water 


Cooler 


Gas  burner 


Mixing 
tank 


Pump 


Fig.  4.  Diagram  of  algae  plant. 


is  a  fat-soluble  gas  that  is  1-67  times  more  soluble  in  lipoid  solutions  such 
as  olive  oil  than  it  is  in  water  (o  ).  This  lipo-solubility  enables  the  free  CO., 
molecule  to  pass  through  a  fatty  cell  wall  when  the  bicarbonate  ion  can  not, 
the  cell  membrane  therefore  acting  as  a  semi-permeable  membrane  that 
distinguishes  between  free  and  bound  CO.,  molecules.  These  facts  are  well 
known  to  plant  physiologists,  for  they  have  found  that  photosynthesizing 
algae  can  only  obtain  the  large  amounts  of  CO2  that  they  require  from 
gaseous  CO2  which  can  penetrate  their  cell  walls  when  the  bicarbonate 
can  not.  Any  algae  farm  therefore  has  two  characteristic  features:  (Fig.  3) 


CELL    DIFFERENTLATION  ^ly 

the  large  flat  culture  beds  that  are  exposed  to  sunlight  and  the  tall  towers 
through  which  ascending  CO.,  gas  is  bubbled  through  the  descending 
culture  medium  that  is  circulated  through  the  culture  beds  (Fig.  4).  If 
the  bicarbonate  ion  were  equivalent  to  free  CO.,,  then  the  photosyn- 
thesizing  troughs  would  not  have  to  be  covered  with  a  transparent  plastic 
cover  that  reduces  the  available  light  but  keeps  in  the  vital  CO.,  gas.  All 
that  would  be  necessary  would  be  to  add  bicarbonate  to  the  culture  media 
and  grow  the  algae  in  direct  sunlight. 

Jacobs  [5]  has  described  an  elegant  experiment  to  show  that  free  CO., 
can  selectively  penetrate  cell  walls  and  produce  intracellular  acidity  e\"en 
in  alkaline  solutions.  He  found  a  flower  whose  colour  changed  reversibly 
from  blue  to  red  when  it  was  dipped  in  alkaline  or  acid  solutions,  an 
internal  type  of  litmus  reaction  that  could  be  used  to  determine  intra- 
cellular pH.  When  this  flower  was  placed  in  an  alkaline  solution  that  had 
a  pCO.,  of  5",,  atm.,  the  colour  of  the  flower  changed  from  blue  to  red, 
indicating  that  the  free  gaseous  COo  in  solution  had  penetrated  the  fatty 
cell  membranes  and  had  ionized  inside  these  cells  to  form  carbonic  acid. 

Accepting  pCO.^  therefore,  as  an  important  but  unfamiliar  biological 
variable,  what  are  the  units  used  to  measure  it  .-  The  easiest  unit  I  believe 
is  as  a  percentage  of  an  atmosphere,  for  almost  all  biological  reactions  are 
carried  out  near  sea-level  and  hence  simple  percentages  mav  be  used  rather 
than  the  more  confusing  pressure  units  such  as  millimetres  of  mercury. 
Take  for  example  the  syringe  described  above  in  which  water  and  pure 
carbon  dioxide  gas  had  been  shaken  together  to  equilibrium.  The  water  in 
this  syringe  has  a  pCOo  of  760  mm  Hg.  but  it  is  just  as  true,  and  more 
vivid  operationally,  to  say  that  it  has  a  pCOo  of  100",,  of  an  atmosphere. 
This  latter  terminology  brings  to  mind  the  method  of  making  such 
solutions,  for  water  shaken  with  50",,  CO2  gas  mixtures  has  a  pCO.^  of 
5o"o  of  i^ii  atmosphere;  that  shaken  with  lo*^,',  CO.^  has  a  pCO.,  of  lo'^'o 
atm. ;  and  that  shaken  with  i",,  CO.,  has  a  pCO._,  of  i",,  atm. 

This,  then,  is  the  simplifying  principle  behind  our  nomenclature  that 
allows  one  to  handle  concentrations  of  free  COo  with  the  same  ease  as  one 
handles  calcium  ion  concentration.  •- 

We  have  recently  devised  a  rapid  means  of  measuring  pCO.,  that  does 
not  involve  pH  and  so  avoids  many  of  the  complications  that  ha\  e  plagued 
this  field  [6].  The  essence  of  the  measurement  is  to  shake  10  ml.  of  the 
sample  of  water  to  be  tested  with  an  equal  volume  of  air  and  then  to 
determine  how  much  CO.,  enters  the  gas  phase  from  the  solution.  The 
results  ot  this  measurement  are  simply  multiplied  bv  a  factor  to  obtain  the 
pCOo  of  the  original  solution.  In  our  laboratorv  this  summer,  my  son  and 
I  had  side-by-side  arrangements  for  measuring  pH  and  pCOo.  This  en- 
abled us  to  examine  all  cultures  from  both  these  angles,  as  well  as  to  vary 
pCO.,,  pH  and  bicarbonate  concentration  relative  to  one  another.  Since  the 


5l8  W.    F.    LOOMIS 

well-known  Henderson-Hasselbalch  equation  states  that  the  concentration 
of  the  H+  ion  varies  both  with  the  pCOg  and  the  bicarbonate  present: 


Concentration  of  H+ 


HCO3 


it  is  clear  that  any  change  in  one  of  these  three  variables  will  affect  at  least 
one  of  the  other  two.  Picture  a  metal  triangle  supported  at  its  centre,  on  a 
table,  its  three  corners  representing  pH,  pCOa  and  bicarbonate  respec- 
tively. If  now  one  of  the  three  corners  of  the  triangle  is  held  firmly  against 
the  table,  it  will  remain  constant  while  the  other  two  vary  reciprocally  like 
a  see-saw.  This  is  the  principle  of  a  triad  of  experiments  that  we  have  used 
to  determine  which  of  these  three  variables  is  the  biologically  active  one 
in  any  given  situation.  Experimental  control  of  pCOg  may  be  effected  by 
(i)  exposing  shallow  Petri  dish  cultures  to  known  concentrations  of  CO, 
within  a  desiccator;  (2)  injecting  closed  containers  with  varying  volumes 
of  a  culture  solution  high  in  pCOo  that  has  been  previously  prepared  by 
shaking  it  in  a  syringe  with  air  containing  the  desired  amount  of  COo  gas; 
and  (3)  bubbling  the  experimental  culture  continuously  with  air  from  a 
tank  containing  the  desired  concentration  of  COg.  In  my  laboratory  in 
Greenwich  I  have  a  series  of  gas  tanks  that  vary  from  o- 1%  to  100%  COg. 
With  their  aid,  almost  any  desired  pCO.,  can  be  easily  and  rapidly  obtained. 

Today,  then,  tissue  pCO.,  represents  an  old-but-new  variable  of  some 
complexity.  Small  wonder  that  most  modern  textbooks  either  ignore  the 
subject  or  else  dangerously  oversimplify  it.  Many  people,  for  example,  have 
been  taught  that  carbonic  acid  is  a  weak  acid  when  in  fact  it  is  as  strong  an 
acid  as  citric,  formic  or  nitrous  acid!  [7].  The  reason  for  this  widespread 
misconception  is  the  little  appreciated  fact  that  99-9%  of  the  CO2  dis- 
solved in  water  does  not  hydrate  to  HoCOg  but  remains  as  free  gaseous 
CO2  [8].  The  o-i%  of  carbonic  acid  that  does  form  is  a  surprisingly 
strong  acid.  Taken  together,  these  two  facts  combine  to  make  gaseous  CO2 
equivalent  to  a  weak  acid,  a  simplification  that  may  be  legitimate  in  certain 
situations  but  not  in  others. 

Summarizing  the  physical-chemical  facts  then,  we  can  say  that  pCOa 
is  a  universal  biological  variable  generated  by  all  respiring  cells.  Being  fat- 
soluble,  COo  can  easily  pass  through  fatty  cell  membranes  and  hence 
unify  a  cellular  aggregate  into  one  overall  "field  of  force".  Highly  reactive 
chemically,  it  enters  into  many  cellular  reactions  as  a  direct  participant  as 
well  as  specifically  affecting  intracellular  pH.  Present  as  a  gas  within  the 
alveoli  of  the  lung,  as  a  dissolved  gas  within  the  tissues  and  finally  as  a 
solid  within  the  matrix  of  a  bone  or  shell,  five  separate  steps  are  needed  to 
connect  all  forms  of  this  one  metabolite,  the  final  complexity  arising  when 
part  of  this  inorganic  chain  is  catalyzed  by  the  zinc-containing  enzyme 
carbonic  anhydrase  and  so  is  subject  to  all  the  variables  that  affect  the 


CELL   DIFFERENTIATION  519 

activity  of  enzymes.  Extremely  difficult  to  measure  until  recently,  it  is 
small  wonder  that  most  biologists  and  biochemists  have  avoided  the  subject 
as  much  as  possible  and  even  dismissed  it  from  existence  with  remarks 
like,  "  Oh,  we  use  COo  to  set  the  pH,  that's  all."  If  this  were  "  all"  in  fact, 
then  the  same  pH  could  far  more  easily  be  obtained  with  one  of  the  many 
excellent  buffers  that  are  not  volatile.  Yet  "setting  the  pH"  with  gaseous 
COo  is  compulsory  in  tissue  culture  (J.  H.  Hanks  [9]) : 

"The  problem  of  pH  control  often  appears  baffling  when  cell  culture 
work  is  first  undertaken.  In  view  of  the  simplicity  of  many  buffer  systems 
it  seems  almost  a  crime  that  a  gas  such  as  COo  in  equilibrium  with  HoCOg 
and  NaHCOg  should  be  a  major  physiological  mechanism  of  pH  control, 
that  this  system  should  be  essential  for  respiration  and  growth,  and  that 
it  should,  at  the  same  time,  afford  such  inefficient  buffering  action  in  the 
working  range  of  pH  7-8.  Until  such  time  as  man  or  Maker  may  provide 
a  substitute,  one  must  be  prepared  to  fight  the  battle  of  CO.^." 

Returning  now  to  cellular  differentiation,  let  us  picture  nature  trying 
endless  experiments  at  the  protozoan  level  in  an  attempt  to  obtain  more 
than  one  kind  of  cell  with  which  to  build  a  metazoan  animal.  Stacking 
identical  protozoa  together  into  larger  and  larger  clusters  would  auto- 
matically expose  the  central  cells  to  higher  and  higher  levels  of  pCO.,. 
Since  fatty  cell  membranes  form  no  appreciable  barrier  to  free  CO2 
molecules,  the  whole  mass  of  respiring  cells  would  form  one  large  "  field  of 
force"  whose  medullary  pCOo  would  be  far  higher  than  the  peripheral 
cortex.  Such  a  unifying  "field  of  force  "  would  represent  a  function  of  the 
whole,  for  if  it  were  cut  in  two,  new  gradients  of  pCOo  would  form  within 
each  half  just  as  they  do  when  a  pile  of  glowing  coals  is  divided  in  two : 
the  centre  of  each  aggregate  soon  is  hotter  than  the  newly  exposed 
periphery. 

Suppose  a  mutant  should  now  arise  among  these  protozoa,  a  mutant 
whose  DNA  behaved  differently  under  high  and  low  levels  of  pCOo:  i.e. 
a  protozoan  with  genetic  material  that  could  not  be  expressed  pheno- 
typically  except  when  grown  under  conditions  of  high  pCOo.  Clearly  such 
a  protistan  would  replicate  until  a  critical  mass  was  formed  in  whose 
centre  the  pCO.,  reached  the  postulated  threshold  for  the  activation  of 
this  additional  set  of  mutant  genes.  Here  then  would  be  a  mechanism  by 
which  replication  could  lead  to  differentiation,  and,  with  further  elabora- 
tion as  during  gastrulation,  Aristotle's  A  could  lead  to  B  and  C. 

Thus,  it  is  known  that  slime-mould  amoebae  lay  down  walls  of  cellulose 
only  when  they  are  buried  deep  inside  the  multicellular  pseudoplasmodial 
aggregate.  Would  it  not  be  a  great  step  forward  if  it  were  found  that  single 
slime-mould  amoebae  form  walls  of  cellulose  when  grown  in  isolation  if 
they  are  exposed  to  increased  levels  of  pCOo  ? 

Many  years  ago  Rache\sky  examined  the  mathematical  relations  that 


W.    F.    LOOMIS 


520 

would  exist  around  a  spherical  cell  that  produced  an  diffusible  meta- 
bolite [10].  Figure  5  presents  his  results  as  applied  to  pCOa-  Clearly  the 
curve  of  pCOo  is  highest  in  the  centre  of  the  postulated  cell  and  drops  in 
hyperbolic  fashion  towards  the  periphery.  A  further  drop  at  the  cell 
membrane  then  occurs  that  is  dependent  on  the  permeability  of  the  meta- 
bolite in  question.  Since  we  know  that  fatty  cell  membranes  are  more 
permeable  to  CO2  than  to  oxygen  or  even  water,  this  second  part  of  the 
curve  may  be  essentially  eliminated  from  consideration  in  the  case  of  COg. 
Outside  the  wall  appears  a  third  gradient  that  I  refer  to  as  the  "blue 


Fig.  5.  Gradient  of  pCO.,  in  a  spherical  cell  or  cell  aggregate:  q  =  respiratory 
rate ;  Z),  =  rate  on  internal  diffusion  (or  cell  streaming) ;  £),  rate  of  external  diffusion 
(or  convectional  streaming) ;  h  =  permeability  of  the  membrane  and  r^  =  radius  of 
cell  or  cell  aggregate.  Modified  from  Rachevsky  [10]. 


haze"  effect,  for  it  makes  me  think  of  the  quiet  lounge  of  some  London 
club  where  three  older  members  are  reading  their  newspapers,  each 
member  surrounded  by  a  blue  haze  of  pipe  smoke  that  he  has  produced 
himself.  Clearly  any  analysis  of  the  smoke  within  the  room  that  first 
allowed  it  to  become  mixed  would  not  give  a  correct  idea  of  the  smoke 
concentration  to  which  each  club  member  had  been  exposed  all  afternoon. 
Rachevsky 's  third  or  blue-haze  gradient  therefore  reflects  the  degree  of 
stagnation  within  the  system.  Whenever  extracellular  currents  exist,  no 
external  gradients  can  form,  while  simple  stagnation  reacts  upon  a  system 
so  as  to  increase  the  final  level  of  pCO.^  existing  at  the  centre  of  the 
respiring  mass. 


CELL    DIFFERENTL\TION  52I 

Rachevsky's  fourth  factor  is  the  level  of  pCOa  in  the  general  back- 
ground. Human  tissues,  for  example,  have  a  background  of  5  •3^0  atm. 
for  this  exact  level  of  pCO.,  is  carefully  maintained  in  the  arterial  blood 
stream  by  the  medullary  centre  of  the  brain.  Most  fresh-water  and  marine 
animals  in  contrast  are  exposed  to  a  background  of  about  0-03",',  atm.  [i  i], 
for  this  is  the  level  of  pCO.,  that  exists  in  water  that  has  been  equilibrated 
with  normal  air. 

Four  separate  factors  then  contribute  to  the  pCO.,  existing  at  the 
centre  of  a  spherical  cell  where  the  chromosomes  usuallv  are  found.  Of 
these,  the  single  most  important  is  probably  the  size  of  the  cell  or  cell 
aggregate  itself,  for  here  the  parabolic  cur\e  rises  as  the  square  of  the 
radius.  Is  this  the  reason  that  a  large  cell  like  an  amoeba  liquefies  its 
central  protoplasm  ?  Certainly  no  amoeba  can  build  a  steep  Rache\  skv 
gradient  on  a  permanent  basis,  for  its  protoplasm  is  continuallv  rolling 
over  and  thus  destroying  the  geometrical  relations  that  caused  it  to  arise 
in  the  first  place. 

Experimental  study  of  amoebae  in  our  laboratory  suggests  that  their 
reversible  solation-gelation  is  pCOo  dependent.  Pantin,  for  example,  has 
used  pH-dependent  vital  dyes  to  show  that  the  cvtoplasm  of  newly 
forming  pseudopods  is  decidedly  more  acid  than  the  older  gelated 
material  [12].  \\'e  ha\e  taken  acti\ely  migrating  amoebae  that  are  extended 
in  thin  strands  like  the  horns  of  a  deer  and  exposed  them  to  20",,  CO.,. 
Almost  immediately,  the  ends  of  such  staghorns  begin  to  soften  and 
"melt"  back  into  the  body  of  the  amoeba  so  that  it  soon  assimies  a  uni- 
formly round  and  spherical  appearance.  Clearly  the  normal  inside-out 
gradient  of  pCO.2  has  been  abolished  by  the  artificial  application  of  20",, 
CO.,  from  the  outside-in.  If  now  this  same  animal  is  examined  20  min. 
later,  it  will  be  found  to  be  moving  around  in  normal  staghorn  fashion 
just  as  if  it  were  not  still  under  20",,  CO.2.  What  has  happened  in  these  20 
min.  .-  Clearly  the  answer  is  that  continued  respiration  on  the  part  of  the 
amoeba's  large  mass  of  cytoplasm  has  allowed  a  new  inside-out  gradient 
to  form  even  against  an  outside  background  of  20",,  CO.2;  the  inside  of 
such  an  adjusted  amoeba  therefore  is  again  softer  than  the  outside  and 
once  again  it  can  begin  to  flow  out  from  a  central  liquid  pool  in  successive 
"  lar\a  flows "  that  cool  and  solidify  as  they  loose  their  excess  CO.,.  Experi- 
mental evidence  for  this  view  is  provided  by  the  fact  that  this  adjustment 
to  20'j'o  COo  does  not  occur  under  anaerobic  conditions.  Further  evidence 
is  provided  by  the  fact  that  dinitrophenol  does  not  inhibit  normal  amoeboid 
movement  even  though  it  is  known  to  uncouple  specifically  oxidation  from 
phosphorylation  [13],  and  hence  inhibits  ATP  production  while  allowing 
CO.2  production  to  continue.  Finally,  it  should  be  mentioned  that  a  pC02 
of  50",,  atm.  permanently  liquefies  such  an  amoeba  and  reduces  it  to  a 
round  sphere  that  is  unable  to  form  pseudopodia.  Bv  this  x'lew,  amoeboid 


522  W.    F.    LOOMIS 

motion  results  secondarily  from  the  protoplasmic  streaming  that  was 
originally  designed  to  aerate  even  the  innermost  cytoplasm  of  a  cell.  With 
a  cell  as  enormous  as  an  amoeba,  this  is,  of  course,  more  necessary  than 
usual. 

Gradients  of  pCOo  are  highly  dependent  on  simple  geometrical  forces 
such  as  those  of  total  mass,  flattened  versus  spherical  shape  and  similar 
changes  in  the  surface /volume  ratio  of  an  aggregate  of  cells.  A  delightful 
description  of  the  fundamental  character  of  such  geometrical  forces  is 
presented  in  John  Bonner's  Morphogenesis  [14].  His  conclusion  is  in- 
escapable: i.e.  nature  uses  these  simplest  of  all  considerations  to  build  up 
progressive  complexities  during  the  development  of  an  embryo.  The 
enormous  difference  between  the  fate  of  a  blastomere  that  is  separated 
from  other  cells  and  one  that  is  left  attached  to  another  blastomere,  be  it 
alive  or  dead,  is  a  case  in  point.  Even  a  dead  blastomere  somehow  affects 
its  living  twin  by  its  mere  physical  presence,  an  effect  that  can  at  least 
speculatively  be  assigned  to  a  distortion  of  the  gradient  fields  of  pCOo 
produced  in  the  living  half. 

If  embryonic  differentiation  is  to  proceed  along  a  pCOg  gradient  of 
the  type  Rachevsky  pictured,  it  must  be  permanent  and  not  upset  by 
cytoplasmic  streaming  as  in  the  body  of  an  amoeba.  One  means  of  stabil- 
izing such  a  gradient  is  to  have  the  egg  cleave  progressively  into  smaller 
and  smaller  cells  so  that  the  protoplasm  at  the  centre  of  the  mass  is  locked 
in  place  along  the  over-all  gradient  that  extends  throughout  the  entire 
mass  of  respiring  cells  (Fig.  5).  Looked  at  from  this  angle,  it  is  not  sur- 
prising that  cell  cleavage  is  the  first  order  of  business  in  the  developing 
embryo,  for  it  prevents  cytoplasmic  streaming  by  the  erection  of  cell 
membranes  through  which  COg  molecules  may  travel  but  behind  which 
the  protoplasmic  contents  of  each  cell  is  locked  in  place.  According  to  this 
view,  a  physico-chemical  gradient  of  pCOg  would  first  form  as  a  result 
of  the  respiration  of  the  cells  themselves.  Only  gradually  would  this 
chemical  gradient  be  transformed  into  structurally  different  cells  that 
differentiated  in  each  location  according  to  the  micro-environmental  level 
of  pCO.,  that  existed  at  that  particular  site. 

John  Bonner  has  emphasized  that  the  embryo  uses  cell  movement  as 
well  as  cell  growth  and  differentiation  to  achieve  its  ends  (Fig.  6).  Here 
we  encounter  such  problems  as  (i)  the  acrasin  phenomenon  in  which 
slime-mould  amoebae  become  mutually  attractive  to  each  other;  (2)  why 
the  dorsal  lip  of  the  blastopore  grows  downward  and  into  the  hollow  sphere 
of  the  blastula  when  it  might  well  grow  outward  into  new  space  as  during 
budding;  (3)  why  certain  epithelial  cells  sink  down  below  the  surface  as 
during  the  formation  of  the  neural  groove. 

If  cell  migration  is  vital  to  embryogenesis,  perhaps  it  is  because  it  takes 
cells  that  have  been  programmed  one  way  and  then  exposes  them  to  another 


CELL    DIFFERENTIATION 


523 


micro-en\ironment  such  that  "outside "cells  now  find  themselves"  inside" 
as  in  the  case  with  buried  neural  tissue  that  once  was  external  epithelium. 
The  three  germ  layers  of  the  embryo,  in  fact,  represent  the  outside  and 
inside  of  a  hollow  sphere  respectively,  together  with  those  most  anaero- 
bically  placed,  the  mesoderm  that  is  literally  sandwiched  between  its 
epidermal  and  endodermal  neighbours.  A  picture  of  sequential  pro- 
gramming thus  arises,  the  order  of  the  programming  being  vital  in  that 
exposure  to  high-then-low  levels  of  micro-environmental  pCOo  should 


morphogenetic 
movement 


differentiation 


Fig.  6.  Diagram  to  illustrate  tht  interrelatiuns  between  growth,  morphogenetic 
movement,  and  cellular  differentiation  (from  Bonner  [14]). 


cause  a  ditferent  type  of  differentiated  cell  from  one  that  has  been  exposed 
to  low-then-high.  As  in  the  education  of  a  young  man,  exposure  to 
different  environments  can  produce  different  final  results. 

Why  do  cells  move  from  one  location  to  another  within  the  developing 
embryo  ?  One  answer  may  be  that  they  become  positively  or  negatively 
chemotactic  to  pCOo.  Thus,  Chlamydomonas  cells  are  known  to  be  chemo- 
tactic  to  pCO.,  [15]  and  even  the  acrasin  phenomenon  in  slime  moulds  may 
be  due  to  the  mutual  attraction  of  the  washed  amoebae  owing  to  their 
positive  chemotaxis  towards  a  high  pCO.,  such  as  that  generated  by  their 
neighbours.  Preliminary  experiments  in  our  laboratory  have  shown  that 


524  W.    F.    LOOMIS 

as  little  as  5%  CO2  reversibly  inhibits  aggregation,  an  inhibition  that  may 
be  due  to  the  failure  of  one  amoeba  to  find  the  COo  generated  by  its 
neighbour  when  a  high  percentage  of  CO2  is  present  everywhere  and  in  no 
relation  to  the  location  of  other  amoebae.  This  possibility  is  further 
supported  by  the  fact  that  slime  mould  amoebae  will  not  aggregate  in  the 
presence  of  bacteria — possibly  because  these  bacteria  also  generate  CO2 
and  so  drown  out  the  amoeba-to-amoeba  message.  Like  fireflies  in  the 
daylight,  the  background  is  too  high  for  the  "message"  to  get  through. 

In  summary,  embryology  seems  surprisingly  hospitable  to  various 
applications  of  the  pCOo  theory.  Not  only  does  it  shed  light,  and  suggest 
experiments,  on  the  meaning  of  egg  cleavage,  but  it  continues  to  do  so 
during  gastrulation  and  the  formation  of  the  three  germ  layers.  Formative 
cell  movements  connected  with  the  formation  of  the  heart  have  been 
shown  by  Ebert  [16]  to  begin  only  after  a  critical  cell  mass  has  been 
achieved.  Looked  at  from  the  PCO2  point  of  view,  such  facts  become 
understandable,  for  the  embryo  seems  to  contain  its  future  structure 
within  itself  much  as  a  fireworks  rocket  exploding  in  the  night  sky  first 
releases  a  burst  of  blue,  then  red,  followed  in  turn  by  yellow  and  green, 
the  fuse  of  each  setting  ofl^  each  subsequent  explosion.  Rather  than  looking 
for  some  structure-giving  external  organizer  such  as  was  pictured  in 
Spemann's  day,  we  can  visualize  a  sequence  of  micro-environments  self- 
created  by  the  developing  egg  as  it  cleaves  into  a  thousand  cells  and  then 
invaginates  to  form  the  three- layered  structure  of  the  gastrula.  Spemann's 
induction  of  a  second  embryo  is  not  so  miraculous  as  it  first  appears,  for 
once  started,  an  inevitable  chain  of  events  would  proceed  as  they  do  when 
activated  by  the  entrance  of  a  sperm.  Indeed  the  facts  of  parthenogenesis 
suggest  that  the  originating  stimulus  is  of  secondary  importance,  for  many 
causes  may  set  oif  the  chain  reaction  that  is  embryogenesis.  The  real 
miracle  is  that  unwanted  embryos  do  not  start  more  often;  perhaps  they 
do  as  teratomas,  those  monstrous  tumours  composed  of  disorganized  bits 
of  hair  and  cartilage,  bone  and  epithelium. 

Space  does  not  permit  consideration  of  the  many  possible  biological 
roles  of  tissue  PCO2  in  tissue  culture,  cytostasis,  neoplastic  growth,  sexual 
maturation  in  both  plants  and  animal  tissues,  limnology,  etc.  Some  of  these 
aspects  were  discussed  at  the  17th  Symposium  of  the  Society  for  the  Study 
of  Development  and  Growth  [3].  Suffice  it  to  say  that  work  for  many  hands 
exists  within  this  difficult  but  rewarding  area,  work  that  can  aim  at  repro- 
ducing on  the  level  of  an  isolated  cell  all  the  conditions  found  w^ithin  a 
developing  tissue,  so  that  such  an  isolated  cell  diff^erentiates  morphologi- 
cally just  as  if  it  were  still  surrounded  by  its  normal  cell  neighbours. 


CELL   DIFFERENTL'ITION  525 

References 

1.  Sonneborn,  T.   M.,   in  "Genetics  in  the   20th   Century",  ed.   L.   C.   Dunn. 
Macmillan  Co.,  New  York,  291-314  (195  i). 

2.  Loomis,  W.  F.,  Science  126,  735-739  (1957). 

3.  Loomis,  W.   F.,  ///  "Cell,  Organism  and  Milieu",  ed.  D.  Rudnick.  Ronald 
Press  Co.,  New  York,  253-294  (1959). 

4.  Chance,  B.,  Fed.  Proc  16,  671-680  (1957). 

5.  Jacobs,  M.  H.,  Amer.J.  Physiol.  53,  457-463  (1920). 

6.  Loomis,  W.  F.,  Anal.  CJiem.  30,  1 865-1 868  (1958). 

7.  Buytendyk,  F.  J.  J.,  Brinkman,  R.,  and  Mook,  H.  W.,  Biochem.J.  2i,  576-584 

(1927)- 

8.  Bull,  H.  B.,  "Physical  Biochemistry".  John  Wiley  and  Sons  Inc.,  New  York, 
III  (1943). 

9.  Hanks,  J.   H.,  in  "An   Introduction   to   Cell   and  Tissue   Culture".   Burgess 
Publishing  Co.,  Minneapolis,  p.  6  (1955). 

ID.   Rachevsky,   N.,   "Mathematical    Biophysics".    Uni\ersity   of  Chicago   Press, 
Chicago,  18  (1938). 

11.  Loomis,  W.  F.,  and  Loomis,  W.  F".,  Jr.,  Biol.  Bull.  119,  295-296  (i960). 

12.  Pantin,  C.  F.  A.,J7.  marine  Biol.  Assoc.  13,  24-69  (1923). 

13.  Loomis,  W.  F.,  and  Lipmann,  F.,_7.  hiol.  Client.  173,  807-808  (1948). 

14.  Bonner,  J.  T.,  "Morphogenesis".  Princeton  LTniversity  Press,  Princeton,  N.J. 
(1952). 

15.  Mayer,  A.  M.,  and  PoljakofF-Mayber,  .\.,  Nature,  Lond.  180,  927  (1957). 

16.  Ebert,  J.  D.,  in  "  Aspects  of  Synthesis  and  Order  in  Cirowth  ",  ed.  D.  Rudnick, 
Princeton  University  Press,  Princeton,  N.J.  (1954). 


RNA  Synthesis  in  the  Nucleus  and  RNA  Transfer  to 
the  Cytoplasm  in  Tetrahymena  pyriformis 

D.  M.  Prescott 

Biology  Division,  Oak  Ridge  National  Laboratory  * 
Oak  Ridge,  Tenn.,  U.S.A. 

Studies  of  pH]-cytidine  labelling  in  Tetrahymena  have  been  made  in 
regard  to  two  problems,  (i)  the  transfer  of  RXA  between  nucleus  and 
cytoplasm,  and  (2)  the  nucleus  as  the  major  or  exclusive  site  of  RNA 
synthesis. 

In  earlier  experiments,  diverse  types  of  cells  have  been  incubated  with 
pH]-cytidine  or  other  labelled  precursors  of  RNA.  The  first  radioactivity 
incorporated  into  RNA  has  consistently  been  localized  in  the  nucleus  [1-7]. 
After  a  measurable  time  lag,  labelled  RNA  begins  to  accumulate  in  the 
cytoplasm.  These  experiments  have  been  interpreted  as  a  demonstration 
of  RNA  synthesis  in  the  nucleus  and  the  transfer  of  this  molecule  to  the 
cytoplasm.  With  this  explanation  it  has  been  implied  or  stated  that  the 
nucleus  is  a  principal  site  of  RNA  synthesis.  The  early  incorporation  of 
activity  into  the  nucleus  certainly  does  not  appear  open  to  any  other 
interpretation  than,  at  the  very  least,  a  rapid  synthesis  of  RNA  in  that 
location. 

The  three  types  of  experiments  were : 

1.  A  time-study  of  RNA  synthesis  in  the  nucleus  and  cytoplasm  with 
[•^H]-cvtidine  continuously  present  in  the  medium. 

2.  A  study  of  the  pattern  of  labelling  in  the  nucleus  and  cytoplasm  after 
a  short  exposure  to  [^H]-cytidine. 

3.  Investigation  of  the  capacity  of  nucleated  and  enucleated  cells  to 
incorporate  [•'^H]-cytidine  into  RNA. 

All  three  experiments  lead  to  one  general  conclusion;  all  RNA  is  syn- 
thesized in  the  nucleus,  and  cytoplasmic  RNA  is  of  nuclear  origin. 

Figure  i  shows  the  pattern  of  [^H]-cytidine  accumulation  into  nuclear 
and  cytoplasmic  RNA  of  Tetrahymena.  In  the  first  group  of  experiments, 
10  fxc.'ml.  of  [^H]-cytidine  were  added  to  an  early  log  phase  culture.  At 
intervals  of  a  few  minutes,  groups  of  cells  were  withdrawn,  dried  on  slides, 

*  Operated  by  Union  Carbide  Corporation  for  the  U.S.  Atomic  Energy 
Commission. 


528  D.    M,    PRESCOTT 

fixed,  extracted  to  remove  acid-soluble  material,  and  autoradiographed. 
Within  only  i  -5  min.  after  addition  to  the  medium,  pH]-cytidine  is  taken 
up,  converted  to  the  appropriate  form  and  incorporated  into  nuclear  RNA 
(Fig.  2(a)).  After  5  min.  the  rate  of  pH]-cytidine  incorporation  into  the 
nuclear  RNA  rises.  Incorporated  activity  is  not  detected  in  the  cytoplasm 
until  about  12  min. ;  in  contrast,  the  nucleus  is  densely  labelled  (Fig.  2(6)). 
After  12  min.,  label  accumulates  steadily  in  cytoplasmic  RNA  but  the  rate 
of  accuitmJation  of  radioactivity  in  the  nucleus  recedes  to  a  lower  value  at 
about  25  or  30  min.  At  35  min.  the  nucleus  and  cytoplasm  are  equally 
labelled  (Fig.  2(r)),  and  at  60  min.  the  cytoplasm  contains  more  than  twice 
as  much  label  as  the  nucleus.  The  nucleus  at  this  time,  however,  is  still 


(60- 


|120- 


40- 


25        30       35 

TlME(min) 

Fig.  I .  The  two  curves  show  the  time  course  of  the  total  amount  of  [^H]-cytidine 
incorporated  into  RNA  of  the  nucleus  and  cytoplasm  of  Tetrahymena  with  the 
isotope  continuously  present  in  the  medium.  Each  point  is  the  mean  grain  count 
for  autoradiographs  for  23  to  26  cells.  The  range  for  each  point  indicates  95% 
confidence  limits. 


more  densely  labelled.  Ribonuclease  digestion  shows  that  DNA  synthesis 
contributes  very  little  to  this  incorporation.  L  nlabelled  deoxycytidine  has 
been  added  to  the  medium  with  the  intention  of  minimizing  pH]-cytidine 
entrance  into  DNA  in  all  experiments.  The  time  of  appearance  of  tritium 
in  cytoplasmic  RNA  varies  from  one  experiment  to  another.  In  one  case  it 
occurred  slightly  earlier  than  13  min.  and  in  another  experiment  did  not 
begin  until  25  min.  In  the  latter  experiment,  the  longer  delay  is  probably 
related  to  a  short  interruption  in  cell  proliferation  imposed  by  transfer  of 
the  log  phase  cells  to  fresh  nutrient  medium  just  before  the  experiment 
was  begun. 


$   • 


* 


•    a 


.»••  ^  • 


•  .  •      * .  -•  I  •  t 


■8     • 

*    ^     •      r      • 

•    :•  •        .  •  ^*^^ 


Fig.  2.  (rt)  Autoradiograph  of  a  Tetra/iytneiia  incubated  in  [^H]-cytidine  for  i  -5 
min.  All  incorporation  is  localized  in  the  nucleus. 

(b)  Autoradiograph  of  a  Tetrahytnena  incubated  in  [-'HJ-cytidine  for  12  min. 
.All  label  is  still  localized  in  the  nucleus. 

(c)  Autoradiograph  of  a  Tetrahytnena  incubated  in  [^H]-cytidine  for  35  min. 
Nucleus  and  cytoplasm  contain  equal  amounts  of  label,  although  the  nuclear  label 
is  still  more  dense. 

(d)  Autoradiograph  of  a  Tetrahymeiia  incubated  in  [^H]-cytidine  for  12  min. 
followed  by  incubation  in  non-radioactive  medium  for  88  min.  The  cytoplasmic 
RNA  is  heavily  labelled,  but  the  nucleus  contains  no  labelled  RNA. 

VOL.  ir. — 2  M 


530  D.    M.    PRESCOTT 

The  delay  in  the  appearance  of  cytoplasmic  labelling  in  each  experiment 
could  conceivably  be  explained  in  one  other  way  besides  the  hypothesized 
transfer  of  RNA  from  the  nucleus  to  the  cytoplasm.  The  nucleus  might  be 
the  exclusive  site  of  some  contribution  to  RNA  synthesis  which  precedes 
polymerization,  i.e.  some  step  in  the  conversion  of  nucleoside  to  nucleoside 
triphosphate.  According  to  this  hypothesis,  the  delay  in  cytoplasmic 
labelling  might  be  considered  as  a  measure  of  time  for  the  labelled  tri- 
phosphate to  be  formed  in  the  nucleus  and  delivered  to  points  of  RNA 
synthesis  in  the  cytoplasm.  No  evidence  has  been  found  in  the  literature 
that  any  such  activities  are  localized  in  the  nucleus. 

These  results  with  Tetrahymena  are  interpreted  as  evidence  that  RNA 
moves  continuously  from  nucleus  to  cytoplasm.  In  view  of  the  very  rapid 
arrival  of  pH]-cytidine  in  the  nucleus,  it  seems  unlikely  that  the  relatively 
long  lag  in  the  appearance  of  RNA  bound  tritium  in  the  cytoplasm  could 
result  from  cytoplasmic  RNA  synthesis  being  delayed  until  some  cytidine- 
derived  precursor  of  RNA  could  first  difi^use  out  of  the  nucleus. 

Initially  the  nucleus  curve  for  pH]-cytidine  incorporation  shows  a 
short  lag,  which  probably  reflects  the  time  required  for  pH]-cytidine  or  a 
cytidine  derivative  to  be  built  up  in  a  precursor  pool.  The  slope  of  the 
nuclear  curve  subsequent  to  the  lag  does  not  represent  the  rate  of  RNA 
synthesis  in  the  nucleus  but  is  a  composite  of  rates  of  several  events. 
During  the  entire  course  of  the  curve  the  average  rate  of  increase  of  radio- 
activity in  the  nucleus  is  decreased  by  cell  division,  which  occurs  con- 
tinuously during  the  experiment.  At  each  cell  division,  the  activity  of  the 
nucleus  is  divided  between  the  two  daughter  nuclei,  thus  lowering  the 
average  amount  of  activity  per  nucleus.  For  most  of  its  course  the  slope  of 
the  curve  is  also  decreased  by  the  shift  of  radioactivity  from  the  nucleus  to 
cytoplasm.  The  slope  of  the  cytoplasmic  curve  is  also  decreased  by  cell 
division  and  possibly  by  some  breakdown  of  RNA,  although  the  occurrence 
of  the  latter  seems  doubtful.  Granting  that  RNA  does  move  from  nucleus 
to  cytoplasm,  the  lag  in  the  cytoplasmic  curve  also  suggests  that  there  is  a 
delay  between  the  fixation  of  pH]-cytidine  into  an  acid-insoluble  polymer, 
very  probably  RNA,  and  the  transfer  of  the  completed  RNA-protein 
molecule  into  the  cytoplasm. 

A  number  of  studies  [47]  have  show^n  that  incorporation  of  radio- 
activity into  nuclear  RNA  during  a  brief  exposure  to  label  is  observed  to 
disappear  from  the  nucleus  with  concomitant  appearance  of  labelled  RNA 
in  the  cytoplasm  when  the  cells  are  subsequently  transferred  to  and 
incubated  in  a  medium  containing  no  label. 

In  the  second  group  of  experiments,  Tetrahymena  were  exposed  to  a 
pulse  of  pH]-cytidine,  and  the  distribution  of  labelled  RNA  followed  after 
removal  of  exogenous  pH] -cytidine.  Figure  3  describes  the  results  of  the 
experiment.  Ten  /xc./ml.  of  ['^H] -cytidine  were  added  at  time  zero.  The 


NUCLEAR   SYNTHESIS    OF    RNA 


531 


Tetrahxmena  were  centrifuged  out  of  the  radioactive  medium  and  re- 
suspended  in  medium  containing  unlabelied  cytidine  at  the  same  concen- 
tration. This  first  washing  resulted  in  a  twenty-five-fold  dilution  of  the 
[^H]-cvtidine  and  was  completed  at  12  min.  At  12  min.  all  incorporated 
cytidine  is  still  localized  in  the  nucleus  (Fig.  2(1^)).  The  washing  procedure 
was  repeated  three  more  times  to  give  a  150000-told  dilution  of  the 
isotope  by  30  min. 

After  the  first  washing  the  incorporation  of  radioactivity  into  nuclear 
RNA  continues  for  another  40  min.,  indicating  a  large  pool  of  PH]- 
cytidine  or  its  derivatives  that  could  not  be  washed  out  of  the  living 
Tetrahymena  with  medium  containing  unlabelied  cytidine.  Cytoplasmic 
label  begins  to  appear  at  about  15  min.  Forty  minutes  after  the  first  washing 

160-1 


(40 


120- 


(r(00 


FOURTH  WASHING 
COMPLETE 


FIRST  WASHING 
COMPLETE 


':^l 


NUCLEUS 


=5^ 


-H 


40  50  60 

TlME(min) 


70 


100 


Fig.  3.  The  two  curves  describe  the  total  amount  of  incorporation  of  [^H]- 
cytidine  into  RNA  of  the  nucleus  and  cytoplasm.  .A.t  12  min.  the  cells  were  washed 
free  of  the  isotope  with  non-labelled  medium.  The  range  for  each  point  indicates 
95  "^'o  confidence  limits. 


the  density  of  label  in  the  nucleus  begins  to  decrease  and  by  100  min.  has 
fallen  to  about  io",j  of  the  peak  level  of  50  min.  Only  a  small  fraction  of 
this  decrease  can  be  ascribed  to  dilution  through  cell  division.  During  this 
decrease  the  cytoplasmic  label  per  cell  increases  until  the  rate  of  cyto- 
plasmic labelling  per  cell  equals  the  rate  of  dilution  by  cell  division.  This 
transient  balance  occurs  at  about  80  min.  In  the  last  20  min.  of  the  experi- 
ment, cytoplasmic  label  per  cell  is  decreased  more  rapidly  by  cell  division 
than  it  is  built  up  by  newly  labelled  RNA.  At  100  min.  approximately 
one-third  of  the  nuclei  contain  no  radioactivity  (Fig.  2{d)).  The  remaining 
two-thirds  of  nuclei  contain  small  amounts  of  activity,  about  half  of  which 
is  RNase  removable.  The  remaining  trace  of  acti\ity  is  presumed  to  be 
incorporated  into  DNA. 


532 


D.    M.    PRESCOTT 


.-    ••*• 


I 


Fig.  4.  An  enucleated  and  a  nucleated  fragment  exposed  to  [^H]-cytidine  for 
2  hr.  The  RNA  of  the  nucleated  fra.gment  is  heavily  labelled.  The  enucleated 
fragment  contains  no  incorporated  label. 


NUCLEAR   SYNTHESIS   OF    RNA  533 

In  this  pulse  experiment  the  densely  labelled  nucleus  in  an  unlabelled 
cytoplasm  found  at  12  min.  contrasts  sharply  with  unlabelled  nucleus 
surrounded  by  heavily  labelled  cytoplasm  at  100  min.  (Figs.  2(6)  and  2(d)). 
The  presence  of  an  unlabelled  nucleus  surrounded  by  heavily  labelled 
cvtoplasm  not  onlv  indicates  a  transfer  of  RXA  from  nucleus  to  cytoplasm 
but  suggests  in  addition  that  the  transfer  of  RXA  in  the  reverse  direction, 
from  cvtoplasm  to  nucleus,  does  not  take  place.  This  distribution  of 
labelling  also  implies  that  if  breakdown  of  cytoplasmic  RXA  does  occur, 
the  products  are  not  used  by  the  nucleus  for  RXA  synthesis.  Similar 
conclusions  were  suggested  bv  nuclear  transplantation  studies  in  amoeba 
by  Goldstein  and  Plant  [S]. 

The  experiment  in  Fig.  3  also  demonstrates  that  the  pool  into  which 
[■^H]-cvtidine  (or  its  derivatives)  enters  must  be  large  since  tritium  becomes 
incorporated  into  RXA  long  after  [-^Hj-cytidine  has  been  eliminated  from 
the  medium.  Because  the  pool  cannot  be  washed  out  of  living  Tetra/iyinena 
with  non-labelled  medium,  it  may  be  that  the  ['^Hj-cytidine  has  been 
converted  to  a  form  which  is  bound  (but  still  acid-soluble)  or  which  does 
not  readilv  pass  through  the  cell  membrane.  The  pool  may  be  in  the  form 
of  mono-,  di-,  or  triphosphates  of  cytidine.  Whether  the  pool  is  localized 
in  the  nucleus  or  cvtoplasm  or  is  present  in  both  places  is  not  known. 

Bv  removing  the  nucleus  from  a  cell,  it  becomes  possible  to  compare 
the  capacities  of  nucleated  and  enucleated  cells  to  synthesize  RXA. 
Tetrahvniena  were  cut  into  nucleated  and  enucleated  fragments  with  a 
glass  needle  controlled  bv  a  micromanipulator.  All  of  the  nucleated  frag- 
ments survive  longer  than  40  hr.  and  most  of  them  regenerate  and  resume 
proliferation.  The  enucleated  fragments  survive  in  the  complete  nutrient 
medium  for  10  to  40  hr.  and  move  about  by  ciliary  activity. 

Fnucleated  and  nucleated  fragments  of  Tetrahymeua  have  been 
incubated  for  20  to  240  min.  immediately  after  cutting,  in  complete 
medium  containing  ['^HJ-cytidine.  The  incorporation  of  activity  into 
nucleated  fragments  is  always  intense  (Fig.  4).  With  short  incubation  (up 
to  90  min.)  the  nucleus  is  more  densely  labelled  than  the  cytoplasm.  After 
that  time,  labelling  is  so  heavy  in  both  nucleus  and  cytoplasm  that  no 
difference  between  the  two  sites  is  apparent.  About  thirty-five  enucleated 
fragments  of  cytoplasm  have  been  studied,  and  none  has  been  found  to  in- 
corporate p^HJ-cytidine  into  RXA  (Fig.  4). 

In  sharp  contrast,  enucleated  Tetraliymena  are  still  capable  oi  in- 
corporation of  [^^C]-amino  acids.  This  activity  occurs  at  a  lower  rate  than 
in  the  nucleated  fragments  of  comparable  size.  The  [^*C]-amino  acids  are 
presumably  incorporated  into  protein  since  they  are  not  removed  by 
10  min.  extraction  with  5",,  TCA  at  90'  and  ether-alcohol  treatment  for 
10  min.  This  capacity  of  enucleated  Tetvahymena  to  incorporate  [^^C]- 
amino  acids  drops  very  rapidlv  after  enucleation.  By  6  hr.  the  incorporation 


534  D.    M.    PRESCOTT 

ceases   completely,   although  the  enucleated  pieces   remain   motile   long 
beyond  this  time. 

The  cutting  experiments  show  that  the  cytoplasm  is  completely 
incapable  of  incorporating  pH]-cytidine  into  RNA  when  the  nucleus  is 
absent  from  the  system.  As  in  other  enucleated  cell  types  cytoplasmic 
incorporation  of  amino  acids  continues.  These  enucleation  experiments 
with  Tetrahymeno,  combined  with  the  time-labelling  study  (Fig.  i)  and 
the  pulse  experiment  (Fig.  3)  substantiate  the  thesis  that  all  RNA  is 
synthesized  in  the  nucleus  and  that  nuclear  RNA  is  the  source  of  cyto- 
plasmic RNA.  The  fate  of  "transfer  RNA"  (soluble  RNA)  in  all  of  these 
experiments  is  not  entirely  certain ;  it  is  possible  that  some  or  all  of  this 
fraction  might  have  been  lost  during  the  extraction  procedures  prior  to 
autoradiography.  For  this  reason,  cytoplasmic  labelling  of  transfer  RNA 
with  [-'HJ-cytidine  might,  therefore,  have  escaped  detection. 


References 

1.  Prescott,  D.  M.,  Exp.  Cell  Res.  12,  196  (1957). 

2.  Woods,  P.  S.,  and  Taylor,  J.  H.,  Lab.  Invest.  8,  309  (1959). 

3.  Goldstein,  L.,  and  Micou,  ].,y.  biophys.  biocheni.  Cytol.  6,  301  (1959). 

4.  Woods,  P.  S.,  Brookhaven  Symp.  Biol.,  Number  12,  153  (1959). 

5.  Zalokar,  M.,  Exp.  Cell  Res.  19,  559  (i960). 

6.  Perry,  R.  P.,  Exp.  Cell  Res.  20,  216  (i960). 

7.  Amano,  M.,  and  Leblond,  C.  P.,  Exp.  Cell  Res.  12,  196  (i960). 

8.  Goldstein,  L.,  and  Plaut,  W.,  Proc.  nat.  Acad.  Set.,  Wash.  41,  874  (1955). 


Discussion 

Davls:  Your  curve  showed  considerable  increase  in  total  counts  in  the  cell  if 
you  add  together  the  nucleus  and  cytoplasm  after  division  has  started.  How  can 
you  account  for  the  source  of  all  that  further  radioactivity  ? 

Prescott  :  We  can  account  for  this  because  the  cells  contain  a  pool  of  cytidine 
and  its  derivatives  that  cannot  be  washed  out  of  living  cells.  Dr.  Bruce  Jacobson  and 
I  have  started  some  studies  on  this  pool  in  Tetrahymena  and  we  already  know  that 
it  is  in  fact  very  large.  We  haven't  really  identified  it  yet  but  suspect  that  it  is  com- 
posed of  CMP,  CDP,  and  CTP. 

Davis  :  I  missed  the  point  of  technique  ;  didn  't  your  method  of  autoradiography 
involve  getting  rid  of  that  pool  first  ? 

Prescott:  Yes,  I  am  sorry  that  I  didn't  make  that  clear.  The  cells  were  pre- 
pared for  autoradiography  in  such  a  way  as  to  remove  all  acid-soluble  material  and 
to  give  a  picture  of  incorporation  only. 

Chargaff  :  Does  Tetrahymena  contain  cytidine  deaminase  ? 

Prescott:  I  don't  know. 

Chargaff  :  Because  if  you  rely  on  grain  counts  you  do  not  know  that  it  is  still 
cytosine  derivatives  that  you  are  still  following. 


NUCLEAR   SYNTHESIS   OF    RNA  535 

Prescott  :  True.  But  we  can  say  that  this  incorporated  radioactivity  is  RNA-ase 
sensitive,  i.e.  that  it  is  96-98  "0  removable  with  RNA-ase  treatment. 

Chargaff  :  In  other  words  it  could  not  have  gone  through  a  cyclic  process  by 
which  deamination  could  give  a  uridine  derivative  which  was  then  methylated  and 
so  on. 

Prescott  :  And  got  into  DNA  ? 

Chargaff:  Yes,  there  are  some  indications  that  ribosides  can  go  into  deoxy- 
ribosides;  we  heard  about  that  from  Dr.  Reichard  the  other  day. 

Prescott  :  We  checked  this  point  and  found  an  appreciable  amount  of  cytidine 
going  over  into  DNA.  We  next  added  unlabelled  deoxycytidine  to  the  medium, 
and  empirically  it  proved  to  be  a  good  preventative  for  cytidine  getting  over 
into  DNA. 

Siekevitz  :  When  you  did  the  washing  experiment  and  the  activity  of  the  RNA 
in  the  nucleus  went  up,  you  made  the  assumption  that  you  were  not  washing  out 
the  precursor  pool  of  radioactivity.  You  could  make  the  same  assumption  about 
the  cytoplasmic  RNA,  that  the  washing  experiment  was  not  washing  out  any 
radioactivity  from  the  pool  there.  This  would  go  against  the  idea  of  nuclear  RNA 
coming  out  into  the  cytoplasm. 

Prescott  :  Except  that  there  is  such  a  long  lag  between  the  presentation  of 
radioactive  cytidine  to  the  cell  and  the  appearance  of  labelled  RNA  in  the  cvto- 
plasm.  You  are  quite  right  about  this  in  one  respect,  however.  One  possible  inter- 
pretation is  that  the  cytoplasm  doesn't  have  the  necessary  kinases  but  the  nucleus 
does  and  that  cytidine  passes  through  the  cytoplasm  into  the  nucleus  where  it  is 
transformed  into  CTP.  This  in  turn  might  leak  into  the  cytoplasm  where  it  would 
be  used  for  cytoplasmic  RNA  synthesis.  I  personally  don't  believe  that  this 
happens,  but  it  is  one  reason  why  we  are  looking  into  the  pool  question,  particu- 
larly whether  the  cytoplasm  has  the  capacity  to  convert  cytidine  into  CTP. 

Herbert:  I  was  wondering  about  the  resolution  of  your  method.  Is  there  not 
the  possibility  that  there  is  a  sizeable  pool  of  soluble  RNA  in  the  cell  into  which 
radioactivity  is  going  which  you  can't  resolve  by  your  technique  ? 

Prescott:  We  are  a  little  uncertain  about  the  fate  of  soluble,  or  transfer,  RNA 
in  these  experiments.  We  have  had  some  help  from  Dr.  Waldo  Cohn  and  hope  to 
settle  the  ([uestion.  There  could  be  terminal  labelling  in  transfer  RNA,  and  then 
loss  of  the  label  during  the  acid-extraction  prior  to  autoradiography.  Approxi- 
mately 5"o  of  the  RNA  in  this  cell  is  probably  of  the  soluble  type.  We  really  don't 
know  for  sure  what  is  happening  to  that  RNA.  We  hope  to  decide  whether  soluble 
RNA  stays  in  the  cell  with  our  treatment  by  labelling  it  with  tritiated  pseudo- 
uridine.  In  this  connection  we  are  also  interested  in  the  question  of  where  the 
soluble  RNA  is  synthesized ;  in  the  nucleus,  cytoplasm  or  both  places  ? 

Herbert  :  If  it  were  floating  free  or  soluble  in  the  cytoplasni  and  not  concen- 
trated as  it  is  in  particles  in  the  nucleus,  then  it  would  be  very  difficult  to  settle 
this.  Is  that  not  true  ? 

Prescott:  We  would  pick  it  up.  These  cells  are  fixed  in  a  manner  which  we 
believe  precipitates  the  soluble  RNA,  but  we  are  not  sure  that  we  are  not  losing 
some. 

Allfrev  :  I  would  like  to  raise  a  point  that  one  must  make  the  distinction 
between  end-group  labelling  of  RNA  and  net  synthesis  of  RNA  as  I  am  sure 


536  D.    M.    PRESCOTT  ' 

Dr.  Prescott  knows ;  but  there  is  another  problem  which  arises  in  experiments  of 
this  sort  and  that  has  to  do  with  your  observed  lag  period,  and  that  may  involve 
the  fact  that  the  concentration  of  precursor  in  the  nucleus  soon  exceeds  that  in 
the  medium  and  far  exceeds  that  in  the  cytoplasm,  so  you  get  an  apparent  synthesis 
first  in  the  nucleus. 

Prescott:  We  are  aware  of  this  possibility.  We  just  simply  prefer  a  more 
positive  conclusion.  I  am  willing  to  consider  these  criticisms  seriously.  We  also 
are  faced  with  the  possibility  that  RNA  synthesis  may  take  place  in  the  nucleus 
and  this  RNA  is  rapidly  broken  down ;  the  breakdown  products  might  leak  into  the 
cytoplasm  and  only  these  might  be  used  for  cytoplasmic  RNA  synthesis.  There 
are  a  number  of  other  alternatives,  but  I  think  that  the  interpretation  I  have  made 
of  these  data  are  more  probably  correct;  they  also  require  the  least  number  of 
additional  assumptions. 

Allfrey  :  If  I  had  vour  results  I  would  draw  the  same  conclusions. 


Cell  Division  and  Protein  Synthesis 

Erik  Zeuthen 

TJie  Biological  Insjitiite  of  the  Carhberg  Fouiuldtio/i, 
Copenhagen,  Denmark 


I.  The  synchronized  Tetrahymena  system 

In  the  course  of  the  7  years  since  Hotchkiss  [4]  for  bacteria  and  we  [10, 
14]  for  a  protozoon  organism  {Tetrahymena  pxriformis)  proposed  tempera- 
ture changes  as  a  tool  for  phasing  or  synchronizing  cell  populations,  this 
field  has  been  rapidlv  expanding. 


3'^°C ^  ^  ^  ^  ^  j_,  j_j  ^^    1                        1 

ZrC                            (0.i3)' 

t 
f 

1.70) 

(+) 

. 

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0      2       'y      6       8      10     /2     /r      f6      /8     20     22     2i 

hours 

Fig.  I.  The  continuous  curve  represents  division  inciex,  and  the  broken  curve 
represents  cell  counts.  (From  Zeuthen  and  Scherbaum  [14]). 

