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METABOLISM  AND   FUNCTION 


HONOR  OF  OTTO  MEYERHOF 


Marine    Biological    Laboratory 


R^,pi,eH        July  10.    1950 


Accession    No. 


^,       „     i^r.  I^avid  Nachmansohn 

Liiven    By 


Columbia  University 
Place, 


METABOLISM  AND 
FUNCTION 


SOLE  DISTRIBUTORS  FOR  THE  U.S.A.  AND  CANADA: 
ELSEVIER  BOOK  COMPANY,  INC.,  215  FOURTH 
AVENUE,  NEW  YORK.  -  FOR  THE  BRITISH  EMPIRE, 
EXCEPT  CANADA:  CLEAVER-HUME  PRESS  LTD.,  42a 
SOUTH    AUDLEY    STREET,     LONDON,    W.I. 


THE     ORIGINAL     EDITION     OF     THIS     BOOK 

APPEARED     AS    AN     ISSUE    OF    BIOCHIMICA    ET    BIOPHYSICA     ACTA 

VOL.   4   (1950)   P-    1-348 


OTTO   MEYERHOF 


%    // 


METABOLISM  AND 
FUNCTION 

A  COLLECTION  OF  PAPERS  DEDICATED  TO 

OTTO    MEYERHOF 

ON   THE    OCCASION    OF   HIS 
65TH   BIRTHDAY 

EDITED   BY 

D.  NACHMANSOHN,  M.D. 


%^HP^ 


ELSEVIER  PUBLISHING  COMPANY,  INC. 

NEW   YORK     AMSTERDAM      LONDON     BRUSSELS 
1950 


ALL  RIGHTS  RESERVED.  NO  PART  OF  THIS 
BOOK  MAY  BE  REPRODUCED  IN  ANY  FORM 
(including  PHOTOSTATIC  OR  MICROFILM 
form)  without  WRITTEN  PERMISSION  OF 
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PRINTED     IN     THE     NETHERLANDS    BY 
MEIJER'S     BOEK-     EN     H  AN  DELSD  RUKKE  R IJ      N.V.,     WORMERVEER 


CONTENTS 


Introduction  by  D.  Nachmansohn,  A^'^a^  Yor/^  Cz^y i 

PART  I.  MUSCLE 

A  challenge  to  biochemists  by  A.  V.  Hill,  London 4 

Muskelproteine  von  H.  H.  Weber,  Tubingen 12 

Modifications  dans  la  structure  physico-chimique  de  I'edifice  contractile  au  cours 

du  cycle  de  la  concentration  musculaire  par  M.  Dubuisson,  Liege 25 

Actomyosin  and  muscular  contraction  by  A.  Szent-Gyorgyi,  Bethesda 38 

Myosin  and  adenosinetriphosphate  in  relation  to  muscle  contraction  by  D.  M. 

Needham,  Cambridge 42 

A  consideration  of  experimental  facts  pertaining  to  the  primary  reactions  in 

muscular  activity  by  W.  F.  H.  M.  Mommaerts,  Durham,  N.C 50 

Some  factors  influencing  the  contractility  of  a  non-conducting  fiber  preparation  by 

S.  KoREY,  New  York  City 58 


PART  II.  NERVE 

Morphology  in  muscle  and  nerve  physiology  by  F.  O.  Schmitt,  Cambridge,  Mass.  .  68 
Studies  on  permeability  in  relation  to  nerve  function  I.  Axonal  conduction  and 

synaptic  transmission  by  D.  Nachmansohn,  A^^z^;  Yo^^  Cz7y 78 

Studies  on  permeability  in  relation  to  nerve  function  II.  Ionic  movements  across 

axonal  membranes  by  M.  A.  Rothenberg,  New  York  City 96 

Nerve  conduction  without  increased  oxygen  consumption;  the  action  of  azide  and 

fiuoroacetate  by  R.  W.  Gerard  and  R.  W.  Doty,  CAicago, /^/ 115 

Some  evidence  on  the  functional  oiganization  of  the  brain  by  H.  E.  Himwich,  x\rmy 