The  synchronous  Tetrahymena  system  is  demonstrated  in  Figs,  i  and  2. 
In  Fig.  I  the  stippled  curve  represents  the  logarithms  of  the  cell  counts  per 
ml.  During  the  first  11  hr.  growth  is  at  29"  C.  which  is  optimum  (28- 
29°  C).  We  observe  first  a  lag-phase,  then  a  log-phase  of  growth.  During 


538  ERIK   ZEUTHEN 

the  subsequent  yl  hr.  the  temperature  is  shifted  eight  times,  and  in  a 
regular  manner,  between  29""  C.  and  34"^  C.  This  blocks  cell  multiplication. 
The  lower  curve  (division  index  =  cytokinetic  cells /total  cells)  shows  that 
in  response  to  the  temperature  changes  cells  in  division  complete  this 
process  while  no  new  cells  enter  into  division.  At  subsequent  constant 
29°  C.  almost  all  cells  divide  at  the  same  time,  but  only  after  a  delay  of 
1 3  hr.  The  cells  complete  this  division  together  and  they  enter  a  second 
and  a  third  division  at  2-hr.  intervals.  This  is  reflected  both  in  the  popula- 
tion counts  and  in  the  division  indices.  In  Fig.  2  the  fully-drawn  curve 
repeats  and  extends  the  broken  curve  from  Fig.   i.  Only  the  counts  are 


5-0 


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2-5 


10 


14 
(hours) 


22 


26 


Fig.  2.  The  continuous  curve  represents  cell  counts,  the  broken  curve  repre- 
sents volume  of  cells  per  unit  volume  of  culture.  (From  Zeuthen  and  Scherbaum 

[14])- 

not  spaced  close  enough  to  describe  the  synchronous  division  steps.  In 
Fig.  2  the  broken  curve  is  for  total  cell  volume  per  aliquot.  During  the 
phase  of  shifting  temperature  the  average  cell  grows  to  about  three  times 
its  average  logarithmic  size.  This  is  reversed  during  the  synchronous 
division  steps  which  occupy  about  5  hr.  as  indicated.  The  relative  position 
and  the  slopes  of  the  two  curves  suggest  complete  reversibility  of  the 
induced  division  synchrony.  Cells  which  through  synchronous  division 
have  reverted  to  logarithmic  growth  may  be  resynchronized  by  the  method 
shown  in  Figs,  i  and  2.  The  cells  which  after  the  heat  shocks  make  ready 
for  their  first  synchronous  division  are  abnormally  large,  not  only  by 
volume  but  also  by  dry  matter,  protein  content,  RNA,  DNA  and  by 
nuclear  volume  [2,  1,7,  14,  12  (review)].  All  measures  increase  more  or 


CELL    DIVISION    AND    PROTEIN    SYNTHESIS  539 

less  in  parallel  during  the  period  of  blocked  cell  division,  and  at  least  by 
more  than  a  factor  of  2.  The  base  ratios  in  both  nucleic  acids  remain 
constant  [7]. 

2.  Studies  with  base  analogues 

While  rather  extended  biochemical  studies  of  the  Tetiahxmena  svstem 
have  vielded  much  valuable  information,  they  have  not  greatlv  helped  us 
to  understand  the  biochemical  mechanisms  bv  which  temperature  changes 
induce  the  di\  ision  synchrony.  Figure  i  shows  that  no  cells  divide  during 
the  first  hour  which  at  constant  29  C.  follows  the  period  of  changing 
temperature.  This  observation,  extended  in  careful  studies  bv  Thormar 
[11],  shows  that  recovery  from  temperature  damage  must  take  place  before 
the  cells  di\  ide.  The  damage  brings  all  cells  into  a  common  situation  with 
respect  to  their  preparation  for  subsequent  division.  We  have  studied  the 
recovery  by  the  use  of  antimetabolites  of  various  sorts.  The  cells  are 
synchronized  in  a  2%  proteose-peptone  medium,  fortified  with  o-i",, 
(more  recently  with  o-4'^o)  hver  extract  L  (Wilson  Labs.).  In  some 
experiments  we  applied  an  extra  period  of  elevated  temperature  (an  extra 
"temperature  shock").  Before  the  shock  the  organic  medium  was  replaced 
with  a  simple  inorganic  medium  [2].  The  cells  divide  in  standard  time  after 
the  last  shock,  whether  this  is  applied  in  the  proteose-peptone  or  in  the 
inorganic  medium.  Cells  in  the  latter  medium  shall  be  referred  to  as 
"washed  cells". 

The  time  of  maximum  engagement  in  divisions  i,  2,  and  3  (proteose- 
peptone  medium)  and  in  division  i,  often  2  ("washed  cells")  can  be 
determined  with  great  accuracy.  Consequently,  it  is  possible  to  quantitize 
the  division-delaying  effect  of  an  antimetabolite  which  is  added  at  a 
defined  time  before  division.  The  changes  in  the  division  index  is  followed 
in  up  to  twenty  dishes  each  of  which  holds  i  ml.  of  the  population.  Fre- 
quent visual  inspection  of  each  dish  is  made  at  noted  times.  The  percentage 
of  cells  in  fission  is  quickly  estimated.  Curves  through  the  estimates  permit 
that  we  fix  accurately  (  ±  <  3  min.)  the  time  when  in  a  dish  a  maximum  of 
cells  show  fission.  Counting  is  not  necessary  for  sound  estimates  to  be 
made.  As  a  check  of  the  method  we  have  established  that  the  results  of 
tw^o  or  more  independent  observers  agree  nicely.  The  effect  of  an  anti- 
metabolite is  given  by  the  delay  of  division  relative  to  the  proper  control. 
Because  many  dishes  are  followed  drug  concentrations  and  other  factors 
are  easily  varied  in  parallel  runs. 

Out  of  a  considerable  number  of  purine-  and  pyrimidine-analogues 
tested  [13]  only  8-azaguanine  and  6-methylpurine  were  found  to  inhibit 
the  first  synchronous  division  in  the  washed  cells.  Furthermore,  the  two 
analogues  were  inhibitors  of  this  division  only  when  added  before  the 
lapse  of  about  half  the  time  which  the  controls  require  to  prepare  division 


540  ERIK    ZEUTHEN 

at  constant  28°  C.  8-Azaguanine  is  nicely  antagonized  by  guanine,  guano- 
sine,  adenine  and  adenosine.  6-Methylpurine  is  antagonized  by  adenine 
and  by  adenosine,  not  by  guanine. 

The  general  picture  invites  the  suggestion  that  base  analogues  interfere 
much  less  readily  with  the  cell's  preparation  for  synchronous  division  at 
the  level  of  synthesis  of  the  two  nucleic  acids  than  at  the  level  of  the  co- 
factors  (GTP  and  ATP)  involved  in  protein  synthesis.  This  leads  to  the 
hypothesis  [13]  that  the  temperature-shocked  cells,  while  overcharged  with 
nucleic  acids  and  proteins,  are  short  of  one  or  several  proteins  which  are 
specifically  related  to  the  process  of  cell  division.  According  to  the 
hypothesis  this  situation  is  corrected  by  new  synthesis  after  the  termination 
of  the  temperature  shocks. 

3.  Studies  on  DNA 

In  view  of  suggestions  made  by  Scherbaum  [8]  it  is  recalled  that  in 
early  work  with  Dr.  E.  Hofi-Jorsensen  we  [14]  found  close  to  constancy 
of  the  ratio  DNA /unit  cell  volume.  As  shown  in  Fig.  3  this  is  for  the  period 
of  shifting  temperature  when  the  average  cell  increases  by  a  factor  2-5-3 
and  for  the  period  of  synchronous  divisions  during  which  the  average  cell 
size  regulates  to  normal.  This  work  gave  no  indication  of  a  special  role 
played  by  DNA  in  the  induction  by  heat  of  the  division  synchrony. 

As  a  continuation  of  this  work  Dr.  Rose  Cerroni  in  our  laboratory 
independently  made  an  observation  also  reported  by  Scherbaum  [8]. 
Tritiated  thymidine  (specific  activity  1-9  c./mM,  5  /xc.  per  ml.  cell 
suspension,  henceforth  to  be  referred  to  as  the  standard  dose)  added  to 
populations  of  50  000-100  000  cells /ml.  label  the  nucleus  of  only  a  fraction 
of  all  cells.  We  used  the  labelled  compound  undiluted  with  cold  thymidine 
externally  added.  The  total  amount  of  thymidine  represented  by  the 
standard  dose  is  of  the  order  of  only  i/io  of  the  amount  of  DNA  in  all 
cells  in  the  sample. 

Whether  logarithmic  cells  are  studied  in  proteose-peptone  (0-4  liver 
extract)  or  whether  they  are  transferred  to  the  inorganic  medium  before 
the  addition  of  the  labelled  thymidine  only  27-37%  of  all  nuclei  take 
the  label.  These  experiments  suggest  that  the  radioactive  compound  is 
soon  removed  by  the  one-third  to  one-half  of  the  cells  in  the  ran- 
domized population  which  can  be  expected  (cf.  [5]  and  [6])  to  synthesize 
DNA  at  any  one  time.  This  view  is  further  supported  by  the  observation 
that  all  (98%)  nuclei  become  labelled  if  three  additional  standard  doses 
of  tritiated  thymidine  are  given  to  the  same  cells  for  4  min.  at  50  min. 
intervals. 

A  standard  dose  of  tritiated  thymidine  ofi^ered  to  the  synchronized  cells 
in  proteose-peptone  again  label  only  a  fraction  of  the  nuclei ;  20%  when 


CELL    DIVISION    AND    PROTEIN    SYNTHESIS  54I 

offered  at  time  EH,*  regularly  dropping  precentages  when  the  compound 
is  added  later,  finally  almost  no  labelling  when  cells  in  maximal  division  i 
are  exposed.  This  can  be  repeated  with  "washed  cells"  (defined  in 
Section  2).  Immersion  of  washed  synchronized  cells  into  a  solution  of 
5-fluoro-2-deoxyuridine  (5  x  10  -^  m)  at  EH,  thus  prior  to  the  addition  of 
the  tritiated  thymidine  reduces  to  6-12"',,  the  average  number  of  tracks 
over  the  nucleus  which  takes  the  label.  It  does  not  significantlv  alter  the 
percentage  of  labelled  cells.  This  is  so  for  all  time  points  studied.  The  long 
exposures  to  this  base  analogue  interfere  neither  with  division  i  nor  with 


OS 


Fk;.  3.  The  open  circles  represent  log  cell  number  per  ml.  culnire.  The  filled 
circles  represent  DXA  (microbiological  assays)  and  the  crosses  (  x  )  packed  cell 
volume  per  ml.  culture.  Increases  in  cell  volume  and  in  DXA  go  parallel,  for  the 
culture  and  for  the  average  cell.  Plotted  from  Table  i  in  Zeuthen  and  Scherbauni 

[14]). 

dixision  2.  It  is  not  vet  proven,  but  it  can  be  suggested  that  two  syn- 
chronous divisions  can  be  developed  without  (^e  novo  synthesis  of  DXA 
after  the  termination  of  the  temperature  shocks. 

4.  Studies  with  amino  acid  analogues 

The  ideas  developed  in  Section  2  have  stood  the  first  test  with  amino 
acid  analogues.  The  experiments  were  performed  in  association  with  cand. 
mag.  Leif  Rasmussen. 

DL-/)-fluorophenylalanine  (/)-FPhe)  is  a  strong  inhibitor  of  cell  division 

*  The  time  when  the  last  temperature  shock  ends,  as  shown  by  a  signal  on  the 
control  watch. 


542  ERIK   ZEUTHEN 

in  Tetrahymena  cells,  synchronized  as  well  as  normal  cells  from  logarithmic 
cultures.  ^-FPhe  is  antagonized  competitively  by  phenylalanine.  In 
principle  we  have  found  no  difference  in  response  between  synchronized 
cells  and  cells  from  a  logarithmic  population.  The  response  of  the  former 
cells  is  only  much  more  easily  analyzed  than  that  of  the  latter,  so  our  work 
on  logarithmic  Tetrahymena  cells  has  mostly  served  as  a  control  on  results 
obtained  with  the  synchronized  cells. 

Figure  4  shows  two  combined  experiments  for  cells  which  have  been 
synchronized  and  remain  in  proteose-peptone  (0-4%  liver  fraction).  The 
two  upper  curves  show  the  three  first  synchronous  divisions  (1,2,  and  3) 


0.5^ 


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180 


120         180         240       300 

Minutes  after  E  H. 


360 


Fig.  4.  Upper  curve:  Division  maxima  1-3  in  control  represented  by  the 
changes  in  time  of  the  division  index.  Lozver  curves:  Delays  of  divisions  1-3  (curves 
I-III)  as  a  function  of  the  time  of  im.mersion  of  the  cells  into/)-FPhe,  16  mM  in 
proteose-peptone. 


in  the  main  controls.  Divisions  appear  as  maxima  on  the  curves  for  the 
division  index.  The  inhibitor  is  16  niM /)-PThe.  It  is  added  to  the  cells  for 
continuous  exposure  at  the  times  (abscissa)  indicated  by  the  position  of 
the  points  on  curves  I,  II  and  III.  The  ordinate  of  a  point  represents  the 
delay  (relative  to  the  parallel  control)  of  division  i  (curve  I),  division  2 
(curve  II),  and  division  3  (curve  III).  The  infinity  sign  indicates  block  of 
the  subsequent  division.  All  observations  are  at  28°  C. 

Obviously,  there  is  a  critical  time  before  a  division  when  a  decision  is 
made  whether  or  not  that  division  can  be  blocked  by  the  amino  acid 
analogue.  This  time  (interpreted  as  shown  in  Fig.  4)  is  42  mins.  before 
division  i,  40  min.  before  division  2,  and  47  min.  before  division  3.  We 


CELL   DIVISION   AND    PROTEIN   SYNTHESIS  543 

have  found  it  to  be  around  60  min.  before  division  in  the  logarithmic  cell 
growing  in  proteose-peptone  plus  0-4%  liver  extract. 

We  have  further  analyzed  this  effect  by  exposing  the  cells  (medium  as 
before)  to  16  mM/)-FPhe  for  only  20  min.  After  that  time  the  inhibitor  is 
removed  by  three  washings  with  fresh  growth  medium,  using  the  hand 
centrifuge.  The  control  is  similarly  treated  in  a  parallel  tube.  The  results 
are  shown  in  Fig.  5.  In  this  case  we  have  only  delay,  never  block  of  division. 
The  delays  are  plotted  against  the  time  of  immersion  into  the  inhibitor. 
Curve  I  represents  the  delay  of  division  i  (lengthening  of  time  interval 
from  EH  to  division  i).  Curve  II  measures  the  delay  of  division  2  in  terms 
of  extended  intervals  between  divisions  i  and  2.  Several  experiments  are 
combined  but  little  attempt  has  been  made  of  keeping  them  apart  because 


60  120 

Minutes  after  E  H. 


180 


Fig.  5.  Delay  of  synchronous  division  in  proteose-ptptone  by  16  niM  p-FPhe 
for  20  min.  The  abscissa  is  the  time  when  the  exposure  is  initiated.  Cnric  I:  Delay 
of  division  i.  Curve  II:  Delay  of  division  2  (lengthening  of  time  interval  between 
divisions  i  and  2). 


both  the  svnchronizations  and  the  responses  to  the  analogue  are  so  nicelv 
reproducible.  Only  the  arrows  which  show  the  times  for  maximal  division  i 
and  2  separate  between  experiments.  The  results  confirm  those  of  Fig.  4 
in  showing  that  there  is  a  critical  time  about  44  min.  before  division  i, 
and  50  min.  before  division  2  when  the  response  to/)-FPhe  drops  sharply. 
The  new  information  conveyed  by  Fig.  5  is  that  the  reaction  to  a  standard 
treatment  with  the  analogue  increases  (cur\e  I)  from  EH  to  reach  a 
maximum  value  (at  t^^  min.)  just  before  the  drop  in  advance  of  division  i. 
Further  (curve  II),  that  this  cvclic  variation  repeats  itself  between  divisions 
I  and  2  and  even  extends  back  in  time  (left  part  of  curve  II)  to  before 
division  i.  Thus,  20  min.  of  exposure  to  the  amino  acid  analogue,  made 
before  division  i  delays  the  preparation,  not  of  this  immediate  division, 
but  of  the  next  one.  However,  this  delay  of  division  2  is  only  slight  and  of 
the  same  order  as  the  exposure  time  (20  min.)  to  the  analogue.  Curve  I 


544 


ERIK    ZEUTHEN 


shows  equally  short  delays  of  division  i  when  exposure  is  immediately 
after  EH.  However,  for  many  time  points  in  the  cell's  cycle  the  delays  of 
division  are  much  longer  than  the  time  for  which  the  cells  were  in  contact 
with  /)-FPhe. 


mM  p-FPhe. 


Fk;.  6.  "  Set-back"  against  concentration  of  ^-FPhe  in  inorganic  medium.  The 
exposure  is  always  for  20  min.  Crosses:  Division  i — Cells  immersed  at  25  min. 
after  EH.  Circles:  Division  2 — Cells  immersed  at  55  min.  after  EH. 

A  complicating  factor  in  the  experiments  described  is  in  the  complexity 
of  the  growth  medium.  For  cells  ("  washed  cells  ")  in  the  inorganic  medium 
the  extracellular  pool  of  amino  acids  is  nil  and  the  sensitivity  of  the  cells 
to  amino  acid  analogues  is  much  increased.  For  the  washed  cells  we  have 


60         90 
Minutes  of  exposure  to  p-FPhe. 

Ekj.  7.  "  Set-back"  (ordinate)  as  a  function  of  the  duration  (abscissa)  for  which 
exposure  is  made  to  o-8  mM  p-FPhe  in  inorganic  medium.  The  curves  are  for 
different  times  after  EH  of  beginning  treatment  with  /)-FPhe.  EH  is  the  time  when 
the  last  temperature  shock  ends. 

been  able  to  define  combinations  of  concentrations  (Fig.  6)  of /)-FPhe  and 
of  exposure  times  (Fig.  7)  w^hich  give  maximal  delaying  efi^ects. 

Figure  6  show^s  the  relation  between  response  of  the  washed  cells  and 
concentration  of  ^-FPhe  added  at  25  (crosses)  and  55  (circles)  min.  after 
EH.  Exposure  is  for  a  standard  time  of  20  min.  The  analogue  is  removed 
by  four  washings  with  the  inorganic  medium.  Washing  is  only  complete  if 
I  mM  DL-phenylalanine  is  added.  So  this  was  done.  The  response  is 
represented  by  the  "set-back",  w^hich  is  equal  to  the  delay  of  the  division, 
minus  the  time  for  which  the  cells  were  in  contact  with  the  inhibitor.  The 
crosses  represent  "set-backs"  for  division  i,  the  circles  relate  similarly  to 
division  2.  For  both  divisions  maximal  effects  are  obtained  with  o- 16  mM 


CELL    DIVISION   AND    PROTEIN   SYNTHESIS  545 

/)-FPhe  and  with  concentrations  above  this.  We  note  once  more  (cf.  Fig.  5) 
that  when^-FPhe  is  added  (at  55  min.)  later  than  at  a  critical  time  before 
division  i  this  division  is  not  delayed.  This  is  not  because  a  permeability 
barrier  to  p-FFhe  is  established  at  this  time.  If  it  were,  division  2  would 
not  be  delayed,  which  it  is. 

Figure  7  shows  the  relation  between  exposure  time  (abscissa)  and  "set- 
back" (delay  of  division,  minus  the  varied  time  for  which  exposure  to  the 
drug  is  made)  of  synchronized,  washed  Tetrahymena  cells  dumped  into 
the  inhibitor  (o-8  mAi/)-FPhe)  at  defined  times  after  EH.  Separate  curves 
are  shown  for  cells  which  are  immersed  at  5,  15,  25,  and  35  min.  after  EH, 
thus  at  intervals  of  10  min.  The  maximal  set-back  takes  time  to  develop 
but  it  is  nicely  defined  in  each  case.  It  becomes  roughly  10  min.  longer 
for  every  10  min.  by  which  we  postpone  the  addition  of  the  analogue. 

From  Fig.  6  we  learned  that  almost  the  same  effect  is  obtained  whether 
exposure  for  20  min.  is  to  o-i6  or  to  4-0  mM /)-FPhe.  We  shall  assume 
that  a  given  intracellular  level  of  ^-FPhe  is  attained  the  earlier  the  higher 
is  the  external  concentration  of  the  analogue.  Then,  with  o-8  mM  outside 
concentration  the  intracellular  concentration  of  the  analogue  should  reach 
a  maximally  inhibitory  concentration  in  a  fraction  of  the  20  min.  for  which 
(in  Fig.  6)  exposure  is  made.  If  this  is  so,  then  the  curves  of  Fig.  7  do  not 
measure  penetration  rates  of  ^-FPhe.  A  different  interpretation  is  based 
on  the  idea  that^-FPhe  penetrates  the  cells  fast  enough  to  produce  a  quick 
block  for  protein  synthesis.  The  washed  cells  are  starving  cells,  so  protein 
synthesis  must  be  from  an  amino  acid  pool  which  is  supplied  continually 
by  catabolism  working  on  cellular  proteins.  The  method  we  use  can  trace 
the  synthesis  only  of  proteins  which  are  related  to  division  and  which  for 
this  reason  we  shall  call  "division  proteins".  If  also  the  "division  pro- 
teins" show  a  turnover  then  they  shall  decay  as  soon  as  we  block  protein 
synthesis  with  /)-FPhe.  Indeed,  the  curves  of  Fig.  7  may  largely  represent 
decay  of  "division  proteins"  piled  before  the  addition  of  p-FFhe. 

Figure  8  is  based  on  the  data  presented  in  Fig.  7.  It  relates  "set-back" 
and  time  of  the  beginningof  the  exposure  to  the  analogue  (o-8  mM^-FPhe). 
Separate  curves  are  given  for  the  six  exposure  times  of  5,  10,  20,  35,  50, 
and  75  min.  All  curves  tend  to  be  linear  so  that  intersection  points  with 
the  time  axis  at  —  6,  —  11,  —  27,  —  29,  —  29,  and  —  30  min.  can  be  defined. 
The  slope  is  close  to  45  .  Exposure  for  35  min.  and  more  gives  maximal 
set-backs  (Fig.  7).  In  the  light  of  our  "block-and-decay "  hypothesis, 
Fig.  8  indicates  that  a  developing  store  of  "division  protein"  decays  fully 
at  any  time  when  /)-FPhe  is  added  to  stay  for  35  min.  or  more.  Irrespective 
of  when  added,  when  the  analogue  is  again  removed,  the  cells  are  empty  of 
"division  protein".  As  a  consequence  the  treated  cells  have  a  standard 
time  to  go  before  they  will  divide.  This  time  is  no  min.  and  equals  the  time 
which  the  controls  take  to  go  from  EH  to  division  i  (80  min.)  plus  those 

VOL.  II. 2N 


546  ERIK    ZEUTHEN 

30  min.  which  is  represented  by  the  distance  from  EH  to  the  most  left 
intersection  point  in  Fig.  8.  This  figure  also  suggests  that  by  the  time  EH 
the  synchronized  cells  contain  some  "division  protein",  though  not 
enough  for  a  division.  How  much  they  contain  we  cannot  say.  Our  graphs 
only  compare  times  measured  in  two  different  ways.  Linearity  of  a  curve 
which  slopes  45°  means  that  time  spent  on  synthesis  before  the  addition 
of /)-FPhe  is  fully  lost  at  the  time  w^hen  the  analogue  is  again  removed. 

Returning  to  the  experiments  recorded  in  Fig.  5  it  is  observed  that 
both  curves  I  and  H  ascend  towards  the  right  in  a  non-linear  fashion.  In 
those  experiments  cells  in  proteose-peptone  were  treated  for  20  min.  with 
16  mM  p-FPhe.  This  combination  of  growth  medium,  exposure  time,  and 


30  60 

Age,  in  minutes  after 


EH. 


Fig.  S.  "Set-back"  against  time  since  Ell  when  the  cells  were  immersed  into 
08   mM   /)-FPhe   in   inorganic  medium.   The  separate   curves   represent  various 
exposure  times  as  indicated.  The  dashed  parts  of  the  curves  are  extrapolations. 
[Error  in  Figure:  for  30  min.,  read  35  min.] 

inhibitor  concentration  failed  to  give  maximal  set-backs  as  defined  for  the 
washed  cells  (Figs.  6  and  7).  For  this  reason  linearity  of  the  rising  limbs 
of  the  curves  in  Fig.  5  could  not  be  expected. 

Other  amino  acid  analogues  have  been  tested  for  comparison  with 
/)-FPhe.  Ethionine,  yS-thienylalanine  and  canavanine  seem  to  act  in  the 
same  way  as  p-FPhe.  So  do  chloramphenicol  and  puromycin,  well- 
established  inhibitors  of  protein  synthesis.  It  is  the  results  with  the  two 
antibiotics  which  have  induced  us  to  ignore  the  possibility  of  a  significant 
synthesis  of  false  proteins  in  the  presence  of  an  amino  acid  analogue.  This 
possibility  is  under  further  study. 


5.  Conclusions 

We  have  no  evidence  from  the  study  of  the  synchronous  cells  that 
before  every  division  Tetrahymena  must  produce  RNA.  With  regard  to 
DNA  we  have  evidence  that  no  new  synthesis  of  this  substance  needs  take 


CELL    DIVISION    AND    PROTEIN    SYNTHESIS  547 

place  after  the  termination  of  the  last  temperature  shock  and  up  to  a  time 
when  two  synchronous  divisions  have  displayed  themsehes.  This  is 
perhaps  not  so  strange  since  Telrahyuuua  is  highly  polyploid  and  probably 
becomes  even  more  so  when  it  is  synchronized. 

According  to  our  interpretations  Tetrahyuufia  cells  }iiiist  produce 
"di\ision  proteins"  before  e\ery  di\ision.  This  is  so  whether  the  cells  are 
in  the  logarithmic  growth  phase,  whether  they  are  synchronized  and  grow 
in  proteose-peptone,  or,  most  importantly,  whether  they  perform  syn- 
chronous divisions  without  growth.  During  the  period  of  cycling  tempera- 
ture used  for  the  synchronization  Tctnihymeiia  grows  large  and  produces 
a  lot  of  proteins.  However,  it  fails  to  produce  "division  proteins"  to  the 
level  required  for  a  single  di\ision.  In  tact  we  consider  that  the  synchrony 
is  induced  because  at  EH  all  cells  are  equally  low  in  "division  proteins" 
which  later,  at  constant  28'  C,  are  produced  synchronously  to  give  rise 
to  the  first  synchronous  di\"ision  in  standard  time. 

For  the  synchronized  cells  in  proteose-peptone  increases  in  respiration, 
in  dry  matter,  and  in  protein  synthesis  are  discontinuous  (review  by 
Zeuthen  [13]).  In  all  these  measures  synthesis  is  slowed  around  division. 
The  synthesis  of  "  di\ision  proteins"  is  likely  to  be  a  small  traction  of  the 
total  since  it  is  obser\ed  also  in  starving  cells.  It  may  or  it  may  not  follow 
total  synthesis  and  it  mav  or  may  not  be  continuous  through  a  series  of 
divisions.  Howex  er,  that  part  of  the  synthesis  which  conditions  a  division 
seems  to  be  sharply  delimited  and  goes  from  40-50  min.  before  one  to 
40-  :;o  min.  before  the  next  synchronous  tission.  The  way  we  interpret  our 
results  is  that  firstly  the  cell  charges  itself  to  a  threshold  level  with 
"division  protein".  Then,  around  40  50  min.  before  synchronous  tission 
all  "division  protein"  changes  from  a  state  in  which  it  will  decay  in  the 
presence  of /)-FPhe,  to  a  state  when  it  will  not.  So  we  think  that  stabiliza- 
tion has  suddenly  taken  place.  And  this  stabilization  would  be  a  condition 
for  all  the  kinetic  phases  of  the  di\  ision  process  later  to  take  place. 

The  drop  before  division  i  from  maximum  to  no  capacity  to  become 
set-back  bv  /)-FPhe  occurs  at  the  time  when  the  anarchic  tield  [3,  9]  is 
just  beginning  to  organize  into  the  definiti\e  oral  membranelles  of  the 
second  mouth.  This  is  personal  information  from  Dr.  Joseph  Frankel 
obtained  under  identical  conditions  in  our  laboratory. 

This  new  orientation  in  space  of  previously  synthesized  kinetosomes 
may  be  one  of  the  earliest  manifestations  of  the  action  of  the  stabilized 
"  division  protein  ". 

The  work  reported  will  be  published  in  the  C.  R.  Lab.  Carhherg  by 
Rose  E.  Cerroni,  Leif  Rasmussen  and  the  author. 

References 

1.  Christensson,  E.,  Acta  pJiysifd.  scaml.  45,  339  (1959). 

2.  Hamburger,  K.,  and  Zeuthen,  E.,  C.  R.  Lab.  Carhhci-i:;  32,  i  ('i960'). 


54^  ERIK   ZEUTHEN 

3.  Holz,  G.  G.,  Jr.,  Scherbaum,  O.  H.,  and  Williams,  N.,  Exp.  Cell  Res.  13,  618 

(1957)- 

4.  Hotchkiss,  R.  D.,  Proc.  fiat.  Acad.  Sci.,  Wash.  40,  49  (1954). 

5.  McDonald,  B.  A.,  Biol.  Bull.  114,  71  (1958). 

6.  Prescott,  D.  M.,  Exp.  Cell  Res.  19,  228  (i960). 

7.  Scherbaum,  O.,  Exp.  Cell  Res.  13,  24  (1957). 

8.  Scherbaum,  O.  H.,  Ann.  N.Y.  Acad.  Sci.  90,  565  (i960). 

9.  Scherbaum,  O.  H.,  and  Williams,  N.,jf.  Etnbryol.  exp.  Morph.  7  (2),  241  (1959). 

10.  Scherbaum,  O.,  and  Zeuthen,  E.,  Exp.  Cell  Res.  6,  221  (1954). 

11.  Thormar,  H.,  C  R.  Lab.  Carlsberg  31  (14),  207  (1957). 

12.  Zeuthen,  E.,  Advanc.  biol.  med.  Phys.  6,  37  (1958). 

13.  Zeuthen,  E.,  in  "Growth  in  Living  Systems".  Proceedings  of  an  International 
Symposium  on  Growth  held  at  Purdue  University,  June  i960.  Basic  Books, 
New  York. 

14.  Zeuthen,  E.,  and  Scherbaum,  O.,  Colston  Pap.  6,  141  (1954). 

Discussion 

Mazia  :  One  point  made  by  Zeuthen  could  be  stressed  because  it  would  apply 
to  division  in  cells  other  than  Tetrahymena.  While  the  "division  protein"  may  be 
crucial,  it  needs  not  represent  very  much  protein.  His  proof  is  that  the  absence  of 
availability  of  external  nutritional  sources  for  proteins  makes  little  or  no  difference 
to  the  division  of  his  synchronized  cell  populations.  It  has  also  been  observed  in 
studies  on  fission  yeasts  by  Faed  in  Mitchison's  laboratory  that  the  cells  may  go 
through  a  complete  division  cycle  or  two  in  the  absence  of  external  nitrogen 
sources,  producing  small  progeny.  The  "division  protein"  may  be  available  in 
small  but  adequate  amounts,  may  be  supplied  by  conversion  of  other  proteins,  or 
may  be  made  from  the  amino  acid  pool.  The  fact  that  it  is  crucial  does  not  imply 
that  it  represents  a  quantitatively  important  fraction  of  the  protein  synthesis  taking 
place  during  the  growth-division  cycle. 

Davis:  Dr.  Zeuthen,  did  I  understand  correctly  that  during  this  period  of 
growth  without  division  DNA  and  RNA  continued  to  be  synthesized  at  normal 
rates  ?  I  wonder  if  your  problem  might  be  a  little  analogous  to  one  observed  with 
bacteria,  where  under  the  influence  of  many  inhibitory  agents  at  border-line 
concentrations  the  cells  continue  to  grow  and  become  tremendously  elongated, 
but  do  not  divide.  The  limitation  appears  to  be  the  completion  of  the  septum  which 
leads  to  division.  Could  yours  be  a  problem  where  the  limiting  factor  is  cell 
membrane  formation  ? 

Zeuthen  :  We  think  we  have  put  our  fingers — very  lightly — on  a  protein  which 
is  specifically  engaged  in  division  but,  as  Dr.  Alazia  said,  may  be  only  a  very  small 
part  of  the  whole  cell.  What  does  this  protein  perform,  where  does  it  sit  ?  We  do 
not  know.  Our  problem  could  be  analogous  to  the  situation  mentioned  by  Dr. 
Davis.  We  have  incubated  the  washed  cells  with  labelled  amino  acids.  In  radio- 
autographs  the  label  seems  to  sit  everywhere.  We  may  now  try  to  fractionate  for 
cell  walls  ("pellicles"),  nuclei,  particles  and  so  on,  to  see  if  the  label  is  attached 
predominantly  to  one  of  these  organelles.  Another  possible  approach  would  be  that 
we  try  to  separate  the  proteins  after  previous  incubation  of  the  cells  for  short  and 
different  times  with  labelled  amino  acids.  One,  or  more,  proteins  might  take  the 
label  in  excessive  amounts. 


Structure  and  Function  in  Amoeboid  Movement* 

Robert  D.  Allen 

Departvient  of  Bio/ogy,  Princeton  Unirers/tv, 
Princeton,  'XJ.,  U.S.A. 

Amoeboid  movement  is  a  process  with  which  every  biologist  and 
student  of  biologv  is  familiar.  Yet,  despite  its  fundamental  importance, 
this  form  of  cellular  motility  has  been  one  of  the  most  poorly  understood 
phenomena  in  cellular  biology.  Nearly  every  generation  of  biologists  over 
the  last  centurv  and  a  quarter  has  produced  a  new  explanatory  theory,  only 
to  have  it  supplanted  in  the  next  generation  by  a  totally  different  one. 
Theories  of  amoeboid  movement  seem  now  to  have  passed  through  a 
complete  cvcle,  for  the  front-  and  tail-contraction  theories,  which  I  shall 
discuss  here,  both  represent  a  return  to  the  general  idea,  first  expressed  by 
Dujardin  [9],  that  amoeboid  mo\'ement  is  basically  a  contractility  phe- 
nomenon. If  one  accepts  this  idea,  then  obviously  the  most  fundamental 
question  is  the  location  of  active  contraction  (i.e.  the  "engine")  in  the 
moving  cell. 

The  streaming  endoplasm  has  in  the  past  been  excluded  as  a  possible 
site  for  this  "engine"  because  this  region  of  the  cell  has  been  assumed  to 
have  the  phvsical  properties  of  a  Newtonian  sol  [12,  14].  Since  structureless 
fluids  can  neither  develop  nor  transm.it  tension,  this  concept  of  endoplasmic 
consistencv  led  inexorably  to  the  tail  contraction  theory  [10,  13],  according 
to  which  the  endoplasm  is  moved  passively  by  a  pressure  gradient  generated 
bv  an  activelv  contracting  ectoplasmic  tube.  The  concept  of  the  endoplasm 
as  a  structureless  sol  also  excluded  any  consideration  of  an  alternative 
mechanism  such  as  will  be  proposed  below. 

Several  recent  developments  ha\e  led  us  to  propose  such  an  alternati\e 
mechanism.  First,  it  has  been  pointed  out  elsewhere  [3,  4]  that  many  of 
the  behavioural  aspects  of  amoeboid  movement  are  incompatible  with  the 
tail  contraction  theory  in  its  present  form.  Second,  it  has  been  shown  that 
amoeba  cytoplasm  will  continue  to  stream  after  it  has  been  dissociated  from 
the  cell  [i,  5].  This  capability  was  neither  predicted  nor  explained  by  the 
tail  contraction  theory. 

*  Supported  by  Research  Grant  C-3022(Ci-C4)  from  the  U.S.  Public  Health 
Service. 


550 


ROBERT    D.    ALLEN 


Third,  a  new  concept  of  amoeba  cytoplasmic  structure  has  emerged 
from  recent  rheological  studies  of  consistency  differences  in  various  parts 
of  the  moving  cell  [2,  3,  7].  In  contrast  to  the  traditional  "sol-gel"  concept 
of  amoeba  structure,  it  has  been  shown  that  the  axial  portion  of  the 
endoplasm  (Mast's  "plasmasol"  [14])  possesses  weak  gel  structure. 
Velocity  profiles  of  endoplasmic  streaming  within  the  ectoplasmic  tubes 
of  narrow,  cylindrical  pseudopodia  of  Chaos  chaos  were  found  to  be 
similar  to  those  found  for  plug  flow  of  a  non-Newtonian  fluid  in  a  tube  [7], 
and  were  also  very  similar  to  velocity  profiles  of  cytoplasmic  streaming  in 


----HYAtlNE    CAP 

-    FOUNTAIN    ZONE 

----   PLASMALEMMA 

AXIAL    ENDOPLASM 
-SHEAR    ZONE 
■  HYALINE    ECTOPLASM 


GRANULAR 
FCTOPLASM 


RECRUITMENT 
ZONE 


TAIL  OR  UROID 


Fig.  I.  A  diagrammatic  representation  of  the  concept  ot  amoeha  pseudopodial 
structure  discussed  in  the  text.  The  superimposed  curves  are  velocity  profiles  from 
the  data  of  Allen  and  Roslansky  [7]. 


myxomycete  channels  [13].  Plug  flow  occurs  when  the  shear  stress  acting 
on  a  fluid  near  the  centre  of  a  stream  is  insufficient  to  cause  significant 
rates  of  deformation  (i.e.  velocity  gradients)  in  the  fluid,  but  when  the 
higher  shear  stresses  near  the  walls  exceed  the  yield  point  of  the  material 
(if  it  is  a  gel)  its  apparent  viscosity  is  reduced  to  the  range  expected  of  true 
sols.  The  velocity  profiles,  by  demonstrating  the  quasi-pseudoplastic 
nature  of  endoplasmic  flow,  have  drawn  attention  to  the  presence  of  weak 
gel  structure  which  might  permit  the  development  and  transmission  of 
tension.  Only  the  tail  endoplasm  and  the  peripheral  endoplasm  of  the 
"shear  zone"  (Fig.  i)  ha\'e  shown  evidence  of  a  low  apparent  viscosity. 
Studies    with    the    centrifuge    microscope    have    recently   confirmed   the 


STRUCTURE    AND    FUNCTION    IN    AMOEBOID    MOVEMENT  55 1 

presence  of  this  weak  gel  structure  in  the  axial  portion  of  the  endoplasm. 
This  region  of  the  cell  was  found  to  offer  \isible  resistance  to  the  displace- 
ment of  accelerated  cytoplasmic  inclusions  [2].  These  centrifugation 
experiments  have  confirmed  the  generally  held  concept  of  a  more  rigid 
consistency  for  the  ectoplasmic  tube  as  a  whole  (Mast's  plasmagel  [14]), 
but  have  revealed  an  unexpected  gradient  of  rigidity  in  both  the  ectoplasm 
and  axial  endoplasm  from  a  high  at  the  front  of  the  cell  to  a  low  in  the  tail. 

The  results  of  these  and  other  rheological  experiments,  which  have  been 
summarized  elsewhere  [3],  are  presented  schematically  in  Fig.  i.  It  is  this 
concept  of  amoeba  cytoplasmic  structure  which  led  to  the  new  front 
contraction  theory.  It  is  perhaps  simplest  to  outline  the  theorv  first,  and 
then  point  out  some  of  the  experimental  evidence  which  supports  it. 

First  let  us  assume  that  the  endoplasm  (especially  the  axial  portion)  is 
uncontracted  or  relaxed  cvtoplasm.  According  to  the  theory,  a  given 
portion  of  this  material  begins  to  contract  just  before  it  splits  and  becomes 
everted  to  form  the  continually  advancing  ectoplasmic  tube.  The  contrac- 
tion is  completed  by  the  time  this  material  has  become  incorporated  into 


(a)  (b)  (c) 

Fig.  2.  A  diagram  to  illustrate  the  fate  of  a  cylindrical  block  of  axial  endoplasm 
as  it  contracts  at  the  part  of  the  cell  as  proposed  by  the  fountain  zone  contraction 
theory  [4]. 

the  ectoplasmic  region.  During  its  passage  through  the  region  of  the  cell 
which  we  have  termed  the  fountain  zone  (Fig.  i),  this  given  portion  of 
cytoplasm  shortens  (along  the  axis  of  the  pseudopod)  and  thickens 
(radially)  (Fig.  2).  At  the  same  time  it  develops  tension,  which  is  trans- 
mitted posteriorly  through  the  axial  endoplasm  to  "pump  out"  the  tail. 
Increased  cross-bonding  during  contraction  in  the  fountain  zone  region 
causes  localized  syneresis,  the  fluid  from  which  appears  periodically  in  the 
hvaline  cap.  The  increase  in  rigidity  in  the  fountain  zone  accounts  for  the 
difference  in  consistency  between  the  ectoplasmic  tube  and  the  endoplasm  ; 
this  change  is  probably  analogous  to  the  increased  rigidity  which  accom- 
panies muscular  contraction.  The  dilute  fluid  of  the  hyaline  cap,  which 
has  been  pressed  out  of  the  cytoplasm  contracting  in  the  fountain  zone,  is 
pumped  tailward  by  the  advance  of  the  granular  cytoplasm  of  the  pseudo- 
pod  within  the  loosely  fitting  plasmalemma.  The  hyaline  ectoplasm  serves 
as  a  channel  between  the  plasmalemma  and  ectoplasmic  tube  through 
which  the  hyaline  fluid  travels  to  the  tail  region,  where  this  fluid  is  returned 
eventually  to  the  endoplasmic  stream.  The  pull  exerted  on  the  axial  endo- 
plasm from  the  front  may  in  part  draw  some  of  this  fluid  through  the  tail 


552  ROBERT   D.    ALLEN 

ectoplasm  so  that  it  can  be  returned  to  the  endoplasmic  stream.  If  the 
contraction  part  of  the  contractility  cycle  involves  syneresis,  then  this  fluid 
must  be  resorbed  in  whatever  part  of  the  cell  the  relaxation  part  of  the 
cycle  occurs.  It  is  also  possible  that  some  of  the  hyaline  fluid  is  squeezed 
backward  from  the  fountain  zone  as  a  counter-current.  The  theory  further 
requires  that  the  contraction  remain  localized  in  the  fountain  zone ;  hence, 
this  front  contraction  theory  has  been  named  the  "fountain  zone  contrac- 
tion theory".  In  order  to  remain  localized  in  the  fountain  zone,  the 
contraction  itself  must  be  propagated  posteriorly  toward  the  axial  endo- 
plasm  at  approximately  the  velocity  of  forward  endoplasmic  displacement 
relative  to  the  advancing  tip. 

The  theory  so  far  explains  only  endoplasmic  displacement  with  respect 
to  the  ectoplasm.  Locomotion  can  occur  only  if  the  ectoplasmic  tube  is 
attached  at  certain  points,  by  means  of  the  plasmalemma,  to  the  sub- 
stratum. The  larger  species  of  amoebae,  such  as  Amoeba  proteiis,  are 
attached  near  the  middle  of  the  cell  at  from  one  to  several  points  [8].  If 
attachment  does  not  take  place,  the  ectoplasm  and  endoplasm  are  indeed 
displaced  in  opposite  directions,  a  situation  which  has  been  called  "foun- 
tain streaming"  [3,  14]. 

The  fact  that  we  are  now  faced  with  two  opposing  contraction  theories 
which  postulate  contractions  localized  at  opposite  poles  of  the  cell  makes 
it  imperative  to  examine  the  question  of  exactly  what  constitutes  evidence 
of  an  active  contraction.  In  muscle,  the  measurement  of  tension  developed 
or  work  performed  removes  all  doubt  as  to  whether  an  observed  shortening 
is  passive  or  active.  In  the  amoeba,  the  situation  is  not  so  simple.  The 
fundamental  observational  basis  for  the  tail  contraction  theory  is  that  the 
tail  shortens  [11];  many  authors  have  uncritically  accepted  this  as  con- 
clusive evidence  for  tail  contraction.  Actually,  this  shortening  is  compatible 
with  the  tail  contraction  hypothesis,  but  is  also  compatible  with  the 
hypothesis  that  the  tail  is  "pumped  out"  as  the  new  theory  proposes. 
Even  the  measurement  of  tension  between  two  points  in  the  shortening 
tail  would  not  settle  the  question,  for  some  of  the  work  done  in  the  fountain 
zone  contraction  would  appear  as  tension  between  two  points  in  the 
shortening  tail  ectoplasm. 

It  seems  reasonable  to  propose  that  only  the  development  of  tension  or 
the  production  of  large  amounts  of  heat  in  a  localized  shortening  region  of 
cytoplasm  should  be  considered  conclusive  evidence  of  an  active  contrac- 
tion. Localized  syneresis  is  probably  also  conclusive  evidence  of  an  active 
contraction,  since  syneresis  is  well  known  to  result  from  increased  cross- 
bonding  in  gels.  Simultaneous  shortening  and  thickening  of  a  body  of 
cytoplasm,  however,  is  by  itself  not  much  more  than  suggestive  evidence 
of  active  contraction  unless  it  is  accompanied  by  localized  syneresis, 
tension  development,  or  heat  production. 


STRUCTURE   AND   FUNCTION    IN   AMOEBOID    MOVEMENT  553 

The  visible  events  in  amoeboid  movement  are  perfectly  compatible 
with  the  fountain  zone  contraction  theory  but  pro\ide  no  conclusive 
evidence  for  it.  On  theoretical  grounds  it  would  be  expected  that  a  cylin- 
drical block  of  endoplasm  should  widen  in  the  fountain  zone,  and  that  in 
becoming  ectoplasmic  tube  it  should  increase  in  cross-sectional  area  and 
shorten  (Fig.  2).  In  fact,  it  does  increase  in  cross-sectional  area  by  a  factor 
of  2  to  3  [is]»  depending  on  en^■ironmental  conditions;  a  compensatory 
shortening  also  occurs  [5].  The  hyaline  cap  fluid,  which  is  known  to  be 
produced  bv  svneresis  [3,  5,  6],  appears  only  when  there  is  forward  flow 
of  cvtoplasm  through  the  fountain  zone.  Hyaline  caps  erupt  in  diflFerent 
pseudopodia  in  the  same  cell  at  difli"erent  frequencies,  suggesting  that 
dift'erent  contractions  in  diflFerent  pseudopodia  are  the  sources  of  this 
fluid,  rather  than  a  contracting  tail  common  to  all  of  the  pseudopodia.  The 
existence  of  a  fluid  channel  in  the  hvaline  ectoplasmic  region  is  shown  bv 
the  fact  that  the  plasmalemma  is  free  to  slide  over  the  ectoplasmic  tube  in 
most  parts  of  the  pseudopod  except  at  limited  points  of  attachment  [10]. 
Entry  of  the  hyaline  cap  fluid  into  the  tail  region  has  not  been  demonstrated 
but  can  be  perhaps  inferred  from  the  "softening"  of  tail  ectoplasmic 
structure  observed  in  the  centrifuge  microscope  [2].  The  development  of 
tension  in  the  axial  endoplasm  would  be  diflicult  to  demonstrate  directly 
by  physical  methods,  but  the  development  of  tension  would  be  perhaps 
the  best  simple  explanation  for  the  fact  that  birefringence  is  highest  in  the 
tail  endoplasm,  despite  the  modest  velocity  gradients  developed  there  [3]. 
So  far,  none  of  these  observations  excludes  the  tail  contraction  theory 
completely. 

The  most  direct  evidence  in  support  of  the  fountain  zone  contraction 
theory  comes  from  a  recent  study  of  streaming  in  cytoplasm  dissociated 
from  the  giant  amoeba.  Chaos  chaos.  About  6  years  ago  while  working  in 
Professor  Runnstrom's  laboratory  at  the  Wenner-Grens  Institute,  I 
discovered  that  amoeba  cytoplasm  could  continue  to  stream  for  periods 
of  up  to  I  hr.  after  it  had  been  dissociated  from  the  intact  cell  [i].  At  that 
time,  it  was  apparent  that  this  phenomenon  was  neither  predicted  nor 
explained  by  the  tail  contraction  theory,  but  there  was  then  no  alternative 
mechanism  that  offered  an  explanation.  It  seemed  clear,  however,  that  the 
cytoplasm  possessed  more  structure  than  prevailing  concepts  allowed,  and 
that  streaming  endoplasm  might  somehow  be  "self-propelled". 

We  have  recently  re-examined  this  phenomenon  in  the  light  of  the 
concept  of  amoeba  structure  illustrated  in  Fig.  i  and  in  the  light  of  the 
predictions  offered  by  the  fountain  zone  contraction  theory  [5].  Our  data 
not  only  strengthen  this  concept  of  amoeba  structure  (Fig.  i)  but  also 
provide  positive  indications  that  the  fountain  zone  contraction  theory 
satisfactorily  explains  streaming  in  dissociated  cytoplasm. 

When  the  plasmalemma  of  an  amoeba  is  ruptured  while  the  amoeba 


554  ROBERT    D.    ALLEN 

is  held  in  a  glass  capillary,  the  cytoplasmic  streaming  organization  of  the 
cell  often  remains  intact;  the  fountain  streaming  pattern  continues  with 
the  capillary  wall  serving  to  replace  the  destroyed  plasmalemma.  When 
the  fountain-streaming  organization  of  the  cytoplasm  breaks  down,  it 
characteristically  is  replaced  by  one  or  more  loops  of  streaming  cytoplasmic 
material.  In  each  streaming  loop,  cytoplasmic  material  moving  toward  the 
bend  of  the  loop  corresponds  in  origin  and  structure  to  the  endoplasm  of 
the  intact  cell ;  similarly,  the  material  moving  away  from  the  bend  corre- 
sponds to  ectoplasm.  The  bend  is  thus  a  two-dimensional  analogue  of  the 
fountain  zone,  which  must  have  dissociated  roughly  into  radial  sections  in 
order  to  form  loops.  There  is  a  marked  consistency  difference  between 
cytoplasm  on  the  two  arms  of  the  loop,  as  can  be  deduced  from  the  velocity 


Fig.  3.  A  velocity  profile  across  a  loop  of  streaming  cytoplasm  dissociated  from 
Chaos  clians.  Note  the  difference  in  velocities  (greater  in  the  cytoplasm  moving 
toward  the  head)  and  in  the  shape  of  the  profile.  Data  of  Allen,  Cooledge  and 
Hall  [5]. 


profiles  in  Fig.  3.  Therefore,  the  consistency  change  occurs  at  the  bend. 
Cytoplasm  moving  toward  the  bend  slows  down,  shortens  (axially),  thickens 
(radially),  and  gives  up  syncretic  fluid  which  can  be  visualized  as  vacuole 
formation  in  the  presence  of  traces  of  calcium  ions.  The  observations 
listed  so  far  are  strongly  suggestive  of  an  active  contraction  at  the  bend  of 
the  loop.  There  is  one  point  of  evidence  which  appears  to  be  conclusive : 
when  cytoplasmic  loops  stream  sporadically,  a  shortening  can  be  seen  to 
occur  a  brief  moment  before  displacement  of  the  endoplasmic  and  ecto- 
plasmic  arms  of  the  loop  toward  and  away  from  the  bend.  Thus  the 
temporal  sequence  of  mechanical  events  provides  a  seemingly  unequivocal 
indication  of  an  active  contraction  at  the  bend  of  the  loops,  and  therefore 
probably  in  the  fountain  zone  of  the  intact  cell  as  well. 

Some  of  the  behavioural  aspects  of  amoeboid  movement  which  led  to 
doubts  concerning  the  correctness  of  the  tail  contraction  theory  appear  to 


STRUCTURE   AND    FUNCTION    IN    AMOEBOID    MOVEMENT  555 

be  quite  compatible  with  this  front  contraction  theory.  For  example,  it  has 
been  pointed  out  that  occasionally  the  endoplasmic  stream  splits  longi- 
tudinally, and  portions  of  the  stream  move  in  opposite  directions  as  if 
pulled  by  opposing  fountain  zones  [2,  3].  The  reversal  of  streaming  also 
occurs  as  if  most  of  the  endoplasm  were  pulled  instead  of  pushed  in  its 
new  direction,  for  reversal  begins  first  at  the  new  advancing  front  and 
stops  last  at  the  old  advancing  front  [3,  4].  Each  front  exhibits  normal 
hyaline  cap  production  cycles  throughout  the  change.  While  it  may  be 
possible  by  means  of  additional  assumptions  to  reconcile  these  facts  with 
the  tail  contraction  theory,  it  is  important  to  realize  that  these  observations 
are  fulfilments  of  the  predictions  one  would  make  from  the  fountain  zone 
contraction  theory  even  if  one  had  never  seen  an  amoeba. 

The  fountain  zone  contraction  theory  is  only  the  first  step  toward  the 
localization,  identification,  and  understanding  of  the  molecular  mechanism 
of  amoeboid  movement.  We  can  hope  that  the  "engine"  of  amoeboid 
cells,  once  localized,  will  be  easier  to  disect  and  characterize  by  physical 
and  chemical  experiments.  As  has  been  pointed  out  elsewhere  [3,  4],  the 
principle  behind  the  theory  mav  have  wider  applications  to  other  systems 
the  mechanisms  of  which  have  been  obscure,  such  as  reticulopodial 
movement  in  foraminifera  and  certain  cases  of  protoplasmic  streaming 
in  plants. 

References 

1.  Allen,  R.  D.,  Bidl.  Bull.  109,  339  (1955). 

2.  Allen,  R.  D.,  7-  biuphys.  biuclum.  Cytol.  8,  379  (i960). 

3.  Allen,  R.  D.,  /;/  "The  Cell",  ed.  J.  Brachet  and  A.  E.  Mirsky.  Academic  Press, 
Xew  York  and  London  (in  press). 

4.  Allen,  R.  D.,  Exp.  Cell  Res.  Sitppl.  (1961)  (in  press). 

5.  Allen,  R.  D.,  Cooledge,  J.,  and  Hall,  P.  J.,  Nature,  Loud.  187,  896  (i960). 

6.  Allen,  R.  D.,  and  Roslansky,  J.  D.,;7.  biuphys.  biochem.  Cytol.  4,  517  (1958). 

7.  Allen,  R.  D.,  and  Roslansky,  J.  D.,  J.  biophys.  biochem.  Cytol.  6,  437  (1959). 

8.  Dellinger,  O.  P.,  J.  e.\p.  Zool.  3,  337  (1906). 

9.  Dujardin,  F.,  Aim.  Sci.  uat.  Zool.  4,  343  (1835). 

10.  Griffin,  J.  L.,  and  Allen,  R.  D.,  Exp.  Cell  Res.  20,  619  (  i960). 

11.  Cioldacre,  R.  J.,  and  Lorch,  I.  J.,  Nature,  Loud.  166,  497  (1950). 

12.  Heilbrunn,  L.  V.,  Protnplasuia  8,  65  (1929). 

13.  Kamiya,  X.,  and  Kuroda,  K.,  Bot.  Mag.,  Tokyo  69,  544  (1956). 

14.  Mast,  S.  0.,y.  Morph.  41,  347  (1926). 

15.  Mast,  S.  O.,  and  Prosscr,  C.  I..,  J.  cell,  couip.  P/iysiol.  i,  t,33  {^93^)- 


Discussion 

CJoldacre:  How  w(juld  you  account  on  your  hypothesis  for  the  fact  that  ATP 
injected  into  the  cell  causes  a  local  contraction  at  the  site  of  injection  which  then 
becomes  the  tail,  not  the  front  ?  The  second  tjuestion :  I  gather  that  your  hypothesis 
rcc]uires  a  propagated  contraction  which  is  held  in  place  by  the  U-shaped  bend  at 


556  ROBERT   D.    ALLEN 

the  end  of  your  capillary  tube.  What  happens  when  you  blow  the  material  out  of 
your  capillary  tube  on  to  a  cover  slip  to  form  a  circular  drop  ?  Do  you  then  find  a 
propagated  contraction  around  the  circle  with  no  streaming  ? 

Allen:  In  answer  to  your  last  question  I  think  I  would  be  restrained  from 
carrying  out  such  an  experiment  as  you  suggest,  by  the  fact  that  the  cytoplasm  of 
the  amoeba  is  so  delicate  that  if  one  takes  it  out  of  the  cell  by  anything  but  the  most 
careful  methods,  it  fails  to  stream.  In  answer  to  your  first  question  I  am  aware  of 
the  ATP  injection  experiments  which  you  reported  in  1950,  but  I  must  remind 
you  that  you  didn't  give  very  much  information  about  the  time  relations  in  these 
experiments  or  state  the  number  of  experiments  that  were  performed.  I  should 
think  it  quite  possible  that  by  poking  an  amoeba  with  a  needle  or  a  pipette  you 
might  obtain  almost  any  kind  of  behavioural  result.  I  don't  regard  the  changing 
of  direction  of  an  amoeba  in  one  experiment  in  response  to  ATP  injection  as  proof 
that  phosphate  energy  from  injected  ATP  has  intervened  in  the  contractile 
mechanism.  There  are  too  many  unknowns.  The  results  you  reported  are  certainly 
compatible  with  this  idea  but  it  would  take  a  great  deal  more  data  to  prove  it. 

GoLDACRE :  Many  repeated  experiments  showed  an  immediate  contraction  at 
the  site  of  injection  of  ATP.  Injection  of  other  substances  (as  we  reported)  had  no 
effect.  Ts'o  and  his  colleagues  reported,  in  1956,  similar  results  to  ours  for  the 
microinjection  of  ATP  into  slime  moulds ;  they  also  demonstrated  a  reversible 
lowering  of  viscosity  of  protein  extracts  from  slime  moulds  each  time  that  ATP 
was  added  to  the  solution ;  after  the  ATP  was  decomposed,  the  viscosity  rose  to 
its  original  value ;  this  could  be  repeated  indefinitely  with  the  same  protein  solution, 
just  as  with  actomyosin.  Loewy  in  1952  also  reported  this  effect  of  ATP  on  slime 
mould  extracts. 

Allen  :  But  again  it  has  to  be  demonstrated  that  the  energy  from  ATP  has  been 
utilized  to  stimulate  the  contractile  mechanism  ;  ATP  may  have  many  more  possible 
effects  than  we  now  realize.  The  contraction  you  speak  of  is  inferred  from  behaviour 
rather  than  directlv  observed. 


Some  Problems  of  Ciliary  Structure  and 
Ciliary  Function 

BjORX  A.  Afzelius 

The  \Veuney-(jyeu  Institute  for  Expeiiuiental  Biology, 
Stockholm,  Su-eden 

The  purpose  of  this  paper  is  to  review  some  of  the  problems  of  cihary 
movement  in  the  hope  that  new  findings  on  cihary  and  flagellar  fine  struc- 
ture mav  shed  light  on  the  mechanisms  responsible  for  the  movement. 
Several  questions  are  still  to  be  answered : 

1.  Bv  what  mechanism  do  cilia  and  flagella  work  ? 

2.  In  what  respects  does  the  fine  structure  of  a  flagellum  differ  from 
that  of  a  cilium  ?  Is  it  possible  to  correlate  such  differences  with  their 
different  modes  of  movement  ? 