Chemical  Center,  Maryland 118 

The  development  of  muscle-chemistry,  a  lesson  in  neurophysiology  by  A.  von 

MuRALT,  Bern 126 


PART  III.  DRUG  ACTION 

Substrate  specificity  of  amino-acid  decarboxylases  by  H.  Blaschko,  Oxford   .    .    .  130 

Glycolysis  in  pharmacology  by  C.  L.  Gemmill,  Charlottesville,  Va 138 

Zur  Charakterisierung  der  Spezifitat  pharmakologischer  Wirkungen  und  des  sie 
bedingenden  Rezeptorsystems  des  Substrates  von  R.  Meier  und  H.  J.  Bein,  Basel  144 


PART  IV.  INTERMEDIATE  METABOLISM 

Free  radicals  derived  from  tocopherol  and  related  substances  by  L.  Michaelis  and 

S.  H.  WoLLMAN,  New  York  City 156 

The  combination  of  diphosphopyridine  nucleotide  with  glyceraldehyde  phosphate 

dehydrogenase  by  C.  F.  Cori,  S.  F.  Velick,  and  G.  T.  Gori,  5^  Louis,  Mo. .  .  .  160 
Garung  und  phytochemische  Reduktion  von  C.  Neuberg,  New  York  City  ....  170 
Essais   de   bilans    de  la  fermentation  alcoolique  due  aux  cellules  de  levures  par 

L.  Genevois,  Bordeaux 179 

Triosephosphorsaure  als  Intermediarprodukt  bei  der  Zuckergarung  mit  intakter 

Hefe  von  W.  Kiessling,  IngelheimjRhein 193 

Configurational  relationships  between  naturally  occurring  cychc  plant  acids  and 

glucose.  Transformation  of  quinic  acid  into  shikimic  acid  by  G.  Dangschat  and 

H.  0.  L.  Fischer,  Berkeley,  Calif 199 

Partial  purification  of  isocitric  dehydrogenase  and  oxalosuccinic  carboxylase  by 

A.  L.  Grafflin  and  S.  Ochoa,  New  York  City 205 

Spectrophotometric  measurements  of  the  enzymatic  formation   of  fumaric  and 

as-aconitic  acids  by  E.  Racker,  ATgzg;  Yor^  Cz7y 211 

The  intercon version  of  the  retinenes  and  vitamins  A  in  vitro  by  G.  Wald,  Cambridge, 

Mass 215 

Experimentelle  Bindung  von  Eiweisskorpern  an  Zellkerne  und  Nukleinsauren  (kurze 

Mitteilung)  von  P.  Ohlmeyer,  Tiibingen 229 

The  biological  incorporation   of  purines   and  pyrimidines  into  nucleosides  and 

nucleic  acid  by  H.  M.  Kalckar,  Copenhagen 232 

L'energie  de  formation  des  complexes  dissociables    enzyme-substrat    et    antigene- 

anticorps  par  R.  Wurmser  et  S.  Filitti-Wurmser,  Paris 238 

Necessite  d'un  coenzyme  pour  le  fonctionnement  de  la  desulfinicase  par  B.  Bergeret, 

F.  Chatagner  et  Cl.  Fromageot,  Paris 244 

Body  size  and  tissue  respiration  by  H.  A.  Krebs,  Sheffield 249 

Synthese  et  utilisation  de  I'amidon  chez  un  flagelle  sans  chlorophylle  incapable 

d'utiliser  les  sucres  par  A.  Lwoff,  H.  Ionesco  et  A.  Gutmann,  Paris 270 

Inhibition  of  the  metabolism  of  nucleated  red  cells  by  intracellular  ions  and  its 

relation  to  intracellular  structural  factors  by  G.  Ashwell  and  Z.  Dische, 