3.  What  is  the  significance  of  the  "  magical  9  +  2  filament  arrangement" 
in  cilia  and  flagella  ?  Are  there  meaningful  variations  in  their 
arrangements  ? 

4.  Is  the  ciliary  beat  (or  the  flagellar  beat)  to  be  regarded  as  a  con- 
traction process  ?  Are  there  significant  similarities  between  these 
movements  and  the  contraction  of,  for  instance,  a  striated  muscle  ? 

The  observations  that  are  presented  here  have  been  made  with  the 
electron  microscope.  The  different  tvpes  of  cilia  and  flagella  that  have  been 
chosen  for  study  have  only  this  in  common :  they  have  been  subjected 
previously  to  a  careful  analysis  with  regard  to  their  mo^■ements.  The  study 
has  thus  been  intended  to  be  an  attack  on  the  second  question  above.  It 
was  hoped  that  some  definite  conclusions  could  be  made  and  that  an 
answer  to  the  second  question  would  at  the  same  time  answer  the  others. 

Before  proceeding  further  it  is  necessarv  to  define  the  words  "cilium" 
and  "flagellum".  A  cilium  is  a  fine  vibratile  thread  projecting  with  many 
others  from  the  surface  of  a  cell.  Cilia  lash  in  an  orderlv  beat  in  a  constant 
direction.  The  beat  consists  of  an  efl'ective  stroke  and  a  recovery  stroke. 
In  the  effective  stroke  the  cilium  is  stiff  and  it  dri\"es  the  water  ahead  of  it ; 
in  the  recovery  stroke  the  cilium  is  more  flexible  and  the  tip  of  the  cilium 
follows  a  lower  curve.  It  is  of  interest  that  the  difference  in  flexibility  may 
be  retained  some  time  after  the  cilium  has  stopped :  when  moved  with  a 


558  BJORN   A.    AFZELIUS 

needle  the  cilium  appears  rigid  if  moved  in  one  direction  and  limp  if 
moved  in  the  opposite  direction  [4J.  The  force  exerted  on  the  water  by 
the  cilium  is  in  a  plane  perpendicular  to  its  length. 

A  flagellum  is  a  fine  vibratile  thread  projecting  from  a  cell ;  there  are 
normally  only  one  or  two  flagella  on  a  cell.  The  flagellar  beat  consists  of 
the  formation  of  waves  that  propagate  along  the  length  of  the  flagellum — 
either  from  the  base  to  the  tip  or  in  the  reverse  direction.  The  water  is 
pushed  along  the  length  of  the  flagellum.  In  some  cases  it  has  been  noted 
that  a  defective  flagellum  is  capable  of  forming  stationary  waves  only, 
these  flagella  will  not  propagate  the  water. 

There  are  many  similarities  between  the  flagellar  beat  and  the  ciliary 
beat.  In  both  cases  the  beat  is  in  one  plane,  although  successive  beats  of  a 
flagellum  may  be  in  planes  that  rotate  along  the  length  axis  of  the  flagellum 
[9].  In  the  cilium  as  well  as  in  most  flagella  the  beat  can  be  described  as  a 
bending  movement  starting  at  the  base  and  transmitted  to  the  tip.  In  the 
flagellum  the  propagated  waves  follow  each  other  closely  and  are  fairly 
symmetrical ;  the  flagellum  might  therefore  at  each  instant  take  the  shape 
of  a  sine-wave  (one  wavelength  long  in  the  case  of  the  sea  urchin  sperm 
tail  [9]).  One  implication  of  this,  among  others,  is  that  the  inner  side  of  the 
cilium  may  be  shorter  than  the  outer  one  at  the  end  of  the  eiTective  stroke ; 
the  two  sides  of  the  flagellum  can  retain  their  resting  length  throughout 
the  movements.  It  should  be  mentioned  that  cilia  and  flagella  are  active 
units  generating  their  own  mechanical  energy,  they  are  not  passively 
moved  by  units  within  the  cell  body  [8]. 

The  comparatively  simple  movements  performed  by  the  cilia  and  the 
flagella  would  not  seem  to  require  a  very  complicated  type  of  machinery. 
Therefore  it  has  been  astonishing  to  find  that  the  machinery  of  cilia  and 
flagella  is  quite  complicated  indeed;  the  reasons  for  this  are  by  no  means 
clear. 

Figure  5  (p.  562)  is  a  cross-section  through  three  sea  urchin  sperm 
tails.  This  figure  gives  us  a  view  into  the  motor  units  of  the  flagellum.  The 
appearance  of  the  nine  peripheral  double  filaments  and  the  two  central 
ones  has  been  described  in  an  earlier  communication  [i].  The  peripheral 
filaments  have  projections  called  "arms"  and  "spokes",  and  these  pro- 
jections belong  to  one  of  the  two  subunits  of  the  filaments.  There  is  a 
complex  bridge  formed  between  two  of  the  peripheral  filaments.  The 
filament  opposite  this  pair  is  called  the  unpaired  filament  (filament  i). 

The  filaments  of  the  sea  urchin  sperm  tail  are  fixed  proximally  at  a 
disc  in  the  basal  bodv,  and  end  freely  distally  as  separated  filaments  at  the 
tip  of  the  tail. 

Figures  i  and  2  are  from  another  type  of  flagellum — the  tail  of  the 
squid  spermatozoon.  When  the  spermatozoon  is  actively  swimming  this 
flagellum  shows  movements  which  are  similar  to  those  of  the  sea  urchin 


SOME    PROBLEMS    OF    CILTARY    STRUCTURE    AND    CILIARY    FUNCTION  559 

sperm  tail,  but  at  the  end  of  the  life  span  of  the  spermatozoon  the  flagelhim 
mav  perform  asymmetrical  twitches  of  a  non-propagating  type.  A  twitch 
consists  of  a  slower  bending  phase  and  a  more  rapid  straightening  phase 
which  is  directed  towards  the  asymmetrical  midpiece  [3].  F'igure  i  shows 
two  squid  spermatozoa  in  which  the  section  includes  the  two  midpieces 


Fig.  I.  Cross-section  of  two  squid  spermatozoa  {Lolign  pea/ii).  The  respective 
midpieces  (m)  and  tail  flagella  (/)  are  included  in  the  section  but  not  the  heads. 
There  is  a  marked  separation  between  the  midpieces  and  the  tail  flagella  owing  to 
an  asymmetrical  position  of  the  midpiece  in  relation  to  the  centre  axis  (i.e.  the  tail). 
It  is  of  interest  that  the  arrangement  of  the  9  +  2  tail  filaments  is  fairly  constant  in 
relation  to  the  micipiece  :  a  line  through  the  two  central  filaments  is  at  right  angles 
to  the  line  connecting  midpiece  and  tail ;  the  outer  nine  filaments  are  unevenly 
spaced  in  the  ring  around  the  central  ones  as  the  two  filaments  located  away  from 
the  midpiece  are  closer  together  than  any  other  two  peripheral  filaments.  Magnifi- 
cation 57  500  X  . 

and  below  them  the  respective  flagella.  It  can  be  noted  that  the  arrange- 
ment of  the  flagellar  filaments  is  fairly  constant  and  that  the  unpaired 
filament  i  is  closest  to  the  midpiece.  It  mav  be  inferred  from  pictures  like 
this  that  the  arrangement  of  the  filaments  is  fixed  in  relation  to  the  mid- 
piece  and  that  thus,  the  direction  of  the  flagellar  twitch  is  fixed  in  relation 
to  the  arrangement  of  the  filaments. 

The  interest  in  Fig.  2  lies  in  the  two  tail  cross-sections  that  are  properly 


560  BJORN   A.   AFZELIUS 

cross-cut.  In  these  flagella  the  peripheral  filaments  can  be  seen  to  contain 
subunits  of  different  electron  density.  The  subunit  provided  with  "arms" 
and  "spokes"  appears  dark,  the  other  one  appears  light.  Similar  findings 
have  been  described  from  observations  on  some  mammalian  (Faw^cett, 
personal  communication)  and  an  avian  [14]  spermatozoon. 

The  next  type  of  flagellum  to  be  described  is  that  of  the  sponge  (M/cro- 
cioua  sp.)  collar  cell  (choanocyte).  According  to  Kilian  [12]  the  choanocyte 


Fig.  2.  Cross-section  through  four  squid  sperm  tails  (/).  The  left  tail  is  sec- 
tioned close  to  the  centriole  and  is  partly  surrounded  by  the  nucleus  («).  In  this 
flagellum  and  in  the  one  to  the  right  a  proper  orientation  of  the  section  has  allowed 
a  detailed  study  of  the  flagellar  filaments.  The  nine  outer  filaments  are  connected 
to  nine  electron-dense  accessory  filaments  that  form  a  circle  outside  the  proper 
flagellar  filaments.  The  nine  flagellar  filaments  are  themselves  double,  in  that 
they  can  be  said  to  consist  of  two  subunits,  one  having  a  light  and  the  other  a  dark 
appearance.  Magnification  81  250  x  . 


flagellum  works  with  a  regular  flagellar  beat  when  the  collar  is  expanded 
but  with  a  beat  similar  to  that  of  cilia  when  the  collar  is  retracted.  P'igure  3 
shows  at  low  magnification  a  section  through  a  chamber  lined  with  collar 
cells.  The  marked  area  in  this  picture  is  further  enlarged  and  is  shown  in 
Fig.  4.  Two  notable  features  characterize  this  flagellum  with  regard  to  its 
fine  structure:  (i)  There  is  a  marked  difference  in  size  between  the  two 
subunits  of  each  of  the  nine  peripheral  filaments.  The  larger  subunit  is 
that  which  is  provided  with  arms ;  a  reverse  proportion  has  been  found  in 
some  multiflagellated  protozoa  [7].  (2)  There  are  thin  indistinct  hairs 
lining  two  sides  of  the  flagellum  (marked  h).  These  hairs  are  roughly  in  a 
line  parallel  to  the  two  central  filaments.  The  appearance  is  similar  to  that 


SOME   PROBLEMS    OF    CILIARY   STRUCTURE   AND    CILIARY    FUNCTION  56 1 


^pi     ip^     nrwrn  -i^ 


Fig.  3.  Section  through  a  flagellated  chamber  of  the  marine  sponge,  Microciona 
sp.  Nine  collar  cells  (choanocytes)  have  been  cross-sectioned.  In  each  of  them  the 
flagellum  (/)  and  the  collar  (r)  can  be  seen.  The  collar  is  composed  of  about  30 
separate  microvilli.  An  enlargement  of  the  marked  area  appears  in  Fig.  4. 
Alagnification  20  000  x  . 
VOL.  ir. — 20 


iusMPh^ 


f^- 


C' 


■  oij- . 


Fig.  4.  An  enlargement  of  a  portion  of  Fig.  3.  The  flagellum  has  hair-like 
appendages  (h)  and  can  thus  be  regarded  as  a  so-called  flimmerflagellum.  The  hairs 
have  lateral  positions  (i.e.  a  line  can  be  drawn  through  the  two  inner  filaments  and 
through  the  hairs).  Magnification  105  000  x  . 

Fig.  5.  Transverse  section  through  three  sea  urchin  sperm  tails  {Psammecliinus 
miliaris).  The  detailed  morphology  of  this  flagellum  has  been  described  in  an 
earlier  communication  [i].  Magnification  80  500  x  . 

Fig.  6.  Longitudinal  section  through  the  basal  parts  of  two  laterofrontal  cilia 
from  mussel  gill  {Myti/iis  edidis).  The  central  filaments  stop  at  a  transverse  basal 
plate.  The  peripheral  filaments  continue  (arrow)  through  this  plate  and  enter  the 
basal  body  of  the  cilium.  The  cell  borders  are  visible  at  b.  Magnification  56  000  x  . 


SOME    PROBLEMS    OF    CILIARY    STRUCTURE    AND    CILIARY    FUNCTION  563 

of  so-called  "  flimmcrfiayella "  which  ha\e  been  described  in  plants  by 
Manton  [13]. 

We  will  now  turn  to  cilia.  One  animal  seems  to  ha\e  been  used  more 
often  than  any  other  in  studies  of  ciliary  movement,  namely  the  mussel, 
Mytilus  edulis.  The  gills  from  this  animal  seemed  suitable  for  examination 
in  the  electron  microscope  although  there  are  several  different  types  of 
cilia  in  a  mussel  gill.  Figures  6,  7  and  8  represent  some  of  the  types 
present.  In  Y\g.  8  the  section  passes  near  the  tips  of  the  gill  cilia,  and  it 
can  be  seen  that  the  peripheral  filaments  are  single.  As  in  sea  urchin  sperm 
tails  there  is  no  evidence  here  that  the  individual  filaments  join  close  to  the 
tip.  In  Fig.  7  which  represents  another  type  of  gill  cilia  there  is  on  the 
other  hand  a  top  plate  in  which  the  eleven  filaments  fuse.  A  similar  distal 
fusion  of  the  peripheral  filaments  have  been  described  by  Rhodin  and 
Dalhamn  [i^]  in  cilia  from  rat  trachea.  Figure  6  represents  a  longitudinal 
section  through  the  basal  parts  of  two  "  laterofrontal  cilia".  The  peripheral 
fibres  can  be  followed  from  their  more  distal  parts  down  through  the 
"basal  plate"  (arrow).  Thev  terminate  at  some  distance  below  this  plate 
(cf.  ref.  [6]). 

The  last  two  figures  (Figs.  9  and  10)  are  cross-sections  of  a  unique  type 
of  cilia  which  constitute  the  ctenophore  swimming-plate  [Miieiiiiops/s 
h'idyi).  The  cilia  are  very  long  and  a  great  number  of  them  are  fused 
together.  Their  fine  morphology  is  equally  unique,  and  the  filament 
pattern  can  be  described  as  9 -I- 3  in  contrast  to  the  usual  9  +  2  (Fig.  10)  [2]. 
There  are  ridges  in  the  cilia  which  join  two  of  the  peripheral  filaments  to 
the  cell  membrane — and  in  many  instances  seem  to  connect  filaments  in 
neighbouring  cilia  through  a  similar  substance  between  the  ciliarv  mem- 
branes (arrows  in  Fig.  9).  These  ridges  presumably  represent  the  morpho- 
logical equivalent  of  the  phenomenon  of  ciliary  fusion. 

We  have  now  some  information  on  the  structure  of  cilia  and  fiagella, 
and  we  have  some  information  on  their  function.  We  have  two  types  of 
information,  but  these  two  types  do  not  seem  to  fit  together  well.  There  is 
no  simple  answer  to  the  question  of  ciliary  movement.  At  the  present  time 
one  is  tempted  to  propose  temporary  working  hvpothesis  by  finding 
analogies  in  other  systems  that  are  better  understood.  The  most  obvious 
analogy  is  the  contraction  of  a  muscle.  Perhaps  the  filament-sliding 
hypothesis  of  Huxley  and  Hanson  [11]  may  ser\"e  as  a  model.  Bio- 
chemically the  work  of  muscles  and  the  work  of  cilia  and  fiagella  appear 
similar  although  not  identical  [8,  10]. 

The  bending  of  a  cilium  or  a  flagellum  must  consist  of  a  contractile 
element  as  well  as  of  an  element  capable  of  resisting  compression,  an 
elastic  backbone  [9].  It  seems  likely  that  the  nine  peripheral  filaments  are 
contractile  units.  The  "arms"  have  a  certain  resemblance  to  projections 
on  the  myofilaments  [i].  As  the  nine  filaments  are  continuous  throughout 


564 


BJORN   A.    AFZELIUS 


Fu;.  7.  Transverse  section  through  ciHa  from  mussel  gill  {Alyti/ns  edulis).  The 
arrows  point  to  cilia  where  part  of  the  "top  plate"  has  been  included.  The  9  +  2 
filaments  seem  to  fuse  in  the  top  plate  in  the  distal  part  of  the  cilium.  Magnifica- 
tion 54  000  X  . 

Fig.  8.  Another  transverse  section  through  cilia  from  mussel  gill  (Mytilus 
edulis).  This  type  of  cilium  diflfers  from  that  in  the  preceding  figure  by  having  no 
top  plate.  The  nine  peripheral  filaments  are  single  in  their  distal  tips  and  do  not 
join  with  each  other.  Note  here  also  the  asymmetrical  position  of  the  two  inner 
filaments  in  most  of  the  cilia.  Magnification  68  000  x  . 


I: 


-.i%  '■■■■•  ^ 


Figs.  9  and  10.  Cross-sections  through  a  small  portion  of  a  swimming-plate 
from  the  ctenophore,  Mnemiopsis  leidyi.  The  filament  arrangement  is  9  +  3  rather 
than  9  4-  2,  as  there  is  a  compact  centre  filament  close  to  the  two  tubular  ones.  Two 
of  the  nine  filaments  in  the  outer  ring  are  connected  to  the  ciliary  membrane  by  a 
ridge,  visible  as  a  line  from  these  lateral  filaments  to  the  ciliary  surface.  The 
arrows  in  P'ig.  9  point  to  places  between  the  ciliary  membranes  where  there  can 
be  seen  a  bridging  substance  joining  the  cilia.  These  bridges  are  close  to  the 
attachments  of  the  ridges.  Magnifications  125  000  and   105  000  x  ,  respectively. 


566  BJORN   A.    AFZELIUS 

the  length  of  the  cilium  a  contraction  by  sliding  would  seem  possible  only 
if  one  of  their  ends  is  fixed  and  the  other  end  free  to  move.  The  findings 
presented  here  show  that  the  free  end  might  be  either  at  the  tip  or  at  the 
base  of  the  cilium,  or  the  flagellum.  There  is  no  correlation  with  the 
direction  of  the  propagated  wave.  As  the  nine  peripheral  filaments  follow 
straight  paths  [5,  15,  16]  the  filaments  will  have  an  unequal  degree  of 
contraction  (or  sliding)  in  the  uniplanar  beat.  In  this  connection  it  is  of 
particular  interest  to  consider  the  possibility  of  lateral  fusion  of  the 
swimming-plate  cilia  by  means  of  ridges  from  the  "lateral"  filaments. 
These  filaments  would  thereby  be  unable  to  contract  or  move  during  the 
ciliary  beat. 

The  two  central  filaments  are  likely  to  be  the  candidates  for  the  function 
of  an  elastic  backbone.  Their  position  and  morphology  indicate  that  they 
have  this  function  and  that  they  may  determine  the  direction  of  the  beat. 
It  has  been  shown  that  the  inner  filaments  are  in  a  line  perpendicular  to 
the  direction  of  the  beat  in  mussel  gill  cilia  [11],  in  ctenophore  cilia  [2], 
and,  as  stated  above,  in  the  flagellum  of  the  squid  sperm.  This  is  probably 
also  true  of  the  choanocyte  flimmerflagellum ;  only  when  the  flagellar  beat 
is  perpendicular  to  the  line  through  the  inner  filaments  will  the  hairs  be 
helpful  in  increasing  the  efl^ective  area  of  the  flimmerflagellum. 

We  are  beginning  to  understand  a  little  of  the  arrangement  of  the 
filaments  in  cilia  and  flagella,  but  when  it  comes  to  an  explanation  of  the 
basic  mechanism  of  ciliary  (and  flagellar)  movement  we  are  still  left 
without  an  answer. 


References 

1.  Afzelius,  B.  A.,jf.  biophys.  biochem.  Cytol.  5,  269  (i960). 

2.  Afzelius,  B.  A.,_7.  biophys.  biochem.  Cytol.  (1961)  (in  press). 

3.  Bishop,  D.  W.,  Nature,  Lorid.  182,  1638  (1958). 

4.  Carter,  G.  S.,  Proc.  ray.  Sac.  B.  96,  115  (1924). 

5.  Fawcett,  D.  W.,  and  Porter,  K.  R.,^.  Morphol.  94,  221  (1954). 

6.  Gibbons,   I.   R.,  "Proceedings  2nd  European  Regional  Conference  Elect. 
Micr.,  Delft,"  i960. 

7.  Gibbons,  I.  R.,  and  Grimstone,  A..,  J.  biophys.  biuchejti.  Cytol.  7,  697  (i960). 

8.  Gray,  J.,"  Ciliary  Movement".  Cambridge  University  Press,  Cambridge  (1928). 

9.  Gray,  ].,y.  exp.  Biol.  32,  775  (1955). 

10.  HoflFmann-Berling,  H.,  Fortschr.  Zool.  Ii,  142  (1958). 

11.  Huxley,  H.  E.,  and  Hanson,  ].,  Ami.  N.Y.  Acad.  Sci.  82,  403  (i960). 

12.  Kilian,  E.  E.,  Z.  vergl.  Physio/.  34,  407  (1952). 

13.  Manton,   I.,  ///  "Cellular  Mechanisms  in  Differentiation  and  Growth",  ed. 
D.  Rudnick.  Princeton  University  Press,  Princeton  (1956). 

14.  Nagano,  'V.,jf.  appl.  Pliys.  (in  press). 

15.  Rhodin,  J.,  and  Dalhamn,  T.,  Z.  Zellforsch.  44,  345  (1956). 

16.  Sjostrand,  E.  S.,  and  Afzelius,  B.  A.,  "Proceedings   ist  European  Regional 
Conference  Elect.  Micr.,  Stockhf)lm".  Almcjvist  and  Wiksell,   164  (1956). 


SOME    PROBLEMS    OF    CILIARY    STRUCTURE    AND    CILIARY    FUNCTION  567 

Discussion 

GoLDACRE :  Is  there  any  direct  evidence  that  these  (9  +  2)  filaments  are  the 
motile  element  ?  Have  they  ever  been  fixed  and  viewed  in  the  contracted  state  ? 

Afzelius:  Xo.  It  is  obviously  important  to  investigate  a  ciliated  epithelium  in 
which  the  metachronal  waves  of  the  cilia  have  been  preserved.  The  diflFerences 
that  would  be  found  with  regard  to  the  dimensions  or  the  mutual  positions  of  the 
filament  would  probably  tell  us  much  of  the  mechanism  of  the  cilia.  This  project 
is,  however,  not  as  simple  as  one  would  expect.  I  have  not  been  able  to  fix  the 
metachronal  waves.  At  present  it  is  not  known  what  is  the  contracted  state  of  a 
cilium,  or  even  whether  the  ternis  contraction  and  relaxation  can  be  applied  to 
certain  phases  in  ciliary  and  flagellar  movements. 

Sheldon:  In  the  light  of  recent  experiments  from  Portugal  and  looking  at  your 
picture  do  you  think  the  cilia  are  oval  or  round  ? 

Afzelius  :  You  are  talking  about  the  paper  by  Serra  in  the  last  issue  of  Exper- 
mental  Cell  Researcli  [20,  395  (i960)].  I  think  the  ciliary  cross-section  is  round. 
The  author  might  however  be  correct  when  he  emphasizes  that  there  are  other 
factors  than  the  mechanical  work  of  the  cilia  that  determine  the  morphology  of 
the  cilium. 

RuxNSTROM  :  What  other  factors  will  there  be  ? 

Afzelius:  According  to  Serra  the  mode  of  duplication  of  the  basal  body 
determines  some  features  of  the  ciliarv  structure. 


SPECIFIC  MEMBRANE  TRANSPORT  AND  ITS 
ADAPTATION 


Chairman's  Introduction 

Bernard  D.  Davis 

Department  of  Bacteriology  and  InuniDiology, 

Harvard  Medical  School, 

Boston,  Mass.,  U.S.A. 

In  analyzing  metabolic  pathways  it  has  long  been  profitable  to  approach 
the  cell  as  though  it  were  merely  a  bag  of  enzymes.  Recent  years,  however, 
have  seen  a  broad  and  rapid  increase  of  interest  in  the  properties  of  cell 
membranes.  Two  of  the  reasons  have  already  been  prominent  in  this 
Symposium :  the  intracellular  detail  revealed  bv  the  electron  microscope, 
and  the  dependence  of  mitochondrial  function  on  an  organized  relation  of 
enzymes  to  membranes.  But  probably  the  most  dramatic  contribution  has 
come  from  bacteria :  not  only  do  these  cells  also  possess  a  variety  of  specific 
transport  systems,  but  these  systems  have  been  found  to  respond,  like  the 
intracellular  enzymes,  to  control  by  induction,  repression,  and  mutation. 
This  development  provides  compelling  further  evidence  for  the  realitv 
and  the  importance  of  specific  transport  svstems;  even  more,  it  offers 
hope  of  a  new  approach  to  their  understanding. 

The  evidence  for  specific  transport  systems  is  rather  indirect,  com- 
pared with  that  available  for  most  biochemical  entities.  Hence  inferences 
involving  them  have  often  met  with  scepticism.  Indeed,  the  various  kinds 
of  evidence  available  for  these  systems  are  each  usually  capable  of  alter- 
native interpretations,  and  it  is  only  the  convergence  of  a  number  of  lines 
of  evidence  that  has  now  led  to  quite  general  acceptance.  Since  these 
various  kinds  of  evidence  may  not  all  be  familiar  to  biochemists,  and  since 
the  unfortunate  absence  of  two  scheduled  speakers  has  given  us  extra  time, 
I  shall  try  to  summarize  the  evidence  briefly. 

But  first  I  would  like  to  devote  a  few  minutes  to  historical  and  to 
comparati\e  considerations.  For  most  of  the  w^ork  on  permeability  in 
bacteria  has  developed  autochthonously,  rather  than  as  a  product  of 
laboratories  concerned  primarily  with  permeability;  hence  it  has  tended 
somewhat  to  neglect  the  unity  of  biology  at  a  molecular  level,  which  so 
dominates  our  thinking  throughout  biochemistry  today.* 

*  In  this  connection,  howevtr,  it  should  be  noted  that  electron  microscopy 
reveals  for  the  cytoplasmic  membrane  of  bacteria  only  a  single  dark  and  a  single 
light  layer,  whereas  the  membranes  of  all  other  organisms  studied  have  shown, 
following  similar  fixation,   a  light  layer  between  two  dark  layers.   Furthermore, 


572  BERNARD    D.    DAVIS 

Early  studies  on  cell  membranes  naturally  focused  on  their  resemblance 
to  the  simple  physicochemical  models  provided  by  artificial  semipermeable 
membranes,  in  which  penetration  took  place  by  diffusion  through  pores. 
And,  indeed,  the  kinetics  of  the  penetration  of  certain  substances  into  cells, 
including  dissolved  gases  and  some  very  small  organic  molecules,  could  be 
accounted  for  by  this  mechanism.  Many  large  molecules,  however, 
exhibited  anomalously  high  values;  and  studies  of  certain  homologous 
series  showed  a  parallelism  between  rate  of  penetration  and  lipid  solubility 
(which  increased  with  size).  Hence  penetration  by  solution  in  lipids,  in 
which  biological  membranes  are  known  to  be  rich,  was  recognized  as 
another  significant  mechanism.  Both  these  mechanisms  were  compatible 
with  the  view  of  a  biological  membrane  as  a  relatively  homogeneous 
undifferentiated  structure — perhaps  a  somewhat  porous  double  layer  of 
protein  and  lipid. 

Nevertheless,  the  behaviour  of  most  of  the  metabolically  important 
substances  that  have  to  penetrate  into  cells,  including  sugars,  amino  acids, 
and  inorganic  electrolytes,  did  not  fit  either  of  these  mechanisms ;  and  a 
major  development  in  the  nineteen-thirties  was  the  recognition  (at  least 
among  the  band  of  specialists  in  this  field)  that  with  most  substances 
penetration  into  cells  involves  specific  transport  systems.  In  contrast  to 
the  diffusion  mechanisms  previously  described,  the  rate  of  transport  by 
these  specific  systems  does  not  increase  indefinitely  as  a  function  of 
permeant  concentration  but  instead  exhibits  saturability,  implying  a  mass- 
law  interaction  between  permeant  and  transport  system.  This  conclusion 
implies  functionally  differentiated  regions  of  the  membrane.  It  is  this 
aspect  of  the  cell  membrane,  rather  than  its  generalized  or  average 
properties,  that  now  seems  to  deserve  most  attention,  much  as  the  study 
of  specific  enzymes  and  intracellular  organelles  has  displaced  the  study  of 
"protoplasm". 

Two  groups  of  specific  transport  systems  have  been  recognized.  The 
first,  of  which  the  sodium  pump  is  an  example,  can  carry  out  active  trans- 
port— that  is,  it  can  move  its  permeant  to  a  region  of  higher  thermodynamic 
potential.  The  second  group,  such  as  those  responsible  for  entry  of  sugars 
into  erythrocytes,  cannot  transport  uphill :  with  a  non-metabolized  sub- 
stance these  systems  can  only  accelerate  the  approach  to  equilibrium  (i.e. 
to  the  same  chemical  potential  on  both  sides  of  the  membrane),  and  with  a 
metabolizable  substance  the  rate  of  utilization  is  accelerated. 

This  second  kind  of  specific  transport  has  been  called  facilitated 
dijfusion  by  Danielli.  Though  this  term  is  widely  used  it  does  not  seem 

bacteria  are  unique  in  lacking  steroids.  It  is  therefore  quite  possible  that  future 
analysis  will  reveal  significant  diflferences  as  well  as  broad  similarities  in  the 
structure  and  in  the  function  of  transport  systems  in  bacterial  compared  with 
other  biological  membranes. 


chairman's  introduction  573 

ideal ;  for  (a)  the  process  does  not  obey  the  kinetics  of  diffusion ;  (h)  while 
any  transport  must  involve  motion  of  something  from  here  to  there,  we 
are  still  so  ignorant  of  the  mechanism  that  any  emphasis  on  its  resemblance 
to  diffusion  may  be  prejudicial ;  and  (c)  the  term  suggests  a  mechanism 
very  different  from  that  of  active  transport,  whereas  it  is  quite  conceivable 
that  the  same  "ferryboat"  may  be  capable  of  either  w^orking  at  active 
transport  or  coasting  along  uncoupled  from  energy  expenditure,  depending 
on  the  concentration  of  permeant.  It  might  therefore  be  worth  considering 
a  classification  of  specific  transport  into  active  transport  and  passive  trans- 
port; these  terms  seem  as  neutral  as  possible  with  respect  to  mechanism, 
and  they  are  clearly  distinguished  from  non-specific  permeability  due  to 
direct  diffusion  (either  through  lipid  or  through  an  aqueous  pore)  rather 
than  to  transport  by  some  sort  of  carrier. 

What  is  the  evidence  for  the  existence  of  specific  transport  systems — 
and  of  the  corollary  impermeability  of  a  membrane  to  substances  for  which 
such  a  system  is  lacking  ? 

I.  Crypticity 

With  the  discovery  of  more  and  more  enzymes  many  cases  have  been 
recognized,  in  all  kinds  of  biological  material,  in  which  cells  showed  little 
or  no  enzyme  activity  when  intact  but  became  active  after  mechanical 
disruption  or  after  chemical  damage  to  the  membrane  (e.g.  by  toluene). 
While  this  crypticity  clearly  suggested  a  permeability  barrier,  the  evidence 
was  not  rigorous;  for  the  phenomenon  could  also  conceivably  be  due  to 
the  presence  of  the  enzyme  in  the  cell  in  a  masked  or  inactive  form. 

Late  developments  with  bacteria  produced  one  case  in  which  the  latter 
alternative  could  be  excluded.  The  well-known  inability  of  many  bacteria 
to  utilize  citrate  (or  certain  related  members  of  the  tricarboxylic  acid 
cycle)  might  be  due  to  a  permeability  barrier  or  to  absence  of  the  required 
enzymes.  However,  citrate  was  shown  to  be  an  obligatory  intermediate  in 
the  biosynthesis  of  glutamate  from  glucose  in  Aerobacter  aerogenes  [i]. 
Since  cells  could  be  shown  to  be  unable  to  utilize  exogeneoiis  citrate  under 
conditions  where  they  must  be  rapidly  metabolizing  endogenous  citrate,  a 
permeability  barrier  to  citrate  could  be  inferred  [2].* 

*  Reliance  on  studies  with  intact  cells,  combined  with  scepticism  concerning 
the  possibility  of  a  permeability  barrier  to  citrate,  was  responsible  for  prolonged 
doubt  among  many  investigators  concerning  the  existence  of  the  tricarboxylic  acid 
cycle  in  microbes.  In  retrospect,  indeed,  it  is  rather  ironical  to  find  Professor  Sir 
Hans  Krebs  himself  among  this  group  [3].  Similar  barriers  did  not  interfere  with 
the  recognition  of  the  cycle  in  mammalian  cells.  Reconsidering  this  difference,  one 
is  led  to  wonder  whether  bacteria,  growing  often  in  highly  dilute  environments, 
might  not  need  to  retain  tenaciously  their  intracellular  pools  of  essential  inter- 
mediates (such  as  those  of  the  tricarboxylic  acid  cycle),  whereas  the  environment 
of  the  mammalian  cell  might  m.ake  this  requirement  unnecessary.   In  a  related 


574  BERNARD    D.    DAVIS 

2.  Active  transport 

This  phenomenon /)r/'  se  imphes  specific  transport;  it  also  imphes  that 
the  remainder  of  the  membrane,  aside  from  the  "pumps",  must  be 
relatively  impermeable  to  the  substance.  But  the  analytical  determination 
of  an  elevated  concentration  of  a  permeant  in  a  cell  does  not  necessarily 
prove  active  transport;  it  could  equally  well  reflect  binding  to  cell  con- 
stituents (which  has  often  been  invoked).  Such  binding,  however,  cannot 
explain  the  high  osmotic  pressure  of  bacteria  and  plant  cells  relative  to 
their  environment.  Neither  can  it  explain  the  striking  difference  in  con- 
centrations of  specific  electrolytes  found  in  intracellular  and  extracellular 
fluids  of  higher  animals,  nor  the  evident  ability  of  all  kinds  of  secretory 
and  excretory  organs  to  perform  osmotic  work.  An  additional  argument 
put  forward  by  Cohen  and  Monod  [8]  is  based  on  the  properties  of 
bacteria  that  can  take  up  but  not  metabolize  lactose :  such  cells  can  reach 
intracellular  levels  of  the  compound  as  high  as  20%  of  the  dry  weight  of 
the  cell.  This  result  could  be  accomplished  by  a  small  number  of  catalytic 
pumps  but  would  require  an  implausibly  large  number  of  stoicheiometric 
"hooks" — all  the  more  implausible  since  the  capacity  for  active  concen- 
tration, as  shown  below,  could  be  entirely  eliminated  (or  made  to  appear) 
by  growth  for  a  few  generations  under  conditions  of  repression  (or  induc- 
tion). It  should  be  noted,  however,  that  while  macromolecular  "hooks" 
are  thus  excluded,  conversion  of  permeant  to  a  labile  low-molecular-weight 
derivative  is  not. 

It  is  of  interest  to  note  that  the  capacity  of  bacteria  to  concentrate 
amino  acids,  discovered  by  Gale  [5],  appeared  to  be  restricted  to  Gram- 
positive  organisms,  such  as  Staphylucocciis.  The  later  work  of  Cohen  and 
Rickenberg  [6]  showed  that  the  same  phenomenon  can  be  observed  also  in 
Gram-negative  organisms  such  as  Escherichia  coli,  but  these  require 
greater  precautions  to  avoid  washing  the  permeant  out  of  the  cells  before 
analysis. 

3.  Kinetics 

As  noted  above,  the  rate  of  initial  penetration  of  most  substances  that 
have  been  studied,  plotted  as  a  function  of  concentration,  yields  the  mass- 
law  curve  that  would  be  expected  if  the  penetration  required  formation  of 
a  carrier-permeant  complex,  and  if  the  rate  of  penetration  was  proportional 
to  the  concentration  of  that  complex.  This  is  precisely  analogous  to  the 
classical  "  Michaelis  "  kinetics  for  enzyme  action.  Observations  of  this  kind 
on  erythrocytes  provided  the  main  basis  for  the  recognition  of  specific 
transport  systems  for  substances  that  were  not  actively  concentrated. 

consideration,  it  has  been  suggested  that  the  need  for  retaining  intermediates  may 
be  responsible  for  the  curious  fact  that  the  biosynthetic  paths  developed  in  the 
course  of  evolution  involve  almost  exclusively  ionized  compounds  [4]. 


CHAIRMAN'S    INTRODUCTION  575 

Studies  on  kinetics  involve  not  only  rates  of  transport  but  steady-state 
levels.  In  the  recent  work  on  uptake  by  bacteria  of  the  non-metabolized 
/S-galactoside  TMG  (^-methylthio-D-galactoside)  it  has  been  shown  that 
the  levels  reached  in  active  transport,  at  various  concentrations  of  perm- 
eant,  appear  to  depend  on  a  steady  state  between  entrance  by  a  specific 
pump  and  exit  by  difi^usion,  or  at  least  by  a  system  exhibiting  the  linear 
concentration  relations  of  difi^usion  [7,  8]. 

4.  Competition 

Studies  with  radioactive  compounds  in  animal  cells  and  in  bacteria 
have  shown  that  certain  structurally  related  permeants  (e.g.  similar  amino 
acids  such  as  isoleucine  and  valine)  interfere  with  each  other's  entry,  in 
terms  both  of  rate  and  of  final  level  reached.  This  finding  is  incompatible 
with  difiusion  through  pores,  but  consistent  with  either  entrv  via  a  common 
carrier  or  adsorption  to  common  intracellular  sites.  With  /S-galactosides  in 
bacteria,  it  has  been  possible  by  further  competition  studies  to  choose 
between  these  two  mechanisms  [7,  8].  The  active  transport  system  for 
/S-galactosides  exhibits  both  ditferent  Alichaelis  constants  and  diff"erent 
rates  of  transport  for  various  members  of  this  class  of  compounds.  When 
the  cells  are  in  equilibrium  with  permeant  A  the  addition  of  B,  with  higher 
affinity  and  slower  transport  than  A,  displaces  several  molecules  of  A  per 
molecule  of  B  taken  up.  This  finding  fits  competition  for  a  transport 
system  but  would  be  difficult  to  reconcile  with  competition  for  intracellular 
binding  sites. 

This  competition  has  clarified  certain  obscure  cases  of  analogue 
inhibition.  Analogues  of  metabolites,  such  as  the  sulphonamide  drugs, 
have  generally  been  considered  to  inhibit  growth  by  competing  with  the 
corresponding  metabolite  at  an  enzyme  site.  However,  some  analogues, 
notably  of  amino  acids,  interfere  with  exogenously  added  metabolite  but 
not  with  the  same  metabolite  endogenously  formed.  Thus  arginine 
inhibits  the  growth  of  mutants  that  require  lysine  but  not  of  the  parent 
strain,  which  synthesizes  its  own  lysine.  The  prolonged  blindness  of  non- 
specialists  to  permeability  problems  is  illustrated  by  the  fact  that  this 
lysine-arginine  problem  perplexed  all  of  us  interested  in  microbial  mutants 
for  a  decade,  until  Alathieson  and  Catcheside  [9]  suggested  the  now 
obvious  explanation  and  supported  it  with  evidence  that  arginine  inter- 
fered with  lysine  uptake. 

5.  Mutation 

A  novel  contribution  of  studies  on  bacteria  was  the  finding  that  the 
formation  of  a  specific  transport  system,  like  that  of  an  enzyme,  was  under 
the  control  of  a  corresponding  gene.  Suggestions  in  this  direction  arose 
from  explorations  of  biosynthetic  pathways  with  auxotrophic  mutants, 


576  BERNARD   D.    DAVIS 

which  provided  many  examples  of  apparent  intermediates  that  could  not 
serve  as  a  growth  factor,  presumably  because  of  a  permeability  barrier.  In 
at  least  two  cases,  involving  mutants  blocked  before  5-dehydroquinate  [10] 
and  citrate  [2],  it  was  possible  to  select  secondary  mutants  that  had  gained 
the  ability  to  grow  on  the  intermediate.  Since  the  required  enzymes  were 
already  present  in  the  cell  before  this  second  mutation,  it  seemed  evident 
that  a  one-step  mutation  had  altered  the  permeability  properties  of  the  cell. 

A  much  more  extensive  exploration  of  mutational  effects  on  a  transport 
system  has  been  provided  by  Cohen  and  Monod  for  the  /3-galactoside 
system  [8].  It  has  been  demonstrated  that  the  gene  controlling  the  forma- 
tion of  the  transport  system  and  that  controlling  formation  of  the  enzyme 
^-galactosidase  are  distinct  though  closely  linked  on  the  chromosome:  a 
mutation  can  prevent  or  restore  the  formation  of  either  without  affecting 
the  other.  Of  particular  interest,  for  present  purposes,  are  two  properties 
of  a  cryptic  mutant,  i.e.  one  which  retains  /S-galactosidase  but  is  transport- 
negative  for  ^-galactosides : 

(i)  Compared  with  a  transport-positive,  the  transport-negative  strain 
metabolized  /S-galactosides  very  much  more  slowly;  and  the  relation  of 
rate  to  substrate  concentration  implied  that  the  rate-limiting  step  in  this 
strain  is  diffusion  rather  than  the  action  of  a  system  characterized  by  a 
Michaelis  constant.  We  thus  see  that  in  the  absence  of  a  specific  transport 
system  the  same  permeant  can  penetrate  slowly,  presumably  via  a  more 
primitive  mechanism. 

(2)  In  addition,  the  transport-negative  strain  had  lost  not  only  the 
capacity  to  metabolize  lactose  rapidly  but  also  the  capacity  to  concentrate  a 
non-metabolized  ^-galactoside  (TMG).  This  finding  is  important  in 
linking  studies  on  active  concentration  and  those  on  rate  of  substrate 
utilization  to  the  same  functional  unit.  F^or  the  loss  of  apparently  active 
concentration  could  conceivably  also  be  due  to  loss  of  ability  to  convert 
the  permeant  into  a  labile  intracellular  derivative,  and  loss  of  rapid 
utilization  could  conceivably  be  due  to  formation  of  the  intracellular 
enzyme  in  a  masked  form;  but  only  loss  of  a  specific  transport  system 
could  singly  account  for  both  effects  of  the  mutation. 

Incidentally,  metabolic  inhibitors  such  as  azide  eliminated  the  active 
concentration  but  not  the  rapid  utilization,  suggesting  that  the  same 
specific  transport  system,  which  requires  an  energy  supply  for  function  in 
active  transport,  may  in  the  absence  of  an  energy  supply  still  function  in 
passive  transport  [8]. 

6.  Induction  and  repression 

Another  novel  contribution  from  bacteria  was  the  finding  that  in  an 
appropriate  cell  the  presence  of  certain  transport  systems,  like  that  of 
certain  enzymes,   requires  induction  by  growth  in  the  presence  of  the 


chairman's  introduction  577 

substrate  (or  an  analogue),  and  can  be  repressed  by  the  presence  of  a 
preferred  foodstuff  such  as  glucose.  Induction  of  citrate  transport  in 
Pseudomonas  [ii,  12],  and  induction  and  repression  of  citrate  transport  in 
Aerobacter  [13]  and  ^-galactoside  transport  in  Escherichia  coli  [7,  14],  were 
discovered  independently  in  four  laboratories.  It  is  of  interest  to  note,  in 
all  these  early  communications,  reluctance  to  trust  the  conclusion,  however 
logically  derived  from  the  evidence,  that  the  properties  of  a  cell  membrane 
could  be  substantially  modified  by  the  nature  of  the  growth  medium. 
Apparently  such  a  flighty  disposition,  responsive  to  suggestions  from  the 
environment,  was  easier  to  ascribe  to  invisible  molecules  in  the  cytoplasm 
than  to  a  solid,  microscopically  visible  structure  like  a  membrane ! 

These,  then,  are  the  major  kinds  of  evidence  available  for  the  existence 
of  specific  transport  systems.  The  control  of  these  systems  that  is  possible 
in  rnicrobes  has,  of  course,  opened  up  new  avenues  of  approach  to  their 
nature.  One  of  the  most  significant  findings  has  been  that  the  formation  of 
the  inducible  transport  systems  for  citrate  [13]  and  /3-galactosides  [7,  8], 
like  the  formation  of  inducible  enzymes,  requires  conditions  that  permit 
protein  synthesis.  We  thus  have  strong  evidence  for  a  proposal  offered  by 
earlier  permeability  workers  on  more  speculative  grounds  (cf.  [15,  16]): 
that  the  specificity  of  transport  systems  must  depend  on  the  presence  of 
proteins  with  a  specificity  similar  to  that  already  familiar  in  enzymes.  It 
has  also  been  possible,  by  varying  the  number  of  "pumps"  per  cell 
(through  partial  induction),  to  analyze  more  deeply  the  kinetics  of  entry 
and  exit  in  a  system  carrying  out  active  transport. 

Monod  has  proposed  the  term  "permease"  for  specific  transport 
systems,  whether  active  or  passive  [7,  8].  This  term  has  the  advantage  of 
focusing  attention  on  a  most  important  property  of  these  systems:  the 
presence  of  elements  which  resemble  enzymes  in  their  specificity,  in  their 
mass-law  relation  to  substrate,  and  in  the  genetic  and  environmental 
factors  influencing  their  formation.  The  term,  however,  has  serious 
disadvantages.  First,  being  based  on  the  historical  discontinuity  between 
studies  on  bacteria  and  those  on  other  cells,  it  has  been  construed  as 
implying  entities  quite  different  from  the  "carriers"  proposed  by  Danielli, 
Le  Fevre,  Widdas,  Wilbrandt,  and  Ussing  (cf.  [17])  to  account  for  specific 
transport  in  animal  cells.  In  fact,  however,  the /z<//r//o//rt/ properties  of  the 
two  systems  are  essentially  indistinguishable ;  the  novel  feature  of  the 
bacterial  systems  has  been  the  possibility  of  controlling  their  formation. 
Until  proved  otherwise,  it  would  seem  wiser  to  assume  that  the  specific 
transport  systems  of  all  cells  are  fundamentally  similar ;  a  unified  termin- 
ology would  thus  be  desirable.  And  while  "permease"  has  been  widely 
used  with  reference  to  bacteria,  it  does  not  seem  to  have  been  extended  to 
other  cells. 

VOL.  II. — 2  P 


578  BERNARD   D.    DAVIS 

A  second,  more  serious  objection  to  the  term  "permease"  is  its  impli- 
cation that  the  transport  system  is  an  enzyme.  Here  a  good  deal  of  history, 
from  zymase  to  methionine  synthase,  has  sensitized  biochemists  to  the 
distinction  between  an  enzyme  and  a  more  complex  system.  Finally,  while 
one  can  argue  w^hether  or  not  the  term  "enzyme"  should  be  restricted  to 
catalysts  that  change  a  covalent  bond  in  a  substrate,  there  is  general  agree- 
ment that  the  term  is  not  usefully  applicable  to  such  proteins  as  haemo- 
globin, which  only  form  a  loose,  reversible  association  with  their  substrate. 
And  we  must  at  present  not  restrict  our  thoughts  on  models  for  specific 
transport  to  those  in  which  the  permeant  is  enzymically  converted  into 
another  compound  at  one  side  of  the  membrane  and  restored  again  at  the 
other  side;  we  must  also  be  willing  to  entertain  models  in  which  the 
permeant  is  only  loosely  associated  with  a  carrier  which  shuttles  or  rotates 
back  and  forth.  The  latter  models,  indeed,  would  better  fit  the  possibility 
that  organic  compounds  and  inorganic  ions  are  transported  by  similar 
systems.  For  these  several  reasons  the  writer  prefers,  instead  of  "permease", 
the  less  committal  term  "transport  system". 

In  closing,  I  would  like  to  list  some  of  the  problems  concerned  with 
permeability  that  now  press  for  analysis  at  a  molecular  level.  What  is  the 
structure  of  transport  systems,  and  how  are  the  specific  carrier  proteins 
related  to  the  lipid  in  these  differentiated  portions  of  the  membrane  ?  What 
is  the  mechanism  of  the  energetic  coupling  required  for  active  transport  ? 
Does  it  involve  change  in  the  structure  of  the  permeant  (more  than  simply 
reversible  adsorption),  or  change  in  the  structure  (and  hence  affinity)  of  the 
carrier,  or  still  another  process  that  will  have  to  be  described  in  as  yet 
unknown  terms  ?  Does  a  system  capable  of  active  transport  become  un- 
coupled from  energy  expenditure  when  transporting  downhill  rather  than 
uphill  ?  Is  the  same  polypeptide  chain,  differently  attached,  responsible  for 
the  specificity  of  a  transport  system  and  that  of  an  enzyme  acting  on  the 
same  compound  ?  What  accounts  for  the  fact  that  exchange  of  external 
and  internal  permeant  is  faster  than  net  transport :  does  a  loaded  ferryboat 
shuttle  faster  than  an  empty  one  ?  How  do  compounds  normally  imperme- 
able from  the  outside  become  readily  excreted  by  mutants  blocked  after 
them  ?  How  much  does  the  non-specific  "leakiness"  of  membranes  vary 
with  physiological  state,  and  what  is  its  relation  to  cellular  function  and 
to  viability  ?  Is  the  site  of  induction  of  a  transport  system  within  the  cell  ? 
Or  is  it  at  the  membrane,  as  suggested  by  the  fact  that  citrate  can  induce  a 
transport  system  for  itself  in  a  cell  which  is  relatively  impermeable  to  it, 
and  which  is  meanwhile  rapidly  synthesizing  and  converting  citrate 
endogenously. 

In  a  sense,  research  on  transport  systems,  despite  its  spurt  during  the 
past  decade,  has  been  frustrating.  Direct  chemical  attack  on  simplified 
systems,  extracted  from  the  cell,  has  been  extending  the  solid  march  of 


CHAIRMAN  S    INTRODUCTION  579 

biochemistry  from  low  molecular  weight  intermediates  to  macromolecule 
biosynthesis  and  even  to  the  structure,  function,  and  synthesis  of  what 
might  be  thought  the  deepest  secret  of  biology — the  gene.  But  with  mem- 
branes function  is  even  more  intimately  related  to  structure.  When  one 
tries  the  usual  biochemical  approach  of  first  chopping  the  material  up, 
normal  function,  which  requires  separation  of  two  aqueous  phases  by  the 
membrane,  disappears.  Here,  more  than  in  most  of  cell  physiology,  Goethe's 
awed  attitude  toward  Nature  still  applies : 

Und  was  sie  deinem  (ieist  nicht  oftenbaren  mag, 

Das  zwingst  du  ihr  nicht  ab  mit  Hebeln  und  mit  Schrauben. 

Nevertheless,  it  is  clear  that  I  have  exaggerated  for  rhetorical  purposes. 
Certain  fruitful  approaches  to  various  aspects  of  the  problem  will  be 
described  by  this  morning's  participants,  and  Dr.  Holter  has  already 
introduced  the  phenomenon  of  pinocytosis.  Among  other  recent  en- 
couraging biochemical  developments,  not  represented  here,  it  has  been 
observed  that  stimulation  of  the  activity  of  secretory  glands  is  associated 
with  increased  phospholipid  turnover  [i8];  and  from  erythrocyte  mem- 
branes there  has  been  separated  an  ATPase  that  is  activated  bv  K+  plus 
Na+  [19].  Finally,  since  the  formation  of  specific  transport  systems  in 
bacteria  is  readily  subject  to  experimental  control,  there  are  as  yet  un- 
exploited  possibilities  for  comparing  directly  the  properties  of  two 
membranes  which  should  differ  only  with  respect  to  a  single  system.  In 
complex  problems  of  biology,  as  genetics  has  particularly  shown,  we  can 
learn  a  great  deal  from  studying  discrete  differences  in  a  single  component 
long  before  we  have  learned  how  to  isolate  it. 


References 

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O.  H.  Gaebler.  Academic  Press,  New  York,  509  (1956). 

3.  Krebs,    H.    A.,    in    "Symposium    sur    le    Cycle   Tricarboxylique,    Deuxieme 
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6.  Cohen,  G.  N.,  and  Rickenberg,  H.  V.,  Ann.  Inst.  Pasteur  91,  693  (1956). 

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12.  Kogut,  M.,  and  Podoski,  E.  P.,  Biochem.jf.  55,  800  (1953). 

13.  Green,  H.,  and  Davis,  B.  D.,  cited  in  ref.  [2]. 


580  BERNARD    D.    DAVIS 

14.  Monod,  J.,  in  "Enzymes:  Units  of  Biological  Structure  and  Function",  ed. 
O.  H.  Gaebler.  Academic  Press,  New  York,  7  (1956). 

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Approaches  to  the  Analysis  of  Specific 
Membrane  Transport 

Peter  Mitchell 

Chemical  Biology  Unit,  Department  of  Zoology, 
University  of  Edinburgh,  Scotland 

Now  that  we  have  reached  the  last  session  of  this  Symposium,  I  notice 
that  the  number  of  delegates  has  somewhat  decreased,  and  this  prompts 
me  to  begin  with  some  remarks  about  a  fundamental  thermodynamic 
concept  known  as  "escaping  tendency".  The  escaping  tendency  of  a 
particle  such  as  a  molecule  or  an  electron  or  a  chemical  group  describes 
the  tendency  of  the  particle  to  escape  from  one  place  and  pass  to  another 
by  the  thermodynamic  process  of  diffusion.  As  a  matter  of  fact,  we  are  not 
accustomed  to  thinking  of  people  as  ha\ing  an  escaping  tendency  in  this 
sense  (at  least,  not  a  measurable  one),  for  escape  in  the  present  context 
can  only  occur  by  thermal  movement,  and  thus  the  escaping  tendency  can 
only  be  measured  when  the  free  energy  necessary  to  move  the  particle  is 
not  very  much  greater  than  the  thermal  vibration  energy  for  each  degree 
of  freedom.  Nevertheless,  every  kind  of  transport  process  not  involving  the 
absorption  of  radiant  energy  is  primarily  caused  by  diffusion.  It  may  be 
the  diffusion  of  the  molecules  of  hot  gas  that  propel  the  piston  or  turbine 
blade  or  air  stream  of  the  internal  combustion  or  other  heat  engines  that 
are  at  this  moment  transporting  some  of  our  colleagues  away  from  this 
lecture  theatre;  it  may  be  the  diffusion  of  the  filaments  of  actin  and 
myosin  over  one  another  in  our  hearts  or  skeletal  muscles;  or  it  may  be 
the  diffusion  of  group  donors  to  a  glycosidase  or  synthetase  located  in  a 
membrane  and  the  vectorial  extrusion  of  a  polysaccharide  or  other  polymer 
chain  from  it,  as  discussed  in  the  session  on  polysaccharides  yesterday 
afternoon.  Except  in  the  case  of  photosynthesis,  wherever  there  is  transport 
in  biological  systems  it  is  the  result  of  a  spontaneous  escape  of  particles 
from  a  higher  to  a  lower  free  energy  state  by  thermally  activated  diffusion 
in  space.  Of  course,  this  fundamental  fact  will  be  very  well  known  to  many 
of  those  present  here,  but  I  feel  the  necessity  to  mention  it  at  the  outset — 
to  make  clearer  what  I  am  going  to  say  later — because  the  concept  of  what 
has  come  to  be  called  "active  transport"  in  biology  has  sometimes  been 
associated  with  the  idea  that  the  substrate  specific  transport  systems  of 
living  organisms  can  possess  a  special  property  that  will  actually  cause 


582  PETER    MITCHELL 

molecules  or  other  particles  to  pass  against  the  natural  direction  of  the 
diffusion  or  escaping  tendency,  or  act,  as  Cohen  and  Monod  [i]  have 
suggested,  as  Maxwell  demons.  This,  we  can  say  at  least  with  the  certainty 
of  the  physicist,  is  not  possible. 

Membrane  structure  and  transport  function  in  bacteria 

The  relative  simplicity  of  the  structure  of  bacteria  makes  them 
especially  suitable  for  the  study  of  transport  processes  at  the  molecular 
level  of  dimensions  [2,  3,  4].  In  this  paper  I  shall  concentrate  attention 
upon  bacterial  membranes,  but  will  attempt  to  develop  a  simple  conception 
of  the  relationship  between  transport  function  and  physicochemical 
structure  that  may  be  of  general  validity  in  biology. 

Broadly  speaking  there  are  four  main  experimental  approaches  to  the 
analysis  of  membrane  transport  which  can  be  summarized  under  the 
following  headings: 

1.  Osmotic  barrier  function  of  the  plasma  membrane:  General 
impermeability  function ;  studied  by  net  permeation  measurements. 

2.  Osmotic  link  function  of  the  plasma  membrane :  Specific  transport 
function;  studied  by  observations  on  the  specificity  and  kinetics  of 
the  transport  process,  interpreted  in  terms  of  the  catalysis  of  mole- 
cular complex-,  molecule-,  ion-,  electron-,  and  group-translocation. 

3.  Structure  of  the  plasma  membrane:  Chemical  and  catalytic  com- 
position ;  studied  by  orthodox  chemical  and  biochemical  methods. 