New  York  City 276 

The  biochemistry  of  abnormalities  in  cell  division  by  E.  Boyland,  London  ....  293 
Lipase  catalysed  condensation  of  fatty  acids  with  hydroxylamine  by  F.  Lipmann 

and  L.  C.  Tuttle,  Boston,  Mass 301 

Acylation  reactions  mediated  by  purified  acetylcholine  esterase  II  by  S.  Hestrin, 

New  York  City 3^0 

Observations  on  a  factor  determining  the  metabolic  rate  of  the  liver  by  E.  Lunds- 

gaard,  Copenhagen 322 

Is  acetaldehyde  an  intermediary  product  in  normal  metabolism?  by  E.  Jacobsen, 

Copenhagen 33^ 

The  quantum  efficiency  of  photosynthesis  by  O.  Warburg,  D.  Burk,  V.  Schocken, 

Bethesda,  Md.,  and  S.  B.  Hendricks,  Beltsville,  Md 335 


INTRODUCTION 


OTTO  MEYERHOF 
A  TRIBUTE  ON  HIS  65th  BIRTDAY  (APRIL  12,   1949) 

by 

DAVID  NACHMANSOHN,  M.D. 
College  of  Physicians  and  Surgeons,  Columbia  University,  New  York,  N.Y.  {U.S. A 


The  scientific  work  of  Otto  Meyerhof  has  profoundly  influenced  the  development 
of  Physiology  and  Biochemistry  of  the  last  three  decades.  By  the  originality  of  his 
approach,  the  elegance  of  his  methods,  and  the  wide  range  of  his  knowledge  and  his 
interests  he  became  a  pioneer  in  many  fields. 

Otto  Meyerhof  received  his  degree  of  Doctor  of  Medicine  from  the  University  of 
Heidelberg  in  1909.  Under  the  influence  of  Otto  Warburg  his  interest  turned  to  cellular 
physiology,  especially  to  aspects  concerning  energy  transformations.  The  association 
of  these  two  great  scientific  figures  was  extremely  fruitful  and  important  for  the  devel- 
opment of  this  field. 

In  1913  Otto  Meyerhof  became  Privatdozent  in  Kiel  and  in  191S  Professor  extra- 
ordinarius.  It  was  there  that  Meyerhof  started  the  brilliant  work  on  muscular  contrac- 
tion with  which  his  name  will  always  remain  connected  and  for  which  he  received  the 
Nobel  prize  in  1923,  jointly  with  A.  V.  Hill.  In  1924  he  moved  to  the  Kaiser  Wilhelm 
Institute  for  Biology  in  Berlin  Dahlem,  and  in  1929  he  became  head  of  the  Department 
of  Physiology  in  the  Kaiser  Wilhelm  Institute  for  medical  research  in  Heidelberg. 

The  outstanding  feature  of  Otto  Meyerhof's  work  on  muscle  is  the  first  really 
successful  attempt  to  correlate  chemical  and  physical  processes  of  cellular  function. 
He  was  able  to  establish  such  correlations  in  a  great  variety  of  ways  and  with  amazing 
ingenuity.  During  these  investigations  he  maintained  a  continuous  exchange  of  views 
and  information  with  A.  V.  Hill.  The  collaboration  between  these  two  men  who  have 
maintained  a  close  personal  friendship  over  decades  was  most  fortunate  and  essential 
for  the  development  of  muscle  physiology.  These  two  names  will  continue  to  be  linked 
in  the  History  of  Science. 

In  the  course  of  his  research  on  intermediary  metabolism  in  active  and  resting 
muscle,  Otto  Meyerhof  discovered  many  fundamental  laws  which  greatly  stimulated 
the  whole  of  Biochemistry  in  general.  Among  his  many  achievements  may  be  reckoned 
the  clarification  of  the  Pasteur  reaction.  He  showed  that  oxygen  consumption  prevented 
3  to  6  times  the  equivalent  amount  of  lactic  acid  formation  in  muscle.  Otto  Warburg 
later  found  the  same  principle  to  be  true  in  the  glycolysis  of  tumor  cells  and  Meyerhof 
in  yeast  fermentation.  Meyerhof's  discovery  thus  proved  and  extended  Pasteur's 
hypothesis  that  fermentation  is  "la  vie  sans  air'",  i.e.,  to  a  certain  extent  substituted 
respiration,  whereas  in  the  absence  of  respiration  fermentation  increases.  Pasteur  has 
proposed  this  assumption  but  was  unable  to  verify  it,  because  he  used  cultivated  yeast 
in  which  respiration  is  negligible  compared  with  fermentation.  This  reaction  in  the 


2  D.  NACHMANSOHN  VOL.  4  (195O) 

carbohydrate  cycle  has  been  called  the  Pasteur-Meyerhof  reaction.  The  carbohydrate 
cycle  was  the  first  one  to  be  demonstrated  but  the  idea  of  cyclic  processes  in  cellular 
mechanisms  has  since  become  more  and  more  generalized.  Today  it  is  familiar  to  every 
biochemist  and  an  integral  part  of  our  thinking. 

The  discovery  of  Otto  Meyerhof  and  his  students  that  some  phosphorylated 
compounds  are  rich  in  energy  led  to  a  revolution,  not  only  of  our  concepts  of  muscular 
contraction,  but  of  the  entire  significance  of  celular  metabolism.  A  continuously  in- 
creasing number  of  enzymatic  reactions  are  becoming  known  in  which  the  energy  of 
adenosine  triphosphate,  the  compound  isolated  by  his  associate  Lohmann,  provides 
the  energy  for  endergonic  synthesis  reactions.  The  importance  of  this  discovery  for  the 
understanding  of  cellular  mechanisms  is  generally  recognized  and  can  hardly  be  over- 
estimated. 

In  1925  Meyerhof  succeeded  in  extracting  the  glycolytic  enzyme  system  from 
muscle,  retracing  a  pathway  which  Buchner  and  Harden  and  Young  had  explored 
in  yeast.  This  proved  to  be  a  decisive  step  for  the  analysis  of  glycolysis.  Meyerhof  and 
his  associates  were  able  to  reconstruct  in  vitro  the  main  steps  of  the  complicated  chain 
of  reactions  leading  from  glycogen  to  lactic  acid.  They  verified  some  and  extended 
other  parts  of  the  scheme  proposed  by  Gustav  Embden  in  1932,  shortly  before  his  death. 

The  few  examples  given  may  suffice  to  indicate  not  only  the  brilliance  but  also  the 
wide  scope  of  his  achievements.  A  real  appreciation  of  his  work  is  impossible  within  a 
few  introductory  remarks.  Meyerhof  has  always  been  driven  by  the  true  pioneer 
spirit.  His  open  and  critical  mind  quickly  grasped  new  developments.  When,  in  1929, 
EiNAR  LuNDSGAARD  found  that  contraction  in  a  monoiodoacetate  poisoned  muscle 
occurs  without  lactic  acid  formation,  Meyerhof  rapidly  accepted  the  evidence  which 
was  built  essentially  on  his  own  line  of  approach.  This  rapid  change  of  his  views 
shows  the  strength  of  his  scientific  personality  and  was  all  the  more  remarkable  since 
for  many  years  he  had  vigorously  supported  the  idea  that  lactic  acid  formation  was 
the  primary  step. 

After  the  rise  to  power  of  the  Nazis,  Meyerhof,  like  other  Jewish  scientists,  had 
to  leave  Germany.  In  1938  he  went  to  Paris  where  he  was  warmly  welcomed  and  well 
received.  By  the  combined  efforts  of  the  late  Jean  Perrin,  Professor  Rene  Wurmser 
and  Professor  Henri  Laugier,  he  was  appointed  Director  of  Research  at  the  University 
of  Paris  and  was  able  to  continue  his  research  in  the  Institut  de  Biologic  Physico- 
Chimique.  When  the  Nazi  hordes  invaded-  France,  he  had  to  flee  again  under  most 
difficult  circumstances,  and  came  to  the  United  States  at  the  end  of  1940.  Here  he  was 
appointed  Research  Professor  of  Physiological  Chemistry  in  the  School  of  Medicine  of 
the  University  of  Pennsylvania,  a  position  he  holds  at  present.  In  spite  of  all  difficulties 
his  creative  spirit  is  unbroken,  as  shown  by  the  great  number  of  his  publications  during 
the  past  few  years,  concerning  especially  intermediary  metabolism,  the  purification 
and  properties  of  adenosine  triphosphate,  the  free  energy  of  phosphorylated  compounds, 
and  various  other  subjects. 