4.  Correlation  of  structure  and  function  in  "  synthetic  "  or  reconstituted 
membrane  systems. 

The  first  three  of  these  approaches  have  been  pursued  in  parallel  in  my 
laboratory.  Our  studies  of  the  osmotic  barrier  function,  beginning  with 
the  introduction  of  the  term  osmotic  barrier  1 1  years  ago  [5],  can  be 
roughly  summarized  by  saying  that  in  general  bacterial  plasma  membranes 
are  permeable  to  small  molecules  carrying  three  water  molecules  or  less 
(e.g.  glycerol),  but  they  are  impermeable  to  molecules  carrying  more  than 
four  water  molecules  (e.g.  glutamate,  phosphate,  succinate,  and  glucose). 
There  are,  of  course,  factors  other  than  the  degree  of  hydration  that 
influence  the  rate  of  permeation  of  different  solutes  into  bacteria.  For 
example,  D-ribose  permeates  much  more  rapidly  than  L-arabinose  and 
other  pentoses,  probably  because  in  the  ribose  molecule  all  the  hydroxyl 
groups  are  on  the  same  side  of  the  ring  so  that  one  side  of  the  molecule  is 
hydrophilic  while  the  other  is  hydrophobic.  There  are  also  differences 
between  the  permeability  of  the  plasma  membrane  of  different  organisms 
to  a  given  solute.  For  example.  Staphylococcus  aureus  and  Micrococcus 
lysodeikticus  are  quite  permeable  to  alkali  thiocyanates  while  Escherichia 


APPROACHES   TO   THE   ANALYSIS   OF   SPECIFIC   MEMBRANE   TRANSPORT        583 

coli  is  not.  In  general,  however,  bacterial  plasma  membranes  behave  in  the 
way  expected  of  the  type  of  thin  hpid  fihii  postulated  by  Overton  [6]  in  his 
lipid  membrane  concept  at  the  beginning  of  this  century  [7-13,  and  see  4]. 

The  question  naturally  arises — and  this  has  been  discussed  ever  since 
the  lipid  membrane  concept  was  introduced — if  the  membrane  is  im- 
permeable to  the  nutrients  of  the  medium,  how  do  they  get  into  the  cell 
during  metabolism  and  growth  ?  The  kinetic  studies  of  glutamate  and 
lysine  transport  in  streptococci  and  staphylococci  in  which  I  was  impli- 
cated in  Dr.  Gale's  laboratory  [14,  15,  5],  and  the  detailed  kinetic  analysis 
of  phosphate  translocation  in  staphylococci  which  I  undertook  shortly 
after  [7,  16-18],  led  us  to  the  conclusion  that  transport  systems  of  high 
substrate  specificity,  exhibiting  kinetic  features  indistinguishable  from 
those  of  the  classical  enzyme  and  carrier  systems  of  biochemistrv,  must  be 
responsible  for  allowing  the  nutrients  to  enter  the  metabolic  svstems  of 
bacteria.  This  conception  of  the  very  close  relationship  between  transport 
and  metabolism  in  bacteria  has  been  confirmed  by  the  more  recent  studies 
in  my  laboratory  on  sugar  and  carboxylic  acid  transport  [see  2,  3,  4,  19,  13], 
some  aspects  of  which  I  shall  describe  later  in  this  paper.  The  kinetic 
studies  of  galactoside  and  amino  acid  uptake  in  Esclierichia  coli  carried  out 
by  Monod  and  his  collaborators  [20-22]  also  support  our  view  of  the 
intimate  relationship  between  the  phenomena  of  transport  and  metabolism 
— although,  the  interpretation  which  Monod  and  his  collaborators  placed 
on  these  studies  was  fundamentally  difl^erent  from  ours  [i].  According  to 
the  recent  work  on  the  kinetics  of  galactoside  uptake  into  Escherichia  coli 
described  by  Kepes  [23],  the  "galactoside  permease"  system  is  identical 
in  principle  to  the  hypothetical  system  originally  suggested  for  the  passage 
of  "  glutamate  "  into  streptococci  through  the  enzvme-catalvzed  conversion 
of  glutamate  to  glutamine  on  the  cell  surface  and  ditfusion  of  glutamine 
through  the  membrane  [5].  This  is  satisfactory  in  demonstrating  the 
present  consensus  of  opinion  as  to  the  most  elementarv  types  of  molecular 
mechanism  that  could  be  involved  in  specific  membrane  transport;  but  it 
also  shows  how  loose  and  unsatisfactory  the  use  of  the  word  "permease" 
has  become,  even  amongst  different  workers  at  the  Pasteur  Institute.  I  feel, 
therefore,  that  I  must  digress  for  a  moment  to  say  that  I  am  inclined  to 
associate  myself  with  the  suggestion  that  Dr.  Davis  made  in  his  introduc- 
tion to  this  session  of  the  Symposium,  that  the  word  "permease"  might 
best  be  abandoned.  I  advocated  in  the  past  that  the  word  "permease" 
should  be  strictly  used  to  mean  a  protein  catalyst  of  facilitated  diffusion 
[2-4],  and  perhaps  this  use  might  still  be  introduced  if  such  a  catalyst 
should  be  found  to  exist. 

As  Dr.  Davis  mentioned  in  his  introduction,  Kogut  and  Podoski  [24], 
Barrett  et  al.  [25],  Green  and  Davis  [26],  and  Monod  and  his  collaborators 
[21]  discovered  that  the  catalysts  of  the  entry  of  certain  carboxylic  acids 


584  PETER   MITCHELL 

and  sugars  into  the  metabolic  systems  of  Escherichia  coli  and  Pseudomonas 
sp.  resemble  enzyme  systems  in  being  inducible,  and  that  the  induction 
can  be  blocked  by  certain  inhibitors  of  protein  synthesis.  These  kinetic 
observations  lend  further  support  to  the  idea  that  the  transport  catalysts 
may  be  normal  enzyme  and  catalytic  carrier  systems.  But  we  must  be 
careful  not  to  imagine,  as  some  microbiologists  have  done,  that  kinetic  and 
inhibitor  studies  of  the  behaviour  of  whole  cells  or  protoplasts  can  reveal 
the  composition  of  the  catalysts  immediately  involved  in  the  transport 
processes — for  example,  whether  they  are  proteins  or  not.  The  only 
unequivocal  way  of  characterizing  the  catalysts  of  transport  is  to  isolate 
and  purify  them,  and  to  examine  their  structure  and  function  by  direct 
analytical  and  kinetic  methods. 

Let  us  now  turn  to  the  third  method  of  attack  on  the  membrane 
transport  problem — the  study  of  the  composition  of  the  plasma  membrane. 
This  phase  of  the  work  has  its  origins  in  the  isolation  of  a  small-particle 
fraction  from  disintegrated  micrococci  which  Dr.  Moyle  and  I  found  to  be 
a  lipoprotein,  just  sufficient  in  amount  to  have  originated  by  the  frag- 
mentation of  the  plasma  membrane,  and  containing  an  acid  phosphatase 
which,  in  intact  cells,  we  knew  to  be  accessible  to  glycerophosphate  from 
outside  [27,  18].  I  should,  perhaps,  say  at  this  point  that  the  problem  of 
isolating  the  plasma  membrane  material  from  bacteria  is  made  com- 
paratively easy  by  the  very  small  size  of  the  cells  and  their  large  ratio  of 
area  to  volume,  a  membrane  only  10  m^  thick  at  the  surface  of  the  proto- 
plast representing  5-10*;' ,,  of  the  dry  weight  of  the  cell. 

The  isolation  of  the  membrane  material  in  a  morphologically  recog- 
nizable state  owes  much  to  Dr.  Weibull's  discovery  that  the  cell  wall  of 
certain  bacteria  could  be  removed  enzymically  without  breaking  the 
plasma  membrane  as  long  as  the  protoplast  was  prevented  from  swelling 
by  the  addition  of  sucrose  or  other  osmotically  effective  solute  to  the 
suspension  medium  [28].  When  the  suspension  medium  was  suddenly 
diluted  after  removing  the  cell  walls  from  the  protoplasts,  the  contents  of 
the  protoplasts  were  thrown  out,  and  the  membranes,  looking  like  little 
bursted  balloons  in  light  and  electron  microscopy,  could  be  collected  on 
the  centrifuge  [29].  In  this  way.  Dr.  Weibull  was  able  to  show  that  the 
cytochrome  pigments  of  Bacillus  megaterium  sedimented  in  the  membrane 
fraction ;  but  as  this  organism  unfortunately  contains  very  many  cytoplas- 
mic particles  which  also  sedimented  with  the  membrane  fraction,  it  was 
not  certain  whether  the  cytochrome  pigments  belonged  to  the  membrane 
or  to  the  adhering  particles.  Dr.  Moyle  and  I  therefore  undertook  a 
similar  type  of  fractionation  on  staphylococci,  which  are  comparatively 
free  of  cytoplasmic  particles  [30].  We  relied  upon  a  controlled  autolytic 
method  to  weaken  the  cell  wall  before  diluting  the  cell  suspension  to  burst 
the  protoplasts  and  liberate  morphologically  recognizable  membranes.  It 


APPROACHES    TO    THE    ANALYSIS    OF    SPECIFIC    MEMBRANE    TRANSPORT         585 

was  found  that  the  weight  of  the  membrane  fraction  obtained  in  this  way 
corresponded  quite  closely  to  the  weight  of  our  small  particle  lipoprotein 
fraction,  and  we  discovered,  as  shown  in  Table  I,  that  both  the  small 
particles  and  the  intact  membranes  (from  which  the  small  particles  were 
evidently  derived)  contained  not  only  cytochrome  pigments,  but  many 
enzyme  activities  [i8,  31].  Similar  observations  were  made  soon  after  by 
Storck  and  Wachsman  [32]  on  the  membrane  fraction  of  Bacillus  mega- 
terium,  and  subsequent  studies  which  Dr.  Weibull  and  his  collaborators 


TABLE  I 

Distribution  of  Enzymes  and  Catalytic  Carriers  in 
Staphylococcus  Aureus 


Enzvnie  or  catalvtic  carrier 


''  Soluble ' 
fraction 


Plasma  membrane  or 

lipoprotein  particle 

fraction 


Cytochromes  (extinction  at  425  m/^i) 

Succinic  dehydrogenase 

Lactic  dehydrogenase 

Malic  enzyme 

Malic  dehydrogenase 

Formic  dehydrogenase 

x-Glycerophosphate  dehydrogenase(s) 

CJlucose-6-phosphate  dehydrogenase 

Glucose-6-phosphatase 

Acid  phosphatase 


<  10 

>  90 

<  10 

>  90 

5-20 

80-95 

<  10 

>  90 

<  10 

>  90 

<  10 

>  90 

30-50 

50-70 

97 

3 

90 

10 

<  10 

>  90 

have  carried  out  on  this  organism  suggest  that  in  this  case,  too,  the  enzyme 
activities  do  actuallv  belong  to  the  membrane  complex  and  are  not  carried 
bv  adhering  particles  [33].  Dr.  Moyle  and  I  extended  our  work  to  Micro- 
coccus lysodeikticus  with  results  similar  to  those  obtained  with  Staphylo- 
coccus aureus  (see  [2]).  In  organisms  such  as  Escherichia  coli  and  Azotohacter 
riue/audii,  where  it  is  very  difficult  to  separate  plasma  membrane  from  cell 
wall  material  [13,  34],  the  evidence  is  perforce  less  unequivocal,  but  it 
seems  probable  that  the  so-called  insoluble  enzymes  in  these  organisms  are 
part  of  the  plasma  membrane  complex  as  in  the  micrococci  and  Bacillus 
megateriuui  [-^z,,  and  see  4]. 


the  concept  of  translocation  catalysis 

The  fact  that  certain  hydrolytic  and  oxido-reductive  enzymes  and 
catalytic  carriers,  including  those  of  the  cytochrome  system,  are  an 
integral  part  of  the  plasma  membrane  complex  of  certain  bacteria  repre- 
sents  the   experimental   foundation    for   our   conception   of  the   plasma 


586  PETER    MITCHELL 

membrane  as  an  active  participant  in  the  metabolism  of  the  cell  as  a  whole 
[2,  4].  To  admit,  however,  that  the  membrane  participates  in  the  intra- 
cellular metabolic  processes  is  to  pose  a  new  question.  What,  we  may  well 
ask,  is  the  function  of  the  enzymes  and  catalytic  carriers  that  are  located  in 
the  membrane  complex  ?  Why  are  these  metabolic  systems  organized  in 
the  surface  of  the  protoplast  instead  of  being  tucked  away  safely  in  the 
cytoplasm  ?  It  occurred  to  me  some  time  ago  that  this  question  might  be 
answered  in  the  following  way.  During  group  transfer  or  substrate  transfer, 
the  group  transfer  enzyme  or  catalytic  carrier  molecules  of  classical  bio- 
chemistry (in  as  much  as  they  are  anisotropic  catalysts)  catalyze  a  micro- 
scopic vectorial  movement  or  translocation  of  substrate  or  chemical  group, 
directed  in  space  relative  to  the  individual  enzyme  or  catalytic  carrier 
molecules.  We  normally  think  of  metabolism  as  a  scalar  substrate  and 
group  transfer  process  (without  direction  in  space)  because  we  think  of  it 
as  though  the  enzyme  and  carrier  molecules  were  orientated  at  random, 
so  that  there  would  be  no  macroscopic  vector  component  of  the  substrate 
and  group  translocation  processes.  But  if,  as  seems  likely,  the  enzyme  and 
catalytic  carrier  molecules  are  specifically  orientated  in  an  organized 
membrane  structure,  the  microscopic  translocations  of  the  substrates  and 
chemical  groups  which  represent  the  normal  metabolic  transfer  processes 
can  show  as  concerted  macroscopic  transports  of  substrates  and  chemical 
groups  (including  ions  and  electrons)  across  the  membrane  [2,  36-38]. 
Thus,  we  might  not  need  to  stretch  our  imagination  very  far  beyond  the 
bounds  of  classical  biochemistry  to  conceive  how  the  metabolic  systems  of 
the  membrane  could  function  as  the  catalysts  and  controllers  of  membrane 
transport. 

You  will  see  now  the  reason  for  my  opening  remarks  about  escaping 
tendency  and  diffusion.  We  are  accustomed  to  thinking  of  the  diffusion  of 
molecules  and  the  chemical  transformation  of  molecules  in  rather  different 
terms,  but  the  processes  involved  in  diffusion  and  chemical  change  are,  in 
fact,  very  similar.  The  diffusion  of  a  solute  particle  such  as  a  molecule  or 
molecular  complex  in  a  biological  system  describes  the  movement  of  the 
particle  by  the  thermally  activated  breaking  and  making  of  the  secondary 
bonds  that  tend  to  prevent  the  displacement  of  the  particle  relative  to  the 
neighbouring  atoms.  The  chemical  transformation  of  a  molecule  or  mole- 
cular complex  describes  the  movement  of  one  of  its  constituent  chemical 
groups  by  the  thermally  activated  breaking  and  making,  not  only  of 
secondary  bonds,  but  also  the  primary  bond  that  tends  to  prevent  the 
detachment  of  the  group  from  its  partner  (or  donor  group)  and  its  transfer 
to  an  acceptor  group.  The  enzymes  and  catalytic  carriers  of  a  membrane 
complex  must  catalyze  the  movement  of  molecular  complexes,  molecules, 
ions,  electrons  or  chemical  groups  in  the  natural  direction  of  the  diffusion 
or  escaping  tendency.  It  is  convenient  to  call  the  catalysis  of  this  natural 


APPROACHES    TO    THE    ANALYSIS    OF    SPECIFIC    MEMBRANE    TRANSPORT         587 

process  of  diffusion  down  the  electrochemical  activity  gradient  "trans- 
location catalysis".  When  we  describe  the  transport  of  a  substance  as 
"active",  it  is  because  we  do  not  know  (or  do  not  w^ish  to  specify)  in  what 
form  the  substance  actuallv  diffuses  across  the  membrane. 


SUBSTR.\TE   AND    SUBSTRATUM    SPECIFICITIES    OF   ENZYMES 
AND    CATALYTIC    CARRIERS 

The  view  that  I  have  been  developing  of  substrate  and  group  trans- 
location as  part  of  the  metabolic  process  in  the  spatially  organized  enzyme 
and  catalytic  carrier  systems  of  the  membrane  complex,  places  the  problem 
of  membrane  transport  in  a  new  light,  for  it  suggests  that  to  picture  the 
process  of  transport  at  the  molecular  level  of  dimensions  we  need  to 
recognize,  not  only  the  substrate  specificities  of  the  enzymes  and  catalytic 
carriers  as  normallv  understood,  but  also  the  locational  or  substratum 
specificities  that  are  responsible  for  bonding  these  components  into  the 
organized  structure  of  the  membrane  complex  [36].  The  locational  bonds 
may  be  said  to  represent  the  articulations  between  the  bones  of  the 
cytoskeleton  [39]. 

The  conception  of  the  bivalent  specificity  (the  substrate  specificity  on 
the  one  hand  and  the  substratum  specificity  on  the  other)  of  the  trans- 
location catalysts  stems  directly  from  considerations  of  the  transport 
process  per  se.  It  is,  perhaps,  significant  that  when  one  considers  the 
transport  process  from  a  different  angle,  that  is,  in  relation  to  growth  and 
adaptation  (as  we  are  asked  to  do  in  this  session  of  the  Symposium),  the 
same  type  of  conception  as  I  have  just  outlined  seems  to  be  required.  For, 
since  it  is  inconceivable  that  the  catalysts  of  translocation  could  all  be 
synthesized  at  the  sites  of  their  activity  in  the  membrane,  the  very  catalytic 
components  of  a  membrane  system  that  are  to  cause  and  control  the 
translocation  of  a  specific  substrate  must,  themselves,  possess  the  speci- 
ficities that  will  cause  them  to  be  transported  to  and  incorporated  in  the 
organized  membrane  structure  during  growth  and  adaptation.  As  I 
pointed  out  some  years  ago  [2,  4],  one  must  take  much  more  care  than  has 
been  customary  in  interpreting  the  results  of  studies  of  mutants  that  lack 
particular  transport  capacities,  for  loss  of  a  transport  capacity  could  as 
easily  be  due  to  a  change  of  locational  specificity  between  a  transport- 
catalyzing  enzyme  or  carrier  and  its  locator  region  (substratum)  in  the 
membrane  as  to  the  loss  of  the  catalytic  function  of  the  free  enzvme  or 
catalytic  carrier  molecule  itself. 

I  propose  now  to  summarize  the  results  of  two  series  of  experiments 
bearing  on  the  problem  of  enzyme  location  and  enzyme-substratum 
specificity  that  we  have  recently  done  in  Edinburgh.  It  has  been  customarv 
to  assume  that   intracellular  enzymes  which  appear  in   solution  in  the 


588  PETER    MITCHELL 

medium,  when  one  breaks  the  cell  wall  and  plasma  membrane  of  bacteria 
by  methods  that  do  not  cause  appreciable  autolysis,  are  located  within  the 
protoplast  of  the  intact  cell.  Further,  it  has  generally  been  assumed  that 

TABLE  II 

Fractionation  of  Escherichia  coli 

Equivalent  of  930  mg.  dry  \vt.  organisms  disrupted  mechanically  in  standard  saline 
(0-17  M  NaCl,  0-017  M  KCl,  0-005  ^i  MgClo)  for  30  min.  using  the  disintegrator  of 
H.  Mickle  as  described  by  Mitchell  and  Aloyle  [27],  washed  into  centrifuge  tubes  with 
c.  30  ml.  standard  saline  at  2    and  centrifuged  at  2000  g  for  30  min. 


Supernatant 
centrifuged  at 
35  000  g 
for  I  hr. 


Supernatant,  Si 
(48  ml.). 


Pad  in  laver,  P3 

(2  ml.). 

Dispersed  in  10  ml. 

standard  saline. 

Morphology :  aggregates 

of  very  small  particles. 


Pad  in  2  layers:  Top,  Pi  (5  ml.). 

Bottom,  P2  (i  ml.). 


Pi,  easily  dispersed,  made  up  to  10  ml. 
in  standard  saline. 

Morphology:    empty   en\elopes,    intact 
and  fragmented. 

P2,  dispersed  with  difficulty,  made  up 
to  10  ml.  in  standard  saline.  Extinction 
at  700  m/x  equivalent  to  93  mg.  dry  wt. 
intact  cells. 
Morphology:  mainly  intact  cells. 


Of  fraction  Pi,  8  ml.  returned  to  sonic 
disintegrator  for  15  min.  Washed  into 
centrifuge  tubes  with  standard  saline 
(total  vol.  14  ml.).  Centrifuged  at 
2000  g  for  60  min. 


Supernatant,  S2  Pad,  P4  (2  ml.). 

(i2  ml.).  Made  up  to  8  ml. 

in  standard  saline. 
Morphology:  as 
Pi,  but  more 
fragmented. 

The  morphology  of  the  fractions  was  exaniined  by  anoptral  contrast  microscopy  of  very 
thin  films  of  the  untreated  aqueous  suspensions  sealed  between  slide  and  coverslip  w  ith  a 
ring  of  vaseline. 


such  "soluble",  extracellular  enzymes — like  ^-galactosidase  in  Escherichia 
coli  [40,  41] — cannot  be  involved  in  catalytic  activity  at  the  surface  of  the 
protoplast,  or  in  membrane  transport,  because  they  are  said  to  be  "  cryptic  ", 
or,  in  other  words,  enclosed  behind  the  osmotic  barrier  component  of  the 
plasma  membrane  [i].  Dr.  Stephen  and  I  have  studied  the  "solubility" 
and    distribution    of   glucose-6-phosphatase    activity    in    Escherichia   coli 


APPROACHES   TO   THE   ANALYSIS    OF   SPECIFIC   MEMBRANE   TRANSPORT        589 

(strain  ML  30)  with  these  assumptions  in  mind.  We  found,  as  illustrated 
in  Tables  II  and  III,  that  after  disintegrating  washed  suspensions  of 
Escherichia  coli  mechanically,  and  fractionating  on  the  centrifuge,  some 
85%  of  the  glucose-6-phosphatase  was  present  in  the  clear  solution  that 
had  been  centrifuged  at  35  000  g  for  i  hr.,  and  some  i2"o  was  initially 
present  in  the  cell  envelope  fraction  which  centrifuged  down  at  2000  g  in 
30  min.  On  re-submitting  the  cell  envelope  fraction  to  the  disintegration 
procedure,  to  release  any  enzyme  that  might  have  been  trapped  by  re- 
closure  of  some  of  the  membranes,  a  further  5"o  of  the  enzyme  was  ob- 
tained in  the  "  soluble  "  form,  bringing  the  amount  of  "  soluble  "  glucose  6- 
phosphatase  recovered  to  some  90"  ^  of  the  whole.  These  and  other  related 

TABLE  III 

Distribution  of  Glucose  6-phosphatase  in  Esrhen'cliia  cnli  (ML  30) 

Glucose  6-phosphatase        ,,    „       , 
T-        .  ^ .      ^  "o   1  otal 

r  raction  activity  .   . 

(/umole  P/g.  min.) 

From  ivhole  cells 

"Soluble",  Si  i-8o  84-7 

Very  small  particles,  P3  0064  3-0 

Cell  envelopes.  Pi  0263  12  3 

From  redisintegrated  cell  envelopes 

"Soluble",  S2  0-098  4-6 

Cell  envelopes,  P4  0-128  6-0 

Intact  untreated  cells  z-  11  99 


experiments  showed  that  although  the  glucose-6-phosphatase  probably  has 
an  affinity  for  a  cell  envelope  component,  according  to  the  usual  standards 
it  would  be  classed  as  a  soluble  enzyme.  We  discovered,  however,  as 
illustrated  at  the  bottom  of  Table  III,  that  the  rate  of  hydrolysis  of 
externally  added  glucose-6-phosphate  by  suspensions  of  intact  cells 
represents  the  full  expression  of  the  "soluble"  enzyme  activity,  and,  as 
shown  in  Table  IV,  the  activity  of  the  intact  cells  was  little  affected  by 
breaking  the  plasma  membrane  with  benzene  (5*^0  v. /v.)  or  by  freezing 
and  thawing.  We  showed  that  glucose-6-phosphate  does  not  penetrate  into 
the  protoplast  of  intact  cells,  for  although  it  could  be  fermented  rapidly  by 
cells  in  which  the  membrane  was  ruptured,  in  intact  cells  it  w'as  fermented 
only  at  a  rate  corresponding  to  that  of  the  liberation  of  free  glucose  by  the 
fully  expressed  glucose-6-phosphatase.  Further,  the  glucose-6-phosphatase 
of  intact  cell  suspensions  was  found  to  liberate  the  inorganic  phosphate  of 
externally  added  glucose-6-phosphate,  not  in  the  protoplasts,  but  in  the 


59°  PETER   MITCHELL 

TABLE  IV 

Glucose  6-phosphatase  Activity  of  Normal  and  Treated  Escherichia 
coli  (ML  30)  Suspensions  and  of  Growth  and  Suspension  Media 


Glucose  6-phosphatase 

0/ 

/O 

Activity 

Material 

activity 

of  normal 

(/xmole  P/g.  min.) 

cells 

Normal  intact  cells 

2-39 

100 

Benzene-treated  cells 

2-77 

116 

Frozen  and  thawed  cells 

2-19 

92 

Suspension  mediuni 

oil 

4-6 

Growth  medium 

0-07 

2-9 

Normal  intact  cells 


(/xmole  P/g.  min.  liberated 

in  suspension  medium  only) 

2-91 


suspension  medium.  As  illustrated  in  Fig.  i,  these  and  other  confirmatory 
observations  forced  us  to  the  conclusion  that  the  glucose-6-phosphatase  of 
intact  Escherichia  coli  is  enclosed  in  a  region  between  the  cell  wall  and  the 
surface  of  the  osmotic  barrier  component  of  the  plasma  membrane  which 

Cell  wall 
(molecular  sieve) 

I  Plasma  membrane 

;  Periplasm  ''(osmotic  barrier) 


Medium 


Endoplasm 


Glucose- 6- phosphate 

Fig.  I.  Diagram  of  cell  wall,  periplasm  and  plasma  membrane  (osmotic  barrier 
component)  in  Escherichia  coli.  The  glucose-6-phosphatase,  partly  adsorbed  on  a 
substratum  in  the  cell  envelope  complex,  is  confined  to  the  periplasm  by  the 
molecular  sieve  function  of  the  cell  wall. 


we  might  appropriately  call  the  "periplasm".  You  may  ask  how  one  can 
show  that  the  effective  pore  size  of  the  cell  wall  of  living  Escherichia  coli  is 
small  enough  to  prevent  the  passage  of  proteins  between  the  periplasm  and 
the  external  medium.  Figure  2{a)  shows  living  Escherichia  coli  (strain 
ML  30)  in  which  the  protoplasts  have  been  made  to  retract  from  the  cell 
wall  by  the  addition  of  0-4  M  NaCl  to  a  suspension  medium  of  0-02  M 
sodium  phosphate  buffer  at  pH  7,  and  Fig.  2(6)  shows  the  same  with  the 


APPROACHES   TO    THE   ANALYSIS    OF   SPECIFIC   MEMBRANE   TRANSPORT        59 1 

further  addition  of  15%  w./v.  human  serum  albumin.  The  brightness  of 
the  anoptral  contrast  image  increases  with  the  refractive  index  of  the 
object.  If  the  serum  albumin  had  penetrated  into  the  periplasm,  enlarged 
by  plasmolysis,  there  would  be  no  more  contrast  between  the  periplasm 
and  the  suspension  medium  in  Fig.  2{b)  than  in  Fig.  2{a).  The  fact  that 
the  periplasm  is  darker  than  the  suspension  medium  in  Fig.  2{b)  shows 


ia)  (b) 

Fig.  2.  Anoptral  contrast  micrographs  (  x  3800)  of  Escherichia  coli  (ML  30) 
plasmolyzed  in  0-4  m  NaCl  {a),  and  the  same  with  addition  of  15%  (w./v.)  serum 
albumin  (b).  The  dark  periplasm  between  cell  wall  and  retracted  protoplasts  in  (6) 
shows  impermeability  of  cell  wall  to  serum  albumin. 

that  the  serum  albumin  (M.W.  70  000)  does  not  penetrate  the  cell  wall. 
It  seems  reasonable  to  conclude  that  the  cell  wall  acts  as  a  molecular  sieve, 
allowing  entry  of  glucose  6-phosphate,  but  preventing  the  escape  of 
glucose  6-phosphatase  from  the  surface  of  the  protoplast.  The  glucose  6- 
phosphatase  is  thus  in  a  position  to  catalyze  the  first  step  in  the  metabolism 
and  uptake  of  glucose  6-phosphate  in  Escherichia  culi. 

The  facts  established  by  this  work  have  far-reaching  implications  tor 


592  PETER   MITCHELL 

the  interpretation  of  observations  on  enzyme  distribution  in  relation  to 
membrane  transport  in  micro-organisms.  When  we  conceive  the  cell  wall 
as  a  molecular  sieve,  preventing  the  loss  of  enzymes  that  may  exist  entirely, 
or  only  partly,  in  the  free  state  at  the  surface  of  the  plasma  membrane  (i.e. 
in  the  periplasm),  we  must  give  serious  consideration  to  the  possibility  that 
the  enzymes  and  catalytic  carriers  of  the  protoplasm  may  be  poised  in  an 
equilibrium  which  may  favour  their  segregation  in  the  periplasm,  in  the 
plasma  membrane,  or  in  the  endoplasm  according  to  the  satisfaction  of 
mutual  affinities.  The  distribution  of  a  given  enzyme,  and  its  status  as  a 
relatively  "soluble"  or  "insoluble"  protein  would  thus  be  seen  as  an 
expression  of  locational  affinities  for  bonding  the  protein  to  compli- 
mentary substratum  sites  in  the  cell.  In  general,  it  would  be  expected  that 
such  bonding  would  be  due  to  secondary  valencies,  but  it  is  also  possible 
that  primary  valencies  might  sometimes  be  involved.  The  studies  of  Keilin 
and  King  [42]  on  the  reversible  bonding  of  the  soluble  succinic  dehydro- 
genase in  the  insoluble  cytochrome  system  of  heart  muscle  lends  support 
to  this  conception.  Such  a  conception  has  obvious  potentialities  for  helping 
to  explain  induction  and  repression  of  enzyme  synthesis  by  a  mass  action 
type  of  effect  [43,  44]  not  only  thought  of  as  being  due  to  equilibration  of 
nascent  enzyme  with  enzyme-substrate  or  enzyme-(substrate  analogue) 
complexes,  but  also  due  to  the  equilibration  of  nascent  enzyme  with 
enzyme-substratum  and  (enzyme-substrate)-substratum  complexes.  These 
attractive  ideas,  which  are  related  to  those  of  Catcheside  [45],  hinge, 
however,  on  the  experimental  demonstration  of  the  locational  affinities 
between  enzymes  and  substratum  sites  in  bacteria.  For  reasons  that  I  shall 
explain  in  a  moment.  Dr.  Moyle  and  I  decided  to  study  the  distribution 
of  the  a-ketoglutarate  dehydrogenase  activity  in  Micrococcus  lysodeikticiis 
as  an  example  of  the  possible  participation  of  locational  affinities  in 
determining  the  cytological  distribution  of  an  enzyme. 

When  Micrococcus  lysodeikticns  is  ruptured  by  shaking  with  glass  beads 
or  by  gentler  enzymic  and  osmotic  methods,  and  the  plasma  membranes 
are  separated  from  the  "soluble"  or  "protoplasm"  fraction  on  the  centri- 
fuge in  the  usual  way  [see  11,  30],  about  half  the  a-ketoglutarate  dehydro- 
genase activity  is  found  in  the  plasma  membrane  fraction.  The  amount  of 
enzyme  activity  attached  to  the  plasma  membranes  is  not  dependent  upon 
the  distribution  of  dialyzable  cofactors,  nor  is  it  appreciably  affected  by 
the  extent  to  which  the  membrane  material  is  diluted  during  its  isolation. 
One  can  therefore  infer  that  the  enzyme  must  be  strongly  bound  to  the 
membrane  structure.  The  fact  that,  nevertheless,  about  half  the  enzyme 
activity  is  present  in  the  "soluble"  fraction  shows  either  that  there  are 
two  a-ketoglutarate  dehydrogenase  proteins  with  different  solubilities  or 
affinities  for  membrane  and  protoplasm  components,  or  that  there  is  only 
one  type  of  a-ketoglutarate  dehydrogenase,  which  is  a  soluble  protein,  and 


APPROACHES   TO   THE   ANALYSIS   OF   SPECIFIC   MEMBRANE   TRANSPORT        593 

that  the  membrane  contains  a  fixed  number  of  substratum  sites  at  which 
this  soluble  enzyme  can  be  specifically  bonded.  We  designed  a  series  of 
experiments  to  decide  between  these  alternatives,  and  I  can  best  indicate 
briefly  our  conclusion  by  the  example  from  part  of  an  experiment  shown 
in  Table  IV.  The  method  of  approach  was  to  inactivate  the  membrane- 
located  enzyme  in  the  intact  cell  with  an  irreversible  inhibitor  (in  this  case, 
iodoacetate  at  pH  8),  and  then  to  determine  whether  the  enzyme  could  be 
replaced  in  vitro  by  the  "soluble"  enzyme  of  the  normal  protoplasm 

TABLE  V 

EQUn.IBRATION    OF    a-KETOGLUTARATE    DEHYDROGENASE   BETWEEN    THE 

Membrane  and  Protoplasm  of  Micrococcus  lysodeikticus 

Membrane  "Protoplasm" 

Normal  cells  55-5  44-5 

lodoacetate-treated  cells  ^  14-6  4-5 

lodoacetate-treated  membrane  (i)  14-6  — 

Normal  "  Protoplasm"  (2)  —  40  o 

After  re-separating  mixture  of  (i)  and  (2)  35 '5  20 'O 

fraction.  The  numbers  in  Table  \"  represent  a-ketoglutarate  dehydro- 
genase activity  expressed  as  a  percentage  of  the  total  activity  of  the  normal 
intact  cells,  which,  in  this  experiment,  had  an  absolute  value  of  0-65  /^mole 
substrate  per  g.  cell  dry  weight  per  minute.  Rather  more  than  half  the 
total  activity  was  initially  present  in  the  membrane  fraction  in  this  batch 
of  cells.  After  reacting  the  intact  cells  with  iodoacetate  and  separating  the 
membrane  and  "protoplasm"  fractions  as  usual,  the  total  a-ketoglutarate 
dehydrogenase  activity  was  reduced  to  about  20"  ,3  of  the  normal.  A  sample 
of  the  inactivated  membrane  fraction,  representing  an  activity  of  14-6,  was 
now  thoroughly  mixed  with  a  sample  of  the  normal  "  protoplasm"  fraction, 
representing  an  activity  of  40,  and  the  two  fractions  were  separated  again 
on  the  centrifuge.  As  shown  in  Table  V,  the  activity  of  the  membrane 
fraction  was  found  to  have  risen  by  20-9  units,  while  that  of  the  "proto- 
plasm" had  fallen  by  20-0  units — an  equivalent  amount  within  experi- 
mental error — showing  a  substantial  transfer  of  enzyme  from  the  "  soluble  " 
state  in  the  "protoplasm"  fraction  to  the  bound  state  in  the  membrane 
complex.  This,  and  other  confirmatory  and  related  experiments  imply  that 
the  distribution  of  a-ketoglutarate  dehydrogenase  activity  in  Micrococcus 
lysodeikticus  does  indeed  depend  upon  the  mutual  satisfaction  of  locational 
affinities  between  a  soluble  a-ketoglutarate  dehydrogenase  and  a  specific 
substratum  in  the  plasma  membrane  complex  of  the  cell, 
vol..  n. — 2  Q 


594 


PETER   MITCHELL 


Special  characteristics  of  vectorial  metabolism  in  anisotropic 

enzyme  systems 

The  studies  that  Dr.  Moyle  and  I  have  recently  been  carrying  out  on 
the  specificity  and  general  kinetics  of  the  entry  of  "  a-ketoglutarate  "and 
"succinate"  into  Micrococcus  lysodeikticiis,  and  the  comparison  of  these 
characteristics  with  those  of  the  a-ketoglutarate  dehydrogenase,  succinic 
dehydrogenase,  succinyl-coenzyme  A  kinosynthetase,  and  other  enzymes 
present  in  the  plasma  membrane  complex  ([19,  46],  and  extensive  unpub- 
lished observations)  has  led  us  to  represent  the  entry  mechanism  by  the 
tentative  scheme  of  Fig.  3.  This  scheme  is  in  accord  with  all  the  experi- 
mental facts  at  present  available  to  us,  but  I  must  emphasize  that  it  is 


Endoplasm 

c-ketoglutarate 
dehydrogenase 


q;-KETOCLUTARATE  -  *■ 


SUCCINATE 


O'-KETOGLUTARATE 


C02-HH2 

(SUCCINYL^) 
ADP  +  P 

ATP 


Succinyl  CoA 
kinosynthetase 


Fig.  3.  Diagram  of  "succinate"  and  "  3^-ketoglutarate  "  translocation  through 
the  membrane  of  Micrococcus  lysodeikticiis.  The  dehydrogenases  are  depicted  as 
part  of  the  plasma  membrane,  anchored  to  each  other  (and  to  other  substrata  not 
drawn)  by  specific  residual  bonds. 


nevertheless  tentative;  for,  when  one  considers  how  unequivocal  the 
interpretation  of  such  observations  can  be  made,  or  in  other  words,  how 
close  a  correspondence  one  would  expect  to  find  between  the  various 
constants  (Michaelis  constants,  inhibitor  constants,  pH  characteristics, 
temperature  coefficients,  etc.)  measured  on  the  one  hand  for  the  intact 
membrane  system  and  on  the  other  hand  for  the  isolated  enzymes  and 
carriers,  it  is  apparent  that  not  only  the  kinetic  "constants"  but  also  the 
apparent  thermodynamic  "constants"  of  the  reactions  can  be  profoundly 
afl^ected  by  the  anisotropic  situation  of  the  catalysts  in  the  membrane.  It 
will,  I  think,  be  appropriate  to  conclude  my  paper  with  a  brief  discussion 
of  this  aspect  of  translocation  catalysis,  for  it  has  an  important  bearing  on 
any  experiments  designed  to  identify  the  catalysts  of  membrane  transport 
by  comparing  their  kinetic  and  thermodynamic  constants  in  situ  in  a 


APPROACHES    TO    THE    ANALYSIS    OF    SPECIFIC    MEMBRANE    TRANSPORT         595 

membrane  or  other  complex  with  the  "corresponding"  constants  deter- 
mined in  homogeneous  sohition  for  the  soluble  enzymes  or  catalytic 
carriers  extracted  from  the  complex.  The  train  of  thought  that  I  propose 
to  follow  wall  incidentally  suggest  a  new  way  in  which  the  function  of  the 
particulate  systems  that  couple  oxidoreductive  or  photon-activated  electron 
transport  to  phosphorylation  could  be  dependent  upon  their  structure. 
But  I  do  not  propose  to  do  more  than  touch  upon  this  incidental  suggestion 
today  as  it  would  lead  us  outside  the  context  of  this  session  of  the 
Symposium. 


SOME    EFFECTS    OF    ANISOTROPY    UPON    AN    ENZYME-CATALYZED 
HYDROLYTIC    REACTION 

For  the  sake  of  simplicity,  let  us  consider  the  effect  of  one  variable,  pH, 
on  an  enzyme  catalyzed  reaction  of  the  hydrolase  type  represented  by  the 
equation, 

AB  +  H.3O  =^  AH  +  BOH  (1) 

Kinetic  considerations 

When  the  enzyme,  E,  is  dissolved  in  a  homogeneous  aqueous  medium, 
the  usual  constants,  such  as  the  maximum  velocity  or  the  Michaelis 
constant,  will  vary  with  the  pH  of  the  medium,  and  the  kinetic  constants 
of  this  variation  will  be  characteristic  of  the  enzyme  because  the  catalytic 
activity  depends  upon  the  degree  of  ionization  of  acidic  and  basic  groups 
in  different  parts  of  the  protein  molecule.  If,  however,  the  enzyme  is 
situated  in  a  membrane  separating  two  aqueous  phases  which  are  poised  at 
different  hydrogen  ion  activities,  the  degree  of  ionization  of  the  acidic  and 
basic  groups  on  the  two  sides  of  the  enzyme  will  be  different,  and  as  the 
state  of  the  enzyme  molecules  will  not  be  defined  by  a  single  pH  value,  we 
cannot  properly  define  the  kinetic  constants  relating  the  characteristics  of 
the  enzyme  activity  to  the  pH.  It  follows  from  the  generalization  of  this 
kind  of  consideration,  that  the  kinetic  constants  of  enzymes  or  catalytic 
carriers  as  usually  defined  in  homogeneous  systems  are  not  strictly  com- 
parable to  the  "corresponding"  constants  for  the  same  catalysts  when 
present  in  natural  membranes  or  other  anisotropic  complexes. 

Thermodyuiunic  considerations :  (Jlieniiosniotic  couplinif 

In  the  homegeneous  system  represented  by  equation  (i),  the  enzyme 
will  catalyze  the  equilibration  of  the  reaction  according  to  the  equation, 

[AH]  X  [BOH] 

[K^ —  =  ^  ^'^ 


596  PETER    MITCHELL 

in  which  the  square  brackets  stand  for  electrochemical  activities  and  K'  is 
a  thermodynamic  "  constant ",  independent  of  the  properties  of  the  enzyme 
and  independent  of  pH,  other  things  being  equal.  In  writing  equation  (2), 
I  have  followed  the  custom  of  omitting  the  activity  of  the  water  in  the 
system  as  this  is  a  constant  in  homogeneous  aqueous  physiological  media, 
and  has  a  value  corresponding  to  55-5  M  water.  In  an  inhomogeneous 
(pseudo-equilibrium)  system,  however,  the  activity  of  the  water  at  the 
site  (e)  of  the  hydrolytic  process  may  not  correspond  to  that  of  physio- 
logical aqueous  media,  and  in  this  case  it  must  be  included  as  follows. 


[AH],  X  [BOH], 
[AB],  X  [H^O], 

[AH],  X  [BOH], 


=  K  (3«) 


=  AlH^O],  '  (3^.) 


It  will  be  seen  that  the  usual  hydrolysis  "constant",  K\  is  a  variable 
which  is  proportional  to  [HoO],,  the  electrochemical  activity  of  the  water 
at  the  active  centre  of  the  enzyme. 

It  is  a  well-known  fact  that  enzyme  reactions — such  as  the  catalysis  of 
the  transfer  of  the  phosphoryl  group  (or  phosphorylium  ion)  by  phospho- 
kinases — can  be  etfectively  anhydrous  even  though  the  enzyme  molecules 
are  surrounded  by  water.  It  would  not,  therefore,  be  unrealistic  to  assume 
that  the  active  centre  of  a  hydrolytic  enzyme,  situated  anisotropically  in  a 
membrane  complex,  could  be  inaccessible  to  water,  but  could  be  accessible 
to  hydrogen  (but  not  hydroxyl)  ions  from  one  side  (phase  I)  and  could  be 
accessible  to  hydroxyl  (but  not  hydrogen)  ions  from  the  other  side  (phase 
II).  For  the  sake  of  simplicity,  we  will  not  make  any  assumptions  about 
the  anisotropy  of  the  enzyme  with  respect  to  the  accessibility  of  AB,  AH, 
and  BOH  to  its  active  centre.  The  electrochemical  activity  of  the  water  at 
the  active  centre,  e,  of  the  enzyme  would  be  given  by  the  dissociation 
constant  of  the  water,  K^^,,  defined  as  follows, 

[H,.0].  ^  [»-]-[""''■■  (4) 

and  smce,  [OH  ],i  =  A„, — •  (5) 

[W  Jii 

equation  (4)  can  be  written, 

.„  OT    _  [Hiix[H,0]i„,„ 

[HP]^  -  ^^^^ (6) 


APPROACHES   TO   THE   ANALYSIS    OF   SPECIFIC    MEMBRANE   TRANSPORT        597 

The  effect  of  the  anisotropic  situation  of  the  hydroiytic  enzyme  on  the 
dissociation  equiHbrium  represented  by  K'  can  consequently  be  expressed 
as  follows,  using  {^h)  and  (6), 

[AH]  X  [BOH]  [H+], 

^    =  [AB] =  ^[H.O].oM,x  p^  (,) 

This  equation  shows  that  the  poise  of  the  dissociation  equilibrium  repre- 
sented by  K'  is  proportional  to  the  ratio  of  the  hydrogen  ion  electro- 
chemical activity  in  phase  I  to  that  in  phase  II.  The  electrochemical 
activity  of  the  hydrogen  ion  in  the  two  phases  may,  of  course,  differ  either 
because  of  a  difference  of  hydrogen  ion  chemical  activity  or  because  of  a 
membrane  potential,  a  potential  of  about  60  mV  being  equivalent  to  a 
hydrogen  ion  chemical  activity  ratio  of  10.  The  membrane  potential  or 
hydrogen  ion  chemical  activity  difference  across  the  membrane  could  be 
generated  by  a  photoelectric  effect  or  by  a  metabolic  oxidoreduction 
involving  a  flow  of  electrons  across  the  membrane.  Equation  (7)  shows 
that  the  work  done  in  creating  the  asymmetry  of  [H^]  across  the  mem- 
brane can  be  coupled  to  synthesis  of  x\B  by  dehydration  of  AH  and  BOH, 
the  hvdrogen  ions  of  the  water  that  is  eliminated  travelling  to  phase  I  and 
the  hydroxyl  ions  travelling  to  phase  II.  Synthesis  of  AB  is,  of  course, 
promoted  by  lowering  the  chemical  activity  of  the  hydrogen  ion  or  by  a 
negative  potential  in  phase  I  relative  to  phase  II. 

It  will  be  helpful  to  consider  the  reaction  catalyzed  by  glucose-6- 
phosphatase  as  a  relevant  and  quantitative  example  of  the  above  principle 
of  chemiosmotic  coupling.  The  equilibrium  constant.  A.'',  for  the  hydrolysis 
of  glucose-6-phosphate  to  glucose  and  inorganic  phosphate  is  approxi- 
mately 250  in  homogeneous  aqueous  solution  at  pH  7  [47],  and  the 
concentration  of  glucose-6-phosphate  in  equihbrium  with  io~^  M  glucose 
and  ID"'-  M  phosphate  would  be  only  4  x  lO""  M.  If  glucose-6-phosphatase 
were  located  in  the  anisotropic  membrane  complex  as  described  above,  a 
pH  difference  of  only  3  units  between  phases  I  and  II,  or  a  potential 
difference  of  60  mV  and  a  pH  difference  of  2  units,  would  lower  the 
dissociation  constant  by  a  factor  of  1000  and  would  raise  the  concentration 
of  glucose-6-phosphate  in  equilibrium  with  iq-'-  m  glucose  and  10  -  M 
phosphate  to  4x10-^  m — a  concentration  high  enough  to  enter  the 
phosphohexose  isomerase  reaction  of  the  glycolytic  pathway  at  near  the 
maximum  rate.  This  fact  is  all  the  more  interesting  since  the  glucose-6- 
phosphatase  of  Escherichia  coli  (as  discussed  above)  and  of  liver  cells  [48] 
appears  to  be  appropriately  situated  in  a  membrane  complex.  I  need 
hardly  point  out  that  a  similar,  but  greater  asymmetry  of  electrochemical 
hydrogen  ion  activity  to  that  considered  in  the  above  example,  could  be 
responsible  for  converting  the  ATPases  of  the  particulate  systems  of 
photosynthetic  and  oxidative  phosphorylation  into  the  x\TP-synthesizing 


598  PETER   MITCHELL 

catalysts.    I    hope  to   develop   this   interesting  and   important   aspect  of 
translocation  catalysis  on  another  occasion. 

My  purpose  in  concluding  with  these  rather  brief  thoughts  on  what  I 
have  called  vectorial  metabolism  was  two-fold.  First,  these  thoughts  add 
something  to  our  conception  of  the  intimate  relationship  between  transport 
and  metabolism;  and  second,  they  pose  a  most  important  experimental 
question.  The  activities  of  the  translocation  catalysts  in  their  natural 
situation  in  membranes  or  other  anisotropic  complexes  are  not  strictly 
comparable  to  their  activities  in  the  homogeneous  solutions  in  which  we 
are  accustomed  to  isolate  and  study  them.  How,  then,  can  we  proceed  to 
identify  the  translocation  catalysts  and  demonstrate  the  molecular  mechan- 
ism of  their  activity  ?  I  believe  that  the  only  satisfactory  answer  to  this 
question  is  to  be  found  in  the  fourth  method  of  approach  to  the  analysis  of 
membrane  transport  that  I  mentioned  at  the  beginning  of  this  paper.  We 
must  strive  to  set  up  "synthetic"  or  reconstituted  membrane  systems  with 
which  we  can  study  directly  both  the  processes  of  transfer  (in  the  normal 
biochemical  sense)  and  the  processes  of  translocation,  catalyzed  by 
enzymes  and  catalytic  carriers  under  anisotropic  conditions  that  can  be 
controlled  and  measured. 

I  am  indebted  to  Dr.  Jennifer  Moyle  for  helpful  general  discussions 
during  the  preparation  of  this  paper,  to  Dr.  J.  Dainty  for  help  in  describing 
the  electrochemical  activity  in  relation  to  escaping  tendency,  and  to  Dr. 
P.  H.  Tuft  for  suggesting  the  word  "substratum"  as  a  synonym  for 
enzyme-locator.  I  am  also  glad  to  acknowledge  grants  from  the  Nuffield 
Foundation  in  aid  of  this  work. 

References 

1.  Cohen,  G.  N.,  and  Monod,  J.,  Bad.  Rev.  2i,  169  (1957). 

2.  Mitchell,  P.,  in  "Structure  and  Function  of  Subcellular  Components", 
ed.  E.  M.  Crook.  Cambridge  University  Press  (i6th  Symp.  Biochem.  Soc, 
published  1959)  p.  73  (1957). 

3.  Mitchell,  P.,  Nature,  Lond.  180,  134  (1957). 

4.  Mitchell,  P.,  Anmi.  Rev.  Microbiol.  13,  407  (1959). 

5.  Mitchell,  P.,  ///  "The  Nature  of  the  Bacterial  Surface",  ed.  A.  A.  Miles  and 
N.  W.  Pirie.  Blackwell,  Oxford,  55  (1949). 

6.  Overton,  E.,  Vjsclir.  naturf.  Ges.  Zurich  44,  88  (1899). 

7.  Mitchell,  P.,jf.  gen.  Microbiol.  9,  273  (1953). 

8.  Weibull,  C,  E.xp.  Cell  Res.  9,  139  (1955). 

9.  Mitchell,  P.,  and  Moyle,  J.,  Faraday  Soc.  Disc.  21,  258  (1956). 

10.  Mitchell,  P.,  and  Moyle,  J.,  Sytnp.  Soc.  gen.  Microbiol.  6,  150  (1956). 

11.  Mitchell,  P.,  and  Moyle,  J.,  J.  gen.  Microbiol.  15,  512  (1956). 

12.  Mitchell,  P.,  and  Moyle,  J.,^.  gen.  Microbiol.  20,  434  (1959). 

13.  Stephen,  B.  P.,  Ph.D.  Thesis,  University  of  Edinburgh  (i960). 

14.  Gale,  E.  F.,  Bull.  Johns  Hopk.  Hosp.  83,  119  (1948). 

15.  Gale,  E.  F.,  and  Mitchell,  P.,jf.  gen.  Microbiol.  I,  299  (1947). 

16.  Mitchell,  F.,jf.  gen.  Microbiol,  il,  73  (1954). 


APPROACHES   TO    THE   ANALYSIS   OF   SPECIFIC   MEMBRANE   TRANSPORT        599 

17.  Mitchell,  P.,  Symp.  Soc.  exp.  Biol.  8,  254  (1954). 

18.  Mitchell,  P.,^.  gen.  Microbiol.  II,  x  (1954). 

19.  Mitchell,  P.,  and  Movie,  J.,  Proc.  R.  phys.  Soc,  Edinh.  28,  19  (1959). 

20.  Monod,  J.,  in  "Enzymes:  Units  of  Biological  Structure  and  Function",  ed. 
O.  H.  Gaebler.  Academic  Press,  New  York,  7  (1956). 

21.  Rickenberg,  H.  V.,  Cohen,  G.  N.,  Buttin,  G.,  and  Monod,  J.,  Ann.  Inst. 
Pasteur,  91,  829  (1956). 

22.  Cohen,  G.  N.,  and  Rickenberg,  H.  V.,  Ann.  I>!st.  Pasteur,  91,  693  (1956). 

23.  Kepes,  A.,  Bioeliim.  biophys.  Acta  40,  70  (i960). 

24.  Kogut,  M.,  and  Podoski,  E.  P.,  Biochem.J.  55,  800  (1953). 

25.  Barrett,  J.  T.,  Larson,  A.  D.,  and  Kallio,  R.  E.,  7-  Bact.  65,  1S7  (1953). 

26.  Green,  H.,  and  Davis,  B.  D.,  see  Davis,  B.  D.,  ///  "Enzymes:  Units  of  Bio- 
logical Structure  and  Function",  ed.  O.  H.  Gaebler.  Academic  Press,  New 
York,  514  (1956). 

27.  Mitchell,  P.,  and  Moyle,  ].,y.  gen.  Microbiol.  5,  981   (1951). 

28.  Weibull,  C.,y.  Bacterial.  66,  688  (1953)- 

29.  Weibull,  C.,y.  Bacteriol.  66,  696  (1953). 

30.  Mitchell,  P.,  and  Moyle,  ].,J.  gen.  Microbiol.  16,  184  (1957). 

31.  Mitchell,  P.,  and  Moyle,  J.,  Biochem.J.  64,  19P  {1956). 

32.  Storck,  R.  L.,  and  Wachsman,  J.  T.,_7.  Bacteriol.  73,  784  (1957). 

33.  Weibull,  C,  and  Bergstrom,  L.,  Biochim.  biophys.  Acta  30,  340  (1938). 

34.  Cota-Robles,  E.  H.,  Marr,  A.  G.,  and  Nilson,  E.  H.,^.  Bact.  75,  243  (1958). 
33.   DeLey,  J.,  and  Dochy,  R.,  Biochim.  biophys.  Acta  40,  277  (i960). 

36.  Mitchell,  P.,  Symposium  on  Membrane  Transport  and  Metabolism,  Prague 
(i960)  (in  press). 

37.  Mitchell,  P.,  and  Moyle,  J.,  Nature,  Loiui.  182,  372  (1958). 

38.  Mitchell,  P.,  and  Moyle,  J.,  Proc.  R.  phys.  Soc,  Edinb.  27,  61  (1958). 

39.  Peters,  R.  A.,  /;/  "Perspectives  in  Biochemistry",  ed.  J.  Needham  and  D.  E. 
Green.  Cambridge  University  Press,  London,  36  (1939). 

40.  Monod,  J.,  and  Cohn,  AL,  Advaiu\  Enzymol.  13,  67  (1932). 

41.  Cohn,  M.,  Bact.  Rev.  21,  140  (1937). 

42.  Keilin,  D.,  and  King,  T.  E.,  Proc.  roy.  Soc  B  152,  163  (i960). 

43.  Yudkin,  J.,  Biol.  Rev.  13,  93  (1938). 

44.  Rickenberg,  H.  V.,  Nature,  Loud.  185,  240  (i960). 

45.  Catcheside,  D.  G.,  C.  R.  Lab.  Carlsberg,  Se'r.  physiol.  26,  31  (1936). 

46.  Mitchell,  P.,  and  Moyle,  J.,  Biochem.J.  72,  21P  (1939). 

47.  Atkinson,  AL  R.,  Johnson,  E.,  and  Morton,  R.  K.,  Nature,  Loud.  184,  1925 

(1959)- 

48.  Siekevitz,  P.,  //;  "  Ciba  Foundation  Symposium  on  the  Regulation  of  Meta- 
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London,  17  (1939). 


Discussion 

DiscHE  :  Is  this  substance  which  is  responsible  for  permeability  and  for  changes 
in  permeability  really  an  enzyme  ?  The  evidence  based  on  such  phenomena  like 
competitive  inhibitions  seems  completely  consistent  with  the  idea  that  the  mem- 
brane structure  depends  on  properties  of  certain  proteins  in  the  membrane  and 
for  the  phenomena  which  are  very  familiar  about  binding  of  certain  substances  to 
the  protein,  and  the  competitive  inhibition  of  penetration  of  substrates  by  analogous 
substances,  would  be  adequately  explained  by  specific  binding  of  these  substances 


6oO  PETER   MITCHELL 

by  proteins.  The  concept  of  an  enzyme  implies  that  the  substance  which  penetrates 
is  chemically  changed  and  I  should  like  to  ask  if  changes  in  penetrating  substances 
occur  ? 