In  spite  of  his  intense  scientific  activity,  Meyerhof's  interests  have  never  been 
limited  to  science.  The  extraordinarily  wide  scope  of  his  nonscientific  activities  shows 
best  his  rich  personality.  From  his  student  years  on  he  had  been  not  only  interested  but 
actively  engaged  in  philosophy.  He  was  closely  associated  with  the  Nelson  group  in 
Gottingen.  He  devoted  much  time  to  a  critical  analysis  of  Goethe's  scientific  work  and 
presented  recently  at  the  Goethe  Bicentennial  Celebration  of  the  Rudolph  Virchow 


VOL.  4  (1950)  INTRODUCTION  3 

Society  in  New  York  a  profound  and  most  lucid  and  critical  evaluation  of  Goethe's 
scientific  ideas  and  concepts,  especially  the  Farbenlehre.  He  always  had  and  still  has  a 
passionate  love  of  art,  literature  and  poetry.  His  interest  in  painting  has  been  greatly 
stimulated  by  his  wife  Hedwig  who  is  a  painter  and  actively  engaged  in  teaching  the 
art  of  painting.  No  matter  which  field  Meyerhof  discusses,  it  is  always  a  great  stimulus 
and  his  views  show  the  originality  of  his  ways  of  thinking  and  his  remarkable  gift  of 
integrating  a  great  variety  of  phenomena. 

Otto  Meyerhof's  65  birthday  offers  a  happy  occasion  for  his  former  associates 
to  express  their  gratitude  and  for  his  friends  their  esteem.  The  contributions  of  this 
anniversary  volume  are  only  a  very  incomplete  indication  of  the  influence  of  his  work 
in  so  many  fields.  They  are  offered  as  a  small  tribute  to  his  creative  genius. 


PART  I 
MUSCLE 


A  CHALLENGE  TO  BIOCHEMISTS 

by 

A.  V.  HILL 

Biophysics  Research  Unit,  University  College,  London  (England) 


Otto  Meyerhof  has  always  been  betwixt  and  between :  a  physiological  chemist  or 
a  chemical  physiologist,  perhaps  we  should  call  him  a  "chemiologist".  On  my  shelves 
are  about  two  hundred  of  his  reprints,  his  and  his  colleagues'.  The  first  of  these,  with 
its  accompanying  letter  addressing  me  as  "Sehr  geehrter  Kerr  Kollege"  dated  1911 
from  Naples,  dealt  with  the  heat  production  of  the  vital  oxidation  process  in  the  eggs 
of  marine  animals.  Next  follow  papers  on  the  energy  exchanges  of  bacteria,  the  heat 
accompanying  chemical  processes  in  living  cells,  the  inhibition  of  enzyme  reactions  by 
narcotics  (1914).  Some  time  in  those  apparently  peaceful  years,  before  the  explosion 
of  1914,  he  visited  us  at  Cambridge.  Then  comes  a  gap,  so  far  at  least  as  my  collection 
of  Otto  Meyerhof's  reprints  is  concerned.  By  1919  he  had  moved  to  Hober's  laboratory 
at  Kiel  and  the  long  succession  of  papers  began  on  the  respiration,  energetics,  and 
chemistry  of  muscle.  And  when  I  say  muscle,  I  mean  muscle:  living  muscle,  resting, 
contracting  and  recovering  from  contraction,  developing  tension  and  doing  work,  pro- 
ducing lactic  acid  and  removing  it  again,  using  oxygen  and  glycogen,  giving  out  CO2  and 
heat,  all  things  which  living  muscles  are  accustomed  to  do.  And  since  I  too  was  working 
on  living  muscle,  we  were  in  frequent  communication  again,  after  the  five  years'  gap. 
In  the  summer  of  1922,  following  a  suggestion  to  Hopkins,  he  visited  Cambridge  and 
gave  lectures  there.  I  remember  "Hoppy"  expressing  concern  lest  some  anti-German 
demonstration  might  take  place,  but  appearing  to  be  satisfied  by  the  comment  that  if 
so  I  should  be  proud  to  remove  the  demonstrator:  nothing  of  course  happened.  Later, 
he  stayed  with  me  at  Manchester  and  I  recall,  as  an  example  of  his  scientific  perspicacity, 
the  complete  disbelief  which  he,  first  of  anyone,  expressed  in  experiments  he  witnessed 
which  six  months  later  were  proved  to  be  fraudulent.  That  was  our  first  reunion  after 
the  War,  there  were  many  others,  in  London,  Plymouth,  Barcelona,  Heidelberg,  Berlin, 
Rome  and  elsewhere.  The  photograph  shows  us  driving  together  to  Stockholm  for  the 
Physiological  Congress  in  1926. 