Mitchell:  Well,  Dr.  Dische,  you  are  asking  rather  a  big  question.  Let  me 
answer  first  of  all  with  an  example  that  stems  from  my  paper.  In  the  utilization  of 
glucose  6-phosphate  by  Bocteriiim  coli,  what  actually  passes  through  the  membrane 
is,  on  the  one  hand  "glucose",  and  on  the  other  hand  "phosphate".  We  know 
this  because  the  rate  of  utilization  of  glucose  6-phosphate  is  the  same  as  the  rate 
at  which  the  externally  available  glucose  6-phosphatase  can  break  it  down.  This  is 
a  clear  example  of  a  case  in  which  the  first  process  in  the  overall  transport  reaction 
is  an  enzymic  one.  We  have  to  be  very  careful  in  speaking,  for  example,  of  the 
transport  of  glucose  6-phosphate  into  the  cell,  because  we  know  that  the  glucose 
6-phosphate  does  not  go  in,  although  later  glucose  6-phosphate  is  found  in  the  cell 
as  a  result  of  the  separate  entry  of  the  " glucose  "  and  the  "phosphate "  by  different 
molecular  pathways.  Moreover,  there  is  evidence  that  the  membrane  is  im- 
permeable to  glucose  and  phosphate  and  that  these  molecules  pass  across  the 
membrane  as  derivatives  or  chemical  groups  (hence  the  inverted  commas).  This 
illustrates  a  general  principle  that  I  am  trying  to  make,  but  I  do  agree  with  you, 
that  we  must  not  jump  to  silly  conclusions;  we  must  not  say  that  we  have  direct 
evidence  for  the  process  of  group  translocation,  and  as  I  pointed  out  in  my  paper, 
this  evidence  may  well  have  to  await  the  successful  reconstruction  of  in  vitro 
membrane  systems.  All  the  same,  the  specificity  and  kinetics,  and  especially  the 
susceptibility  to  inhibitors,  of  a  number  of  transport  processes  do  strongly  suggest 
that  they  represent  enzyme-catalyzed  chemical  reactions.  Let  us  consider,  for 
example,  the  entry  of  "  succinate  "  into  micrococci.  In  this  case  we  can  examine  the 
catalytic  system  in  rather  an  elegant  way.  Some  years  ago.  Dr.  Moyle  and  I 
thought  that  the  entry  might  be  directly  through  succinic  oxidase,  and  we  argued 
that  as  succinic  oxidase  is  part  of  the  membrane  it  could  have  its  active  centre 
exposed  on  the  outside.  As  we  knew  that  the  membrane  is  impermeable  to  suc- 
cinate and  malonate  in  the  normal  sense,  we  were  able  to  investigate  this  possibility 
by  seeing  whether  external  malonate  would  inhibit  the  succinic  oxidase  of  intact 
cells.  In  fact,  malonate  was  found  to  have  no  effect  at  all  unless  at  first  you 
depressed  the  pH  to  5  to  let  the  malonate  in  and  then  brought  it  back  to  7.  After 
that  the  external  malonate  could  be  washed  away,  and  succinate  oxidation  con- 
tinued to  be  inhibited.  This  shows  that  the  succinic  oxidase  is  inhibitable  by 
malonate,  but  the  active  centre  is  facing  inwards.  It  also  shows  that  the  specificity 
of  the  process  giving  rise  to  "succinate"  entry  is  such  as  to  discriminate  between 
malonate  and  succinate.  This  is  one  of  the  reasons  why  we  think  that  the  kino- 
synthetase  may  catalyze  the  first  reaction  for  succinate  entry,  for  this  enzyme, 
unlike  succinic  oxidase,  does  distinguish  between  succinate  and  malonate  and  is 
not  inhibited  by  the  latter.  Further,  the  substance  that  passes  into  the  cyto- 
plasm of  the  micrococcus  while  succinate  passes  into  the  outer  surface  of  the 
plasma  membrane  is  not  succinate  !  This  illustrates  the  background  of  my 
approach  to  the  closely  related  problems  of  transport  and  metabolism  in  whole 
cells. 

In    kinetic    studies,    the    evidence    obtained    is    circumstantial.    One    creates 
hypotheses  in  order  to  disprove  them.  Our  aim  has  been  in  the  past  to  try  to 


APPROACHES   TO   THE   ANALYSIS   OF    SPECIFIC   MEMBRANE   TRANSPORT        6oi 

formulate  hypotheses  in  accord  with  as  much  of  the  circumstantial  evidence  as 
possible,  and  I  think  that  our  aim  now  must  be  to  try  also  to  develop  in  vitro 
membrane  systems  in  which  these  hypotheses  can  be  put  to  more  crucial  tests. 

Frenkel  :  In  the  scheme  which  you  showed  on  your  slide  glucose  6-phosphatase 
appears  to  be  present  in  the  cell  wall  and  it  is  thus  difficult  to  see  how  it  can  be 
solubilized  readily. 

Mitchell:  I  did  not  say  that  it  was  in  the  cell  wall.  What  I  said  was  that  I 
believed  it  must  be  present  in  the  space  between  the  inner  margin  of  the  wall, 
which  we  know  to  be  impermeable  to  proteins,  and  the  outer  limit  of  the  osmotic 
barrier  component  of  the  plasma  membrane.  I  agree,  I  think,  with  the  implication 
of  Dr.  Frenkel 's  remark — that  we  have  to  be  a  little  careful  about  the  words 
describing  the  cytological  structures  when  we  get  to  such  molecular  dimensions. 
The  plasma  membrane  is  quite  thick — thick  enough  to  be  regarded  in  some  respects 
as  a  separate  phase.  There  is  one  functional  component  of  the  plasma  membrane 
(we  are  not  sure  whether  it  is  in  the  middle,  or  near  its  outer  or  inner  surface), 
which  is  mostly  a  hydrophobic  sheet,  part  probably  being  protein  and  part  lipid. 
This  hydrophobic  sheet  is  called  the  osmotic  barrier.  If  we  find  a  catalytic  activity 
exhibited  only  on  the  outside  of  this,  the  active  centre  of  the  catalyst  responsible 
for  that  activity  must  be  situated  somewhere  outside  the  osmotic  barrier,  or  must 
be  in  a  crevice  accessible  to  the  substrate  only  from  the  outside.  Now,  the  situation 
could  be  that  the  glucose  6-phosphatase  is  tucked  into  or  attached  to  the  osmotic 
barrier,  its  active  centre  being  accessible  to  glucose  6-phosphate  from  outside. 
When  you  break  the  cell,  because  of  the  great  changes  in  ionic  environment,  etc., 
the  enzyme  might  well  become  dissociated  from  the  membrane  complex  and  appear 
as  a  soluble  protein — ^just  as  we  find  it. 

DoRFMAN :  I  wonder  if  we  are  not  prisoners  of  our  conventional  definitions  of 
enzymes  and  specific  proteins.  I  am  thinking  of  what  Dr.  Davis  said  about  the 
analogy  of  a  permease  and  haemoglobins.  In  a  sense  when  oxygen  is  bound  it  is 
chemically  changed  but  it  is  released  as  the  same  substance ;  in  the  same  way  as 
the  transport  of  glucose  as  glucose  6-phosphate  might  occur.  I  wonder  whether  we 
shouldn't  think  more  in  terms  of  protein  specificity  and  less  of  an  enzyme  as  a 
catalyst  which  must  bring  about  a  chemical  change  in  the  more  conventional 
organic  chemical  sense  ? 

Mitchell:  I  agree  with  that  remark,  but  I  take  exception  to  the  suggestion 
that  we  are  being  blinded  by  a  conventional  and  old  attitude  towards  enzymes. 
After  all,  enzjTne  kinetics  is  a  growing  subject.  I  agree  with  Dr.  Dorfman  that  we 
need  to  try  to  obtain  a  more  biochemical  view  of  transport  processes,  but  I  vise  the 
word  biochemical  to  mean  conceived  in  the  most  up-to-date  organic  and  physical 
chemical  terms.  I  have  no  preconceptions  as  to  whether  the  catalysts  of  molecule, 
ion,  group,  and  electron  translocation  will  turn  out  to  be  enzymes  in  the  sense  that 
may  currently  be  in  use.  But  I  would  point  out  that  in  the  enzyme  field  we  have  a 
number  of  catalytic  carriers,  such  as  the  flavoproteins  and  the  haem  proteins  of  the 
cytochrome  system,  and  one  would  not  say  that  the  cytochrome  system  caused  a 
chemical  change  in  the  electrons  which  passed  through  it.  Nevertheless,  we  gen- 
erally regard  these  catalysts  as  part  of  the  overall  enzyme  system.  It  is  in  this  sort 
of  context  that  I  am  trying  to  speak.  I  suppose  that  if  we  found  proteins  with 
quite  new  capacities,  which  were  unlike  haemoglobin,  unlike  the  catalysts  of  the 


602  PETER   MITCHELL 

cytochrome  system,  and  unlike  the  proteins  universally  accepted  as  enzymes,  then 
we  would  be  justified  in  inventing  a  new  name  to  describe  them.  But  I  would  hesi- 
tate to  consider  such  special  proteins  until  we  have  isolated  at  least  one. 

Davis  :  I  would  like  to  ask  Dr.  Porter  to  comment  on  another  aspect  of  this 
problem.  Dr.  Mitchell  has  stressed  the  advantages  of  bacteria  arising  from  their 
smallness  and  simplicity,  but  of  course  we  are  all  aware  of  disadvantages  which 
also  arise  from  smallness,  such  as  the  difficulty  of  recognizing  morphological  sub- 
units.  Now  electron  microscopists  until  very  recently  have  all  agreed  that  there  is 
no  endoplasmic  reticulum  in  bacteria;  but  a  few  months  ago  Glauert  published  in 
the  Journal  of  Biochemical  and  Biophysical  Cytology  some  pictures  showing  with 
new  methods  of  fixation  what  appears  to  be  a  very  fine  reticulum  in  parallel 
lamellae  in  an  actomycete  (which  is  fundamentally  an  elongated  bacterium).  I 
wonder  whether  Dr.  Porter  would  care  to  comment  on  the  generalization  that 
bacteria  may  or  may  not  have  such  a  reticulum. 

Porter:  Audrey  Glauert  at  the  Strangeways  Laboratories  in  Cambridge, 
England,  has  taken  and  published  very  informative  micrographs  of  complex  mem- 
brane systems  in  Streptomyces  and  some  suggestion  has  been  made  that  these  may 
be  analagous  to  the  endoplasmic  reticulum  of  other  cells.  Actually  the  bacterial 
cytoplasmic  membranes  seem  to  be  infoldings  of  the  membrane  limiting  the  pro- 
toplast and  could  represent  an  attempt  on  the  part  of  the  cell  to  increase  a  surface 
available  to  diflfusible  metabolites.  Certain  blue-green  algae  show  a  similar  com- 
plex infolding  of  the  plasma  membrane.  They  are  evidently  common  to  lower 
forms  which  do  not  possess  a  nuclear  envelope  and  associated  ER.  Whether  such 
complex  infoldings  of  the  surface  membrane  have  evolved  into  the  surface- 
independent  endoplasmic  reticulum  of  higher  cell  forms  is  an  interesting  topic  for 
speculation. 

Davis:  Well,  1  wonder,  Dr.  Mitchell,  whether  you  have  any  evidence  as  to 
whether  or  not  certain  activities  which  you  observed  in  the  centrifugable  fraction, 
particularly  those  of  the  cytochrome  system  and  the  TCA  cycle,  could  be  in  small 
particles  attached  to  the  membrane  rather  than  in  the  substance  of  the  membrane 
itself. 

Mitchell:  Yes,  I  think  this  query  is  a  very  difl^icult  thing  to  resolve  experi- 
mentally. It  is  also  difficult  to  speak  about.  The  succinic  dehydrogenase  active 
centre  is  certainly  inside  the  osmotic  barrier.  If  you  think  of  the  succinic  oxidase 
as  a  particle,  you  may  imagine  that  this  piece  of  the  cytochrome,  as  well  as  the 
succinic  dehydrogenase  attached  to  it,  is  under  the  osmotic  barrier.  You  may,  if 
you  are  a  biochemist,  regard  the  plasma  membrane  as  a  hydrophobic  sheet  with 
various  activities  attached  to  it  as  particles,  which  can  be  isolated  and  characterized 
biochemically.  But,  if  you  are  a  cytologist,  you  will  be  impressed  by  the  fact  that 
under  certain  conditions  the  plasma  membrane  complex  behaves  as  a  single 
mechanical  unit  containing  the  enzyme  activities  of  all  its  so-called  particulate 
constituents.  I  think  that  the  work  in  Dr.  Weibull's  laboratory  and  in  my  own 
laboratory  has  now  established  this  fact  in  the  case  of  Bacillus  megaterium,  Staphy- 
lococcus aureus  and  Micrococcus  lysodeikticus  beyond  any  reasonable  doubt.  One 
would  presume  that  the  residual  bonding  that  is  holding  the  various  parts  of  the 
membrane  complex  system  together  is  stronger  in  some  places  than  in  others;  so 
that  if  you  treat  it  very  kindly,  as  you  must  do  if  you  wish  to  isolate  cytologically 


APPROACHES    TO    THE    ANALYSIS    OF    SPECIFIC    MEMBRANE    TRANSPORT         603 

recognizable  membranes,  it  may  remain  more  or  less  intact,  but  rougher  treatment 
makes  it  fall  to  pieces.  Standing  midway  between  the  cytologist  and  the  biochemist, 
we  may  presume  that  the  "pieces "  (of  the  cytologist)  are  the  "particles "  or  groups 
of  "particles"  (of  the  biochemist).  I  think  that  we  need  to  become  as  interested 
in  the  substratum  (or  locational)  specificities  of  enzymes  as  we  have  been  in  their 
substrate  specificities;  and  the  elegant  work  on  mitochondrial  structure  which 
Dr.  Lehninger  described  earlier  in  this  symposium  is  clearly  moving  in  this 
direction.  To  take  the  analysis  of  the  jig-saw  organization  of  the  plasma  membrane 
and  other  membrane  complexes  further  will  require  a  great  collaborative  effort 
between  biochemist  and  cytologist.  Perhaps  one  of  the  most  important  problems 
to  settle  at  the  present  stage  is  whether  there  is  a  general  substratum  substance 
which  represents  the  mechanical  matrix  of  the  membrane  and  acts  as  the  locator 
for  the  enzymes  and  carrier  proteins,  or  whether  the  individual  molecules  of  the 
enzymes,  carriers,  lipids  and  other  components  of  the  complex  represent  the 
substrata  for  each  other  and  share  the  responsibility  for  structural  (locational)  and 
catalytic  properties  of  the  membrane  fabric. 

This  is  a  very  difficult  problem,  and  as  I  have  already  pointed  out,  it  may  well 
be  impossible  to  make  real  progress  until  we  have  learned  to  study  the  material  of 
natural  membranes  in  anisotropic  ///  vitro  systems.  Perhaps  Dr.  Albertsson's 
pohTner  systems  would  be  helpful  for  this  purpose.  We  discussed  this  privately 
the  other  day,  for  it  struck  me  that  the  anisotropic  properties  of  the  interface 
between  the  two  aqueous  polymer  phases  might  be  varied  at  will  over  quite  a  wide 
range.  Such  a  system  might  prove  to  be  useful  for  studying  the  behaviour  of 
biologically  active  molecules  under  anisotropic  conditions,  and  might  serve  as  a 
starting  point  for  setting  up  reconstituted  membrane  systems  in  vitro. 


Protein  Uptake  by  Pinocytosis  in  Amoebae :  Studies 
on  Ferritin  and  Methylated  Ferritin* 

V.  T.  Nachmias  and  J.  M.  Marshall,  jR.f 

Department  of  Anatomy,  School  of  I\Iedhine, 
University  of  Pennsylvania,  Philadelphia,  Penn.,  U.S.A. 

It  has  been  suggested  that  pinocytosis  may  be  the  underlying  process  in 
many  transport  phenomena.  The  idea  has  been  reviewed  by  Dr.  Holter,  in 
this  Symposium  and  elsewhere  [i].  Such  a  view  seems  to  have  been 
especially  attractive  to  electron  microscopists,  who  have  found  evidence 
for  vesicle  formation  in  a  variety  of  cells.  Physiologists,  however,  who  have 
studied  and  defined  active  transport  by  other  methods,  have  felt  that  the 
engulfment  of  droplets  of  the  cell's  environment  is  too  indiscriminate  a 
process  to  account  for  the  highly  specific  effects  which,  as  Dr.  Davis  has 
pointed  out  in  his  review  for  this  Symposium,  are  characteristic  of  active 
transport.  There  are  other  equally  serious  inadequacies  in  the  notion  that 
pinocytosis  is  simply  the  morphological  equivalent  of  active  transport. 

The  chief  difficulty  in  this  debate  is  a  familiar  one ;  so  long  as  we  do 
not  understand  the  mechanisms  of  pinocytosis,  on  the  one  hand,  or  of 
active  transport,  on  the  other,  we  are  free  to  launch  hypotheses  which  can 
soar  quite  freely.  The  purpose  of  this  communication  is  therefore  to 
describe  experiments  which  were  designed  to  answer  two  questions  about 
protein  uptake  by  pinocytosis  in  amoebae.  First,  what  is  the  physical 
mechanism  of  protein  binding  to  the  cell  surface,  the  binding  which  is 
known  to  set  off  the  pinocytosis  response  ?  And  second,  what  happens  to 
the  pinocytosis  vesicle,  and  to  its  contents,  after  it  is  taken  into  the  cell  ? 
Note  that  these  questions  concern  only  the  first  and  last  stages  of  the 
pinocytosis  response;  between  these  there  occurs  the  actual  process  of 
invagination,  the  formation  of  tunnels  and  vesicles.  This  must  also  be 
studied  experimentally,  but  it  will  be  seen  from  the  results  that  the  first 
and  last  stages  are  those  most  directly  related  to  the  problem  of  transport 
between  the  environment  and  the  cytoplasm. 

The  studies  of  Schumaker  [2]  and  of  Brandt  [3]  supplied  the  first 
evidence  that  protein  uptake  by  amoebae  began  with  some  type  of  binding 

*  This  investigation  was  supported  by  Grant  C-1957  from  the  National  Cancer 
Institute  of  the  National  Institutes  of  Health,  United  States  Public  Health  Service. 
t  Scholar  in  Cancer  Research  of  the  American  Cancer  Society,  Inc. 


6o6  V.    T.    NACHMIAS    AND   J.    M.    MARSHALL,   JR. 

reaction  to  the  cell  surface,  and  was  not,  as  had  been  assumed  previously, 
a  simple  matter  of  the  cell  engulfing  droplets  of  the  medium.  Schumaker's 
kinetic  studies  showed  also  that  the  binding  reaction  was  not  affected  by 
cooling  or  by  metabolic  inhibitors,  although  the  later  stages  of  vesicle 
formation  were  readily  blocked.  Additional  evidence  from  several  sources 
suggested  that  the  mechanism  of  the  binding  reaction  w'as  electrostatic  [4, 
5,  6]  and  that  the  receptor  substance  was  the  mucous  coat  which  covers 
the  amoeba.  In  the  work  here  reported,  the  mechanism  of  the  reaction  was 
studied  by  comparing  the  pH  dependence  of  binding  for  two  closely 
related  proteins,  ferritin  and  methylated  ferritin.  Binding  studies  were 
done  on  living  amoebae  {Chaos  chaos  or  Pelomyxa  carolmensis)  and  the 
results  were  confirmed  by  electron  microscopy.  The  same  proteins  were 
used  in  further  studies  on  the  changes  which  occur  within  the  cell  after 
uptake. 

Methods 

At  the  outset,  it  was  found  that  protein  binding  by  the  cell  surface 
could  be  "uncoupled"  from  the  remaining  stages  of  pinocytosis  by 
working  at  5°  [2].  Starving  amoebae  were  rinsed  in  cold  water.  By  this 
treatment,  the  cells  were  rounded  up,  cytoplasmic  motion  was  suppressed, 
and  contaminating  ions  were  removed.  The  amoebae  were  then  pipetted  into 
the  cold  protein  solution,  left  for  3  to  5  min.,  and  washed  in  the  cold  to 
remove  all  unbound  protein.  The  washing  procedure  made  it  possible  to 
test  the  reversibility  of  the  surface  binding  at  different  pH  values  [5].  Some 
cells  were  fixed  at  this  point  in  buffered  osmium  tetroxide,  and  were 
embedded  and  sectioned  in  epoxy  resin  for  electron  microscopy.  Others 
were  allowed  to  warm  to  room  temperature ;  in  such  cells,  the  complete 
pinocytosis  sequence  occurred  despite  the  delay,  and  the  protein  carried 
into  the  pinocytosis  vesicles  was  only  that  previously  bound  to  the  cell 
surface  and  not  removed  by  washing.  As  a  result,  it  was  possible  to  follow 
the  changes  which  occurred  within  the  vesicles  more  clearly  than  when 
"  bulk"  pinocytosis  was  induced  at  room  temperature.  Cells  treated  in  this 
way  were  kept  in  their  normal  medium  for  i  to  48  hr.  after  uptake,  and  were 
then  fixed,  embedded,  and  sectioned. 


PROPERTIES    OF    FERRITIN    AND    METHYLATED    FERRITIN 

Ferritin  was  isolated  from  horse  spleen  by  ammonium  sulphate 
precipitation  followed  by  crystallization  in  the  presence  of  cadmium 
sulphate  [7,  8].  It  has  some  unusual  properties  [9,  10],  which  made  it 
especially  suitable  for  this  study.  The  unit  particle  of  ferritin,  which  is 
94  A  in  diameter,  consists  of  a  protein  coat  surrounding  an  ordered  cluster 


PROTEIN    UPTAKE    IN    AMOEBAE  607 

of  micelles  of  ferric  hydroxide  and  ferric  phosphate.  The  particles  are  of 
uniform  size  and  shape,  and  the  internal  cluster  of  dense  iron  micelles 
makes  it  possible  to  identify  in  high  resolution  electron  micrographs  even 
a  single  particle  [ii,  12].  The  protein  coat  is  responsible  for  the  electro- 
chemical properties  of  ferritin,  such  as  electrophoretic  mobility  and 
solubility.  Ferritin  behaves,  therefore,  as  a  typical  protein  ampholyte,  with 
an  isoelectric  point  of  4-4  [13]. 

A  methylated  deriyative  of  ferritin  was  prepared  by  esterifying  the 
protein  in  acid  methanol  [14].  The  effect  of  methylation  was  judged  by 
comparing  at  several  pH  \alues  the  solubility  of  the  product  with  that  of 
the  original  protein.  Solubility  (in  optical  density  units)  was  measured  by 
determining  the  absorbance  at  280  m/i  of  supernatant  solutions  in 
equilibrium  with  precipitated  proteins  (Fig.  i). 


pH 

Fig.  I.  Solubility  of  ferritin  (curve  A)  and  of  methylated  ferritin  (cur\e  B),  as 
a  function  of  pH.  Ordinate:  solubility  in  optical  density  units,  at  280  m/t. 


No  direct  measure  of  the  percentage  of  carboxyl  groups  blocked  was 
attempted,  but  the  solubility  curves  demonstrated  that  the  charge  proper- 
ties were  significantly  modified  by  methylation.  The  solubility  of  normal 
ferritin  above  pH  5  is  almost  entirely  due  to  the  ionization  of  carboxyl 
groups.  When  these  are  blocked  by  extensive  methylation,  the  solubility  at 
pH  6  to  7  drops  to  a  very  low  value  (about  io~^",)  by  weight).  In  electron 
micrographs,  the  unit  particles  of  methyl  ferritin  appeared  the  same  as 
those  of  ferritin.  It  seemed  safe  to  conclude  that  methylation  did  not 
grossly  alter  the  structure  of  the  ferritin  particle,  and  that  any  difference 
in  binding  or  uptake  bv  amoebae  could  be  correlated  with  the  specific 
charge  effect  of  blocking  carboxyl  groups. 


6o8  V.    T.    NACHMIAS   AND   J.    M.    MARSHALL,   JR. 

EFFECTS    OF    PH    ON    BINDING 

The  effects  of  pH  on  the  binding  of  ferritin  and  of  methyl  ferritin  to 
the  cell  surface  were  studied.  Because  the  proteins  had  an  intense  orange 
colour,  the  results  of  binding  and  washing  experiments  could  be  followed 
by  direct  observation  of  the  living  cells.  The  results  were  confirmed  by 
electron  microscopy. 

At  pH  4,  when  both  proteins  were  positively  charged,  both  were  bound, 
and  both  invoked  the  pinocytosis  response.  At  neutral  or  slightly  acid  pH, 


Fig.  2.  Binding  of  ferritin  to  surfacL-  coat  ot  amoeba  C/kius  cJiaos,  at  pH  4.  The 
binding  persists  after  washing  at  pH  4  for  5  to  10  min. 

when  its  particles  carried  a  net  negative  charge,  ferritin  was  not  bound 
and  did  not  invoke  the  pinocytosis  response. 

Methyl  ferritin  was  too  insoluble  at  neutral  pH  to  permit  a  direct 
comparison  of  its  binding  with  that  of  ferritin,  but  a  clear  difference  in  the 
reversibility  of  binding  was  seen  in  washing  experiments.  Once  bound  to 
the  cell  surface  at  pH  4,  neither  protein  was  removed  by  washing  in  the 
cold  at  pH  4.  When  the  washing  was  done  above  pH  5,  ferritin  was  quickly 
removed,  but  methyl  ferritin  was  not. 

Figure  2  is  an  electron  micrograph  which  shows  ferritin  bound  to  the 
cell  at  pH  4,  and  remaining  bound  after  washing  at  the  same  pH.  Note  that 


\ 


\ 


Fig.  3.  Binding  of  ferritin  to  surface  coat  at  pf  1  4.   I'he  specimen  w  as  washed 
for  2  min.  at  pH  6  •  5  ;  most  of  the  ferritin  has  been  removed. 


^.^ 


^^ 


M 
^P^ 


M 


i 


Fig.  4.  Binding  of  methylated  ferritin  to  surface  coat  at  pH  4.  The  binding 
persists  after  washing  at  pH  for  5  to  10  min. 
VOL.  11. — 2R 


6lO  V.    T.    NACHMIAS   AND   J.    M.    MARSHALL,   JR. 

the  particles  are  held  in  a  layer  about  looo  Angstroms  thick  just  outside 
the  cell  membrane,  which  is  not  itself  stained  by  osmium. 

Figure  3,  again  of  a  specimen  treated  with  ferritin,  shows  the  effect  of 
2  min.  of  washing  at  pH  6-5.  Most  of  the  ferritin  has  disappeared.  Longer 
washing  (for  3  to  4  min.)  completely  removed  the  protein.  In  this  instance 
the  layer  which  binds  protein  is  seen  as  a  faintly  osmiophilic,  fibrillar 
substance  [15].  Usually  this  substance,  the  mucous  coat,  is  not  well 
stained  by  osmium.  Its  fibrillar  character  is  variable,  and  may  well  be  an 
artifact  of  fixation. 


0 


Fig.  5.  Binding  of  methylated  ferritin  persists  after  washing  at  pH  6-4  for 
5  to  10  min. 

Figure  4  is  a  micrograph  of  a  specimen  treated  with  methyl  ferritin  at 
pH  4,  and  washed  at  the  same  pH.  The  binding  persists,  whether  the 
specimen  is  washed  at  pH  4  or  pH  6-4  (Fig.  5). 

These  comparative  studies  demonstrated  that  binding  depends  upon 
the  net  charge  carried  by  the  protein,  but  gave  no  direct  information  about 
the  chemical  nature  of  the  mucous  coat.  By  working  with  mass  cultures  of 
Chaos  chaos  in  10  to  50  g.  lots,  it  has  been  possible  to  isolate,  after  tryptic 
digestion,  an  acidic,  metachromatic  polysaccharide.  A  preliminary  analysis 
suggests  that  it  is  a  sulphated  polyglucose,  but  this  must  be  confirmed  by 
further  investigation. 


PROTEIN   UPTAKE   IN    AMOEBAE  6x1 

CHANGES    WITHIN    VESICLES    AFTER    UPTAKE    OF    FERRITIN    OR    METHYL 

FERRITIN 

The  second  question  asked  at  the  beginnnig  of  this  work  was:  what 
happens  to  the  pinocytosis  vesicle,  and  to  its  contents,  after  it  is  taken  into 
the  cell  ?  Amoebae  bearing  bound  ferritin  or  methyl  ferritin  were  warmed 
to  room  temperature  in  the  wash  medium  at  pH  4.  The  cells  began  to 
change  shape,  to  move,  and  the  orange-stained  coat  substance  was  seen 
to  collect  in  smaller  regions  of  the  surface,  most  commonly  in  the  tail 
region.  Pinocvtosis  occurred  rapidly.  The  cells  were  kept  at  neutral  pH, 


'%  ■ 


■^ 
J4 


Fig.  6. 

and  individual  specimens  were  fixed  at  intervals  from  i  hr.  to  48  hr.  after 
uptake.  When  sections  were  examined  by  electron  microscopy,  a  complex 
sequence  of  morphological  changes  was  found  to  have  occurred.  Five 
features  of  the  process  may  be  described  in  summary  form : 

I,  Within  an  hour  after  a  pinocytosis  vesicle  is  formed,  ferritin  is 
released  from  the  carrier  substance,  and  collects  in  irregular  masses, 
usually  in  the  centre  of  the  vesicle.  Methyl  ferritin,  by  contrast,  remains 
bound  to  the  carrier  substance  for  the  entire  48  hr.  period  of  observation. 
This  difference,  because  it  parallels  the  effects  already  described  in  washing 
experiments  at  different  pH  values,  is  thought  to  indicate  a  rise  in  pH 
within  the  \esicle. 


l»*%*->p-  .^ 


Fig.  7. 


U.^^'v'^-^  %, 


.;-,'»  !^r 

/   p 

«^: . 

'••>V«^ 

< 

Fig.  8. 

Figs.  6,  7  and  8.  Successive  stages  in  the  evolution  of  vesicles  containing 
ferritin,  from  i  to  48  hr.  after  uptake.  See  text  for  description  of  changes  which 
occur. 


PROTEIN    UPTAKE    IN    AMOEBAE  613 

2.  In  vesicles  containina;  ferritin,  the  carrier  substance  appears  to 
become  detached  from  the  membrane  proper,  and  to  break  up  into 
irregular  masses.  When  the  carrier  substance  is  bound  to  methyl  ferritin, 
however,  the  entire  layer  maintains  its  structural  integritv,  is  detached 
from  the  membrane  proper,  and  becomes  highly  folded  and  crumpled  by 
48  hr. 

3.  The  osmiophilic  membrane  which  limits  the  vesicle  remains  intact. 
No  pores  or  holes  are  seen,  but  many  microvesicles  appear  attached  to  the 


fe' 


outer  or  cytoplasmic  side  of  the  limiting  membrane.  Also,  complex 
membranous  structures,  resembling  myelin  figures,  accumulate  within  the 
original  vesicle. 

4.  Neither  ferritin  nor  methyl  ferritin  particles  are  seen  in  any  part  of 
the  cell  except  the  pinocytosis  \'esicles.  Even  in  amoebae  which  have 
carried  large  numbers  of  vesicles  for  48  hr.,  the  ground  cytoplasm,  mito- 
chondria, contractile  vacuoles  and  nuclei  are  consistently  free  of  ferritin  or 
methyl  ferritin.  The  microvesicles,  a  few  hundred  Angstrom  units  in 
diameter,  which  fill  the  cytoplasm  and  are  intimately  attached  to  the 
pinocytosis  vesicles,  never  have  been  found  to  contain  ferritin  or  methyl 
ferritin. 


i"v^'^T 


Fig.   io. 


Fig.   II. 
Figs.  9,   10  and   ii.  Successive  stages  in  the  evolution  of  vesicles  containing 
methylated  ferritin,  from   i   to  48  hr.  after  uptake.   Sec  text  for  description  of 
changes. 


PROTEIN    UPTAKE    IN    AMOEBAE  615 

1^.  In  48  hr.  many  of  the  ferritin  particles  within  the  vesicles  lose  their 
discrete  character  and  form  dense  amorphous  masses,  as  though  their 
protein  coats  were  removed. 

These  features  are  illustrated  in  Figs.  6,  7,  and  8,  which  show  successive 
stages  encountered  in  vesicles  containing  ferritin,  and  in  Figs.  9,  10,  and  11, 
of  comparable  stages  in  vesicles  containing  methyl  ferritin. 


Conclusion 

These  studies  on  ferritin  and  methylated  ferritin  demonstrate  that  the 
initial  reaction  of  proteins  with  the  cell  surface  depends  upon  net  charge 
effects,  that  the  binding  step  may  be  temporarily  uncoupled  from  the 
subsequent  stages  of  pinocytosis,  and  that  neither  free  ferritin  nor  bound 
methvl  ferritin  escapes  in  recognizable  form  from  the  pinocytosis  vesicle. 
Such  observations,  when  considered  with  the  experimental  evidence 
alreadv  available  from  other  studies,  lead  to  the  following  view  of  pino- 
cytosis in  amoebae : 

Pinocytosis  is  a  co-ordinated  sequence  of  three  main  processes  or 
stages.  The  initial  binding  is  an  ion  exchange  reaction,  which  is  capable  of 
some  selectivity  and  of  concentrating  positively  charged  substances  from 
the  environment.  The  binding  reaction,  under  normal  conditions,  sets  off 
the  active  process  of  vesicle  formation.  In  this  stage,  both  the  membrane 
proper  and  the  coat  substance,  with  some  free  fluid  as  well,  are  carried  into 
the  cell  as  vesicles  are  pinched  off.  This  process  is  metabolicallv  linked,  as 
the  first  is  not,  and  appears  to  depend  on  cytoplasmic  contractilitv  in  a 
way  which  as  yet  has  not  been  studied  adequately. 

The  third  stage  comprises  a  complex  series  of  morphological  and 
chemical  events  within  the  cell.  The  lipoprotein  membrane,  the  muco- 
polysaccharide carrier  substance,  and  the  ingested  substances  are  all 
modified,  each  in  a  different  way.  The  evidence,  though  by  no  means 
complete,  suggests  that  the  changes  include  the  digestion  of  protein  and  the 
breaking  up  or  partial  digestion  of  the  mucopolysaccharide  carrier.  This 
implies  the  accumulation  of  hydrolytic  enzymes  within  the  vesicle,  and 
supports  the  idea,  for  which  there  is  as  well  morphological  evidence,  that 
the  pinocytosis  vesicle  is  fundamentally  the  same  as  the  normal  food 
vacuole  in  which  the  amoeba  digests  his  prey  [16]. 

From  what  is  known  of  the  fate  of  smaller  molecules,  such  as  [^^C]- 
glucose  [17]  and  ribonuclease  [18],  it  seems  that  such  substances  do  pass 
readily  from  the  primary  vesicle  into  the  ground  cytoplasm,  or  into  the 
microvesicles  which  are  formed  in  great  numbers  from  the  primary  vesicle. 
Since  neither  free  ferritin  nor  bound  methyl  ferritin  escapes  the  vesicle, 
such  exchanges  cannot  be  the  result  of  a  gross  breakdown  of  membrane 


6l6  V.    T.    NACHMIAS   AND   J.    M.    MARSHALL,   JR. 

Structure  or  function.  The  cell  does  not  at  any  stage  relinquish  its  control 
over  permeability;  there  must  be,  therefore,  highly  selective  exchange 
mechanisms  operating  in  both  directions  across  the  vesicle  membrane,  or 
between  the  primary  vesicle  and  the  microvesicles. 

If  this  general  view  is  correct,  it  follows  that  active  transport  mechan- 
isms are  essential  elements  within  the  overall  process  of  pinocytosis,  but 
the  two  phenomena  are  not  strictly  equivalent.  The  debate  between 
physiologist  and  morphologist  may  be  resolved  by  considering  pinocytosis 
(in  this  instance,  of  protein)  to  include  the  entire  sequence  from  the  initial 
binding  reaction  to  the  point  of  metabolic  utilization.  Alternatively,  it  may 
be  desirable  to  retain  the  somewhat  arbitrary  morphological  definition  of 
pinocytosis  as  extending  Only  to  the  formation  of  vesicles,  and  to  speak  of 
digestion  and  assimilation  as  subsequent  processes.  In  either  case,  the 
mechanisms  of  trans-membrane  exchange,  and  the  role  of  the  microvesicles 
within  the  cytoplasm,  remain  to  be  investigated. 


Acknowledgments 

The  authors  are  indebted  to  Miss  Marianne  Pieren  and  to  Mrs.  Diane 
Evans  for  technical  assistance. 


References 

1.  Holter,  H.,  Ann.  N.Y.  Acad.  Sci.  78,  524  (1959);  Int.  Rev.  Cytol.  8,  481  (1959). 

2.  Schumaker,  V.  N.,  Exp.  Cell  Res.  15,  314  (1958). 

3.  Brandt,  P.  W.,  Exp.  Cell  Res.  15,  300  (1958). 

4.  Marshall,  J.  M.,  Schumaker,  V.  N.,  and  Brandt,  P.  W.,  Ann.  N.Y.  Acad.  Sci. 

78,  515  (1959)- 

5.  Chapman-Andresen,  C,  and  Holter,  H.,^.  biophys.  biochein.  Cytol.  (in  press). 

6.  Rustad,  R.  C,  Nature,  Lond.  183,  1058  (1959). 

7.  Granick,  S.,^.  biol.  Chetn.  146,  451  (1942). 

8.  Laufberger,  M.  L.,  Bidl.  Soc.  Chim.  biol.,  Paris  19,  1575  (1937). 

9.  Farrant,  J.  L.,  Biochim.  biophys.  Acta  13,  569  (1954). 

10.  Granick,  S.,  Physiol.  Rev.  31,  489  (195 1). 

11.  Kuff,  E.  L.,  and  Dalton,  A.  J.,^.  Ultrastructure  Res.  I,  62  (1957). 

12.  Muir,  A.  R.,  Quart.  J.  exp.  Physiol.  45,  192  (i960). 

13.  Mazur,  A.,  Litt,  I.,  and  Shorr,  E.,  J.  biol.  Chem.  187,  473  (1950). 

14.  Fraenkel-Conrat,  H.,  and  Olcott,  H.  S.,  7-  biol.  Chem.  l6l,  259  (1945). 

15.  Pappas,  G.  D.,  Ann.  N.Y.  Acad.  Sci.  78,  448  (1959). 

16.  Roth,  L.  E.,^.  Protozool.  7,  176  (i960). 

17.  Chapman-Andresen,  C,  and  Holter,  H.,  Exp.  Cell  Res.  Siippl.  3,  52  (1955). 

18.  Brachet,  J.,  "Biochemical  Cytology".  Academic  Press,  New  York  (1957). 


PROTEIN    UPTAKE    IN   AMOEBAE  617 

Discussion 

Allen:  In  the  case  of  tissue  cells  it  is  possible  to  see  especially  in  time  lapse 
movies  that  the  formation  of  pinocytotic  vesicles  is  dependent  upon  the  formation 
of  pseudopodia,  what  appears  to  be  a  short  of  "chewing"  movement  of  the 
hyaloplasmic  ruffles  of  tissue  cells.  I  wonder  if  there  is  the  possibility  that  the 
same  might  actually  take  place  in  amoeba  and  have  escaped  notice.  Have  you 
looked  into  this  ? 

Marshall:  We  have  looked  into  this,  but  have  not  seen  in  amoebae  quite  the 
process  you  describe,  nor  have  others  who  have  studied  this  more  thoroughly,  I 
believe.  I  would  only  agree  with  the  point  made  by  Dr.  Holter  in  his  review: 
different  cell  types  show  different  morphological  patterns  of  uptake.  And  even  in 
one  cell  type,  the  amoeba,  different  agents  invoke  different  responses  (as  Chapman- 
Andresen  has  shown).  I  don't  know  whether  we  can  equate  all  forms  of  pinocytosis ; 
differences  exist,  but  these  may  be  less  important  than  the  general  similarities. 

Porter:  What  do  you  regard  as  the  source  of  the  hydrolytic  enz\TTies  acting  in 
these  vesicles  ?  Is  it  possible  that  they  are  contained  in  some  of  the  smaller  vesicles 
that  you  see  associated  with  the  surface  of  the  microvesicles  ? 

Marshall:  It  is  possible.  We  don't  really  know  which  way  the  microvesicles 
are  going.  All  those  we  see  clustered  around  a  big  vesicle,  in  micrographs,  contain 
something  which  in  density  and  texture  closely  resembles  the  substance  within 
the  larger  vesicle.  Also,  the  microvesicles  are  found  sometimes  in  a  row,  like  a 
string  of  pearls  attached  at  the  end  to  the  larger  vesicle.  From  these  points,  it 
seems  more  likely  that  they  are  being  detached  from  the  larger  vesicle,  but  I  agree 
that  this  sort  of  evidence  by  no  means  settles  the  question.  We  must  still  say  that 
transport  in  and  out  of  this  chamber  may  be  "transmembrane"  transport  in  the 
strict  sense,  or  may  be  achieved  by  the  addition  or  subtraction  of  microvesicles. 

GoLD.'XCRE :  I  am  interested  to  see  that  there  is  no  evidence  of  very  tight  packing 
of  your  ferritin  molecules  on  the  outside  of  the  membrane  which  might  indicate 
a  tendency  to  expand  the  outside  and  thus  cause  a  mechanical  invagination  of  the 
vesicles.  I  wonder  if  you  can  see  in  any  of  your  pictures  evidence  of  that  or  anything 
else  which  might  suggest  a  mechanism? 

M.^rshall:  There  is  evidence  suggesting  expansion  of  the  coat  substance. 
Schumaker's  kinetic  studies,  you  may  recall,  showed  a  brief  second  stage  of 
protein  uptake,  as  though  the  first  binding  led  to  the  appearance  of  new  binding 
sites.  The  ferritin  work  points  in  the  same  direction ;  when  an  amoeba  is  treated 
in  the  cold  and  washed,  the  entire  surface  is  covered  by  ferritin  initially.  As  the 
cell  warms  up  it  changes  shape,  the  coloured  material  accumulates  in  smaller 
regions,  and  the  greater  part  of  the  cell  surface  becomes  clear.  A  few  minutes  after 
clearing,  the  new  surface  will  again  bind  ferritin  or  methylferritin.  This  implies 
that  new  surface  material,  and  probably  new  membrane  as  well,  is  formed  very 
rapidly.  The  amoeba  surface  is  a  dynamic,  continually  renewed  structure,  and  we 
think  this  is  particularly  so  at  the  tips  of  advancing  pseudopodia.  All  this  can  be 
seen  with  living  cells  in  the  light  microscope.  At  the  lev'el  of  fine  structure,  it  may 
be  explained  by  a  fusion  of  microvesicles  into  the  original  membrane,  the  contents 
of  the  microvesicles  becoming  new  coat  substance.  We  have  seen  occasionally 
something  to  suggest  this  in  electron  micrographs,  and  hope  to  find  out  if  it  is  true. 


6l8  V.    T.    NACHMIAS   AND   J.    M.    MARSHALL,    JR. 

Mitchell:  I  should  like  to  congratulate  Dr.  Marshall  on  a  beautiful  piece  of 
work.  It  makes  one  feel  that  the  phrase  "membrane  transport"  which  we  all  keep 
on  using  has  a  double  meaning.  We  are  speaking  on  the  one  hand  of  "membrane- 
transport"  and  on  the  other  hand  of  "transport  membrane".  Dr.  Marshall  has 
just  used  the  expression  "transmembrane  transport",  and  this  is  very  descriptive 
of  what  we  usually  mean  by  membrane  transport  (although  we  should  more 
logically  say  "transmembrane  port").  The  other  kind  of  transport  in  which  the 
membrane  itself  is  transporting  and  transported  we  ought,  perhaps,  to  call  "cis- 
membrane  transport " ! 

Holter:  Just  very  briefly  I  would  like  to  answer  Dr.  Allen's  question  from 
before.  While  we  have  never  seen  in  amoebae  movements  comparable  with  the 
undulating  movement  that  occurs  in  tissue  culture  cells,  we  have  seen  something 
else  that  might  be  related  to  it  and  that  is  a  peristaltic  movement  down  along  the 
invagination  of  the  amoeba  surface.  This  peristaltic  movement  has  been  observed 
in  a  time  lapse  film  that  Mrs.  Chapman-Andresen  made  in  Glasgow  some  years 
ago.  Unfortunately  the  film  is  technically  not  good  enough  to  be  published  or 
shown,  but  this  special  feature  was  rather  distinct  in  several  of  the  sequences. 

Allen  :  I  would  like  to  introduce  one  word  of  caution  regarding  the  question 
of  the  lability  of  the  membrane  in  the  amoeba.  It  may  be  quite  true  that  under 
special  circumstances,  such  as  during  feeding  and  during  pinocytosis,  membrane 
is  indeed  formed ;  the  fact  that  the  cell  can  change  from  almost  a  sphere  into  a  long 
cylinder  in  a  matter  of  a  few  minutes  indicates  that  the  cell  can  form  a  new  mem- 
brane. However,  there  is  a  vast  amount  of  evidence  in  the  literature  showing  that 
during  normal  locomotion  there  is  no  membrane  formed  at  the  front  end  of  an 
amoeba.  This  evidence  was  gathered  chiefly  by  Schaefl^er  and  by  Mast.  They 
showed  essentially  that  a  particle  placed  on  the  surface  of  an  amoeba,  let  us  say 
a  quarter  of  the  way  back  from  the  tip,  remains  in  a  constant  position  with  regard 
to  the  tip  as  an  A.  proteus  type  amoeba  advances.  However,  if  you  watch  particles 
on  the  tail  surface,  their  behaviour  is  not  quite  according  to  expectation,  in  terms 
of  the  membrane  being  pulled  forward  on  the  surface  of  the  amoeba.  By  and  large, 
it  can  be  said  that  during  normal  locomotion  there  is  no  mass  formation  of 
membrane. 

Holter  :  This  fits  very  well  with  the  fact  that  in  amoebae  pinocytosis  and  loco- 
motion are  antagonistic  features.  A  amoeba  that  crawls  will  not  pinocytose,  and 
vice  versa. 

Marshall:  I  think  there  are  many  unsettled  questions  in  this.  But  we  are  still 
left  with  the  finding  that  amoebae  can  rapidly  form  new  binding  material.  We 
don't  know  what  is  happening  to  the  membrane  proper,  but  at  any  rate  there  is 
movement  and  renewal  of  the  surface  coat,  and  in  a  way  it  seems  simpler  to  think 
of  membrane  and  coat  as  moving  together.  We  have  no  direct  evidence,  so  I  will 
have  to  leave  it  very  open  as  to  how  this  is  done  by  the  amoeba. 

Goldacre:  With  regard  to  the  point  made  by  Dr.  Allen  about  the  forward 
motion  of  the  membrane  as  indicated  by  carbon  particles,  I  think  there  may  be  a 
quite  different  interpretation  of  this  motion,  and  that  they  are  not  strongly  attached. 
If  one  attaches  a  series  of  oil  drops  to  the  membrane,  which  can  be  seen  to  be 
firmly  attached  because  of  their  contact  angle  of  90,  they  do  not  move  forward. 
They  are  overtaken  by  the  tail  and  are  squeezed  off  eventually  at  the  rear;  so  that  I 


PROTEIN    UPTAKE    IN    AMOEBAE  619 

think  that  the  movement  of  carbon  and  carmine  particles  apparently  on  the 
membrane  does  not  indicate  beyond  doubt,  the  movement  of  the  membrane. 

Allen  :  If  you  place  a  micro-needle  through  an  amoeba,  carbon  particles 
attached  to  the  membrane  behind  the  needle  will  move  around  the  needle.  On  the 
other  hand,  when  you  put  an  oil  droplet  on  the  surface,  it  apparently  makes  a  firm 
contact  causing  the  membrane  to  adhere  to  the  ectoplasmic  tube  at  this  point,  in 
the  same  way  that  the  membrane  adheres  to  the  ectoplasm  tube  on  the  bottom  of  a 
pseudopod.  Thus  I  still  think  that  the  membrane  slides  forward  over  newly  form- 
ing ectoplasmic  tubes. 

Davis  :  I  think  it  is  fair  to  say  that  the  fact  that  papers  on  pinocytosis  were 
placed  in  different  parts  of  this  Symposium  is  perhaps  an  indication  that  we  arc 
not  yet  quite  clear  on  the  function  of  pinocytosis  in  the  cell. 


Comparative  Study  of  Membrane  Permeability 

E.    SCHOFFENIELS* 

Institiit  Leon  Fredeiicq,  Laboiatoires  de  Biochimie, 
I'niversite  de  Liege,  Belgium 

The  ultimate  purpose  of  studies  on  the  permeabihty  of  Hving  mem- 
branes is  to  obtain  information  concerning  the  organization  and  the 
chemistry  of  such  membranes.  It  is  a  well-known  fact  that  cellular  mem- 
branes actively  transport  not  only  inorganic  ions  but  also  small  organic 
molecules.  In  fact  the  number  of  functions  attributed  to  cellular  mem- 
branes is  increasing  rapidly  with  the  constant  progress  in  that  field.  One 
wonders  therefore  how  these  various  mechanisms  are  organized  at  the  cell 
surface. 

Thus  I  should  like  to  discuss  what  could  be  called  the  functional 
structure  of  a  living  membrane,  i.e.  the  way  some  of  the  functions  so  far 
identified  are  organized  and  distributed  at  the  cell  boundary.  As  I  will 
show  later,  it  is  possible  to  identify  a  living  membrane,  knowing  its 
permeability  properties  in  exactly  the  same  way  as  a  svstematician  identifies 
a  species  using  morphological  features.  This  is  the  reason  why  I  suggest 
defining  what  we  could  call  the  permeability  characters  of  a  living  mem- 
brane. As  far  as  inorganic  ions  are  concerned,  the  active  transport  of  Na, 
CI,  etc.,  for  instance,  are  permeability  characters.  It  is  the  same  for  the 
properties  of  passive  permeability  to  Na,  K,  CI  and  so  on. 

The  comparative  study  of  membrane  permeability  ofiers  many  interest- 
ing aspects  since  it  may  help  us  to  establish  not  only  the  distribution  of  the 
permeability  characters  in  the  animal  kingdom  but  also  their  organization 
within  the  cell  membrane.  Last  but  not  least,  having  established  the 
functional  organization  of  a  living  membrane  one  still  has  to  define  the 
chemical  nature  of  the  molecular  architectures  responsible  for  the  various 
permeability  characters. 

I  should  like  to  illustrate  these  various  points  by  discussing  some  of  the 
recent  work  we  have  been  doing.  Miss  INI.  Baillien  and  I,  in  Professor 
Florkin's  laboratory. 

Let  us  first  examine  the  results  of  potential  difi'erence  measurements 
performed  at  various  levels  of  the  digestive  tract  in  some  animal  species. 

*  Chercheur  qualifie  du  Fonds  National  de  la  Recherche  Scientitique. 


622 


E.   SCHOFFENIELS 


The  potential  difference  is  measured  on  isolated  segment  using  the  method 
of  Wilson  and  Wiseman  [i]  or  a  new  method  described  elsewhere  [2].  In 
the  latter  case,  the  fragment  of  organ  is  opened  flat  and  mounted  between 
two  chambers.  When  anatomically  possible,  the  muscle  layers  are  stripped 
off  the  epithelium. 

It  can  be  seen  (Table  I)  that: 

(a)  the  lumen  of  the  digestive  tract  is  negative  with  respect  to  the 
serosal  fluid, 


TABLE  I 

Potential  Difference  across  the  Epithelium  of  the  Digestive  Tract 
IN  Various  Animal  Species 

The  potential  difference  is  recorded  on  isolated  preparations :  isolated  sac  (A) 
or  method  of  Baillien  and  Schoffeniels  [2]  (B)  with  (S)  or  without  stripping  of  the 
muscle  layers.  Both  sides  of  the  preparation  are  bathed  with  physiological  saline. 
The  sign  refers  to  the  serosal  fluid. 


Species 

Organ 

Potential 

difference 

in  mV 

Methods 

Guinea-pig 

{Caiid  porcelhis  L.) 

jejenum 

ileum 
colon 

0-5-2 

0  •  5-2 
17-20 

A 

Rat 

(Rattus  norvegiciis  Exl.) 

ileum 
colon 

0-5-2-5 
30-40 

A 

Rabbit 

{Oryctolagits  ciini cuius  L.) 

ileum 
colon 

1-5-2 

5-10 

A 

Goldfish 

(Carassius  auratus  L.) 

small  intestine 

3-5 

B 

Carp 

{Cyprinus  carpiu  iiudus  L.) 

small  intestine 

3-5 

B 

Trout 

(Salmo  irrideus  Gibbons). 

small  intestine 

0-5 

B 

Terrestrial  turtle 

{Testudu  herynanni  d.  P.  Gmelin) 


gastric  mucosa 

37 

small  intestine 

2-4 

caecum 

10-15 

colon 

20-50 

BS 


Water  turtle 

(Emvs  orbicularis  L.) 


small  intestine 
colon 


I -5-2 -5 
12-20 


BS 


Frog 

{Rana  tempororia  L.) 


gastric  mucosa 
rectum 


30-50 
10-50 


BS 


COMPARATIVE   STUDY    OF   MEMBRANE    PERMEABILITY  623 

(h)  while  the  potential  difference  is  around  30  mV  across  the  gastric 
mucosa,  the  caecum  and  the  colon,  it  is  only  a  few  m\'  across  the 
epithelium  of  the  small  intestine. 

These  observations  raise  two  important  points :  what  is  the  origin  of  the 
potential  difference  recorded  and  why  is  the  potential  difference  across  the 
small  intestine  so  low  ?  We  have  therefore  measured  the  fluxes  of  Na  in  the 
small  intestine  and  in  the  colon  of  Testudo  hermanni  G.  F.  Gmelin,  using 
the  double  labelling  and  the  short-circuit  current  techniques  [3].  We  have 
chosen  the  turtle  because  in  this  species  it  is  quite  easy  to  strip  the  muscle 
layers  from  the  epithelium  [2]. 

TABLE  II 

Influx  and  Outflux  of  Na  across  the  Isolated  Epithelium  of  the  Small 
Intestine  and  the  Colon  in  the  Turtle   Testudo  hermanni  G.   F.  Gmelin 

The  epithelium  is  bathed  with  physiological  saline  on  both  sides.  Results 
obtained  when  2-4,dinitrophenol  (DXP)  is  applied  are  also  given.  The  fluxes,  the 
net  flux  and  the  short-circuit  current  are  expressed  in  mcoul.  cm"-.H^^.  C  =  con- 
trol. DNP  concentration:  o-i  m^L  Experimental  periods:  i  hr. 


Influx 

Outflux 

Current 

Xet  Flux 

smn 

ill  intestine 

c 

492 

401    4 

41-6 

90-6 

c 

540  ■  4 

437-1 

40-1 

103-3 

DXP 

492 

329 

32-2 

163 

419 

314 

colon 

28-5 

105 

C 

174 

42-4 

104 

131-6 

C 

121  -6 

34-7 

61 

86-9 

DXP 

424 

28-9 

33 

13-5 

50-2 

50-2 

13 

0 

Table  II  gives  the  results  obtained.  In  this  Table  influx  means  flux 
from  mucosal  to  serosal  side  while  outflux  means  the  flux  in  the  opposite 
direction. 

It  is  clear  from  the  results  given  that  there  is  an  active  transport  of  Na 
from  the  mucosal  to  the  serosal  side  in  both  small  intestine  and  colon.  The 
values  of  the  short-circuit  current  are  always  smaller  than  the  correspond- 
ing values  of  net  flux.  This  means  that  a  cation  must  be  transported  from 
the  serosal  to  the  mucosal  side  or  that  an  anion  is  transported  in  the 
opposite  direction.  The  flux  values  are  higher  in  the  small  intestine  than 
in  the  colon.  Since  the  DNP  inhibits  the  influx  in  the  colon  while  it 
inhibits,  at  least  partly,  both  influx  and  outflux  in  the  small  intestine,  this 
could  mean  that  part  of  the  outflux,  in  the  latter  case,  is  due  to  active 
transport. 


624  E.    SCHOFFENIELS 

As  shown  in  Table  II  the  extent  of  the  inhibition  is  very  different 
depending  on  whether  we  consider  the  colon  or  the  intestine.  This  could 
be  explained  by  a  very  high  passive  permeability  to  Na  in  the  intestine. 


TABLE  III 

Effect  of  Na  and  K  Concentrations  on  the  Potential  Difference  across 

THE   Isolated  Epithelium  of  the  Colon  and  the  Small   Intestine  in  the 

Turtle   Testudo  Jientionni  G.  G.  Gmelin 

M  =  mucosal  side;  S  —  serosal  side.  Potential  difference  in  mV;  the  sign  refers 
to  the  serosal  side.  Explanations  in  the  text. 


Exp. 

Small  intestine 

Colon 

M 

S 

PD 

(mV) 

M 

S 

PD 

(mV) 

A 

RCl 

rso. 

RCl 
RSO4 

I 
I 

RCl 
RSO4 

RCl 
RSO4 

IS 

34 

B 

RCl 

RSO4 

R.SO.-Na/io 

RCl 

RSO4 

RSO4 

I 
-26 

RCl 

RSO4 

R.S04-Na/io 

RCl 

RSO4 

RSO4 

20 

45 
25 

C 

RSO, 
RSO, 

RSO, 
RSO,-Na/io 

I 
30 

RSO4 
RSO4 

RSO4 

RS04-Na/io 

30 
60 

D 

RCl 
RCl-Na/io 

RCl 
RCl 

3 
-18 

RCl 

RCl-Na/io 

RCl 
RCl 

II 

S 

E 

RCl 
RCl 

RCl 

RCl-Na/io 

05 
20 

RCl 
RCl 

RCl 
RCl-Na/io 

38 
40 

f 

RSOi 
RSG.-K  ■  10 

RSO4 
RSO4 

I 
1 

RSO 

RSO4-KX10 

RSO4 
RSO4 

20 
20 

G 

RSO4 
RSO, 

RSO4 
RSO,-K     10 

I 

—  2 

RSO4 
RSO4 

RSO4 
RSOi-Kxio 

22 
14 

RS04 

RS04-Na/io 
RSOj-Na/io-K     10 


RSO4 
RSO4 
RSO4 


RSO4 

RSG.-Na/io 

RS04-Na/io-K;-  10 


RSO4 
RSO4 
RSO4 


RSO4 
RSO4 
RSO4 


RSO. 

RSO.-Na/io 

RS04-Xa/io-K     10 


RSO, 
RSO, 
RSO, 


RSO4 

RSO,-Na/io 

RS04-Na/io-K  •  10 


57 
48 
48 


J 

RSO4 
RSO4 

RSO4 
RSO4-K  ■  10 

0-5 
I 

RSO4 
RSO4-K  -  10 

RSO4 
RSO, 

35 
35 

K 

RSO, 
RSO4-K  ■  10 

RSO4 
RSO4 

05 
0-5 

RSO, 
RSO4 

RSO, 
RSO4-K  X  10 

23   S 
22   5 

L 

RSO4 

RSO4 

RSO4-K     10 

RSO4 
RS04-Na/io 
RS04-Na/io 

0-5 
21 
24 

RSO, 

RSO, 

RSO4-K  ,■  10 

RSO4 

RSOi-Na/io 
RS04-Na/io 

32 
40 

40 

M 

RSO4 
RSO,-Na/io 
RSO,-Na/io 

RSO, 

RSO, 

RSO4-K     10 

0 
-32 
-26 

RSO, 
RSO,-Xa'io 
RSO,-N.i/io 

RSO4 

RSO4 

RSO,-K,    10 

43 

30 
12-5 

Let  us  consider  now  the  effects  of  a  modification  in  Na  and  K  concen- 
trations on  the  potential  difference  across  both  intestine  and  colon  (Table 
III).  In  these  experiments  CI  is  generally  replaced  by  an  equivalent  amount 
of  the  non-penetrating  anion  SO4.  When  Na  or  K  are  removed,  they  are 
replaced  by  an  iso-osmotic  amount  of  sucrose.  RCl  means  a  physiological 


COMPARATIVE    STUDY    OF    MEMBRANE    PERMEABILITY  625 

saline  having  the  following  composition:  113  niM  NaCl;  1-9  mM  KCl; 
0-45  niM  CaCU;  phosphate  buffer  pH  7-0.  RSO4  means  the  same  saline 
in  which  CI  has  been  replaced  by  an  equivalent  amount  of  SO4.  RSO^- 
Na/io  means  sulphate  saline  containing  ten  times  less  Xa  than  the  physio- 
logical saline.  RSO4-K  x  10  means  sulphate  saline  containing  ten  times 
more  K  than  the  physiological  saline.  RSOj-Xa/io-K  x  10  is  a  sulphate 
saline  containing  ten  times  less  Xa  and  ten  times  more  K  than  the  physio- 
logical saline.  RSO4-K  10  is  a  sulphate  saline  containing  ten  times  less  K 
than  the  physiological  saline. 
It  can  be  seen  that : 

(i)  The  replacement  of  CI  by  SO4  is  without  any  effect  on  the  potential 
difference  across  the  small  intestine,  contrary  to  the  situation  in  the  colon 
where  the  potential  difference  increases  (expt.  A). 

(2)  In  a  sulphate  saline,  a  decrease  in  Xa  concentration  in  the  mucosal 
solution  results  in  the  inversion  of  the  potential  difference  across  the  small 
intestine.  In  the  colon,  we  observe  a  decrease  in  potential  difference 
(expt.  B). 

(3)  In  sulphate  saline,  a  decrease  in  X*a  concentration  in  the  serosal 
solution  increases  the  potential  difference  across  both  small  intestine  and 
colon  (expt.  C). 

(4)  In  normal  saline,  alterations  in  Xa  concentrations  in  the  mucosal 
or  serosal  solutions,  result  in  the  same  variations  as  in  SO4  saline.  The 
variations  are  nevertheless  not  so  important  (expts.  D  and  E). 

(5)  In  sulphate  saline,  if  the  concentration  in  K  is  increased  in  the 
mucosal  solution,  the  potential  difference  across  the  colon  and  the  small 
intestine  is  unaffected  (expt.  F). 

(6)  However,  it  is  possible  to  show  that  the  K  concentration  in  the 
mucosal  solution  affects  the  potential  difference  in  the  intestine  if  we 
decrease  first  the  Xa  concentration.  The  result  is  an  inversion  of  the 
potential  difference.  If  one  then  increases  the  K  concentration,  the  poten- 
tial difference  decreases  in  the  small  intestine  while  it  stavs  constant  in  the 
colon  (expt.  H). 

(7)  In  sulphate  saline,  an  increase  in  K  concentration  in  the  serosal 
solution  results  in  an  inversion  of  the  potential  difference  across  the  small 
intestine  and  in  a  decrease  of  the  potential  difference  in  the  colon  (expt.  G). 

(8)  In  sulphate  saline,  a  decrease  in  Xa  concentration  in  the  serosal 
solution  increases  the  potential  difference  across  the  small  intestine  and 
the  colon.  If  the  K  concentration  is  increased,  the  potential  difference 
decreases  (expt.  I). 

(9)  In  sulphate  saline,  a  decrease  in  K  concentration  in  the  mucosal  or 
in  the  serosal  solution  is  without  appreciable  effect  on  the  potential 
difference  across  both  the  colon  and  the  small  intestine  (exts.  J.  and  K). 

VOL.  n. 2S 


626  E.    SCHOFFENIELS 

(10)  In  sulphate  saline,  a  decrease  in  Na  concentration  in  the  serosal 
fluid  increases  the  potential  difference  in  the  colon  and  in  the  small 
intestine.  An  increase  in  K  concentration  in  the  mucosal  solution  increases 
the  potential  difference  across  the  small  intestine  while  it  does  not  modify 
the  potential  difference  in  the  colon  (expt.  L). 

(11)  In  sulphate  saline  a  decrease  in  Na  concentration  in  the  musocal 
solution  produces  an  inversion  of  the  potential  difference  in  the  small 
intestine,  while  it  decreases  the  potential  difference  in  the  colon.  An 
increase  in  K  concentration  decreases  then  the  potential  difference  in  both 
tissues  (expt.  M). 

The  above  observations  suggest  the  following  conclusions.  Since  the 
replacement  of  CI  by  SO4  in  the  solutions  bathing  both  sides  of  the  colon 
produces  an  increase  in  potential  difference,  it  is  clear  that  this  epithelium 
is  relatively  impermeable*  to  SO4.  The  epithelium  of  the  small  intestine  is 
equally  permeable  (or  impermeable)  to  CI  and  SO4  since  the  replacement 
of  CI  by  SO4  has  no  effect  on  the  potential  difference.  This  result  could  also 
be  explained  if  one  postulates  that  the  epithelium,  although  impermeable 
to  SO4,  is  permeable  to  K  and  Na.  This  possibility  is  most  likely  to  be 
correct  since  the  other  results  demonstrate  that  the  epithelium  is  permeable 
to  Na  and  K.  Moreover,  using  ^'S  as  tracer,  we  have  been  able  to  show 
that  the  permeability  coefficient  for  SO4  in  frog  skin,  turtle  colon  and 
intestine  are  of  the  same  order  of  magnitude  (unpublished  results).  Never- 
theless the  fact  that  the  replacement  of  CI  by  SO4  does  not  much  affect  the 
magnitude  of  the  potential  difference  when  the  Na  concentration  is 
modified,  seems  to  suggest  that  the  passive  permeability  of  the  small 
intestine  to  CI  is  low. 

The  small  spontaneous  potential  difference  existing  across  the  small 
intestine  is  related  to  the  concentrations  of  Na  and  K  in  the  mucosal  and 
serosal  solutions :  it  is  thus  clear  that  both  mucosal  and  serosal  sides  of  the 
epithelium  are  permeable  to  these  ions.  On  the  other  hand,  in  the  colon  the 
mucosal  side  is  permeable  to  Na,  but  impermeable  to  K,  while  the  serosal 
side  is  permeable  to  both  ions.  The  conclusions  may  be  summarized  in  the 
following  scheme  (Fig.  i). 

This  is  a  schematic  representation  for  the  permeability  characters  of 
the  cells  forming  the  turtle  intestine  epithelium  (Fig.  i,  A  and  B).  The 
permeability  characters  of  the  frog  skin  are  also  given,  for  comparison 
(Fig.  I,  C).  The  outer  membrane  is  in  contact  with  the  mucosal  or  outside 
solution  while  the  inner  membrane  is  in  contact  with  the  serosal  or  inside 
solution. 

The  outward  facing  membrane  is  Na  and  K  selective  in  the  small 

*  Impermeability  must  be  considered  in  terms  of  relativity  or  in  statistical 
terms  of  probability.  Given  a  highly  sensitive  method,  any  substance  can  be  shown 
to  cross  a  membrane. 


COMPARATIVE   STUDY   OF   MEMBRANE    PERMEABILITY  627 

intestine  and  Xa  selective  in  the  colon  and  frog  skin.  The  inner  membrane 
is  Xa  and  K  selective  in  the  colon  and  small  intestine  while  it  is  K  selective 
in  the  frog  skin.  The  active  transport  mechanism  for  Xa  is  located  at  both 
outer  and  inner  faces  in  the  small  intestine  while  it  is  located  at  the  inner 
membrane  in  the  colon  and  the  frog  skin. 

Koefoed-Johnsen  and  Ussing  [4]  have  demonstrated  that  in  frog  skin, 
the  active  transport  of  Xa  is  in  fact  a  forced  exchange  for  K.  This  has  not 
yet  been  demonstrated  in  the  intestinal  epithelium.  Such  a  picture 
describes  satisfactorily  how  the  potential  difference  develops  under  a 
wide  varietv  of  conditions.  Let  us  first  consider  the  small  intestine  cell  in 
the  presence  of  a  non-penetrating  anion  (Fig.  i,  A).  Xa  diffuses  into  the 


Mucosal 

or 
outside 


Serosal 

or 

inside 


OK 


K(7)  Small 

intestine 


Colon 


Skin 


F 

V 

-0 
0 

-0 

UJ 

F 

0) 

0 

-0 

u 

Fig.    I.   Scheniatic  representation  of  the  permeability  characters  of  epithelial 
cells  from 

A  =  turtle  small  intestine 
B  =  turtle  colon 
R  =  frog  skin 

The  oblique  arrows  indicate  passive  diffusion.  AT  is  the  mechanism  of  active 
transport  with  a  one-to-one  exchange  for  K  and  Xa  in  frog  skin ;  this  type  of 
exchange  has  not  yet  been  demonstrated  in  intestinal  cells.  The  level  of  the 
chemical  symbols  designate  the  concentration  levels  of  the  cations.  Explanations 
in  text. 


cell  from  the  outside  border  and  gives  rise  to  a  diffusion  potential  which 
makes  the  cell  positive  in  relation  to  the  outside.  K  diffuses  out  in  the 
opposite  direction.  The  magnitude  of  the  potential  difference  across  the 
outer  border  is  given  bv  the  Goldman  equation  [21] : 


(I) 


628  E.    SCHOFFENIELS 

where  Em  —  Ec  is  the  potential  difference  between  the  mucosal  solution  and 
the  intracellular  fluid,  P  is  the  coefficients  of  relative  permeability  for  Na 
and  K  of  the  outer  (w)  membrane.  The  other  symbols  have  their  usual 
meaning. 

It  is  evident  from  this  equation  that  P'^  and  P'^.^  being  equal  as  well  as 
the  ratio  of  concentration  for  Na  and  K,  there  will  be  no  potential  difference 
across  the  cell  membrane.  The  potential  at  the  inner  border  is : 

The  total  potential  difference  across  the  epithelium  is  then 

E  =  {Em-Ec)  +  {Ec-Es) 

E  will  depend  not  only  on  the  relative  magnitude  of  the  concentration 
ratios  but  also  on  the  relative  values  of  the  P's.  This  is  well  demonstrated 
in  Table  III.  Equations  (i)  and  (2)  show  also  why  the  results  of  experi- 
ments J  and  K  (Table  III)  are  not  in  contradiction  with  the  conclusion 
that  both  mucosal  and  serosal  faces  of  the  small  intestine  are  permeable  to 
K.  It  is  indeed  evident  that  a  decrease  in  the  extracellular  K  concentration 
will  lead  to  a  smaller  overall  change  in  potential  than  an  increase. 

The  situation  is  more  complex  if  we  consider  the  behaviour  of  CI. 
Since  in  most  cells,  the  CI  distribution  is  generally  thought  to  be  entirely 
passive,  we  would  have  to  assume  that  in  the  specific  case  of  the  intestinal 
cell,  the  cellular  CI  concentration  is  equal  to  that  in  the  extracellular  fluid. 
But  low  intracellular  CI  concentration  could  nevertheless  be  found  if  the 
cell  possesses  a  mechanism  of  active  extrusion  for  this  anion  located  at  the 
inner  border.  This  is  certainly  the  case  since  we  have  found  (Table  II) 
that  the  short  circuit  current  is  smaller  than  the  net  flux  of  Na.  More 
direct  evidence  may  be  found  in  the  results  of  Durbin  et  al.  [5],  showing 
that  in  the  rat  small  intestine  CI  is  actively  transported  from  the  mucosal 
to  the  serosal  side  (see  also  [6]).  This  question  will  be  settled  as  soon  as  we 
have  not  only  measured  the  flux  of  CI  with  tracers,  but  also  determined 
with  micro-electrodes  the  exact  magnitude  of  (Etn  —  Ec)  and  {Ec  —  Es). 

As  far  as  the  colon  is  concerned,  the  total  potential  difference  may  also 
be  related  to  the  sum  of  two  potential  differences  arising  at  the  outer  and 
inner  borders  of  the  cell.  Since  the  spontaneous  potential  difference 
increases  if  SO4  replaces  CI,  we  have  to  introduce,  in  the  Goldman 
equation  [22],  the  concentration    ratio  for  CI.  Thus 

E  =  {Em-Ec)  +  {Ec-Es) 
and 

^^     P^A^^\n  +  P'MC\)         Pk(K).  +  Pk(Na).  +  P^i(Ca 
zF  ''P?^a(Na),  +  PS(Cl).        Pk{Kl  +  PUA^^l  +  Pa{C\\-    ^^^ 
Equation  (3)  is  in  agreement  with  the  results  given  in  Table  III. 


COMPARATIVE    STUDY    OF    MEMBRANE    PERMEABILITY  629 

It  is  obvious  from  the  results  reported  here,  that  the  distribution  of  the 
permeabihty  characters  in  the  small  intestine  is  different  from  that  in  the 
colon  or  in  the  frog  skin.  The  organization  and  the  distribution  of  the 
permeability  characters  is  thus  an  important  aspect  of  cell  differentiation. 
A  purely  speculative  scheme  has  been  proposed  to  explain  the  possible 
evolution  of  the  permeability  characters  in  the  epithelial  tissues  ([7],  p.  136). 
According  to  this  hypothesis  the  primitive  state  should  be  characterized  by 
a  mechanism  of  active  transport  located  at  both  inner  and  outer  border  of 
the  cell.  The  cell  would  then  evolve  toward  an  asymmetrv  bv  losing  the 
active  transport  mechanism  at  one  of  the  borders.  The  results  reported 
here  give  some  experimental  support  to  this  conception. 

It  is  also  worth  noting  that  the  small  intestine  and  colon  are  differ- 
entiated from  the  same  embryonic  layer,  the  endoderm,  while  the  frog 
skin  comes  from  the  ectoderm.  Cells  originating  in  two  different  embry- 
ologic  layers  (colon  and  frog  skin)  may  thus  evolve  in  the  same  direction 
(convergent  differentiation). 

Without  going  further  into  a  detailed  comparative  studv  of  membrane 
permeability  it  is  apparent  that  epithelial  cells,  such  as  those  of  frog  skin 
and  turtle  intestine,  possess  in  principle  the  same  permeability  characters 
as  those  found  in  conducting  cells,  such  as  electroplax,  nerve  fibre  or 
muscle,  or  those  found  in  red  blood  corpuscles.  But  one  advantage  of  the 
epithelial  cells  studied  in  the  present  paper  lies  in  the  fact  that  some  of  the 
permeability  characters,  e.g.  passive  permeability  to  Na  and  K  in  frog  skin 
and  turtle  colon,  are  spatially  separated  at  a  microscopic  level  or  are  found 
together  in  the  same  membrane  (turtle  small  intestine),  while  in  the  nerve 
fibre  for  instance,  one  of  them,  the  Na  selective  character,  appears  only 
for  short  periods  of  time  (action  potential). 

Further  investigations  are  now  necessary  to  answer  the  question 
whether  or  not  the  characters  of  passive  permeability  to  Na  and  K  are 
spatially  separated  in  the  small  intestine  cells.  One  attempt  to  solve  this 
problem  has  been  made  with  the  isolated  electroplax  of  the  electric  eel 
Electrophonis  electricus  L.  and  the  reader  is  referred  to  another  publication 
for  a  more  thorough  analysis  of  this  matter  [7]. 

Another  interesting  point  raised  by  the  above  considerations  is  the 
question  of  the  chemical  nature  of  the  molecular  architectures  responsible 
for  the  various  permeability  characters.  One  may  obtain  information  on 
this  subject  by  studying  the  effect  of  various  classes  of  compounds  known 
to  affect  the  permeability  of  living  membranes.  It  is  a  well-known  fact  that 
ouabain  and  some  ammonium  derivatives  affect  ionic  movement  in  a 
variety  of  cells  functionally  different  or  belonging  to  species  situated  far 
apart  on  the  evolutionary  scale  [7  19].  These  results  suggest  that  a  com- 
mon biochemical  system  could  be  responsible  for  all  the  permeability 
characters  ot  living  membranes.  This  would  thus  mean  that  we  could 


630  E.    SCHOFFENIELS 

consider  the  permeability  characters  as  being  heterotypic  expression  of  a 
common  biochemical  system,  the  concept  of  heterotypy  being  used  here 
in  the  sense  defined  by  Mason  [20]. 

An  argument  in  favour  of  this  view  is  offered  by  the  observation  of 
Tosteson  and  Hoffman  [21]  concerning  the  movement  of  cations  in  sheep 
erythrocytes.  It  is  known  that  some  individual  sheep  have  red  cells  with 
high  K  (HK)  and  low^  Na  concentrations,  while  other  sheep  have  red  cells 
wath  high  Na  and  low  K  (LK)  concentrations.  Recent  evidence  given  by 
Evans  (cited  in  [21])  suggests  that  the  LK  character  is  inherited  as  a  Men- 
delian  dominant.  Tosteson  and  Hoffman  found  that  the  active  transport  of 
K  is  four  times  greater  in  HK  cells  than  in  LK  cells,  while  active  trans- 
port of  Na  has  not  been  identified  in  LK  cells.  Moreover,  LK  cells  have 
a  greater  passive  permeability  to  K  and  smaller  passive  permeability  to 
Na  than  HK  cells.  Therefore  it  appears  that  a  single  gene  controls  the  per- 
meability characters  responsible  for  both  active  transport  and  passive 
diffusion  of  K  and  Na. 

Now  to  conclude.  The  results  reported  here  show  that  the  various 
permeability  characters  are  organized  or  distributed  differently  in  the  cells. 
This  distribution  is  in  fact  an  essential  aspect  of  cell  differentiation. 
Another  interesting  aspect  of  the  comparative  study  of  membrane  per- 
meability is  the  fact  that  a  common  biochemical  system  seems  to  be 
responsible  for  the  permeability  characters  found  in  living  membranes. 
Such  study  may  help  us  to  solve  the  problem  of  the  biochemical  mechanism 
responsible  for  the  cellular  differentiation  and  also  to  narrow  the  gaps  in 
our  knowledge  concerning  the  structure-function  relationships. 

Acknowledgments 

The  author  is  greatly  indebted  to  Professor  M.  Florkin  for  his  continued 
interest  and  stimulating  discussions  in  the  course  of  this  work. 

References 

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3.  Ussing,  H.  H.,  and  Zerahn,  K.,  Acta  physiol.  scatid.  23,  no  (1951). 

4.  Koefoed-Johnsen,  V.,  and  Ussing,  H.  H.,  Acta  physiol.  scand.  42,  298  (1958). 

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9.  Kirschner,  L.  B.,  Nature,  Loud.  172,  348  (1953). 
10.   Brink,  F.,  Pharmacol.  Rev.  6,  107  (1954). 


COMPARATIVE   STUDY   OF    MEMBRANE   PERMEABILITY  631 

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12.  Matchett,  P.  A.,  and  Johnson,  J.  A.,  Fed.  Proc.  13,  384  (1954). 

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15.  Glynn,  I.  M.,J.  Physiol.  136,  148  (1957). 

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Discussion 

^  Davis  :  I  might  say  that  in  one  aspect  of  active  membrane  transport  I  see  no 
basis  for  believing  that  the  fundamental  mechanism  of  transport  of  electrolytes, 
including  mineral  ions,  is  going  to  be  very  different  from  that  of  stereospecific 
organic  molecules.  This  is  one  of  the  reasons  why  I  am  reluctant  to  use  the  word 
"enzyme"  in  relation  to  these  carriers,  because  we  cannot  very  well  speak  of  an 
enzymic  conversion  of  potassium  to  some  product  on  the  way  in. 


Active  Transport  and  Membrane  Expansion- 
Contraction  Cycles 

R.    J.    GOLDACRE 

Chester  Beatty  Research  Institute,  Institute  of  Cancer  Research, 
Royal  Cancer  Hospital,  London,  England 

I.  Introduction 

That  a  rhythmically  expanding  and  contracting  surface  might,  by 
adsorbing  and  subsequently  desorbing  yarious  substances,  be  a  mechanism 
used  by  the  cell  to  concentrate  substances  ^yithin  it  \yas  suggested  by 
Goldacre  and  Lorch  [4]  and  Goldacre  [5].  Chemical  model  experiments 
sho\yed  that  substances  could  indeed  be  concentrated  in  this  \yay,  and 
tests  \yith  amoebae  in  yarious  dye  solutions,  such  as  neutral  red,  methylene 
blue  and  other  basic  dyes,  indicated  that  dyes  accumulated  at  that  part  of 
the  cell  \yhere  contraction  took  place,  namely  the  rear  end  or  "tail".  This 
accumulation  \yas  sho\yn  not  to  be  due  to  a  pH  difference  bet\yeen  the 
amoeba's  tail  and  the  external  medium,  for  it  occurred  equally  well  in 
media  from  pH  4  to  8  [6].  Further  support  was  giyen  by  Prescott  [i  i],  who 
not  only  confirmed  the  original  obseryations  but,  by  preyenting  plasmagel 
contraction  by  conyerting  it  all  into  plasmasol  by  the  application  of  a 
hydrostatic  pressure  of  seyeral  hundred  atmospheres,  preyented  the 
accumulation  of  neutral  red  by  the  cell  which,  howeyer,  began  to  accumu- 
late it  when  the  pressure  was  released  and  plasmagel  contractions  were 
restored. 

The  contractile  protein  hypothesis  has  been  modified  and  extended  by 
Danielli  [3]  who  suggested  that  protein  chains  lying  on  the  outside  of  the 
cell  membrane  with  adsorbed  substances  on  them  might  actually  be  pulled 
inside  through  a  micropore  by  an  internal  contraction ;  he  further  showed 
that,  on  the  assumption  that  ATP  proyided  the  energy  for  the  contraction 
(which  ATP  is  known  to  cause  when  injected  into  amoebae  [4]  and  slime 
moulds  [13])  the  distribution  of  phosphatases  in  yarious  secretory  tissues 
is  appropriate  to  the  direction  of  secretion. 

In  amoebae,  the  rhythmic  contraction-expansion  cycle  is  obyious 
microscopically,  but  other  cells,  such  as  red  cells,  which  do  not  reyeal 
marked  rhythmic  moyements  eyen  in  time-lapse  cinematography,  haye 
actiye  transport  mechanisms.  It  is  interesting  in  this  connection  that  a 


634  R-   J-    GOLDACRE 

"  shimmering  "  or  vibratory  activity  of  the  red  cell  membrane  was  reported 
by  Pulvertaft  [12],  using  a  special  optical  technique,  which  may  represent 
minute  contractions  of  various  small  regions  of  the  cell  membrane.  This 
"shimmering"  could  be  abolished  by  o- 1  m  fluoride,  which  also  abolished 
the  uptake  of  potassium  ions,  indicating  a  possible  connection  between 
the  two  [i].  Recently,  also,  ATP  has  been  implicated  in  the  maintenance 
of  the  biconcave  shape  of  the  red  cell  [10]. 

In  plant  cells,  a  contraction  occurring  in  the  membrane  at  one  end  of 
the  vacuole  in  root  hairs  appeared  to  be  associated  with  the  release  of 
neutral  red  into  the  vacuole  at  that  place  [5],  when  the  cells  were  immersed 
in  the  dye  solution.  That  the  vacuole  is  implicated  in  the  accumulatory 
mechanism  is  indicated  by  the  observations  of  Brown  [2]  who  showed  that 
active  uptake  of  potassium  occurred  in  the  roots  of  higher  plants  only  at 
and  above  the  zone  of  elongation,  where  vacuoles  were  present. 

In  this  communication  I  should  like  to  describe  an  expansion-contrac- 
tion cycle  in  the  vacuolar  membrane  of  giant  NiteUa  cells,  which  appears  to 
be  associated  with  an  accumulatory  mechanism. 

In  order  for  an  accumulatory  mechanism  to  be  efi^ective,  there  should 
ideally  be  two  processes:  {a)  concentration  of  material,  and  {b)  a  valve 
action  or  one-way  movement,  so  that  accumulated  material  does  not  leak 
away.  However,  (6)  is  not  absolutely  necessary,  if  the  rate  of  accumulation 
is  high,  and  the  rate  of  leakage  lower ;  some  accumulation  would  still  occur 
and  this  appears  to  be  the  situation  in  the  accumulation  by  amoebae  of 
neutral  red,  which  can  in  time  be  washed  out  of  the  cells  by  water.  Nitella 
cells  appear  to  provide  both  these  mechanisms. 

2.  Internodal  cells 

The  large  chloroplast-lined  internodal  cells  of  NiteUa  are  not  particu- 
larly active  in  accumulating  neutral  red,  accumulating  about  as  much  in  a 
few  hours  as  the  more  active  rhizoids  accumulate  in  a  few  minutes  from 
the  same  solution.  A  casual  inspection  of  the  cyclosis  reveals  no  obvious 
regions  of  cytoplasmic  contraction,  the  cytoplasm  circulating  at  a  uniform 
speed  of  about  100  microns  per  second  in  a  slow  spiral  path  up  one  side 
of  the  cell  and  down  the  other.  In  some  of  the  cells,  however,  interesting 
phenomena  occur  at  the  ends  where  the  cells  are  attached  to  their  neigh- 
bours. Usually,  groups  of  small  cells  cover  and  obscure  the  junction  and 
it  is  necessary  to  search  through  many  specimens  until  a  cell  favourably 
placed  for  observation  can  be  found  (Fig.  i,  B). 

In  some  of  these,  it  can  be  observed  that  the  layer  of  cytoplasm  at  the 
end  is  thicker  (^3,  Fig.  i)  than  that  running  into  it  {d^),  and  this  thicker 
layer  moves  more  slowly.  This  means  that  the  surface  area  of  the  vacuolar 
membrane  is  contracting  at  this  point.  For  example,  consider  unit  volume 


ACTIVE   TRANSPORT   AND    MEMBRANE   EXPANSION-CONTRACTION    CYCLES      635 

of  cytoplasm  of  thickness  d^.  This  has  an  area  of  i  jd-^^.  Similarly,  when  the 
thickness  increases  to  d^,  the  area  decreases  to  i  /V/3,  and  the  area  contracts 
to  dijd^  of  its  original  area.  Measurements  of  the  ratio  d-^^jd^  varied  from 
1/3  to  I /id  in  different  cells.  The  thickness  do  of  cytoplasm  flowing  away 
from  the  junction  was  also  usually  greater  than  that  flowing  in,  and  its 
speed  consequently  less.  Measurements  of  the  ratio  of  the  speeds  were 
approximately  equal  to  the  measured  ratio  of  the  thicknesses,  and  changed 
from  time  to  time  in  a  given  cell,  and  varied  from  cell  to  cell. 

The  cause  of  this  contraction  seems  to  be  as  follows :  the  cytoplasm 
moves  fastest  near  the  cell  membrane  and  slowest  near  the  vacuolar 
membrane  as  particles  on  the  outside  of  the  stream  can  be  seen  continually 
overtaking  particles  on  the  inside  (see  also  [8]).  A  tangential  force  appears 
to  be  acting  on  the  cytoplasm  in  contact  with  the  membrane.  This  force 
also  appears  to  be  partly  associated  with  the  chloroplasts  in  this  kind  of 
cell,  for  {a)  if  some  of  the  chloroplasts  are  removed  from  their  rows  by 
micromanipulation,  streaming  stops  at  the  gap,  and  {h)  detached  chloro- 
plasts can  often  be  seen  spinning  in  the  cytoplasm  at  about  23  revolutions 
per  second — even  in  stationary  cytoplasm  squeezed  out  of  the  cell  under 
oil — indicating  a  tangential  force  between  chloroplast  and  cytoplasm  (see 
also  [9]). 

Chloroplasts  are  absent  from  the  wall  joining  two  cells,  so  that  the  cyto- 
plasm would  tend  to  accumulate  here,  being  driven  into  an  inactive  region. 

Chloroplasts,  however,  cannot  be  the  motive  force,  or  the  sole  motive 
force,  since  the  rhizoids  have  no  chloroplasts  and  the  cytoplasmic  move- 
ment seems  very  similar,  though  the  thickness  of  the  moving  cytoplasmic 
layer  is  in  the  region  of  1-2  microns  compared  with  10-70  microns  in  the 
internodal  cell. 

3.  Rhizoids 

Several  active  regions  in  the  rhizoid  cells  where  membrane  contraction 
occurred  were  found,  and  these  regions  were,  in  addition  to  increase  in 
thickness,  characterized  by  conspicuous  wrinkling  and  vacuole  formation. 

(a)  rhizoid  tip  (fig.   I,  a) 

The  thickness  of  the  cytoplasm  at  the  tip  of  the  rhizoid  cells  is  about 
100  times  greater  than  the  thickness  of  the  rapidly  moving  layer  on  the 
rest  of  the  cell.  As  the  rapid  thin  stream  feeds  into  this  deep  layer,  it  slows 
greatly  and  masses  of  wrinkles  appear  at  the  point  of  entrv. 

(b)  jun'ction  between  rhizoid  cells  (fig.   I,  c) 

The  peculiar  discontinuity  in  the  shape  of  rhizoid  cells  where  thev  join 
one  another  (rather  like  a  hand-clasp)  gives  rise  to  a  thick  pool  of  more 
slowly  moving,  wrinkling,  cytoplasm. 


636  R.   J.    GOLDACRE 

(c)    INERT    BAND    IN    MID-REGION    OF    RHIZOID    CELL    (PIG.    I,    d) 

More  seldom,  a  sudden  increase  in  cytoplasm  thickness,  accompanied 
by  wrinkling  of  tonoplast  and  vacuole  formation,  can  be  observed  in  the 
mid-region  of  rhizoid  cells.  Close  inspection  indicates  that  the  tangential 
force   on   the   cytoplasm   here   ceases   to   operate — cytoplasmic   particles 


O 


^ 


D 


Fig.  I.  Diagram  showing  regions  of  tonoplast  contraction  in  Nitella.  A,  tip  of 
rhizoid  cell;  B.  elongated  internodal  cell,  at  junction  with  neighbouring  cells 
(chloroplasts  are  omitted  for  clarity);  C,  junction  of  two  cells  in  rhizoid;  D,  active 
patch  in  mid-region  of  rhizoid.  Note  formation  of  small  vacuoles  within  some 
wrinkles  (C,  D).  These  subsequently  unite  with  the  central  vacuole. 

which  elsewhere  move  fastest  near  the  cell  membrane  here  move  slowest 
near  the  cell  membrane,  so  that  the  cytoplasm  rides  up  over  itself  and 
forms  wrinkles. 

4.  Discussion 

Close  inspection  of  the  wrinkles  indicated  that  their  area  decreased 
rapidly  with  time,  as  if  dissolving  in  the  cytoplasm,  in  a  manner  similar  to 
the  wrinkles  in  the  contracting  tail  region  of  the  amoeba  [4,  5,  7]. 

The  disappearance  of  surface  area  of  the  vacuolar  membrane  at  these 
regions  of  increased  thickness  of  the  cytoplasm  would  be  accompanied  by 
the  simultaneous  release  there  of  anything  adsorbed  on  the  membrane. 

The  small  vacuoles  which  usually  accompanied  the  wrinkles  and  which 
suddenly  formed  as  the  depth  of  cytoplasm  increased  could  be  observed 
growing  in  size,  then  moving  to  coalesce  with  the  large  vacuole  of  the  cell. 
In  the  presence  of  neutral  red,  concentrated  dye  could  be  observed  in  these 
small  vacuoles. 


ACTIVE    TRANSPORT    AND    MEMBRANE    EXPANSION-CONTRACTION    CYCLES      637 

These  small  vacuoles  are  probably  induced  to  form  in  the  cytoplasm 
by  the  sudden  release  into  it  of  a  high  concentration  of  accumulated 
material :  it  was  observed,  during  work  on  amoebae,  that  neutral  red 
injected  into  the  cytoplasm  with  a  micropipette  soon  induced  vacuoles 
containing  the  neutral  red  to  form  in  the  cytoplasm ;  similarly,  the  dye 
accumulating  naturally  in  the  amoeba,  when  present  in  the  external 
medium,  at  first  formed  a  diffuse  cloud  in  the  tail  and  shortly  after  became 
segregated  into  vacuoles  [4,  5].  Vacuoles  appear  to  form,  by  some  unknown 
mechanism,  as  a  result  of  high  concentrations  of  various  substances. 

This  coalescence  of  the  induced  vacuoles  with  the  large  vacuole 
occupying  most  of  the  cell  would  provide  a  valve  action  or  one-way  effect 
for  the  accumulated  dye,  for  the  reverse  phenomenon — the  pinching  off  of 
small  pieces  of  the  central  vacuole — was  not  observed ;  so  that  dye  released 
locally  in  high  concentration  by  a  contraction  of  the  vacuole  membrane, 
on  which  it  was  adsorbed,  would  be  captured  in  these  small  induced 
vacuoles,  and,  by  their  coalescence  with  the  large  vacuole,  be  prevented 
from  returning  to  the  external  medium. 

It  might  be  wondered  whether  the  amount  of  material  capable  of  being 
adsorbed  on  the  vacuolar  membrane  could  make  a  significant  contribution 
to  the  cell's  content  when  desorbed  in  a  contraction.  Suppose  a  monolayer 
of  adsorbed  substance  weighed  one-tenth  milligram  per  square  metre  (this 
is  about  one-tenth  of  the  weight  of  a  protein  monolayer).  Then  in  a  stream 
of  cytoplasm  travelling  at  100  microns  per  second  and  30  microns  wide, 
3000  square  microns  will  disappear  per  second,  releasing  3  x  io~^''  mg.  to 
the  vacuole  per  second,  and  increasing  the  sap  concentration  by  0-3  p. p.m. 
per  second,  or  2°o  per  day.  Hence,  if  any  substances,  adsorbable  on  the 
vacuolar  membrane,  succeeded  in  diffusing  passively  into  the  cell  in  low 
concentration,  they  would  be  concentrated  by  the  local  contractions  and 
ultimately  captured  by  the  central  vacuole  by  the  coalescence  with  it  of 
the  small  vacuoles  they  induced. 

5.  Summary 

In  Nitella  cells  exhibiting  cyclosis,  small  localized  regions  exist  where 
a  continuous  contraction  of  area  of  the  vacuolar  membrane  occurs.  These 
are  regions  where  a  sudden  fall  in  speed  of  streaming  occurs,  owing  to  an 
increase  in  depth  of  the  stream  produced  by  some  discontinuity  in  the 
cells,  resulting  in  accumulation  of  membrane,  with  wrinkling  and  its 
ultimate  dissolving  into  the  cytoplasm.  Any  adsorbed  substances  would  be 
shed  there.  A  valve  action  appears  to  be  provided  by  the  formation  of  small 
vacuoles  in  the  cytoplasm,  apparently  induced  by  the  high  concentration 
of  substances  shed,  and  containing  the  substances  in  high  concentration ; 
these  small  vacuoles  coalesce  with  the  large  vacuole  in  the  cell. 


638  R.   J,    GOLDACRE 

Acknowledgments 

This  investigation  has  been  supported  by  grants  to  the  Chester  Beatty 
Research  Institute  (Institute  of  Cancer  Research :  Royal  Cancer  Hospital) 
from  the  Medical  Research  Council,  the  British  Empire  Cancer  Campaign, 
the  Jane  Coffin  Childs  Memorial  Fund  for  Medical  Research,  the  Anna 
Fuller  Fund,  and  the  National  Cancer  Institute  of  the  National  Institutes 
of  Health,  U.S.  Public  Health  Service. 


References 

1.  Blowers,  R.,  Clarkson,  E.  M.,  and  Maizels,  M.,jf.  Physiol.  113,  228  (1951). 

2.  Brown,  R.,y.  exp.  Bot.  4,  197  (1953). 

3.  Danielli,  J.  F.,  Proc.  roy.  Soc.  B  142,   146  (1954);  see  also  Danielli,  J.  P., 
Symp.  Soc.  exp.  Biol.  6,  i  (1952). 

4.  Goldacre,  R.  J.,  and  Lorch,  I.  J.,  Nature,  Loud.  166,  497  (1950). 

5.  Goldacre,  R.  J.,  Int.  Rev.  Cytol.  I,  135  (1952). 

6.  Goldacre,  R.  J.,  Nature,  Lond.  172,  594  (1953). 

7.  Goldacre,  R.  J.,  Exp.  Cell  Res.  Suppl.  8,  i  (1961). 

8.  Kamiya,  N.,  and  Kuroda,  K.,  Bot.  Mag.,  Tokyo  69,  545  (1956). 

9.  Kamiya,  N.,  and  Kuroda,  K.,  Proc.  imp.  Acad.  Japan  33,  201  (1957). 

10.  Nakao,  M.,  Nakao,  T.,  and  Yamazoe,  S.,  Nature,  Lond.  187,  945  (i960). 

11.  Prescott,  D.  M.,  Nature,  Lond.  172,  593  (1953). 

12.  Pulvertaft,  R.  J.  V.,  7-  d'"-  Path.  2,  281  (1949). 

13.  Ts'o,  P.  O.  P.,  Bonner,  J.,  Eggman,  L.,  and  Vinograd,  ].,y.  gen.  Physiol.  39, 


325  (1956). 


Discussion 


ScHOFFENiELS :  As  an  extension  of  what  you  have  said  in  your  introduction 
about  some  models  of  active  transport  presented  by  Danielli,  I  would  like  to 
consider  the  following  mechanical  model  we  have  been  working  with  lately.  It  is 
made  of  two  chambers  separated  by  a  hole  shaped  like  a  funnel.  Now  if  we  place 
small  steel  balls  in  both  chambers  and  if  we  shake  the  whole  apparatus  we  get  an 
accumulation  of  balls  in  the  chamber  facing  the  large  apperture  of  the  funnel. 
This  could  well  be,  among  many  others  proposed,  a  model  for  a  cellular  mechanism 
of  active  transport.  This  would  imply  the  existence  of  a  contractile  structure  in  the 
cellular  men.brane  as  well  as  the  presence  of  a  funnel-like  pore  exhibiting 
specificity  toward  an  ion  species. 

Allen:  I  don't  think  there  is  any  doubt  that  Dr.  Goldacre 's  observations  on 
dye  accumvilation  in  the  amoeba  are  correct.  It  was  described  also  independently 
by  Dr.  Okada  in  Japan,  1930,  but  dye  accumulation  is  subject  to  alternative 
interpretations.  In  fact,  one  might  say  that  the  dye  accumulation  phenomenon 
is  compatible  with  at  least  three  hypotheses.  First,  as  Dr.  Goldacre  pointed  out  in 
his  paper  in  1950,  the  entire  surface  of  the  amoeba  (i.e.  the  plasmalemma  and  the 
outer  layer  of  ectoplasm)  adsorbs  quite  a  bit  of  dye ;  by  the  time  the  amoeba  has 
moved  one  cell  length,  this  adsorbed  dye  has  accumulated  in  the  tail.  Now  if  you 


ACTIVE   TRANSPORT   AND   MEMBRANE   EXPANSION-CONTRACTION    CYCLES      639 

follow  cytoplasmic  inclusions  in  the  outer  region  of  the  ectoplasm  as  markers,  you 
find  that  they  also  accumulate  over  a  two-minute  period  in  the  same  tail  region  in 
which  the  dye  has  been  found  to  accumulate.  Therefore,  one  expects  that  the 
normal  pattern  in  which  cytoplasm  circulates  in  the  amoeba  will  cause  dye 
adsorbed  to  any  part  of  the  surface  underneath  the  membrane  to  accumulate  in  the 
tail.  There  is  a  second  good  reason  why  this  accumulation  should  occur;  if  you 
look  carefully  at  the  tail  of  an  amoeba  you  find  it  contains  a  great  deal  of  the  surface 
area.  The  membrane  looks  superficially  like  a  bunch  of  grapes,  or  the  surface  of 
large  lobose  villi.  The  greater  surface  area  in  the  tail  would  lead  one  to  expect 
greater  penetration  of  dye.  A  third  factor  which  should  be  taken  into  account  is 
that  when  the  dye  penetrates,  it  enters  small  vacuoles  which  look  very  similar  to 
injury  vacuoles.  These  form  not  only  in  amoeba  but  in  other  cells  as  well  as  a 
result  of  neutral  red  treatment.  Nowland  in  1957  showed  not  only  that  neutral  red 
accumulates  in  vacuoles,  but  that  the  cytoplasm  in  the  region  of  large  accumula- 
tions of  neutral  red  vacuoles  is  in  an  injured  state.  Goldacre  has  once  reported 
finding  an  accumulation  of  stained  particles  which  so  altered  the  consistency  of  the 
cytoplasm  that  they  could  be  felt  as  a  lump  with  a  micromanipulator  needle.  If  the 
dye  is  sealed  in  vacuoles,  it  seems  unlikely  that  it  would  tell  very  much  about 
molecular  events  in  the  ground  cytoplasm.  In  view  of  these  difficulties  and 
alternative  interpretations  of  the  dye  accumulation  phenomenon  I  believe  that 
one  really  can't  draw  any  conclusions  from  it.  I  would  also  like  to  point  out  again 
that  Dr.  Goldacre 's  views  on  membrane  formation  and  destruction  just  do  not 
conform  with  the  results  of  many  published  experiments. 

Goldacre:  I  should  like  first  to  comment  on  the  wrinkles  in  the  amoeba's  tail: 
the  area  of  membrane  per  unit  mass  of  cytoplasm  there  would  be  several  times  that 
in  most  other  parts  of  the  cell,  but  the  amount  of  neutral  red  which  accumulates 
in  the  tail  may  be  a  hundred  or  a  thousand  times  as  much  as  you  get  elsewhere, 
increasing  as  time  goes  on;  so  I  don't  think  that  passive  diffusion  through  the 
extra  area  of  the  wrinkles  could  account  for  the  dye  accumulation  in  the  tail,  by 
several  powers  of  ten.  If  it  were  a  passive  diffusion,  as  Dr.  Allen  suggests,  the 
amoeba  would  tend  to  become  more  uniformly  coloured  with  time,  say,  after  the 
first  few  minutes;  instead,  the  opposite  occurs — the  concentration  in  the  tail 
continues  to  mount,  and  the  cytoplasm  in  the  rest  of  the  amoeba  remains  relatively 
free  of  dye. 

With  regard  to  Dr.  Allen's  statement  that  all  the  ectoplasmic  particles  accumu- 
late in  the  tail  region :  that  is  not  in  accordance  with  my  experience  nor  with  the 
published  accounts  of  Mast  and  other  careful  observers;  the  particles  circulate 
indefinitely  around  the  cell  and  do  not  accumulate  anywhere. 

I  don't  think  I  can  agree  with  Dr.  Allen's  third  point  because  there  are  many 
experiments  which  show  that  the  membrane  does  not  move  forwards,  including 
those  with  oil  drops  which  are  attached  to  the  membrane  and  when  the  contraction 
of  the  tail  advances  up  to  them  they  are  squeezed  off"  into  the  external  medium; 
carmine  particles  have  indeed  been  shown  to  move  forwards  and  I  have  seen  that 
myself,  but  I  don't  think  they  are  properly  attached  to  the  membrane;  you  can 
even  see  them  moving  forwards  in  the  external  medium  separated  by  10  or  20 
microns  from  the  surface  of  the  cell,  but  with  oil  drops  there  is  no  doubt  that  the 
attachment  is  firm,  for  the  angle  of  contact  is  90',  I  think  the  movement  of  carmine 


640 


R.   J.    GOLDACRE 


is  probably  an  electrical  phenomenon  of  some  kind.  If  you  reverse  the  electric 
charge  by  pre-treating  them  with  polyethylene  imine  solution,  they  move  in  the 
opposite  direction  and  form  a  clump  on  the  amoeba's  tail. 

Another  experiment  which  shows  that  the  membrane  does  not  move  forwards 
is  as  follows :  if  you  put  an  amoeba  into  a  narrow  capillary  tube  so  that  it  is  squeezed 
on  all  sides,  becoming  thereby  about  twice  its  natural  length,  it  continues  to  crawl 
through  the  tube.  If  the  membrane  was  moving  forwards  this  would  not  happen. 
Also,  if  you  put  parallel  glass  fibres  on  the  surface  of  the  amoeba  with  a  micro- 
manipulator, so  that  one  end  rests  on  the  amoeba  and  the  other  on  the  slide  (see 
Fig.  Ai),  then  as  the  amoeba  moves  forwards  those  in  the  rear  are  pulled  together 
whereas  those  in  the  front  remain  unmoved.  Contraction  therefore  appears  to  occur 
in  the  tail  region,  with  the  membrane  remaining  stationary  in  the  middle  portion 
of  the  amoeba,  and  forming  de  novo  in  the  front. 


1. 


'tii) 


Allen:  In  the  next  issue  of  Experimental  Cell  Research  Dr.  J.  L.  Griffin  and  I 
are  publishing  pictures  of  a  monopodial  amoeba  which  is  turning  to  the  left.  A 
carbon  particle  attached  to  the  left  side  advances  toward  the  front  of  the  cell,  over 
the  hyaline  cap  and  comes  to  lie  on  the  right  side  of  the  pseudopod.  I  think  this 
experiment  very  nicely  invalidates  the  idea  that  a  bioelectric  phenomenon  might 
be  the  cause  of  forward  movement  of  particles  attached  to  the  plasmalemma. 
While  an  amoeba  can  form  a  new  membrane  (e.g.  to  replace  that  lost  during  phago- 
cytosis), all  available  evidence  suggests  that  there  is  not  much  membrane  turnover 
during  active  locomotion. 

Porter:  I  understood  that  you  take  the  foldings  in  the  tonoplast  membrane 
or  the  vacuolar  membrane  to  be  indicative  of  contraction,  is  that  so  ?  Could  those 
not  also  represent  a  production  of  excessive  membrane  at  8  sites  on  the  surface  of 
the  tonoplast  ? 

GoLDACRE :  Perhaps,  but  since  the  motion  is  towards  the  site  of  the  deeper 
region  of  cytoplasm  you  would  expect  that  the  surface  membrane  would  inevitably 


ACTIVE   TRANSPORT   AND    MEMBRANE   EXPANSION-CONTRACTION    CYCLES      64 1 

accumulate  there  and  fold  or  wrinkle  because  of  the  reduced  surface  area  available. 
If  you  have  a  stream  of  water  running  into  a  lake,  the  natural  surface  film  carried 
down  by  the  streani  collapses  at  the  point  of  entry  and  you  can  see  this  collapse  in 
myriads  of  parallel  striations  usually  over  several  metres  near  the  point  of  entry. 
In  a  stream  of  cytoplasm  or  water  running  into  a  deeper  region,  unit  volume  will 
have  an  area,  if  the  depth  is  D^  in  the  shallow  part,  of  1/-D1,  and  if  the  depth  is  D.y 
in  the  deep  part,  of  i!D.,,  so  the  ratios  of  the  surface  areas  will  be  D./Dj ;  the  ratio 
of  these  thicknesses  may  be  10  to  i  or  a  100  to  i  in  the  cell,  which  means  that 
when  cytoplasm  with  its  associated  membrane  reaches  the  end  of  the  cell  at  these 
turning  points,  90  to  99",,  of  the  area  disappears  giving  almost  complete  desorption 
of  anything  that  would  be  adsorbed  on  it. 

Allen  :  I  am  very  curious  to  ask  Dr.  Goldacre  exactly  how  he  changed  the 
charge  of  the  particles  in  the  experiments  he  just  described  a  moment  or  two  ago. 

Gold.acre:  Carmine  particles  were  suspended  in  a  solution  of  polyethylene 
imine  hydrochloride  (a  cationic  polymer,  which  is  adsorbed  by  the  carmine)  and 
then  washed,  and  it  was  shown  that  the  charge  had  been  reversed  with  wires  and  a 
battery;  the  particles  moved  in  the  opposite  direction. 

Davis  :  It  seems  to  me  that  the  possible  mechanism  of  active  transport  involving 
a  contractile  element  does  not  critically  depend  on  the  kind  of  contractions  that  are 
grossly  visible.  Are  these  not  more  of  a  model  system  for  the  micro-contractions 
which  would  change  the  affinity  of  the  substance  for  the  carrier  ? 

Goldacre:  Yes,  there  might  well  be  submicroscopical  or  near  submicroscopical 
contractions  in  cells  such  as  the  red  cell ;  in  the  red  cell  I  imagine  you  might 
possibly  have  something  like  a  protein  chain  or  fibre  perhaps  running  along  the 
surface  and  absorbing  things  and  then  folding  up  in  the  middle  of  the  cell  some- 
where. There  is  not  much  evidence  of  that,  but  the  shimmering  movements 
associated  with  active  transport,  which  can  be  inhibited  by  various  chemicals 
(described  by  Pulvertaft  and  Maizels)  indicate  that  something  must  be  going 
on  there. 

Mitchell:  I  was  worried  a  bit  by  Dr.  Schoffeniels'  model.  Just  as  we  can  be 
too  easily  convinced  by  what  we  see  as  cytologists  when  we  look  down  the  micro- 
scope and  are  confronted  by  a  very  attractive  picture ;  in  the  same  way,  we  must 
be  very  careful  indeed  in  drawing  conclusions  about  an  attractive  everyday  macro- 
scopic model  and  imagining  that  what  happens  in  the  model  can  happen  at  the 
molecular  level.  The  model  described  by  Dr.  Schoffeniels,  as  it  is  stated  without 
all  sorts  of  additional  specifications,  is  really  a  Maxwell  demon  and  can't  work  at 
the  molecular  level.  And  perhaps  it  would  be  appropriate  to  say  that  this  is  true 
of  most  of  the  contractile  mechanisms  of  transport  that  have  been  proposed.  I  am 
not  sure  that  all  of  those  proposed  so  far  are  Maxwell  demons,  but  certainly 
nearly  all. 

You  could  legitimately  say  that  the  contraction  itself  can  increase  the  rate  at 
which  the  transport  takes  place :  but  it  simply  cannot  be  responsible  for  the  change 
of  free-energy  of  the  molecule  which  is  eventually  regenerated  after  carriage  of  the 
molecule  through  the  membrane  as  a  result  of  its  formation  of  a  compound  or 
complex.  It  simply  cannot  be  responsible  for  that  change. 

Goldacre:  Schoffeniel's  model  would  require  a  membrane  with  a  valve-like 
action  on  the  molecular  scale.  Such  asymmetry  does  not  appear  to  exist  in  non- 

VOL.  n. — 2  T 


642  R.    J.    GOLDACRE 

living  membranes,  though  Hving  membranes,  using  metabolic  energy,  usually 
have  it  in  their  active  transport  mechanisms  ;  passive  diffusion  remains  symmetrical, 
otherwise  thermodynamic  laws  would  be  violated,  as  Mitchell  indicates.  On  the 
other  hand  expansion-contraction  cycles  can  in  fact  concentrate  adsorbable 
substances  (i.e.  change  their  free-energy  state).  This  happens  in  the  working 
model  I  described  [Internat.  Rev.  Cytology,  I,  135  (1952)] — "  the  inversion  tube  " — 
which  by  adsorbing  substances  on  a  large  surface  and  subsequently  desorbing 
them  by  contracting  the  surface  mechanically,  in  a  small  volume,  can  give  you  a 
very  large  increase  in  concentration.  The  energy  for  the  concentration  comes  from 
the  unequal  energies  of  expansion  and  contraction  of  the  surface — in  contraction, 
there  are  adsorbed  molecules  to  squeeze  off  the  surface,  and  work  must  be  done 
against  the  energy  of  adsorption.  In  the  mechanical  model,  the  energy  provided 
is  mechanical ;  in  the  living  cell,  the  energy  source  of  the  contraction-expansion 
cycle  would  be  metabolic  (probably  ATP)  as  in  muscle. 

Davis  :  May  I  direct  a  comment  to  Dr.  Mitchell  ?  Let  us  consider  a  model  in 
which  a  site  with  a  certain  affinity  for  the  permeant  is  also  attached  to  something 
contractile ;  the  contraction  would  distort  the  site  and  thereby  decrease  its  affinity. 
You  are  not  getting  work  for  nothing,  as  ATP  energy  would  be  expended  in  the 
contraction.  Are  you  sure  that  Maxwell's  demon  is  invoked  in  this  kind  of  model  ? 

Mitchell  :  This  is  evidently  a  difficult  matter  to  discuss,  for  it  has  been 
chewed  over  many  times  and  there  is  not  yet  general  agreement  about  the  con- 
clusions— especially,  I  believe,  since  the  concepts  that  we  must  depend  upon  are 
still  in  process  of  formulation.  I  think  1  would  look  at  it  like  this.  When  you 
postulate  a  macroscopic  model  of  the  propulsion  process  in  membrane  transport 
by  considering  something  like  a  piece  of  elastic  and  how  you  can  manipulate  it  as 
a  catapult,  you  are  likely  to  run  straight  into  a  conceptual  difficulty.  Of  course, 
when  you  let  go  a  piece  of  elastic  it  goes  flip  straight  away  and  one  does  not  think 
of  the  thermal  activation  of  this  contraction  process.  If  a  protein  molecule  becomes 
"stretched"  or  unrolled,  the  regaining  of  the  configuration  that  it  originally  had 
is,  in  fact,  a  diffusion  process  which  must  occur  by  the  making  and  breaking  of 
residual  valencies.  Moreover,  the  "stretching"  during  the  phase  of  the  process 
when  it  actually  occurs  must  also  be  a  diffusion  (or  down  hill)  process,  since  it 
would  not  otherwise  happen.  In  this  sense  a  "contraction"  and  "expansion" 
process  can  be  associated  with  the  movement  of  an  ion  or  other  particle  through 
a  membrane;  but  if  we  are  going  to  "take"  the  ion  and  "put  it  on"  to  such  a 
system  we  have  to  change  the  ion  to  "put  it  on  ".  It  has  to  be  attached,  not  by  hand 
(as  a  stone  may  be  put  in  a  catapult),  but  by  a  chemical  bond.  When  the  elastic 
has  got  through  the  membrane  (in  the  ion  or  molecule  type  of  transport  that  we 
are  considering  here,  but  not,  of  course,  in  group  transport)  the  carried  particle 
has  to  be  detached  again — the  bond  must  be  broken.  The  actual  work  done  on  the 
particle  in  the  transport  is  determined  by  the  difference  in  free  energy  in  attaching 
and  detaching,  and  it  has  no  necessary  connection  with  the  change  of  configuration 
of  the  elastic  which  happened  in  between.  You  may  say,  as  Dr.  Davis  has  just 
done,  that  the  change  of  configuration  is  related  in  some  way  to  the  affinity  between 
the  carrier  and  the  particle,  but  this  does  not  make  the  actual  change  of  configura- 
tion responsible  for  the  work  of  transport.  Although  the  difficulty  with  which  we 
are  confronted  is  undoubtedly  partly  conceptual,  it  is  not,  as  has  sometimes  been 


ACTIVE   TRANSPORT   AND   MEMBRANE   EXPANSION- CONTRACTION    CYCLES      643 

suggested,  only  a  matter  of  words ;  for,  when  you  come  to  analyse  the  transport 
process  you  can  often  distinguish  between  the  free-energy  changes  invoh-ed  in 
making  and  breaking  the  intermediate  which  is  going  to  travel,  and  the  free-energy 
changes  associated  with  the  change  of  configuration  that  must  happen  in  between : 
for  example,  in  the  transport  of  oxygen  by  the  heart  and  circulatory  system.  You 
have  two  energy  considerations.  One  is  the  overall  free-energy  step,  up  which  (or 
down  which)  the  carried  particle  must  travel — and  this  simply  represents  the  free- 
energy  difference  between  the  carried  species  of  particles  in  the  phases  on  either 
side  of  the  membrane.  The  other  is  the  activation  energy  for  diffusion  (or  for 
"contraction-expansion")  of  the  carrier-carried  particle  complex — and  this  will 
determine  the  rate  of  the  process.  The  elastic  process  can  represent,  as  it  were,  a 
local  heating,  due  to  a  locally  catalyzed  exothermic  reaction,  and  this,  as  I  said 
before,  might  facilitate  the  transport.  But  the  elastic  process — being  no  more  than 
a  diffusion  process  at  the  molecular  level  of  dimensions — cannot  actually  drive  the 
transport  in  the  normal  energetic  sense.  My  criticism  of  Dr.  Schoffeniels'  model, 
and  my  statement  that  it  represents,  as  it  stands,  a  Maxwell  demon  is  based  on  the 
fact  that  it  does  not  include  an  adequate  description  of  the  vital  part  which  must 
be  played  by  primary  or  secondary  chemical  bonding  in  any  ion  selective  active 
transport  system:  he  has  substituted  demons  for  bonds. 

Schoffeniels  :  Just  a  few  words  about  Dr.  Mitchell's  comment.  I  do  not  believe 
that  the  mechanical  model  I  have  drawn  on  the  board,  or  any  model  presented  so 
far,  really  tells  us  what  is  happening  in  the  cell.  As  a  working  hypothesis  I  prefer 
the  carrier  hypothesis,  but  I  want  to  point  out  that  the  mechanical  model  proposed 
could  work  at  the  cellular  level.  We  have  first  to  postulate  that  the  pore  is  vibrating 
by  means  of  some  kind  of  contractile  machinery,  and  this  would  account  for  the 
energy  spent  by  the  cell  in  the  process  of  active  transport.  Then  of  course  the  pore 
must  exhibit  specificity  towards  a  certain  ionic  species.  This  model  could  be  taken 
as  a  working  hypothesis  for  active  transport,  as  well  as  the  carrier  hypothesis  or 
any  other  kind  of  hypothesis  since  we  really  do  not  know  anything  about  the 
molecular  mechanism  underlying  active  transport.  Any  model  thermodynamically 
sound  is  then  just  as  good  as  any  other.  We  nevertheless  must  be  aware  of  the  fact 
that  it  is  possible  to  fit  the  results  obtained  experimentally  with  living  cells  in  a 
great  variety  of  models.  This  is  especially  true  with  regards  to  the  energy  require- 
ments of  a  given  model  since  it  is  always  possible  to  make  ad  hoc  hypothesis  as  far 
as  efficiency  is  concerned. 

Davis:  I  am  not  altogether  convinced  that  something  involving  a  contractile 
element  may  not  be  a  satisfactory  model,  and  one  could  even  support  the  proposi- 
tion that  at  small  enough  dimensions  a  contractile  element  that  changes  the  shape 
of  a  molecule  and  a  group  transfer  that  changes  the  surface  of  that  molecule 
converge  and  become  essentially  the  same  thing.  It  may  well  be  that  our  thorough 
understanding  of  active  transport  is  going  to  depend  on  our  study  of  the  physical 
chemistry  of  macromolecules  in  such  a  subtle  way  that  we  can  really  recognize 
such  a  convergent  phenomenon  if  it  occurs.  ^^"^  i/»^~"~^ 


AUTHOR  INDEX 


Numbers  in  brackets  are  reference  numbers  and  are  included  to  assist  in  locating 
references  in  which  authors'  names  are  not  mentioned  in  the  text.  Numbers  in  italics 
indicate  the  page  on  \\hich  the  reference  is  listed. 


A 

Abelin,  1.,  4,  -'6 

Abrams,  A.,  48  [52],  50 

Aebi,  H.,  4,  -'6 

Afzelius,  B.,  5s8[i],  s62[i],  s63[i,  2],  s66 
[2,  16],  566 

Akabori,  S.,  383  [156],  4i)j 

Albright,  C.  D.,  sygfig],  ^So 

Alfert,  M.,  478  [11],  481  [11],  494 

Allen,  M.  B.,  285  [42],  288  [53],  -'9J,  308 
[2,  3].  .>-J.  340 [13,  15.  22,  23],  343 
[38,  39],  345  [13.  50.  51.  52,  53I. 
346[i3].  347[i3,  22,  23,  73],  348[i3. 
38,  39,  51.  52,  52,]^  349 [86,  88,  8q], 
351  [50,  88,  89],  352  [13,  95],  358  [50I, 
363  [38,  39],  366[38,  39,  95]-  369[95l. 
384,  385  [104,  158],  388  [13],  391  [89], 
394.  395.  398  [13,  15],  400  [95].  402 
[86],  403  [95].  403,  404,  405,  407,  411 
[3],  413  [6-8],  4i4[3.  8],  415  [8].  416 
[34].  7-'7,  4-'S,  431  [i].  44",  451  [7]. 
453,  455  [i].  459 

Allen,  R.  D.,  466,  473,  549  [i,  3,  4,  5,  10], 
55o[2,  3.  7].  551  [2,  3,  4],  552[3],  553 
[i,  2,  3,  5,  6,  10],  554,  555  [2,  3,  4], 
555 

Amano,  M.,  527 [7],  530 [7],  _5,?.^ 

Anderson,  I.  C,  315  [i,  23,  24],  J-'J,  ,?-'-/, 
■      347,  3bb[bSl403 

Anderson,  N.  G.,  48o[i5,  16],  4(^4 

Anderson,  W.  W.,  149  [31],  16 j 

Anfinsen,  C.  B.,  184,  i'^9 

Appclmans,  F".,  19  [92],  -^*^ 

Arison,  B.  H.,  349  [81],  705 

Arnold,  W.,  360,  406 

Arnon,  D.  I..  285  [42],  288,  291,  -^93,  -''y-/, 
303  [21,  22,  23,  24],  3<^4,  307.  308, 
323  [5].  J-'i.  339[i.  5].  34o[i3.  14.  15- 
22,  23],  342  [34-36,  37],  343  [37.  38, 
39].  344[5.  37.  46],  345  [13.  i4,  50,  51. 
52,  53].  346,  347  [22,  23,  73],  348 
[13.  35.  37,  38,  39.  52,  53].  349[84. 
86,  88,  89],  350,  351  [50,  88,  89],  352 
[94.  95].  353  [94].  354 [94].  355  [94. 
100],  356 [103],  357,  338,  359,  360, 
361,  362,  363  [38,  39,  94,  118],  364, 
365,  366,  367[i2i],  368,  369[95,  121], 
370,  372,  373  [i,  91,  114,  136],  374 
[i.  136],  375.  376,  377,  378[i36,  I54, 


155].  379[55.  154].  38o[i54,  155], 
381,  382,  383,  384,  385  [i,  103,  104, 
136,  158],  386,  387,  388[i3,  92],  389. 
390,  391,  394.  395.  396,  397.  398,  399 
[35,  94,  121],  40o[95,  121],  401,  402 
[86],  403  [95],  40J,  404,  40t,  406, 
407,  411.  4i3[6-8],  4i4[3.  8,  13,  15, 
17].  415  [8],  4i6[34].  418,  4-'7,  V-'-^', 
431,  440,  451  [7].  453,  455.  459 

Aronoff,  S.,  400,  41  iS 

Arwidsson,  B.,  431  [7],  433  [7],  437[i8], 
438  [7].  44",  44i 

Ash,  O.  K.,  300,  301  [13],  302,  30J,  J04, 

373,407' 

Atchison,  A.,  4 [45],  -7 

Atkinson,  AI.  R.,  597 [47],  599 

Aubert,  J.  P.,  341,  399 [33].  4^4 

Avron,  M.,  346,  347,  348,  349  [74].  360  [74], 
388  [55],  390,  404,  40  =i,  415  [26-28], 
417  [40,  41],  418  [26-28],  42S,  446, 
44^,  455  [2,  3.  6],  459 

Azzone,  G.  F.,  5  [50,  51,  52,  54,  58],  24 
[51],  25[50,  51,  52],  27,  76[i2],  77, 
80,  .Vj,  1 39  [6,  7,  8],  151,  152  [7,  8,  34], 
153  [34].  i56[43].  165  [52,  53].  ^66, 
167,  193  [5.  6],  i94[6].  195  [5.  12], 
i96[6],  i97[6],  i99[i2],  201  [11],  Jy-% 
227  [5],  -\?9 


B 

Baessler,  K.  H.,  119  [9],  134 

Baillien,  1\I.,  622  [2],  623  [2],  630 

Bain,  J.  A.,  4,  -'6 

Ball,  E.  G.,  184,  1S9 

Ballentine,  R.  B.,  459,  .^59 

Baltscheffsky,  H.,  i29[i6],  134,  157,  i6j, 
215.  --5,  431  [3-7].  432  [5,  11],  433 
[7.  14].  434[5].  435  [15],  436[3].  437 
[3,  18],  438  [3.  7].  439  [3].  440  [6,  24], 
440,  441 

Baltscheffsky,  AI.,  98,  100,  215,  J-'5,  358, 
406,  4i4[io],  42S,  431  [4,  7],  432[8, 
10,  11],  433[7].  435[i5].  436[i6], 
438  [7.  10],  440  [24],  44",  44^ 

Baranov,  \'.  I.,  347,  _/"5 

Barkulis,  S.  S.,  i7[S8],'-'^' 

Barrett,  J.  T.,  577  [11],  579,  583.  499 


646 

Bartley,  W.,  42,  50,  119  [21],  I34,  208,  209, 

224 
Bartsch,  R.  G.,  281  [26],  289 [26],  -'pj,  358, 

406 
Bassham,  J.  A.,  344 [45],  347  [69]-  404,  4^5 
Basso,  N.,  25,  2(j 
Beechey,  R.  B.,  43  [38],  50,  87,  92,  95  [3], 

99 
Begg,  R.  W.,  433,  440 
Beinert,  H.,  255,  260 
Bellamy,  D.,  74[7],  <^'-' 
Bendali,   F.,   288,   291,   293,   4i4[i6],   41s 

[i6],  ^2,V 
Benson,  A.  A.,  281,  2<j2,  340 [10],  341  [28], 

344 [45],  403,  404 
Bergeron,  J.  A.,  308 [6,  7],  309 [6,  7],  315 

[i,  6,  8,  24],  321  [6],  322  [6],  J-'j,  324 
Bergmann,  R.,  45  [45],  50 
Bergstrom,  L.,  585  [33],  .599 
Bermath,   P.,  2S3I4].   2S4[4l,   2S7U1.   2sS 

[4],  260 
Bernard,  W.,  482  [24],  -/97 
Bernath,  P.,  io4[8],  117 
Beyer,    R.    E.,    4 [36],    6 [62],    2y,    43,    =^0, 

149  [32],  167 
Beyer,  T.,  4 [36],  -'7,  43.  5<J 
Bibring,  T.,  482  [23],  494 
Bieri,  J.  G.,  183  [8],  iSg 
Birch-Andersen,  A.,  313  [9,  32],  3-3,  3-4 
Birt,  L.  M.,  42,  50,  ii9[2i],  ^34,  208,  209, 

224 
Bishop,  D.  W.,  559 [3].  566 
Bishop,  N.   I.,  323  [10],  3-^3,  349  [78,  79l. 

405,  415  [19],  427[i9],  4-''-^ 
Bloch,  D.,  480 [13],  494 
Blowers,  R.,  634[i],  6,j,V 
Bond,  H.  E.,  48o[i6],  494 
Bonner,  J.  T.,  522,  523,  5-5,  633  [13I,  63S 
Borst,  P.,  74[6]-  76 [9],  ^^,  <^'i,  218 [23,  24], 

221  [23,  27],  225 
Bove,  C,  356,  357,  384,  386,  406 
Bove,  J.,  356,  357,  384,  386,  406 
Bowyer,  P.,  577  [16],  580 
Bovchenko,  E.  A.,  347,  705 
Bover,  P.  D.,  11  [82],  23  [11 3,  144],  -'.V,  -'9, 

34[i2],  4^,  266 [5],  267 
Bovle,  F.  P.,  423,  42g 
Brachet,  J.,  47i  [3],  473,  47^  [3]-  487  [38], 

494,  493,  615  [18],  6/6 
Bradfield,  J.  R.  G.,  313  [n,  12],  3^3 
Brandt,  P.  W.,  605,  606  [4],  6/6 
Brawerman,  G.,  278  [si,  280,  292 
Bregoff,  H.  M.,  378 [141.  i43]-  4^^7 
Brenner-Holzach,  O.,  42  [3 1],  _5'->,  86,  97 
Bridgers,  W.  F.,  4 [45],  27 
Briggs,  R.,  472,  473 
Brill,  A.  S.,  286 [48],  29.? 
Brink,  F.,  629 [10],  630 
Brinkman,  R.,  5 18  [7],  523 
Brodie,  A.  F.,  41  [23],  50,  149  [3°].  i'^7 
Bronk,  J.  R.,  16,  24,  2S 
Brown,  A.  H.,  303  [28,  29,  30],    304,   340 

[9],  403 
Brown,  R.,  634,  6  j.V 
Brown,  T.  E.,  289 [56],  29J 


AUTHOR    INDEX 


Broyer,  T.  C,  356,  406 
Brummond,  D.  O.,  341  [30],  404 
Bucher,   T.,    ii9[5],    121  [5],    133  [5],   i34, 
215,   223,   227 [8],   23o[i3],   2J9,   302, 

303  [19],  304,  339>  403 
Bull,  H.  B.,  518 [8],  323 
Burkhard,  R.  K.,  256,  259 [9],  260 
Burnett,  G.,  46  [47],  30 
Buttin,  G.,  575  [7],  577  [7].  579,  583  [21], 

599 
Buytendyk,  F.  J.  J.,  5i8[7],  325 


Callan,  H.  G.,  477 [4],  494 

Calo,  N.,  340 [19],  403 

Calvin,    M.,   286,    29J,    303  [26],    304,   340 

[10].  341,  344[45],  347[69,  7i],  399, 
400,  40J,  404,  403,  407,  40S 

Capindale,  J.  B.,  34° [15],  343  [38,  39l, 
348  [38,  39],  366  [38,  39],  398  [15],  4'>3, 
404 

Carlat,  L.,  4  [40],  27,  42  [30],  30 

Carlton,  A.  B.,  356 [loi],  706 

Caro,  L.  G.,  313  [13],  J2j 

Carter,  G.  S.,  558 [4],  366 

Catcheside,  D.  G.,  575,  579,  592,  599 

Cavallini,  D.,  492[47],  -/9  5 

Chalfin,  D.,  628  [6],  bjo 

Chance,  B.,  5  [57],  24,  27,  33,  49,  72  [2], 
S2,  85  [i],  87,  88,  89,  91,  92,  96,  98, 
99  [6,  7],  99,  100,  108  [16],  118,  119 
[i,  2,  3,  4],  i2o[3],  121  [2],  I23[i4], 
i24[i5],  i28[i,  18,  19,  20],  i29[i6], 
134,  139,  153,  i56[io,  42],  159,  161, 
162,  166,  167,  174,  179,  185,  i86[i9, 
20],  187,  /.V9,  190,  193,  200 [2],  202, 
208,  212,  214,  215,  217,  223,  224,  223, 
227  [2,  3,  6,  7,  9I,  235  [16],  236,  2J9, 
283,  286,  289 [35,  36,  54,  55],  293, 
301  [10-12],  303,  357,  406,  438,  441, 
5 14  [4],  5-'_5 

Chantrenne,  H.,  471  [3],  473 

Chapman-Andresen,  C,  606 [s],  6is[i7], 
6j6 

Chappell,  J.  B.,  4[43],  -7,  42 [32],  43[37, 
43],  50,  56[(>,  7],  ^J;,  72[i],  76[ii], 
82,  83,  87,  91 

Chargaff,  E.,  472 [5],  473 

Chayen,  J.,  481  [19],  494 

Chiga,  M.,  35,  ,50 

Chmielewska,  L,  188  [25],  190 

Choules,  G.  L.,  459,  -^59 

Chow,  C.  T.,  280,  292,  347 [63],  403 

Christensson,  E.,  538  [i],  347 

Christie,  G.  S.,  208,  224 

Ciotti,  M.  M.,  74[4],  82 

Clark,  V.  M.,  363,  406,  415  [22,  23],  428 

Clarkson,  E.  M.,  634 [i],  638 

Clayton,  R.  K.,  360,  406 

Cleiand,  K.  W.,  5  [59],  -'7,  87,  91 

Clendenning,  K.  A.,  281  [24],  292 

Cohen,  G.  N.,  574,  575  [7,  8],  576  [8],  577 
[7,  8],  379,  582,  583  [I,  21,  22],  588[i], 
59S,  599 


AUTHOR    INDEX 


647 


Cohen-Bazire,   Cj.,   278  [6],   -'9-',   295,   299, 

303 
Cohn,  E.  J.,  331  [12],  337 
Cohn,  M.,  266 [3],  267,  588 [40,  41],  59'y 
Colpa-Boonstra,  J.  P.,  142,  /66,  172,  ij'j 
Conover,  T.  E.,  24[io9,  no,  in,  112],  -'<V, 

-'9.    1 39  [5].    142,    145  [28],    147,    166, 

Tf^7,    172  [5],    177  [4],    179,    255,    260, 

440-  441 
Conti,   S.   P.,   3i9[i4],   32o[26],  323,   324, 

J-'-/ 
ContopouloLi,  R.,  369[i2s],  4'J'^ 
Cook,  T.  M.,  1 84,  iSg 
Cooledge,  J.,  549[5].  553  [5].  554,  555 
Coomber,  J.,  325  [2],  334[i5],  33^,  337 
Cooper,   C,  4[i8,    19,   20],   7  [70],   26,   2j, 

32[2],    34[2,    13,    14],    35  [2],    49,    50, 

91,  Cj2 
Cooper,  ().,  184,  iSg 
Cooperstein,  I.  L.,  628 [6],  630 
Copenhaver,  J.  H.,  162,  i6j 
Cori,  C.  P.,  207 [i],  224 
Cori,  G.  T.,  207  [i],  224 
Cota-Robles,  E.  H.,  585,  599 
Crane,  F.  L.,  104,  108,  io9[ii],  11  j,  254 

[6],  260,  279,  280  [12],  2g2,  323  [15], 

i-'J,  349.  4'>5,  415  [20],  427  [20],  ^-'.v 
Crane,  R.  K.,  3  [4],  4 [4],  26 
Curran,  P.  P.,  628  [5],  630 
Cynkin,  M.  A.,  340 [17],  403 


D 


Dalhamn,  T.,  563,  566 [15],  566 

Dallam,  R.  D.,  24,  -'V,  i49[3i],  i6j 

Dalton,  A.  J.,  607  [i  i],  6/6 

Dam,  H.,  349 [77].  4^5 

Dan,  K.,  481  [20],  483,  487[35.   36],  488, 

489.  493  [36].  494,  495 
Daniel,  H.,  281  [23],  2g2 
Danielli,  J.  P.,  577[i5].  5S0,  633,  63S 
Danielson,    L.,    2o[g8],    21  [98,    103],    2S^ 

1 15  [23].   -f-f"^',    i4o[i5],    141  [15].    144. 

145,   j66,    i69[io],    i7o[io],    i72[io], 

177  [6,  10],  779 
Das,  N.,478[ii],  48i[ii],  494 
Das,  V.  S.,  344 [49],  404 
Datta,    A.,  241  [2,   3,    4],    243  [4],    244I4], 

245[3.   4].   246 [3,  4],   248 [4],    249 [4], 

250 
Davenport,  H.  E.,  282  [89],  -'9,?,  385  [159], 

387  [162],  407,  416  [33],  417  [33].  -/-'•^■, 

449.  450  [0],  453 
Davies,  B.  H.,  275  [6],  J76 
Davies,  R.  E.,  4  [41,  42],  27,  43  [41],  ^o 
Davis,  B.  D.,  573  [i,  2],  574  [4].  57f>[2,  lol, 

577  [13].  579,  583.  599 
Davis,  J.,  4[4o],  27,  42 [30],  30 
de  Duve,  C.,  ig[92],  24 [92],  2S 
DeHarven,  E.,  482  [24],  4g4 
den  Hartog,  H.,  286  [48],  2g3 
DeLey,  J.,  585  [35].  599 
Dellinger,  O.  P.,  552  [8],  555 


DeAIarco,  C,  492  [47],  -/9  5 

Deul,  D.,  182,  iSg 

Devlin,  T.  M.,  32  [2],  34  [2],  35  [2],  49 

Dickens,  P.,  i6g,  J79 

Dieterle,  B.  D.,  387,  401,  402,  40 j 

Di  Sabato,  G.,  4 [44],  27 

Dochy,  R.,  585  [35].  599 

Doeg,  K.  A.,  103  [3],  104 [3],  117,  253  [i,  2, 

5].  254[5],  257 [5,  12],  258 [5],  260 
Donaldson,  K.  O.,  182  [4,  5],  1^9 
Doudoroff,  AI.,  369  [125],  406 
Douglas,  H.  C,  309  [40],  324 
Drews,  G.,  309 [16],  32^ 
Drysdale,  G.  R.,  266 [3],  267 
Dujardin,  P.,  549,  335 
Durhin,  R.  P.,  628,  630 
Durham,  L.  J.,  34o[i5],  39S[i5].  4"3 
Duysens,   L.    X.    AE,    283  [33],    J'a?,    357, 

406 
Dwyer,  P.  P.,  284 [40],  286,  2g3 


E 


Ivbert,  J.  D.,  S24,  ,5-' ,5 

Pdsall,  J.T.,  331  [12],  337 

Eeg-OIofsson,  ().,  5  [58],  27,  156  [43],  767 

Eggman,  L.,  633  [13],  63S 

Ehrenberg,  A.,  286 [47,  48],  2g3 

Ehrlich,  B.,  323  [is],  72? 

Eiler,  J.  J.,  7[69].25[69],  -7 

Elbers,  P.  P.,  279  [15].  287  [15],  289  [15], 
2g2 

Eldjarn,  L.,  492  [46],  -/95 

Elowe,  D.  G.,  ii6[25],  117,  iiS 

Elson,  D.,  472  [5],  473 

Elvehjem,  C.  A.,  244 [8],  248 [8],  230 

Emerson,  R.  L.,  308,  324,  399,  40S,  425, 
4^9 

P>ickson,  R.  E.,  349 [81],  403 

Plrnster,  B.  B.,  175  [15],  ^79 

Ernster,  L.,  4 [35-39.  47].  5  [35.  37.  50,  51, 
53,  54.  58],  6  [61,  62],  7  [75],  9  [84], 
"[75].  i2[75].  13  [84],  15  [85],  i6[75], 
i8[53,  85.  90],  i9[38,  39.  47.  93.  95]. 
2o[53,  96,  97,  98],  21  [98,  103],  24 
[38,  39.  47.  51.  53.  85,  93.  95,  i09, 
no,  in],  25[50,  51,  53,  85,  115,  118], 
27,  2,V,  jg,  43  [42],  30,  76,  77,  80,  S3, 
87,  g2,  99,  gg,  115  [23],  //<'^,  121  [11], 
134,  i39[6,  7],  i4o[i3,  14,  15].  141. 
142,  144,  145,  147,  i5o[33],  151, 
1 52  [7.  34].  153  [34.  38,  40],  1 56  [43], 
i65[52,  53],  166,  167,  i69[io],  170 
[5,  9.  10],  174,  175  [15],  i77[4,  6,  9, 
10],  J79,  193  [5,  6],  i94[6],  195  [5,  12], 
i96[6],  i97[6],  I99[i2],  201  [n],  202, 
227  [5].  -'J9.  255,  260,  440,  441 

Esau,  K.,  344 [48],  404 

Estabrook,  R.,  79  [13],  S^,  106  [13],  109 
[17].  iiS 

Evans,  AL,  347  [61],  403 

Eyring,  H.,  34o[i2],  403 

Eyster,  H.  C,  289  [56],  29J 


648 


AUTHOR   INDEX 


Falcone,  A.  B.,  25  [113,  114],  2y,  34[i2], 
50,  266 [3,  5],  26y 

Farr,  A.  L.,  53  [4],  6y 

Farrant,  J.  L.,  606 [9],  616 

Fawcett,  D.  W.,  566[5],  566 

Feldott,  G.,  5,  26 

Ferrari,  R.  A.,  281  [23],  292 

Ficq,  A.,  476  [3],  494 

Fink,  J.,  4[46],  27,  53  [i],  56  [i],  61  [i], 
67,  87[ii],  91 

Fisher,  W.  D.,  480  [16],  494 

Fluharty,  A.  L.,  257  [13],  260 

Fogg,  G.  E.,  378  [152,  153],  407 

Folkers,  K.,  349  [81],  405 

Fonnesu,  A.,  4  [41,  42,  44],  -'7,  43  [41],  50 

Forster,  Th.,  327  [6],  337 

Ford,  L.,  4 [34].  ~7 

Forro,  F.,  Jr.,  313  [13].  .)-i 

Fraenkel-Conrat,  H.,  607  [14],  616 

Franck,  J.,  340 [9],  403 

French,  C.  S.,  321  [18],  3^4 

Frenkel,  A.  W.,  279  [9,  10],  285  [43],  -'9J, 
293,  295,  298  [3,  31],  299 [6,  8],  301 
[8,  14,  15],  302[i5,  16,  18],  303,  304, 
307,  308,  309  [22,  25],  3-'4,  346,  347, 
371  [135],  373.  378[i5i].  404,  405, 
40-j,  411,  413  [4,  9].  4-7.  4-S,  431. 
437.  438  [17],  440,  441 

Frey-WyssHng,  A.  J.,  403,  40S 

Fromageot,  C,  333  [14],  337 

Friedkin,  M.,  46,  50 

Fuller,  R.  C,  3o8i7],  309[7].  3i5[i.  8,  23, 
24],  323,  324,  34o[2o],  341  [28],  347. 
348  [20I,  366  [68],  403,  404,  405 

Fynn,  G.  H.,  182  [11],  183  [11],  184,  iSc, 


Gaflfron,  H.,  347[72].  37i[i33.  i34].  378, 

405,  407 
Gale,  E.  F.,  574,  .579.  583  [i4,  15].  59'^' 
Gamble,  J.  L.,  Jr.,  32  [2],  33.  34[2],  35  [2], 

49,  89,  9-' 
Garrett,  R.  H.,  182  [5],  iSg 
Gav,  H.,  481  [17],  494 
Geiler,  D.  M.,  278  [2,  4],  292,   347,   349, 

351  [66],   361,   405,  4i4[ii],  4i7[ii]. 

428,  432  [9],  437,  440,  441 
George,  P.,  285,  291,  29J,  294 
Gerretsen,  F.  C.,  29J 
Gest,   H.,    369[i3i,    132],    374[i38].    378, 

379[i49],  383  [132,  157],  407 
Gewitz,  H.-S.,  4i5[3i].  4i7[3i].  42o[3i], 

421  [31],  425  [31].  42S 
Gianetto,  R.,  19  [92],  24 [92],  2S 
Gibbons,  I.  R.,  560 [7],  563  [6],  ,566 
Gibbs,  M.,  303  [20],  304,  34o[i7,  19],  403 
Gibson,  J.  F.,  285,  293 
Gilvarg,  C,  573  [i],  579 
Giovanella,  B.,  492  [47],  495 
Giuditta,  A.,   106 [14],   ii4[2o],   JiS,    140, 

166 


Glenn,  J.  L.,  104,   io8[ii],   i09[ii],  117, 

254 [6],  260 
Glock,  G.  E.,   121  [12],  134,   i69[7],   177, 

179,  208,  209,  212, 224 
Glynn,  I.  M.,  629[i5],  631 
Godman,  G.  C,  48o[i3],  494 
Goedheer,  J.  C,  327 [S,  8],  329[8],  337 
Goldacre,    R.   J.,    552 [11],    555,   633,    634 

[5].  636[4.  5.  7].  637[4,  5]>6j5 
Golder,  R.  H.,  88,  92 
Goldman,  D.  E.,  628,  631 
Goldstein,  L.,  527 [3],  533,  534 
Good,  N.,  417 [46],  429 
Goodwin,  T.  W.,  271,  272,  274[3,  4], 275 

[5.  6],  2y6,  28o[i6,  17],  292,  326,  33(> 
Gotterer,  G.  S.,  5  [48],  27,  47  [49],  48  [53], 

5",  51 
Govindjee,  R.,  425  [59,  60],  429 
Grabe,  B.,  25  [117,  118],  29,  153  [39,  40], 

167 
Granick,  S.,  315,  337,  606 [7,  10],  616 
Gray,  J.,  558[8,'9],  563  [8,  9].  5^6 
Green,  D.  E.,  i03[3],  io4[3,  11],  io8[ii], 

i09[ii],    117,    i85[i4,    24],    i88[24], 

1S9,    190,    207,    208,    214,    215,    224, 

241,  250,  253  [i],  254[6],  256,  257[9], 

260 
Green,  H.,  577  [13],  579,  583,  599 
Gregg,  C.  W.,  38 [20],  50 
Greig,  M.  E.,  629 [8],  630 
Greville,  G.  D.,  4 [43],  27,  42  [32],  43  [37], 

50,  56 [6,  7],  67,  76 [11],  83,  87,  91 
Griffin,  J.  L.,  549  [10],  553  [10],  555 
Grimstone,  A.,  560 [7],  566 
Gross,  P.  R.,  481  [22],  485  [22],  488,  494, 

495 
Guerra,  F.,  4[46].  27,  53  [i],  56[i],  61  [i], 

67,  87[ii],  91 
Gustafson,    T.,   472 [5],    473,   498 [i],    501 

[i,  2,  3,  6],  502 [2,  4],  506  [5],  507 [2], 

507 

H 

Haans,  A.  J.  M.,  340 [16],  347,  403,  405 
Haavik,  A.  G.,  256 [10],  257 [10],  260 
Hagihara,  B.,  119,  I23[i4],  134,  159,  167, 

227 [6],  239,  301  [11],  303 
Hagstrom,  B.,  467,  468  [27],  474 
Hall,  D.  O.,  346,  361,  362,  388[92],  389, 

390,  391,  406 
Hall,  P.  J.,  549  [5I.  553  [5].  554.  555 
Hall,  W.T.,  374 [137].  ■/'>7 
Hamburger,  K.,  538  [2],  539  [2],  547 
Hammett,  F.  S.,  487,  495 
Hanahan,  D.  J.,  183  [9],  189 
Hanks,  J.  H.,  519,  525 
Hanson,  J.,  563,  566 [11],  566 
Harris,  A.  Z.,  344 [45],  404 
Harris,  D.  L.,  42  [27],  50 
Harris,  P.  J.,  482  [23],  494 
Harris,  R.  S.,  348 [76],  405 
Harrison,  K.,  188  [23],  190,  363,  406 
Harrison,    W.    H,    25  [113],    29,    266    [3], 

267 


AUTHOR    INDEX 


649 


Hartree,  E.  F.,  130,  134 

Harvey,  D.  T.,  281  [23],  -'9-' 

Hase,  E.,  487  [39],  495 

Hassan,  M.,  438  [22],  441 

Hatefi,  Y.,   162,  16-,   188,   /90,  210,  -'-'5, 

256  [10],  257  [10],  2bo 
Heath,  O.  V.  S.,  369 [127],  406 
Heber,  U.,  427  [6,  63],  4^<^ 
Hecht,  L.,  1 1  [81],  -'.V 
Heilbrunn,  L.  V.,  466,  -/;.?,  S49[i2],  555 
Hendlin,  D.,  184,  iSg 
Held,  H.  W.,  227  [11,  12],  23<) 
Hess,  B.,  3,  4[8,  12],  -^6,  88,  89,  9J,   174, 

179 
Hickman,    D.    D.,    279  [9,    10],    -^9-%    295, 
298 [3,  31],  299  [6],  joj,  jo_/,  309  [22, 

25I  .?-V 
Hill,  R.,  282[29,  31],  288,  291,  -'9,),  340, 

353  [97],  357 [97].  387  [162],  7'A?,  ^"7, 

411,    4i4[i6],    4i5[i6,    18],    417U2]. 

4^7,  42S,  449,  ^,5.? 
Hixon,  W.  S.,  163,  ibj 
Hjorth,  E.,  349  [77],  405 
Hoch,  F.  L.,  4,  6,  26 
Horstadius,  S.,  472 [9],  473 
Hoffman,  J.  F.,  627 [21],  630,  631 
Hoffmann-Berling,  H.,  ^63  [10],  566 
Hogben,  C.  A.  M.,  628 [6],  630 
Hogeboom,  G.  H.,  186,  iSg 
Hokin,  L.  E.,  S79[i8],  ^So 
Hokin,  M.  R.,  S79[i8],  ^!^o 
Holland,  W.  C,  629  [8],  630 
Hollunger,  G.,  24,  2S,  119,  i2o[3],  121  [2], 

^.?7,  139,  153,  i56[io],  161,  162,  j66, 

193,  200 [2],  202,  215,  217,  223,  22^, 

227[7,  9],  23Q,  301  [10],  3>>3 
Holter,  H.,  605  [i],  606  [5],  615  [17],  6/6 
Holton,   F.  A.,  43,  50,  87,  92,  95  [3],  96, 

98[5],    99,    100,    208,    209,    210,    215, 

224 
Holz,  G.  G.,  Jr.,  547 [3].  54''^ 
Holzer,  H.,  175  [12],  J79 
Horecker,  B.  L.,  341,  397  [166],  399 [166], 

404,  407 
Horio,  T.,  281  [27],  287[27],  289[27],  291 

[27],  -'93 
Hoshikawa,  H.,  48  [51],  30 
Hotchkiss,  R.  D.,  537,  i-/**' 
Howard,  A.,  48o[i2],  404 
Howard,  R.  B.,  24,  ^.S" 
Howard,  R.  L.,  104 [7],    109  [7],    116,    117, 

117,  iiS 
Hiilsmann,  W.  C,  98[5],  99,  209[i5],  210 

[15],  215  [15],  216,  224,  223 
Huennekens,    F.   M.,   207,   208,   214,    215, 

--'4 
Huff,  J.  \V.,  66 [13],  67 
Hulcher,  E.  H.,  320 [26],  324,  324 
Hunter,  F.  E.,  4[34,  40],  27,  163,  167 
Hunter,   E.  E.,  Jr.,  4 [45,  46],   7  [65!,   27, 

33  [11],    42  [30],    50,    53  [i].    56  [i],    61 

[i],  67,  87,  91,  221  [25],   -'-'5,  244[9], 

248  [9],  -50 
Hurwitz,  A.,  4 [46],  27,  53  [i],  56  [i],  61  [i], 

67,  87 [II],  9r 


Hurwitz,  J.,  341  [29],  404 
Hutchinson,  D.  W.,  415  [23],  42S 
Huxley,  H.  E.,  563,  566 [11],  566 


I 

Igo,  R.  P.,  183  [9],  /.V9 
Ikkos,  D.,  6 [63],  27 
Ingram,  D.  J.  F.,  285,  -'9J 
hnmers,  J.,  472  [10],  473 
Inoue,  S.,  481  [21],  482,  404 
Irvine,  D.  H.,  285,  291,  2^3,  2<j4 


J 

Jacob,  F.,  472  [11,  20],  473,  474 

Jacobs,  M.  H.,  517,  323 

Jacobson,  K.  B.,  121  [13],  134,  i77[i3], 
170,  208,  209,  210,  212,  224 

Jarnefelt,  J.,  185  [24],  188  [24],  J90 

Jagendorf,  A.  T.,  346,  347,  348,  349  [74], 
356.  360 [74],  384 [104],  385  [104], 
388 [55],  390,  392,  4'J4,  405,  406,  407, 
4i4[i4],  415  [26-29],  417U0.  41. 
43],  418  [26-29],  -/-'.V,  432,  440,  446, 
446,  451,  433,  455  [2,  3,  4,  5,  8],  459 

Jakoby,  E.  B.,  341  [30],  404 

Jailing,  O.,  19 [93].  24 [93].  -'**'.  12.1  [i\],  134 

James,  T.  W.,  487 [40],  493 

James,  W.  O.,  344[49],  365  [120],  404,  406 

Jensen,  E.  V.,  488,  492,  493 

Johnson,  C.  M.,  356[ioi],  706 

Johnson,  E.,  597  [47],  599 

Johnson,  D.,  53,  67,  76  [10],  77  [10],  S3, 
1 59 [46],  167,  265  [i],  266  [i],  -'67 

Johnson,  J.  A.,  629 [12],  631 

Johnston,  J.  A.,  •?o-?[28],  304 

Jones,  L.  C,  18  [90],  -'^' 

Judah,  J.  D.,  3,  26,  208,  221  [26],  224,  223 

Judis,  J.,  378 [144],  407 

Jurtshuk,  P.,  256 [10],  257 [10],  260 


K 

Kadenbach,  B.,  24[iii],  2S 

Kahn,  J.  S.,  455  [8],  459 

Kallio,  R.  E.,  s77[ii],  579<  s83[25],  399 

Kamen,  M.  D.,  278[3],  28o[i8,  19],  281 
[26,  27],  282[i9,  28],  283[3,  37,  38], 
287  [27,  37],  289 [26,  27],  290 [19,  60], 
291  [27],  292,  293,  323  [27],  3-'4,  347, 
349,  358,  374[i38],  378[i38,  141,  142, 
143,  149],  379  [149].  405<  406,  407, 
4i4[i2],  4i7[i2],  425  [57],  4^''',  4-^9, 
438  [23],  441 

Kameyama,  T.,  383  [156],  407 

Kamiva,  N.,  q49[ii],  5So[i3],  333,  635 
[8,  9],  6j.v 

Kaplan,  N.  O.,  5,  27,  74 [4],  S2,  119  [8], 
121  [13],  134,  I77[i3],  ^79,  208 [5,  14], 
209,  210,  212,  214,  224 

Karunairatnam,  M.  C,  37o[i32],  383 
[132],  407 


650 


Kates,  M.,  281  [25],  293 
Katoh,  S.,  282 [30],  2g3 
Kaufman,  B.  T.,  119  [8],  134,  208  [5],  214, 

224 
Kaufmann,  B.  P.,  481  [17],  4<^4 
Kaufmann,  B.  T.,  5,  2y 
Kawamura,  N.,  487  [36],  488,  493  [36].  4'J5 
Kay,  L.  D.,  344 [45].  404 
Kearney,  E.  B.,  io4[8],  ii4[2i],  117,  iiS, 

198,     202,     253  [4],     254  [4],     257  [4], 

258 [4],  -'60 
Kegel,  L.  P.,  323  [15],  323 
Keilin,  D.,  592,  5gg 
Kellenberger,  E.,  313  [28],  324 
Kendrew,  J.  C,  329 [10],  337 
Kennedy,  E.  P.,  3  [4],  4[4],  -'6,  46[47],  .5''' 
Kepes,  A.,  583,  599 
Kessler,  E.,  289,  -'9J 
Kielley,  R.  K.,  7 [64],  8  [80],  11  [80],  16,  17, 

19,  20,  27,  28,  144,  j66 
Kielley,  W.  W.,  7  [64],  8  [80],   1 1  [80],   16, 

17,  19,  20,  27,  28,  144,  166 
Kilian,  E.  F.,  560,  566 
Kimura,  T.,  109  [17],  iiS,  440,  441 
King,  N.  K.,  286,  2g3 
King,  T.  E.,  i04[7],  109 [7],  116,  117,  117, 

iiS,  592,  599 
King,  T.  J.,  472,  473 
Kinnander,  H.,  498[i],  501  [i,  2,  6],   502 

[2],  507  [2],  507 
Kinsolving,  C.  R.,  579  [19],  ,5''*''^' 
Kirby,  G.  W.,  363  [117],  406,  415  [22],  42S 
Kirschner,  L.  B.,  629 [9],  630 
Klemperer,  H.  G.,  4,  26 
Klingcnberg,   M.,   5  [51].   25  [51,    108],   25 

[51],   27,   2S,    ii9[5,   6],    121,    133  [5]. 

134,  139.  152,  153.  -^66,  167,  193,  201, 

202,  208,  209,  210,  212,  215,  217,  222, 

224,  224,  225,  227 [4,  5,  8,  10,  ir,  12], 

23o[4,   13,   14,   15],  231  [15],  238[i9], 

23g,  302,  303  [19],  304 
Knox,  W.  E.,  66[ii],  67 
Koch,  H.,  629 [11],  ^31 
Koefoed-Johnsen,     V.,     627  [4],     629  [16], 

6jo,  631 
Kogut,  M.,  577 [12],  579,  583,  599 
Kondo,  Y.,  383  [156],  407 
Korkes,  S.,  369 [130],  407 
Kornberg,  A.,  339,  347  [3].  403 
Kornberg,  H.  L.,   169  [14],  i7g,  238  [18], 

239 
Krebs,  H.  A.,  74[7],  82,  169,  179,  238,  2J9, 

573.  579 
Krippahl,   G.,  415  [31],   4i7[3i],  42o[3il, 

421  [31.    52,    53,    54],    422,    425  [31I, 

426  [54],  -/2cV,  729 
Kriszat,  G.,  474 
Krogmann,  D.  W.,  289 [59],  29 j,  346,  347 

[56!,  388  [56],  390  [56],  393  [165],  404, 

407,  415  [26,  28,  29],  4i7[36,  37].  418 

[26,  28,  29],  419U8-51],  428,  42g,  455 

[2],  459 
Krotkov,  G.,  340 [21],  403 
Krueger,  S.,  253  [2],  260 
Kruse,  I.,  349 [77],  4'^5 


AUTHOR    INDEX 


Krygier,  A.,  487  [37],  495 
Kuff,  E.  L.,  6o7[ii],  6x6 
Kunisawa,  R.,  295,  299,  303,  369  [125],  406 
Kupke,  D.  W.,  325  [i],  Jj6 
Kuroda,    K.,    549[i3],    55o[i3],    555,    635 
[8,  9],  6ji' 


L 

LaCour,  L.  F.,  481  [19],  797 

Land,  D.  G.,  28o[i6],  292 

Lang,  H.  M.,  285,  29J,  302 [17],  304,  402 

[148],  407,  416 [32],  42S,  449,  453 
Lansing,  A.  L,  468,  473 
Lardy,  H.  A.,  3,  4[9,   10,   11],  7 [67,  79], 

26,  27,  28,  56 [9],  67,  76  [8,  10],  77 [10], 

82,    83,     159,     162,     167,    227[l],     2J9, 

244[8,  10],  248[8,  10],  250,  265  [i,  2], 
266 [i],  267,  438  [21],  44^ 

Larsen,  H.,  308  [29,  30,  31],  324 

Larson,  A.  D.,  577[ii],  579,  583  [25],  599 

Lascelles,  J.,  275,  276 

Latimer,  Paul,  327  [7],  337 

Laufberger,  M.  L.,  606 [8],  6/6 

Lavollay,  J.,  356,  406 

Leblond,  C.  P.,  527[7],  53o[7],  534 

Lee,  K.-H.,  7  [69],  25  [69],  27 

Leech,  M.  R.,  365,  406 

Lehman,  L  R.,  182,  i8g 

Lehninger,  A.  L.,  4[i8,  21,  22],  5  [29,  48, 
55,  56,  60],  6 [29],  7 [70],  15,  16,  17 
[88],  19 [91,  94],  24 [91,  94],  25  [26,  29], 
26,  27,  28,  31  [i],  32[2],  33,  34[2,  13, 
14,  15,  16],  35[2,  16],  36[i6,  19],  38 
[20],  39  [16,  21],  42  [26,  28,  29],  43 
[29,  34,  35,  40,  44],  44 [29,  34,  35,  4°], 
45  [29,  35,  40,  45],  46  [26,  34,  46], 
47  [48,  49],  48  [50,  53,  54],  49  [55,  56], 
49,  50,  51,  53,  56  [5,  8],  61  [10],  67, 
86,  87,  90,  gi,  145  [26],  1 53  [26],  166, 
207,  224,  266 [4],  267,  344 [44].  353 
[44],  404,  438  [22],  441,  458,  459 

Leone,  V.,  470,  473 

LePage,  M.,  281  [23],  2g2 

Lester,  R.  L.,  185  [14],  i8g,  210,  225,  241 
[5],  250,  279,  280(12],  2g2 

Levine,  L.,  278 [8],  292 

Levitt,  L.  S.,  283  [32],  297 

Levy,  J.  F.,  4[46],  27,  53  [i],  56  [i],  61  [i], 
67,  87[ii],  gi,  92 

Lewin,  R.,  371  [i 35],  407 

Lewis,  G.  N.,  352[96],  354[96],  355[96], 
406 

Lillie,  F.  R.,  468,  473 

Lindahl,  P.  E.,  472  [15],  473 

Lindberg,  O.,  4 [47],  7 [75],  9[84],  "[75], 
i2[75],  i3[84],  i5[85],  i6[75],  i8[85], 
i9[47,  93],  24[47,  85,  93],  25[85,  115, 
118],  27,  28,  2g,  121  [11],  134,  145, 
1 53  [38,  40],  167 

Lindstrom,  E.  S.,  378  [150],  407 

Linnane,  A.  W.,  41  [24],  49,  50,  242,  250, 
257  [11],  258  [11],  260 

Lipkin,  D.,  352  [96],  354,  355  [96],  4^6 


Lipmann,  F.,  3  [4],  4,  6,  _'6,  278  [4],  2g2, 
341,  404,  4i4[ii],  417  [11],  428, 
437,  43S[i9],  44^,  521  [3].  5-5 

Litt,  I.,  6o7[i3],  6/6 

Livingston,  R.,  352  [93].  7'^^ 

Ljunggren,  M.,  2o[98],  21  [98,  103],  25 
[115],  -'<V,  J9,  1 15  [23],  11^.  1 40  [15]. 
141  [15].  144,  145,  -^66,  i69[io],  170 
[10],  172  [10],  177  [6,  10],  J79 

Low,  H.,  4 [35,  36].  5  [35],  6  [61,  62],  7  [75, 
76,  77,  78],  8  [76],  9  [84],  "[75,  76, 
77],  12  [75,  76,  77],  13  [84],  15  [85], 
i6[75-78],  i8[76-78,  85,  89],  i9[93], 
24  [76-78,  85,  93],  25  [85,  118],  -'7, 
^8,  -'9,  43[42],  50,  121  [11],  134,  145 
[28],  150,  151,  1 53  [38,  40],  1^7,  467. 
474 

Loomis,  W.  F.,  512,  513  [3],  5 17  [6],  521 
[11,  13],  524b],  5^5 

Loomis,  W.  F.,  Jr.,  521  [11],  5^^ 

Lorch,  L  J.,  552 [11],  555,  633,  636 [4], 
637U],  6J.V 

Losada,  M.,  342  [34-36],  348  [35],  366 
[121],  367[i2i],  368,  369[i2i],  370 
[121],  372,  373[i36],  374[i36],  376, 
377,  378[i36,  i54,  155],  380,  381, 
382,  385[i36],  386[i2i],  396,  397, 
398,  399[35,  121],  40o[i2i],  401,  402, 
404,  406,  40 J 

Lowry,  O.  H.,  53,  67 

Luchsinger,  W.  E.,  23  [i  14], -V,  ■!4[i2],  5". 
266  [5],  -'67 

Lucke,  B.,  89,  Q-' 

Luft,  R.,  5  [58],  6  [63],  --;,  1 56  [43],  ^^- 

Lumry,  R.,  340 [12],  4" J 

Lundbiad,  G.,  467,  4JJ 

Lundegardh,  H.,  130,  1J4,  357,  406 

Lusty,  C.  J.,  107  [15],  ttS 

M 

Alaaloe,  O.,  313  [32],  7--/ 
McDonald,  B.  A.,  540 [5],  57^ 
]\IcDonald,  M.,  481  [17],  404 
Macfarlane,  AL  G.,  4[32],  -7 
Alacgregor,  H.  C,  477  [4],  404 
Mackler,  B.,  183  [9],  -r-S'9 
Maclachlan,  G.  A.,  369,  399  [128],  40 j 
McLean,    P.,    121  [12],    134,    i69[7],    177, 

170,  208,  209,  212,  -'-'-/ 
McLeod,  G.  C,  334,  33J 
McMurrav,  W.  C,  76  [10],  77  [10],  v  j,  i  :^g 

[46I,  767,  265  [i,  2],  266  [i],  J67,  433, 

44" 
Marki,  F.,  2o[ioo,  102],  -''V,  115  [24],  iiS, 

I39[2],      I40[2,     21],      141  [2,     21],      ISO 

[21],  166 
Mahler,  H.  R.,    103,    115  [2],    ii6[25,   26], 

1 17,  II J,  iiS 
Maizels,  M.,  634 [i],  63S 
Maley,  G.  F.,  4[9,  10,  1 1],  7  [10],  j6,  56  [9], 

67,  438[2i],  44^ 
Malison,  R.,  4 [45],  -7 
Manton,  L,  478  [8],  404,  563,  .566 
Margulies,  \l.,  41 7  [43],  42S 


AUTHOR    INDEX  65 1 

Markman,  B.,  471,  472[i7,  18],  473,  474 

Marr,  A.  G.,  585  [34],  599 

Marre,  E.,  349  [87],  .^05,  446,  446 

Marshall,  J.  M.,  606 [4],  6x6 

Martin,  E.  M.,  280,  Jgj 

Martin,  G.,  356,  406 

Martius,  C,  3,  4,  2o[99,  100,  loi,  102],  -'6, 

-'V,   ii5[24],  i^S,  i39[i-4],   140,   141, 

149,   i5o[2i,  29],  166,  i6y,  349,  40J, 

415  [25],  ^(J.V 
Mason,  H.  S.,  630,  6j2 
Massey,  \'.,   103,   104,   115,  ii~,  256,  j6o, 

287,   Jyj,   349  [90],   354,   362  [90],   393 

[90],  403 
Mast,  S.  O.,  549  [14],   550,   551,   552  [14], 

553  [15],  555 

Matchett,  P.  A.,  629  [12],  631 

Mathieson,  M.  J.,  575,  579 

Mayer,  A.  ^L,  523  [15],  5-^5 

Mayne,  B.,  417  [36],  42S 

Mazia,  D.,  475  [i],  476  [2],  477  [5,  7],  478  [i] 
48o[i],  481  [18,  20],  482[23],  483  [20, 
25],  486,  487,  488  [30],  489 [43],  490 
[30,  30a],  491  [i],  404,  495 

Mazur,  A.,  607  [13],  6j6 

Mehler,  A.  H.,  417,  42S 

Mercer,  E.  H.,  468,  474 

Merritt,  C.  R.,  579[i9],  .r'^o 

Micou,  J.,  527 [3],  534 

Mihara,  S.,  487  [39],  ^9.5 

Milhaud,  G.,  341  [33],  399[33],  4"4 

Miller,  J.  A.,  281  [23],  -^9-' 

Miller,  S.  L.,  400,  402  [170],  40S 

Millet,  J.,  341  [33],  404 

Minakami,  S.,  104,  105  [12],  117,  iiS 

Minnaert,  K.,  279[i5],  287[i5],  289[i5], 
-9- 

Mitchell,  P..  582[2,  3,  4,  5],  583  [2,  3,  4,  5, 
7,  9,  10,  II,  12,  IS,  16-18,  19],  584 
[18,  27,  30],  585  [2,  4,  18,  31],  586 
[2,  4,  36-38].  587 [2,  4,  36],  588,  592 
[11,  30],  594[i9,  46],  59''',  599 

Miyachi,  S.,  303  [25],  304 

Mondovi,  B.,  492  [47],  4<)5 

Monod,  J.,  472 [11,  20],  473,  474,  S74, 
575  [7,  8],  576  [8],  577  [7,  8,  14],  579, 
5S0,  582,  583  [i,  20,  21],  585  [31], 
588 [i,  40],  3qS,  599 

Mook,  H.  W.,  5i8[7],  323 

Moraw,  R.,  418  [47],  420 

Morimura,  Y.,  487  [39],  ^95 

Morton,  R.  A.,  279 [13],  2<)j 

Morton,  R.  K.,  280,  -"9-',  597  [47],  599 

Moses,  v.,  303  [26],  304 

Moyle,  J.,  583  [9-12,  19],  584[27,  30],  586 
[37,  38],  588,  592  [i  I,  30],  504[i9, 
46],  59''*',  599 

Mudd,  S.  H.,  4,  26 

Miihlethaler,  K.,  403,  40S 

Muller,  A.,  418 [47],  4-'9 

Miiller,  F.  AL,  368 [123],  371  [12^],  406 

Muller,  H.R.,  363  [118],  364,  365,  366,  406 

Muir,  A.  R.,  607  [12],  616 

Myers,  D.  K.,  7  [72-74],  2S,  98  [5],  99, 
209[i5],  2io[i5],  215  [15],  224 


652 


AUTHOR    INDEX 


N 


Nagano,  T.,  56o[i4],  566 

Nakamoto,  T.,  346,  347  [56],  388  [56],  390, 

392,    404,   407,   417E35,    36,    37,    38], 

422  [35],  4^^ 
Nakamura,  H.,  280,  292 
Nakao,  M.,  48 [51],  50,  634[io],  6,38 
Nakao,  T.,  48[5i],  50,  634[io],  638 
Nason,  A.,  i4o[i6-i8],  166,  182,  i8g 
Nass,  S.,  481  [22],  485  [22],  494 
Navazio,  F.,  20 [97],  2S,  140 [14],  166,  175 

[15],  179 

Neubert,  D.,  48  [50],  50 

Neufeld,  E.,  489 [43],  495 

Newton,  G.  A.,  279,  280,  281  [11],  282, 
292 

Newton,  J.  W.,  278 [3,  7,  8],  279,  280,  281 
[11],  282,  283  [3],  292,  347,  349,  374 
[139],  378[i39,  150].  405,  407,  414 
[12],  417  [12],  ^-^V 

Nichols,  P.,  285,  293 

Niemeyer,  H.,  3,  4 [4],  26 

Nilson,  E.  H.,  585  [34],  599 

Nishimura,  M.,  283  [35],  286,  289  [35],  -'yj, 
357,  406,  438,  441 

Nitz-Litzow,  D.,  149 [29],  150 [29],  267 

Northcote,  D.  H.,  449 [4],  453 

Nossal,  P.  M.,  241,  -'50 

Novikoff,  A.  B.,  ii[8i],  --.-V 

Nozaki,  M.,  350,  358,  359,  360,  370,  372, 
373[9i,  114,  136],  374[i36],  375,  376, 
377,  378[i36,  i54,  iSS],  379[i54, 
155],  38o[i54,  155],  381,  382,  383, 
385  [136],  401,  402,  405,  406,  407 


O 

Ochoa,  S.,  341,  344 [42,  47],  384 [42],  397 

[166],  399 [166],  404,  407 
O'Connor,  C.  M.,  88[i8],  92,  99[8],  99 
Ogata,   S.,   350,   358,   359,   360,   366 [121], 

367[i2i],    368,    369[i2i],    370,     373 

[91,    114],    375,    383,    386[i2i],    399 

[121],  40o[i2i],  405,  406 
Oh-hama,  T.,  303  [2s],  304 
Olcott,  H.  S.,  607  [14],  616 
Olson,  J.  M.,  283  [36],  286,  289  [36],  293, 

357,  406,  435,  44^ 
Oparin,  A.  I.,  400,  402  [169],  40S 
Orchard,  B.,  369[i27],  406 
Ottolenghi,  A.,  66  [12],  67 
Overton,  E.,  583,  39S 


Packer,  L.,  43,  50,  86[-j],  87[i2,   15,   i6], 

88[i5,    19],    89[24],    9^,    9-',    95,    98, 

99,  100,  io9[i7],  118 
Pantin,  C.  F.,  521  [12],  525 
Papenberg,  K.,  227  [12I,  239 
Pappas,  G.  D.,  6io[i5],  616 
Pardee,  A.  B.,  198,  202,  279,  28o[i4],  292, 

295  [i],   303,    307,    315  [35],   3-^4,   472 

[20],  474 


Park,  J.H.,  4[i5],  -^6 
Parpart,  A.  K.,  89,  92 
Pavan,  C,  476  [3],  494 

Peck.H.D.,  369[i3i],  378[i44],  383[i57], 
407 

Pelc,  S.  R.,  48o[i2],  494 

Penefsky,  H.,  41,  48  [25],  49 [25],  50,  241 
[i,  2,  3,  4],  243  [4],  244 [4],  245  [3,  4], 
246 [3,  4],  248  [4],  249 [4],  23U 

Perlmann,  P.,  468  [27],  474 

Perry,  R.  P.,  527  [6],  53© [6],  534 

Perry,  S.  V.,  43,  50,  91,  92 

Peters,  R.  A.,  587  [39],  599 

Petrack,  B.,  347,  403 

Pfeffer,  W.,  340 [7],  403 

Phagpolngarm,  S.,  326,  336 

Philpott,  D.  E.,  481  [22],  485  [22],  494 

Pihl,  A.,  492  [46],  -/95 

Pinchot,  G.  B.,  41  [22],  50 

Plaut,  G.  W.  E.,  35,  50 

Plaut,  W.,  533,  534 

Plesner,  P.,  491,  495 

Podber,  E.,  11  [81],  2<S' 

Podoski,  E.  P.,  577  [12],  579,  583,  599 

Polis,  D.  B.,  131,  134 

PoljakoflF-Mavber,  A.,  523  [15],  5-' 5 

Pollard,  C.  J.',  183  [8],  189 

Porter,  H.  K.,  369,  399[i28],  407 

Porter,  K.  R.,  566  [5],  566 

Post,  R.  L.,  579  [19],  580 

Potter,  V.  R\  7  [66,  68],  27,  198,  202 

Pratt,  D.  C,  295  [6,  7],  299 [6],  ,?oj,  378 

[151],  407 
Prescott,  D.  M.,  527  [i],  534,  54o[6],  548, 

633,  6j.S' 
Pressman,   B.  C,    19 [92],   24 [92],   2<S',    119 

[9],  134,  162,  167 
Price,  C.  A.,  4U1],  -'7,  43,  5" 
Prosser,  C.  L.,  553  [15],  555 
Pullman,  M.  E.,  41,  48,  49,  50,  170,  179, 

241  [i,  2,  3,  4],  243  [4],  244U],  245  [3, 

4],  246  [3,  4],  248  [4],  249  [4],  -50 
Pulvertaft,  R.  J.  V.,  634,  638 
Pumphrev,  A.  M.,  181  [i],  i82[7,  11],  183, 

184,    185  [15],    i86[is,    17],   189,   253 

[3],  -'60 
Purvis,  J.  L.,  74[3],  ''^'-,   H9[7],  U4,  208 

[10,  11],  209,  215,  216,  224 

Q 

Quayle,  J.  R.,  341  [28],  404 

R 

Raaflaub,  J.,  4[30,  31,  33],  26,  2j,  42  [31], 
50,  86,  91 

Rabinowitch,  E.  I.,  308 [34],  324,  339,  34° 
[11],  348[75],  399[75].  403,  405,  412 
[5],  425  [59,  60],  4^S,  429 

Rachevsky,  N.,  520 [10],  525 

Racket,  E.,  41,  48[25],  49[25],  50,  89,  92, 
170,  179,  241  [i,  3,  4],  243  [4],  244M, 
245  [3,  4],  246  [3,  4],  248  [4],  249  [4], 
-50,  341,  344U0,  41],  369 [40],  404 


Ramirez,  J.,  358,  -^06 

Randall,  R.  J-.'ssW,  <^7 

Rapkine,  L.,  487,  489,  41)4 

Ray,   B.   L.,  4  [24,  25],   -'6,  42  [28,   29],  43 

[29],  44[2q],  45I29].  5",  86[6],  90,  y/, 

9-' 
Recknagel,  R.  ().,  7 [66],  2-j 
Redfearn,    E.    R.,    181  [i],    182,    183,    184, 

i85[is,  16],  i86[is,  16,  17],  /V'A  2S1 

[3],  -'60 
Reed,  J.  Al.,  24,  2S 
Remmert,  L.  F.,  4 [27],  5  [55,  56],  26,  jj, 

33  [10].  49  [55,  56],  49,  51 
Rendina,  G.,  109  [18],  iiS 
Rhodin,  J.,  563,  566[i5],  566 
Rickenberg,' H.    V.,  574,    575[7],    577[7]. 

579.  583  [21,  22],  592  [44],  599 
Ringler,  R.  L.,  i04[9,  10],  105  [12],  109  [9], 

ii^[igl  117,  iiS 

Ris,H.,47»[gl  494 

Ritt,  E.,  1 39  [12],  153  [12],  -r66,  193  [4],  -'oj, 

2o8[9],     2io[9],     217,     222  [9],     2J4, 

227  [10],  JJQ 
Robertson,  H.  E.,  11  [82],  -'^ 
Robertson,  J.  D.,  23,  49 
Roelofsen,  P.  A.,  369 [129],  7^7 
Root,  R.  K.,  48  [so],  v> 
Rose,  T.  H.,  48 [50],  ju 
Rosebrough,  N.  J.,  54 [4],  67 
Rosenberg,  L.    L.,    303  [22,    23],    304,    34-? 

[38,  39],  348  [38,  39],  366  [38,  39],  404 
Rosenthal,  T.  B.,  468,  473 
Roslansky,  J.   D.,  466,  473,  486  [29],   4Q4, 

550 [7l.  553  [6],  555 
Roth,  L.  E.,  615  [16],  6i6 
Ruben,  S.,  341,  404 
Rubin,  J.,  378,  407 
Runnstrom,  J.,  466 [23],  467,  468  [24,  27], 

470  [21],    472  [21,    25],    473  [22],    474, 

503  [7],  507  [7].  507 
Rustad,  R.  C,  606  [6],  6/6 
Ryan,  J.,  11  [81],  -',V 
Ryter,  A.,  313  [28],  3-'4 


Sachs,  J.,  340 [6],  403 

Sakai,  H.,  487  [35].  489.  495 

Sanadi,  D.  R.,  257  [13],  -'60,  287,  2g3 

Sandritter,  W.,  487  [37],  -/95 

Sands,  R.  H.,  255,  260 

San   Pietro,   A.,   285,    J9J,    302,   304,   402 

[148],  407,  416 [32],  t(-'^',  449.  453 
Sarkar,    N.    K.,    103  [2],    115  [2],    116  [26], 

117,  iiS 
Schachman,  H.  K.,  279,  280 [14],  292,  295, 

303,  307[33.  35].  315  [35].  3-4 
Scherbaum,  O.  H.,  537  [10,   14],   538,   539 

[7].  540,  541.  547  [3.  9].  54^ 
Schmukler,  H.  W.,  131  [17],  134 
Schneider,    M.,   4[25],    5  [60],    26,    27,   42 

[29],   43  [29].   44  [29],   45  [29].   47  [48], 

50,  53  [3].  56  [5],    67,    86  [6],    91,    186, 

iSg 
Schneider,  S.,  175  [12],  179 


AUTHOR    INDEX  653 

Schnek,  G.,  333  [14].  337 

Schoffeniels,  E.,  622  [2],  623  [2],  629  [7,  18, 

19],  630,  631 
Schollmeyer,  P.,  24  [108],  28,  227  [11],  230 

[15],  231  [15],  238[i9],  239 
Schreiber,  S.  S.,  629 [13],  631 
Schroder,  W.,  429 
Schultz,  A.  R.,  4S5,  459 
Schulz,  H.,  6[6i]V-^7  ' 
Schumaker,  V.  N.,  605,  606 [2,  4],  616 
Schutz,  B.,  4[45,  46],  27,  53  [i],  s6[i],  67, 

87[ii],  9^ 

Searls,  R.  L.,  287,  29] 

Sebrell,  W.  H.,  348  [76],  405 

Servettaz,  O.,  349 [87],  -/"5.  446 [3],  446 

Sharon,  X.,  455,  439 

Shorr,  E.,  607  [13],  6/6 

Shunk,  C.  H.,  349 [81],  403 

Siegel,  J.  M.,  378 [142],  407 

Siekevitz,  P.,  7  [68,  75],  11  [75!.  12  [75].  15 
[85],  i6[75l,  18  [85].  24[85],  25  [85, 
118],  27,  2S,  29,  153  [38,  40],  /67,  597 
[48],  599 

Simonson,  H.  C,  7  [68],  27 

Singer,  T.  P.,  I04[8,  9,  10],  105  [12],  106 
[14],  io7[i5],  i09[9,  17,  18],  //;, 
118,  198,  202,  253,  254 [4],  257  [4], 
258  [4],  260,  440,  441 

Sistrom,  W.  R.,  278[6],  292,  295  [4],  303 

Sjostrand,  F.  S.,  6  [61],  27,  566  [16],  566 

Skou,  J.  C.,  629[i7],  631 

Slater,  E.  C,  5  [59],  7  [72-74].  -'7.  -■>'.  33, 
35.  49,  74[6],  76[9],  79.  ■>-'.  '''3,  85  [2], 
87,  9/,  98  [5],  99,  1 19 [10],  /66,  /67, 
172,  /79,  i82[6],  1S9,  209[i5],  210 
[15],  2i5[i5],  216,  2i8[23,  24],  221 
[23,  27],  224,  223,  415  [24],  v-""' 

Slein,  M.  W.,  207 [i],  224 

Slenczka,  W.,  I39[ii,  12],  i53[ii,  12], 
^66,    193  [3,   4],   202,   208  [8,   9],   209, 

2I0[9],     212,     215,     217,     222[9],     224, 

224,  227  [10],  239 

Smillie,  R.  j\I.,  34o[20,  21],  348 [20],  4"3 
Smith,    J.   H.    C,    3o8[37],    3i9[37].    321 

[36],  3-^4,  325  [i.  2,  3].  327  [3.  7.  8,  9]. 

329  [8],  336,  337 
Smith,    L.,    283  [34],    289  [55],    293,    358, 

406,    4i4[io],    4-'^,    432 [8,    lo],    438 

[10],  440 
Solomon,  A.  K.,  628  [5],  630 
Sonneborn,  T.  M.,  510,  ,5-5 
Spencer,  A.  G.,  4 [32],  27 
Spikes,  J.  D.,  34o[i2],  403 
Spindel,  \V.,  488  [42],  795 
Stalfelt,  M.  G.,  369 [126],  406 
Stanier,  R.  Y.,  271,  274,  276,  278  [6],  279, 

28o[i4],  292,  295  [i,  4],  303,  307[33. 

35],   3o8[37].    3i5[35l    3i9[37],    J-V. 

369,  406 
Stauffer,  J.  F.,  308,  3-'4,  399 [167],  4oS 
Steere,  R.  L.,  363  [118,  1 19],  364[ii8,  1 19], 

365,  366,  406 
Steffenson,  D.,  478 [10],  494 
Stein,  A.  M.,  74 [4],  S2 
Stein,  \V.  D.,  577  [15],  5S0 


654  AUTHOR    INDEX 

Stephen,  B.  P.,  583  [13],  585  [13],  59^ 
Stern,   B.  K.,  4i7[35>   44],  422,  424 [SSl- 

428,  429 
Stern,  H.,  487,  495 
Storck,  R.  L.,  585,  599 
Stout,  P.  R.,  356'[ioi],  406 
Stracher,  A.,  331,  337 
Straub,  F.  B.,  103,  I04[i],  117 
Strecker,  H.  J.,  140,  166 
Strufe,    R.,    20 [99],    2S,    139  [i],     140 [i], 

141  [i],  166 
Suddath,  H.,  56 [8],  67,  438  [22],  441 
Swann,  M.  M.,  487,  490,  494 
Swanson,  M.  A.,  7  [71],  ii[83],  25  [83],  -'6' 
Szabolcsi,  G.,  5  [58],  27,  67,  156  [43] 
Szent-Gyorgyi,  A.,  355  [98],  706 


T 

Tagavva,  K.,  378  [i 54-  155].  379 [i 54,  155], 
38o[i54,  155],  381,  382,  401,  402, 
407 

Talalay,  P.,  175  [17],  179 

Tanner,  H.  A.,  2891:56],  -'9J 

Tamiya,  N.,  383  [156],  4'^7,  487  [39],  495 

Tapley,  D.  F.,  4,  25,  26,  29,  43  [36],  30, 
90,  92 

Tappel,  A.  L.,  86  [7],  9 J 

Tatibana,  M.,  48  [51],  jo 

Taube,  H.,  284 [39],  293 

Taylor,  E.  W.,  48o[i4],  482  [14],  494 

Taylor,  J.  F.,  207  [i],  224 

Taylor,  J.  H.,  477 [6],  494,  527 [2],  534 

Tedeschi,  H.,  42  [27],  5" 

Tevrell,  A.  J.,  i4o[i7],  166 

Than-Tun,  378  [152,  153],  4<'7 

Theorell,  H.,  286^7],  293 

Thiele,  E.  H.,  66[i3],  67 

Thimann,  K.  V.,  341,  403 

Thomas,  J.  B.,  279[i5],  287[i5],  289[i5], 
292,  307.  324,  34o[i6],  347,  403,  405, 
425  [60],  429 

Thompson,  T.  E.,  48 [53],  51 

Thormar,  H.,  539  [11],  54S 

Thome,  C.  J.  R.,  74[s]',  81  [s],  S2 

Tisdale,  H.  D.,  185  [24],  188  [24],  190 

Titchener,  E.  B.,  41  [24],  49,  50 

Todd,  A.,  363  [117],  4o(>,  415  [22,  23], 
428 

Tolbert,  N.  E.,  340 [18],  403 

Tolmach,  L.  J.,  344 [43],  384 [43],  404 

Tosteson,  D.  C,  627  [21],  630,  631 

Treadwell,  F.  P.,  374 [137],  407 

Trebst,  A.  V.,  34o[22,  23],  342 [34-36,  37], 
343  [37],  344[37],  347[22,  23],  348 
[35,  37],  366[37,  121],  367[i2i],  368, 
369[i2i],  37o[i2i],  386[i2i],  396, 
397,  398,  399[35,  121],  40o[i2i],  403, 
404,  406 

Trehahne,  R.  W.,  289,  293 

Trenner,  N.  R.,  349  [81],  405 

Trudinger,  P.  A.,  341,  399  [32],  404 

Ts'o,  P.  O.  P.,  633  [13],  63S 


Tsujimoto,  H.  Y.,  342  [37],  343  [37],  344 
[37],  346,  348[37],  361,  362,  366[37], 
388,  389,  390,  391,  404,  406 

Tyler,  D.  B.,  198,  202 


U 

ul  Hussan,  M.,  56 [8],  67 

Umbreit,  W.  W.,  308  [17],  324,  399  [167], 

40S 
Urey,  H.  C,  400,  402  [170],  408 
Ussing,  H.  H.,  627  [4],  623  [3],  630 


V 

Vacum,  E.  C,  308  [30],  324 

Van  der  Kloot,  W.  G.,  629  [14],  631 

Van  der  Leun,  A.  A.,  340 [16],  403 

Van  der  Veen,  R.,  417  [45],  429 

van  Niel,  C.  B.,  303  [27],  304,  308  [30],  324, 
368[i22,  124],  371  [122],  406 

van  Tubergen,  R.  P.,  313  [13],  J- J 

Vatter,  A.  E.,  295,  303,  309 [39,  4°],  3^4 

Veeger,  C,  287  [50],  293 

Veldstra,  L.,  182 [6],  189 

Velick,  S.  F.,  207 [i],  224 

Vennesland,  B.,  280,  292,  346,  347,  388 
[56],  39o[56],  392,  393  [165].  404, 
405,  407,  417 [35,  37,  38,  44],  419 
[49],  422  [35,  55],  424 [55],  4^^,  429 

Vernon,    L.    P.,    103  [2],    115  [2],    116  [26], 

117,    118,    282[28],    293,    300,    301  [13], 

302,    303,    304,    373,    386,    387  [160], 

401  [160],  407,  438  [23],  441,  446,  44^^ 
Vinograd,  J.,  633  [13],  638 
Vishniac,  W.,   3i9[i4],  J-i,   344[42,  47], 

384[42],  397[i66],  399[i66],  404,  407 
Volker,  V.,  415  [31],  4i7[3i],  42o[3i],  421 

[31],  425  [31],  ■^-'^' 


Wachsman,  J.  T.,  585,  599 

Waddington,  C.  H.,  472,  474 

Wadkins,  C.  L.,  5  [56],  15,  16,  27,  28,  32 

[2],  33  [10],  34[i5,   16],  35  [2,  16],  36 

[16,    18,    19],   38[2o],   39[i6,   21],   49, 

50,  266  [4],  267,  458,  459 
Walker,  D.  A.,  417 [42],  42S 
Walker,  D.  E.,  45  [45],  49 
Walker,  P.  G.,  156,  167 
Warburg,  O.,  355,  406,  415  [31],  4i7[3i], 

420,  421,  422,  425,  426[54],  427[64], 

428,  429 
Warringa,  M.  G.  P.  J.,  ii4[2o],  iiS 
Watanabe,  S.,  89 [24],  92 
Wattiaux,  R.,  19 [92],  24[92],  28 
Weber,  F.,  184,  189 
Weber,  H.  H.,  485,  494 
Weibull,  C,  583  [8],  584[28,  29],  585  [33], 

59-'^,  599 
Weis,  D.,  303  [30],  304 


Weiss,  U.,  576 [lo],  579 

Weissbach,  A.,  341  [29],  404 

Wellman,  H.,  7  [67],  27,  76  [8],  ,v.',  227  [i], 
2J9,  244 [10],  248 [10],  2 so 

Wenner,  C.  E.,  178,  ij(j 

Went,  H.  A.,  486  [28],  487,  494 

Wessels,  J.  S.  C,  346,  347,  349 [85],  363, 
388  [54],  390,  392 [62],  404,  405,  415 
[21,  30],  416  [30],  417  [45].  42S,  420, 
444 [i],  446 

Whatley,  F.  R.,  285  [42],  288  [53],  29.?,  303 
[22],  304,  3o8[2,  3],  J2J,  34o[i3,  15, 
22,  23],  343[38,  39].  345[i3.  50.  5i- 
52,  53].  346[i3],  347[i3,  22,  23,  73], 
348[i3,  38,  39,  51,  52,  53],  349[86, 
88,  89],  351  [50,  88,  89],  352[95],  355 
[100],  356  [103],  357,  358  [50],  363 
[38,  39],  366 [38,  39,  95],  369  [95], 
384,  385  [103,  104.  158],  386,  387, 
388[i3],  391  [89].  394,  395,  398[i3, 
15],  400 [95],  401,  402  [86],  403  [95], 
403,  404,  405,  40^,  407,  41 1  [3],  413 
[6-8],  4i4[3,  8],  415  [8],  416 [34],  4-7. 
4-'''^,  431  [i],  44'^,  449  [2],  451  [7],  453. 
455  [i],  459 

Widdas,  W.  F.,  577  [16],  ,rSo 

Williams,  A.  M.,  320,  324,  347,  705 

Williams,  G.  R.,  33  [8],  49,  72  [2],  S2,  85  [i], 
87,  97,  96,  99,  153  [36],  767,  185,  186 
[19,  20],  1S9,  190,  208,  212,  214,  215, 
224,  22s,  227 [2,  3],  235  [16],  236, 
^39 

Williams,  X.,  547  [3,  9],  54S 

Williams,  R.  J.  P.,  290,  291,  293,  294 

Williams-Ashman,  H.  G.,  3,  26,   175  [17], 

Willmer,  E.  X.,  ^07,  507 
Wilson,  A.  T.,  344 [45],  ^o^ 


AUTHOR    INDEX  655 

Wilson,    P.   W.,    374[i39],    378[i39.    150!, 

407 
Wilson,  T.  H.,  622,  630 
Winfield,  M.  E.,  286,  293 
Wintermaus,  J.  F.  G.,  281  [22],  292 
Wiseman,  G.,  622,  630 
Wiser,  R.,  281  [22],  292 
Wiss,  O.,  184,  1S9 
Witt,  H.  T.,  418,  429 
Wolf,  D.  E.,  349  [81],  40s 
Wolfe,  R.  S.,  295,  303,  309 [39,  40],  324 
Wolpert,    L.,    468,    474,    501  [3],    502  [4], 

506  [5],  507 
Wolstenholme,  G.  E.  W.,  88  [18],  92,  99 [8], 

99 
Woods,  P.  S.,  527  [2,  4],  530 [4],  534 
Wosilait,  W.  D.,  115,  //^,  140,  166 
Wu,  R.,  89  [22],  9-'  ' 
Wyman,  Jeffries,  Jr.,  329  [11],  337 


Yamazoe,  S.,  634 [lo],  63S 
Yudkin,  J.,  592  [43],  599 


Zalokar,  AL,  527 [5],  530 [5],  534 
Zaugj^,  W.  S.,  386,  387  [160],  401  [160],  407 
Zerahn,  K.,  623  [3],  629  [17],  630,  631 
Zeuthen,   E.,   490,   495,   537  [10,    14],    538, 

539  [2,  13],  54o[i3,  14],  541,  547,  547, 

54-^' 
Zieyler,  D.  'SL,   103  [3],  104,  117,   241  [5], 

250,  253  [1,2,  5],  254  [5],  257  [5,  II,  12], 

258  [5,  1 1],  260 
Zimmerman,  A.  M.,  486 [27],  404 


SUBJECT  INDEX 


Acetate,  conversion  into  isopentenyl  pyro- 
phosphate, 271-272 
photoassimilation  of,  368-369 
Acetoacetate,  endergonic  reduction  ot,  hy 

succinate  in  mitochondria,   155-165 
Adenosine    diphosphate    (ADP),    effect   on 
mitochondrial  structure,  97-99 
stimulation    of  glutamate    oxidation    by, 

72 
stimulation    of    isocitrate    oxidation    by, 

72-73 
Adenosine  triphosphatase  (ATPase), 

of  liver  mitochondria,   effect   of  amytal 
on,  12-13 
effect  of  atebrin  on,  1 1-16 
effect  of  azide  on,  11    14,  16 
effect  of  chlorpromazine  on,  11-15 
effect  of  desaminothyroxine  on,  8-15, 

16-17,  20 
effect     of    sodium     fluoride     on,      16- 

17 
effect  of  thyroxine  on,  7-10 
effect  of  triiodothyronine  on,  8-9 
properties  of,  244-249 
role  in  oxidative  phosphorylation,   241- 
249 
Adenosine    triphosphate    (ATP),    and    the 
mitotic  apparatus,  490-492 
effect   on   cytochromes   and    respiration, 

233-237 
effect     on     mitochondrial      contraction, 

43-45  ,    ^      ■ 

effect  on  pyridme  and  na\ m  nucleo- 
tides', 230-233 

effect  on  succinate-linked  reduction  ot 
acetoacetate,  157-162 

effect  on  succinate  oxidation  and  pyridine 
nucleotide    reduction,    1 51-154 

equivalence  with  light  in  acetate  assimila- 
tion, 368 

formation  by  spinach  chloroplasts,  455- 

459 

generation  in  photosynthesis,  344-345 

role   in   reduction   of  a-ketoglutarate   by 
succinate,  163-164 
Adenvlate    kinase    reaction,    effect    of    de- 
saminothyroxine on,  17 

effect  of  sodium  fluoride  on,  17 
Albumin,    serum,    effect    on    flavoprotein, 
233 

effect  on  redox  state  of  mitochondrial 
DPN,  232-233 

penetration  through  cell  wall  of  E.  coli, 

591 
VOL.  II. — 2  U 


Amoeba,     movement     of,     structure     and 
function  in,  549-555 

protein  uptake  in,  605-616 

re\ersible   solation-gelation   in,   effect  of 
carbon  dioxide  tension  on,  521—522 
Amytal,  effect  on  ascorbate-induced  lysis, 
56,  60 

effect  on  DPNH-cytochrome  r  reductase, 
19-20 

effect  on  DPNH  oxidase,  18-20 

effect  on  liver  ATPase  activity,  12-13 

effect     on     mitochondrial     oxidation     of 
extramitochondrial    TPNH,     170-172 

effect    on     mitochondrial     vitamin     K3- 
induced  respiration,  142-144 

effect  on  reduction  of  a-ketoglutarate  by 
succinate,  164 

effect  on  respiration  of  liver  slices,   173— 
176 

effect  on  soluble  DPNH  dehydrogenase, 
107-109 

effect  on   succinate-linked    DPN   reduc- 
tion, 125-126 

effect   on   succinate-linked    reduction   of 
acetoacetate,  155-156 

effect  on  succinate  oxidation,  77-78,  80, 
196-198 

effect  on   \itamin    Kj-stimulated   oxida- 
tion    of     glucose-6-phosphate,      170- 

'71 
Anisotropy,  and  specific  membrane  trans- 
port, 594-598 
Antimycin  A,  effect  on  ascorbate-induced 
lysis,  56,  59-60 
effect    on    DPNH-cytochrome    c    reduc- 
tase, 19-20 
effect  on  DPNH  oxidase,  18-20 
effect  on  flavoprotein,  233 
effect  on  mitochondrial  oxidation  of  ex- 
tramitochondrial  TPNH,    170-172 
effect      on      photophosphorylation,      by 

Chromatiwn  particles,  349-350 
effect   on    redox   state    of  mitochondrial 

DPN,  232-233 
effect   on   succinate-linked    reduction   of 

acetoacetate,  1 61-163 
effect  on   vitamin   Kg-stimulated   oxida- 
tion   of    glucose     6-phosphate,     170- 
171 
Apyrase,    potato,    effect    of    desaminothy- 
roxine on,  18 
Arsenate,    effect    on    succinate    oxidation, 

76-78,  80,  151-153,  193-201 
Arsenite,    effect    on    absorbancy    of   mito- 
chondria suspensions,  61-62 


658  SUBJECT   INDEX 

Ascorbate,  as  electron  donor  in  non-cyclic 
photophosphorylation,  386-387 
effect  on  light-induced  phosphorylation 

in  R.  rubrum,  437-438 
lytic    effects    on    isolated    mitochondria, 
53-67 
Atebrin,  effect  on  ATP-ADP  exchange,  16 

effect  on  liver  ATPase  activity,  11-16 
ATP-ADP  exchange,  effect  of  atebrin  on, 
16 
effect  of  azide  on,  15-16 
effect  of  desaminothyroxine  on,  16-17 
effect    of   dinitrophenol    on,    15,    34-37, 

39-41 
effect  of  sodium  fluoride  on,  16-17 
ATP-ADP    exchange    enzyme,    separation 
of,  35 
soluble,  effect  of  dinitrophenol  on,  35-37 
recombination     with    digitonin     frag- 
ments, 35-38 
recoupling  of  oxidative   phosphoryla- 
tion by,  38-39 
Aurovertin,  and  oxidative  phosphorylation, 

265-267 
8-Azaguanine,      effect      on      synchronized 

Tetrahyniena  system,  539-540 
Azide,    effect    on    ascorbate-induced    lysis, 
56,  60 
effect  on  ATP-ADP  exchange,  15-16 
effect  on  li\er  ATPase  activity,  1 1-14,  16 


B 

Bacillus  megateriitm,  isolation  of  membrane 

fraction  of,  584-585 
Bacteria, 

cell  membrane  of,  structure  and  trans- 
port function  of,  582-593 

photophosphorylation  in,  345-348 
cofactors  of,  349-351 

photoreductant  in,  371-383 

photosynthesis  in,  "non-cyclic"  electron 
flow  mechanism  in,  372-374,  378 


Carbon  dioxide, 

assimilation,  role  of  light  in,  341-344 
tension,   effect   in   selective   gene  activa- 
tion, 509-524 
effect  on  reversible  solation-gelation  in 

amoebae,  521-522 
effect     on     sexual     differentiation     in 
Hydra,  512-513 
Carotenoids,     in     protochlorophyll     holo- 
chrome,  325-327 
synthesis  of,  271-276 
Catalase,  effect  on  ascorbate-induced  lysis, 

59-61 
Catechol,  effect  on  ascorbate-induced  lysis, 

58-59 
Cell     differentiation,     and     selective     gene 
activation,  509-524 
division,     and     protein    synthesis,    537- 
547 


Cell   differentiation,    and    thiol    chemistrv', 
487-490 
biochemistry  of,  490—492 
structure  and  differentiation,  control  of, 

465-473 
C-factor,      and      mitochondrial     swelling- 
contraction,  47-48 
Chaos  chaos,  in  studies  of  amoeboid  move- 
ment, 550,  553-555 
surface  coat  of,  binding  of  ferritin  and 
methylated  ferritin  to,  608-610 
Chloride,     effect     on     cyclic     photophos- 
phorylation, 355-356 
effect  on   non-cyclic   photophosphoryla- 
tion, 385-386 
/)-Chloromercuribenzoate, 

effect    on    absorbancy    of  mitochondrial 
suspensions,  compared  with  ascorbate- 
induced  lysis,  61 
effect  on  photophosphorylation,  in  Chro- 

matium  particles,  289-521 
effect   on   swelling— contraction   of  mito- 
chondria, 90-91 
Chlorpromazine,    effect    on    liver    ATPase 

activity,  1 1-15 
Chlorobitim    thiosiilfatophilum,    analysis    of 
crude  extracts  of,  313-317 
characteristics  of,  308-313 
photosynthetic  macromolecules  of,  307— 

323 
purified       pigmented       component      of, 
characterization  of,  317-320 
/)-Chloromercuribenzene  sulphonate,  effect 

on  ATP-ADP  exchange  enzyme,  35 
/)-Chlorophenyldimethylurea,  effect  on  cy- 
clic photophosphorylation,  390-391 
Chlorophyll,     light-induced     changes     in, 
360 
structural    association    with    photophos- 
phorylating  system,  363-366 
Chloroplasts, 

isolated,  ATP  formation  by,  455-459 
carbon    dioxide    assimilation    in    340- 

344,  396-398 
cyclic  photophosphorylation   in,   345- 
348,  389-393 
effect  of  chloride  on,  355-356 
effect  of  ferricyanide  on,  356-357 
relation    to    non-cyclic    photophos- 
phorylation, 393-398 
light-induced  phosphorylation  (photo- 
phosphorylation) in,  431-440 
reduction   of  dinitrophenol    by,    443- 
446 
non-cvclic       photophosphorvlation       in, 
385-388 
^-Chlorovinylarsenious     oxide,     effect     on 
ADP-stimulated  glutamate  oxidation, 
75-76 
effect   on   ADP-stimulated    a-ketogluta- 

rate  oxidation,  75 
effect  on  ADP-stimulated  proline  oxida- 
tion, 75 
effect     on     ADP-stimulated     succinate 
oxidation,  75 


Chromatium, 

cell-free  preparations,  effect  of  cofactors 
on    photophosphorylation    in,    349- 

351. 
light-induced       oxidations       of    cyto- 
chromes in,  357-359 
nitrogen  fixation  by,  381-383 
photoassimilation  of  acetate   in,   366- 
369 
with  Ho  as  reductant,  369-371 
reduction  by  succinate  in,  371-374 
reduction  of  cytochromes  in,  effect  of 

vitamin  K  on,  359-360 
carbon   dioxide   fixation   by,    effect   of 
nitrogen    and    ammonia    on,     380- 

photofixation  of  X.,  by,  378-381 
photoproduction  of  Ho  by,  374-378 
pyridine    nucleotide    reductase    from, 
401-402 
Chromatophores,  molecular  composition 
and  function  of,  279-283,  286,  289-290 
Chromatophores, 

bacterial,  light-induced  pyridine  nucleo- 
tide reduction  by,  301-303 
relation  of  haem   protein  and   photo- 
chemical processes  in,  277-292 
cyclic  photophosphorylation  in,  effect  of 
ferricyanide  on,  356-357 
effect  of  vitamin  K  on,  359-360 
influence  of  chloride  on,  355-356 
of  Rhodospirillinn    nibrum   formation    in 
the  dark,  295-300 
light-induced  phosphorylation  in,  43  i- 
440 
Chromosomes,   in   the   mitotic  cycle,   480- 
481 
reproduction  of,  477-480 
Cilia,  structure  and  function  of,  557-566 
Citrate,  effect  on  ascorbate-induced   Ivsis, 

57-58 

Clark  oxygen  electrode,  71 

Coenzyme  Q  (see  Ubiquinone) 

Ctenophore  (Mnemiopsis leidyi),  swimming- 
plate  cilia  of,  structure  and  function 
of,  563,  565 

Cyanide,  effect  on  ascorbate-induced  lysis, 

56,  59 

effect    on    mitochondrial    oxidations    of 

extramitochondrial    TPNH,     170-172 

effect   on   succinate-linked    reduction   of 

acetoacetate,  161-163 
effect  on  vitamin   Kj-stimulated   oxida- 
tion of  glucose  6-phosphate,   1 70-1 71 

Cysteine,  swellmg  effect  on  isolated  mito- 
chondria, 53-54 

Cysteine  sulphinate,  and  mhibition  of  suc- 
cinate oxidation,  198-201 

Cytidine,  tritiated,  incorporation  into  RXA 
in   Tetrahymoia,  527-534 

Cytochrome, 

concentrations     in     mitochondrial     pre- 
parations, compared  with  ubiquinone 
concentrations,  185-186 
effect  of  ATP  on,  233-237 

2U2 


SUBJECT  INDEX  659 

Cytochrome, 

energy-linked     oxidation     of,     general 
features,  1 28-1 31 
light-induced   oxidations   of,    357-359 

Cytochrome  oxidase,  activities  in  pig  heart 
muscle  preparations,  effect  of  extrac- 
tion with  organic  sohent,    182-184 


D 

Dehydroascorbate,  effect  on  absorbancy  of 
mitochondrial  suspensions,  63-64 

effect  on  ascorbate-induced  lysis,  64 
Deoxyribonucleic     acid     (DNA),     role     in 
synchronized      Tetrahymena     system, 
540-541 

synthesis,  and  cell  division,  476-477 
and    reproduction    of    chromosomes, 
477-480 
Desaminothyroxine,     effect    on     adenylate 
kinase  reaction,  17 

effect  on  ATP-ADP  exchange,   16-17 

effect  on  AT  Pases,  18 

effect    on    DPXH-cytochrome    c    reduc- 
tase, 20 

effect  on  DPNH  oxidase,  19-20 

effect  on  DT  diaphorase,  20-21 

effect  on  liver  ATPase   reactions,   8-15, 
16-17,  20 

effect    on    li\er    mitochondrial    P^-ATP 
exchange,  i 5 

effect  on  liver  respiration  and  phosphon.-- 
lation,  21-24 
Diaphorase,   comparison   with   DPNH   de- 
hydrogenase, 1 1  5-1 1 6 

reactions,    effects   of   thyroxine    and    re- 
lated compounds  on,  18-21 
Dicoumarol, 

and  effect  of  ATP  on,  pyridine  nucleo- 
tide reduction,  1 51-153 
succinate  oxidation,  151-153 

effect    on     mitochondrial     oxidation     of 
extramitochondrial  TPNH,  170-172 

effect    on    mitochondrial    respiration    in 
presence  of  amytal,  141-142 

effect  on  respiration  of  liver  slices,   174- 
176 

effect    on    submitochondrial    diaphorase 
activity,  144-145,  150 

effect  on  succinate  oxidation,  194-195 

effect   on   vitamin    Ks-stimulated  oxida- 
tion of  glucose  6-phosphate,   170-171 
Dihydroxyfumarate,   effect   on   absorbancy 
of  mitochondria  suspensions,  63-64 

effect  on  ascorbate-induced  lysis,  64 
Dihydroxymaleate,  effect  on  absorbancy  of 

mitochondria  suspensions,  63-64 
2  :  3-Dimercaptopropanol  (BAL),  effect  on 
inhibition  of  glutamate  oxidation,  75- 
76 
2  :4-Dinitrophenol   (DNP),  as  catalyst  for 
cyclic  photophosphorylation,  443-446 

effect  on  ascorbate-induced  lysis,  57 

effect  on  ATP-ADP  exchange,    15,   34- 
47 


66o 


SUBJECT  INDEX 


2:4-Dinitrophenol  (DNP),  effect  on  forms 

of    DPN    in    rat    liver    mitochondria, 

218-221 
effect    on    mitochondrial    swelling,    43, 

47-48,  56. 
effect  on  oxidative  phosphorylation,  3-4, 

7-13,    24-25,    246-247 
effect  on  P'-ATP  exchange,  15,  33-34 
effect     on      photophosphorylation,      by 

Chromatium  particles,  349-350 
effect  on   soluble   ATP-ADP  exchange 

enzyme,  35-37 
effect  on  succinate-linked   reduction  of 

acetoacetate,  156-157 
effect  on  succinate  oxidation,  76-78,  80, 

reduction  of,  by  chloroplasts,  443-446 
stimulation    of   isocitrate    oxidation    by, 

.73 
Diphosphopyridine  nucleotide  (DPN), 
energy-linked      reduction      of,      general 

features,  11 9-1 24 
"extra",  in  rat  liver  mitochondria,  214- 
222 

in  sarcosomes,  222-223 
forms  in  mitochondria,  effect  of  added 

substrate  on,  216-223 
succinate-linked    DPN    reduction,    and 

activation  of  succinate  oxidation,  150- 

165 
pathway  of,  125-128 
Dithiodiglycol  (DTDG),  method  for  isola- 
tion   of   mitotic    apparatus,    483-485 
DPNH,  mitochondrial  oxidation  of,   rela- 
tion of  DT  diaphorase  to,  148-150 
DPNH-cytochrome   c   reductase,   effect  of 
amytal  on,  19-20 
effect  of  antimycin  A  on,   19—20 
effect  of  desaminothyroxine  on,  20 
DPNH-dehydrogenase,  assay  of,  105-108, 

115 

isolation  of,  104 

linkage  with  respiratory  chain,  104 

properties  of,  110-117 

purification  of,  no 

solubilization  of,  107-110 

soluble,  effects  of  amytal  on,   107-109 
DPNH-diaphorase,   comparison   with  DT 

diaphorase,  177 
DPNH  oxidase, 

preparation,  use  for  DPNH  dehydroge- 
nase isolation,  104-109 

separation    from    DT   diaphorase,    144- 
146 

system,  effect  of  amytal  on,  18-20 
effect  of  antimycin  on,  18-20 
effect    of   desaminothyroxine  on,    19- 
20 
DT  diaphorase,  and  the  oxidation  of  extra- 
mitochondrial    reduced    pyridine    nu- 
cleotides,   169-179 

effect    of   thyroxine    and    analogues    on, 
20-21 

properties    and    functional    aspects    of, 
140-150 


E 

P^hrlich  ascites  tumour  cells,  C-factor  in,  48 

Electron    transport,    and    phosphorylation 

in    photophosphorylation,    431-440 

functions  of  flavoenzymes  in,  139-166 

Enzymes, 

and    catalytic    carriers,    distribution    in 
Staphylococcus  aureus,  585 
substrate  specificities  of,  587-593 
anisotropic  systems,  vectorial  metabolism 
in,  594-598 
Epithelium, 

of   digestive    tract,    potential    difference 
across,  622-623 
effect   of  sodium   and   potassium   on, 
624-628 
Erythrocytes,  C-factor  in,  48 
Escherichia  coli,  cell  wall  of,  590-591 
electron  micrograph  of  section  of,  312- 

313 
extracts  of,  C-factor  in,  48 
fractionation  of,  588 
^-galactosidase,  activity  of,    588 
galactoside   and   amino   acid   uptake   of, 

583-584 
glucose  6-phosphatase  activity  of,  588- 
591,  597 
I'thylenediaminetetraacetate    (EDTA),    ef- 
fect on  ascorbate-induced  Ivsis,  57-58, 
60 

F 
Farnesyl     pyrophosphate,     conversion     of 
isopentenyl  pyrophosphate  into,  272— 
274 
Ferricyanide,   effect  on   cyclic   photophos- 
phorylation,   356-357 
in  assay  of  DPNH  dehydrogenase,  105- 

109 
photoreduction  of,  418-419 

effect  of  carbon  dioxide  on,  423-425 
effect     of     trichlorophenolindophenol 
on,  423-425 
Ferritin,   binding  to  cell  surface,  608-610 
effect  on  pinocytotic  vesicles,  611-615 
properties  of,  606-607 
Flagella,   structure   and    function   of,    557- 

566 
Fla\in  mononucleotide  (FMN),  effect  on 
photophosphorylation,   345-346,   360- 
366,   389-398 
P'lavin    nucleotides,    effect    of    exogenous 

ATP  on,  230-233 
Flavoenzymes,  functions  in  electron  trans- 
port   and    oxidative    phosphorylation, 
139-166 
Fluoromalate,   effect   on   integrated   oxida- 
tions in  isolated  mitochondria,  81-82 
i)i.-/)-Huorophenylalanine     (P-FPhe),     in- 
hibition of  Tetraliymena  cell  division 
by,  541-546 

G 
^-Galactosidase,  of  E.  coli,  588 
Gene,  selective  gene  activation,  and  carbon 
dioxide  tension,  509-524 


SUBJECT  INDEX 


66i 


Glucose,  influence  on  succinate-linked 
reduction   of  acetoacetate,    157-161 

Cjlucose  oxidase,  effect  on  ascorbate- 
induced  lysis,  60-61 

Glucose  6-phosphatase,  in  E.  coli,  588-591, 

597 
Glucose    6-phosphate,    \itamin    Ks-stimu- 

lated  oxidation  of,  170-172 
Glutamate,    effect    on    forms    of   DPN    in 
heart  sarcosomes,  222-223 
oxidation  of,  comparison  with  isocitratc 
oxidation,  74-76 
effect   of  2:3-diniercaptopropanol    on 

inhibition  of,  75-76 
stimulation  by  ADP,  72 

effect       of      /^-chlorovinyiarsenious 
oxide  on,  75 
Cjlutathione,    swelling    effect    on    isolated 

mitochondria,  53-56 
Cjulonolactone,  effect  on  ascorbate-induced 
lysis,  65 


H 
Haem  protein,  in  bacterial  chromatophores, 
content  and  function  of,  in  relation  to 
structure  and  photochemical  processes, 

277-2Qi 

2  -n-Heptyl-4-hydrosyquinoline-N -oxide, 
effect  on  photophosphorylation  in 
R.  rubriim  chromatophores,  436-440 

Hexokinase, 

yeast,  effect  of  desaminothyroxine  on,  18 
influence  in  succinate-linked  reduction 
of  acetoacetate,  1 57-161 

Hill  reaction,  carbon  dioxide  requirement 
of,  421-427 
mechanism    of,    relation    to    photophos- 
phorylation,   411-428 

Hydra,  sexual  differentiation,  effect  of 
carbon    dioxide    tension    on,    512-513 

Hydrogen,  photoproduction  hy  Chromatiuni 
cells,  374-378 

Hydrogen  peroxide,  effect  on  ascorbate- 
induced  lysis,  59-61 

Hydroquinone,  effect  on  ascorbate-induced 
lysis,  59 

/3-Hydroxybutyrate,  effect  on  absorbancy  of 
mitochondria  suspensions,   54 

/)-Hydroxymercuribenzoate,   effect   on   ab- 
sorbancy of  mitochondria  suspensions, 
61-62 
effect  on  ascorbate-induced  lysis,  62-63 

8-Hydroxyquinoline,  effect  on  ascorbate- 
induced  lysis,  57-58 


I 

Insulin,  structure,  510-51 1 
Internodal  cells,  of  Nitella,  634-636 
lodoacetamide,    effect    on    absorbancy    of 

mitochondria  suspensions,  61-62 
Isoascorbate,  effect  on  absorbancy  of  mito- 
chondria  suspensions,    63-64 


Isocitrate,  oxidation  of,  72-76 

comparison    with    glutamate    oxidation, 

74-76 
inhibition  by  /3-chlorovinylarsenious  ox- 

inhibition  by  malonate,  74 
stimulation  by  ADP,  72-73 
stimulation  by  DNP,  73 
stimulation  by  malate,  73-74 
Isopentenyl      pyrophosphate,      conversion 
into  farnesyl  pyrophosphate,  272,  274 
conversion  of  acetate  into,  271-272 


a-Ketoglutarate, 

ADP-stimulated   oxidation   of,    effect  of 

^-chlorovinylarsenious  oxide  on,  75 
aminative     reduction     by    succinate    of, 

163-165 
effect  on  ascorbate-induced  lysis,  65 
translocation    through    cell    membrane, 

,594 
a-Ketoglutarate  dehydrogenase,  activity  in 
^licrococcus  lysodelkticus,  592-594 

L 
Lipoate,    effect    on    absorbancy    of    mito- 
chondrial suspensions,  61-62 
Lycopene,  con\ersion  into  spirilloxanthin, 
274 
conversion      of      C40     polyenes     into, 
273-274 

M 
A'lagnesium  chloride,  effect  on  mitochon- 
dria  structure,   97-99 
Magnesium   ions,   influence  on  succinate- 
linked  reduction  of  acetoacetate,  157- 
158 
Malate, 

oxidation,  81-82 

effect  of  fluoromalate  on,  81 
stimulation    of    isocitrate    oxidation    by, 

73-74 
Malonate,  effect  on  succinate  oxidation,  79 
inhibition  of  isocitrate  oxidation  by,   74 
Membrane, 

acti\e  transport,  574 

and  membrane  expansion-contraction 
cycles,  633-637 
permeability,    comparative    study,    621- 

630 
specific  transport,  analysis  of,  581-598 
competition  in,  575 
"crypticity  "  of,  573 
induction  and  repression  of,  576-579 
kinetics  of,  574-575 
mutational  effects  in,  575-576 
structure,     and     transport     function     in 
bacteria,  582-593 
Menadione  (see  Vitamin  K3) 
Methaemoglobin  reducing  factor,  as  cata- 
lyst for  TPN  reduction,  450 
relation    with     photosvnthetic    pyridine 
nucleotide    reductase,    449-452 


662 


SUBJECT  INDKX 


Methylated  ferritin,  binding  to  cell  surface, 
608-610 
effect  on  pinocytosis  \esicles,  611-615 
properties  of,  606-607 
6-Methylpurine,    effect    on    synchronized 

Tetrahymena   system,    539-540 
Metmyoglobin, 

as    hvdrogen    acceptor,    compared    with 

TPN,  450-451 
oxidation  complex,  285-286 
M-factor,  of  mitochondria,  5-6,  39-41 
Micrococcus    lysodeikticus,     a-ketoglutarate 
dehydrogenase  activity  in,  592-594 
membrane  of,  composition  and  function 
of,  592-594 
Mitochondria, 

isolated,      ascorbate-induced      lysis      in, 

53-67 
integrated  oxidations  in,  71-82 
pyridine   nucleotide   content  of,    208- 
211 
lipids   of,    extraction   with   organic   sol- 
vents, 182-184 
nature,  181-182 
liver,  effect  of  thyroxine  and  related  com- 
pounds on  3-26,  42-43,  47-48 
M-factor  of,  5-6 
R-factor  of  5-6 
membranes    of    molecular    organization 

of,  32-33 
nucleotide  systems  in,  227-230 
structure,  and  energy  coupling  mechan- 
ism, 31-49 
stable  states  of,  95-99 
swelling    of,    protein    distribution    after, 

65-66 
swelling-contraction  ot,  42-49 
metabolic  control  of,  85-91 
Mitotic  apparatus,  and  ATP,  490-4(;2 
bonding  of,  488 
chemistry  of,  485-486 
general,  481-482 
isolation  of,  482-485 
origin  of,  486-487 
structure,  492-493 
Mitotic  cycle,  chromosomes  in,  480-481 
Morphogenesis,     in     sea     urchin,     cellular 

basis  of,  497-507 
Mussel  (Mytilus  eciulis),  gill  cilia  of,  struc- 
ture and  function  of,  562-564 
Myokinase,    muscle,    effect    of    desamino- 

thyroxine  on,  18 
Myosin,   ATPase    of,    effect   of  desamino- 
thyroxine  on,  18 


N 

Nitella,  internodal  cells  of,  634-636 

rhizoid  cells  of,  635-636 
Nitrite,  effect  on  ascorbate-induced  Ivsis, 

58-59 
Nitrogen,  fixation  by  cell-free  Chromatmm 
preparations,  381-383 
photofixation  bv  Chromatimu  cells,  379- 
381 


Nucleotide     systems,     intramitochondrial, 
227-230 

0 
Oligomycin  A,  and  the  study  of  reactions 
in  oxidative  phosphorylation,  265-267 
effect   on   succinate-linked   reduction   of 
acetoacetate,  158-161 
Oxaloacetate,    effect    on    photofixation    of 
nitrogen,  379 
effect    on    succinate    oxidation,    78-81, 
198-201 
Oxidative   phosphorylation,   effect  of  2:4- 
dinitrophenol  on  3-4,  7-13,  24-25 
effect  of  thyroxine  on,  3-26 
functions  of  flavoenzymes  in,  139-166 
mechanism  of,  33-35 

by  studies  with  antibiotics,  265-267 
recoupling      by      ATP— ADP     exchange 

enzyme,  38-39 
role  of  ATPase  in,  241-249 


Pj-ATP    exchange,    eflFect    of    desamino- 
thyroxine  on,  15 
eflfect  of  dinitrophenol  on,  33-34 
Penicillamine,  effect  on  ascorbate-induced 

lysis,  57 
o-Phenanthroline,      eflFect     on     ascorbate- 
induced  lysis,  57-58 
effect      on      photophosphorylation,      in 

chloroplasts,  390-391 
in  Cliromatiuw  particles,  349-350 
Phenazine  methosulphate, 

effect  on  photophosphorylation,  360-366, 
395.  431-432 
in    cell-free    preparations    ot   Chroma- 

liitni,  349-350 
in  chromatophores  of  R.  nihrum,  433- 
440 
Phenols,  as  catalysts  for  cyclic  photophos- 
phorylation, 444-446 
Phloridzine,  effect  on  mitochondrial  struc- 
ture, 5 
Phosphate,  effect  on  absorbancy  of  mito- 
chondria suspensions,  54,  95-98 
inorganic,    effect    on    ascorbate-induced 
lysis,  57-58 
Phospholipase  A,  solubilization  of  DPNH 

dehydrogenase  with,  107-110 
Phosphorylation, 

in  liver,  effects  of  thyroxine  and  related 

compounds  on,  21-24 
photosynthetic    (photophosphorylation), 
and  the  energy  conversion  process  in 
photosynthesis,  339-403 
catalysts  of,  348-351 
electron  flowmechanismof,  351-355 
Photophosphorylation    (photosynthetic 
phosphorylation), 
cyclic,  3527355 

as   primitive   photosynthesis,   366-369 
dinitrophenol  as  catalyst  for,  443-446 
evidence  for  electron  flow  mechanism 
of,  355-360 


SUBJECT  INDEX 


66^ 


Phntophosphorylation   ( photosynthetic 
phosphorylation , 
cyclic,  multiple  sites  in,  360-3(13 
oxygen-catalyzed,  388-393,  417-418 
relation  to  non-cyclic  photophospho- 
rylation,  393-398 
electron   transport   and   phosphorylation 

in,  431-440 
non-cyclic,  384-388 
"oxidati\e",  41Q-421 

relationship   with   mechanism   of  Hill 
reaction,  411-428 
stimulation  of  TPX  reduction  by,  451- 

system,  structural  association  of  chloro- 
phyll with,  363-366 
Photoreductant,  in  bacteria,  371-383 

in  plants,  384-388 
Photosynthesis,     and     biochemical     evolu- 
tion, 400-403 
energy  conversion  process  in,  and  photo- 

phosphor\'lation,  339-403 
outside  living  cell,  339-341 
Pinocytosis,  and  protein  uptake  in  Amoe- 
bae, 605-616 
vesicles,  effect  of  ferritin  and  methylated 
ferritin  on,  611-615 
Potassium,    effect    on    potential    difference 

across  epithelium,   624—629 
Proline,     ADP-stimulated     oxidation     of, 
effect    of    /3-vinylarsenious    oxide    on, 

.  75 
Protein, 

synthesis,  and  cell  division,  537-547 

uptake,  in  Amoebae,  605-616 
Protochlorophyll  holochrome,  physical  and 

chemical  properties  of,  325-336 
Pyridine  nucleotide  reductase,  from  Cliio- 
matium,  401-402 

relationship  with  methaemoglobin  reduc- 
ing factor,  449-452 
Pyridine  nucleotides  (DPN,  DPNH,  TPX, 
TPNH),    content    in    isolated    mito- 
chondria, 208-211 

effect  of  exogenous  ATP  on,  230-233 

light-induced  reduction  of,  by  bacterial 
chromatophores,  301-303 

mitochondrial,  oxidation  and  reduction 
of,  212-214 

reduction  by  hydrogenase  in  the  dark, 
369-371 

Q 

Quinone,  effect  on  ascorbate-induced  lysis, 

59 

effect  on  mitochondrial  oxidation  of 
extramitochondrial  TPNH  in  presence 
of  DT  diaphorase,  172—174 

-specificity  of  DT  diaphorase,    146-148 


R 

Rat  tissues,  extracts  of,  C-factor  in,  48 
Rat  liver  mitochondria,  pyridine  nucleotide 
content  of,  208-211 


Respiration,  in  liver,  effects  of  thyroxine 
and  related  compounds  on,  21-24 

Respiratory  chain,  coupling  of  reduced 
pyridine  nucleotide  oxidation  to,  169- 

,    '79 

functional  link  of  succinic  dehydrogenase 

with,  193-202 
functions  of  mitochondrial  lipids  in,  181- 

189 
influence  of  ATP  on,  227-238 
relation    to    DT    diaphorase,     141-144, 

146-147 
reversal    of    electron    transfer    in,     119- 

134 
"R  factor",  of  mitochondria,  5-6 
Rhizoid  cells,  of  Nitclla,  635-636 
Rhodospirillum     riihrum,     carotenoid     syn- 
thesis in,  274-276 
chromatophores  of,  light-induced  phos- 
phorylation   in,    scheme    for,    431- 
-1-40 
light-induced  pyridine  nucleotide   re- 
duction by,  301-303 
dark    grown    cells,    growth    and    chloro- 
phyll  formation    by,   effect   of  oxygen 

tension  on,  295-299 
electron  microscope  structure  of,  299 
photochemical    activities    of,    299-300 
photoassimilation    of    acetate     in,     368- 

3^9 
"RHP",    haem   protein    in    purple    photo- 
synthetic  bacteria,  290-292 
RXA,  in  mitotic  apparatus,  485-486 

synthesis    in    nucleus,    and    transfer    to 
cytoplasm,  527-534 
Sarcosomes,     heart,     pyridine     nucleotide 

content  of,  208-211 
Sea  urchin, 

eggs,  effect  of  pretreatment  with  trypsin, 
466-469 
mitotic  apparatus  of,  484-485 
morphogenesis  in,  cellular  basis  of,  497- 

5°7 
sperm,  structure  and  function  of  tails  of, 

SX     5949,     effect     on     ascorbate-induced 

lysis,  56,  59 
Sodium,  influx  and  outflux  across  isolated 

epithelium,  623-624 
Sodium  dithionite,  influence  on  ATPase  in- 
hibition, 12-14 
Sodium  fluoride,  effect  on  adenylate  kinase 
reaction,  17 
effect  on  ATP-ADP  exchange,  16-17 
effect  on  liver  ATPase  activity,  16-17 
Spinach, 

chloroplasts,   ATP   formation   by,    455- 

459 
electron     transport     in     light-induced 
phosphorylation  in,  431-440 
Spirilloxanthin,    synthesis    in    R.    ruhnini, 

274-276 
Sponge   {Microciona),   collar   cell   (choano- 
cyte)    of,    structure    and    function    of, 
560-561 


664  SUBJECT  INDEX 

Squid,  spermatozoa,  structure  and  function 

of  tails  of,  558-560,  562 
Staphylococcus  aureus,  distribution  of  en- 
zymes and  catalytic  carriers  in,  585 
Succinate, 

ADP-stimulated   oxidation  of,   effect  of 
/3-vinylarsenious  oxide  on,  75 

aminative    reduction    of    a-ketoglutarate 
by,  163-165 

effect  on   forms   of  DPN    in   heart   sar- 
cosomes,  222-223 

effect    on    forms    of    DPN    in    rat    li\er 
mitochondria,  216-218 

effect  on  photoiixation  of  nitrogen  gas, 
378-380 

effect  on  stable  structural  states  of  mito- 
chondria, 95-98 

endergonic  reduction  of  acetoacetate  by, 
^ 55-165 

in  reduction  by  photosynthetic  bacteria, 

.371-374 
-linked    pyridine    nucleotide    reduction, 
effect  of  amytal  on,  125-126 
general  features,  1 19-125 
pathway  of,  125-128 
oxidation  of,  activation  of,  150-165,  195, 
197-202 
effect  of  amytal  on,  77-78,  80 
effect  of  arsenate  on,  76-78,  80,  193- 

201 
effect    of    dinitrophenol     on,     76-78, 

80 
effect  of  oxaloacetate  on,  78-81 
inhibition  of,  and  depletion  of  mito- 
chondrial   high    energy    phosphate, 
193-202 
translocation    through    cell    membrane, 

594 

Succinic-CoQ  reductase,  activity  and  pro- 
perties of,  253-260 

Succinic    dehydrogenase,     functional     link 
with  respiratory  chain,  193-202 

Succinoxidase  activity, 

of  pig  heart  muscle,  effect  of  extraction 

with  organic  sohents,  182-183 
of    rat     liver     mitochondria,     effect     of 
ageing  on,  150-151 


T 

Tetrahymena    pyriformis, 

RNA  synthesis  in  nucleus  of,  and  transfer 

to  cytoplasm,  527-534 
synchronized  system  in,  537-539 
and  studies  on  DNA,  540-541 
and  studies  with  amino  and  analogues, 

541-546 
effect      of     purine      and      pyrimidine 
analogues  on,  539-540 
2-Thenoyltrifluoroacetone,    effect    on    suc- 
cinic  CoQ-reductase   activity,   258 
Thiobacillus    denitrificaus,    carbon    dioxide 

assimilation  in,  341 
Thiol  chemistry,  and  cell  division,  487-490 


Thiosulphate,    and     reduction     in    photo- 
synthetic  bacteria,  371-374 
effect  on  photofixation  of  nitrogen  gas, 

378-381 
photoproduction  of  hydrogen  gas  from, 
3747378 
Thymidine,    tritiated,    in    studies   on    syn- 
chronized   Tetrahymena  system,    540- 

541 
Thyroxine,  effect  on  ATPase  reactions  in 
liver,  7-10 
effect  on  diaphorase  reactions,  18-21 
effect  on  liver  mitochondria,  3-26 
effect   on   mitochondrial   structure,   4-6, 

42-43,  47-48 
effect  on  oxidative  phosphorylation,  3—26 
effect   on   respiration   and   phosphoryla- 
tion in  liver,  21-24 
Translocation    catalysis,    concept   of,    585- 

.587 
Trichlorophenol      indophenol,     effect     on 
photoreduction   of  ferricyanide,   423- 

.    425     .       .      _, 

in     "oxidative"     photophosphorylation, 

419-421 
photoreduction  of,  418-419 
Triiodothyroxine,  effect  on  DT  diaphorase, 
20-21 
effect  on  liver  ATPase  reaction,  8-9 
Triphosphopyridine  nucleotide  (TPN), 
photochemical  reduction  of,  384-388 
reduction    of,    metmyoglobin    reducing 
factor  as  catalyst  for,  450 
stimulation  by  photophosphorylation, 
451-452 
TPNH,  extramitochondrial,  mitochondrial 

oxidation  of,  169-173 
Trypsin,  effect  on  sea  urchin  eggs,  466-469 
Turtle  (Testudo  hermamii), 

epithelium   of  digestive   tract   of,    influx 
and  outflux  of  sodium  across,  623- 
624 
potential  difference  across,  622 

effect  of  sodium  and  potassium  on, 
624-629 


U 
Ubiquinone  (Coenzyme  Q), 

in      mitochondrial      preparations,      con- 
centrations in,  185-186 
steady-state  oxidation-reduction  levels 
in,  186-189 


\'alinomycin, 

effect    on    photophosphorylation,    in    R. 
ruhrum  chromatophores,  433-434 
in  spinach  chloroplasts,  433-434 
Vitamin  K,  role  in  DPN-linked  respiration 

and  phosphorylation,  148-149 
Vitamin  K3,  and  DT  diaphorase  quinone 
specificity,  146-148 


SUBJECT  INDEX                                                                    665 

Vitamin    K3,    effect   on   mitochondrial   res-  Vitamin  K3,   effect  on  respiration  of  liver 

piration  in  presence  of  amytal,  141-146  slices,   174-176 

effect  on  photophosphorylation,  345-346,  influence  on  mitochondrial  oxidation  of 

360-366,  389-397  extramitochondrial    TPNH,     169-173 

in    cell-free    preparations    of  Chroma-  influence    on    oxidation    of    glucose    6- 

tiurn,  349-350,  359-360  phosphate,  170-172 

in   R.   rubriim   chromatophores,    438-  \"itamin  K  reductase,  comparison  with  DT 

440  diaphorase,  1 40-1 41