The  results  of  his  researches,  and  those  of  his  colleagues,  are  a  part  of  scientific 
history.  They  are  linked  with  most  that  is  known  of  the  chemistry  of  muscle  and  with 
much  that  is  established  of  changes  involving  phosphate  and  carbohydrate  in  the  cell. 
For  some  years  his  investigations  were  concerned  mainly  with  muscle  —  living  muscle : 
more  recently  they  followed  the  trend  in  biochemistry,  perhaps  even  they  helped  to 
establish  the  fashion,  of  dealing  in  vitro  with  the  enzyme  systems  of  muscle.  As  late, 
however,  as  1935,  he  was  working  on  the  volufhe  changes  of  living  muscle  during 
contraction  and  relaxation  and  relating  them  to  the  underlying  chemical  cause.  I 
read  these  papers  again  recently,  very  carefully,  having  come  to  the  conclusion  that  the 

References  p.  11.  4 


VOL.  4  (1950) 


A   CHALLENGE   TO   BIOCHEMISTS 


reversible  part  of  the  volume  change  is  attributable  mainly  or  wholly  to  pressure  set 
up  by  contraction.  The  elegance  and  clarity  of  Meyerhof's  work  and  its  description 
impressed  itself  again  as  it  had  done  in  earlier  days.  One  might  criticize  some  of  the 
conclusions,  but  not  the  methods  or  results.  To  read  these  papers  once  more  was  a  sudden 
pleasure,  after  so  many  in  which  one  could  not  be  sure  what  an  author  had  really  done  I 


My  last  reprint  from  Heidelberg  is  dated  1938.  Perhaps  if  Hitler  had  not  driven  him 
from  the  beautiful  Institute  and  the  excellent  colleagues  and  facilities  he  had  there,  the 
succession  of  papers  on  muscle — living  muscle — might  have  continued.  Alas  that  they 
could  not!  This  paper,  however,  is  to  challenge  him  and  his  disciples  to  make  a  few 
more  chemical  investigations  on  living  muscle,  to  see  how  far  the  chemistry  in  vitro 
of  muscle  extracts  can  be  fitted  to  the  physical  facts  of  muscular  contraction. 

It  is  customary  for  biochemists   {e.g.,  Baldwin^,  p.  341)  to  describe  "The  probable 
course  of  events  in  normal  muscular  contraction"  is  some  such  terms  as  these: 
References  p.  11. 


6  A.  V.  HILL  VOL.  4  (1950) 

"On  the  arrival  of  a  nerve  impulse,  ATP  is  broken  down,  giving  rise  to  ADP  and 
inorganic  phosphate,  furnishing  at  the  same  time  the  contraction  energ}-.  The  ADP  is 
promptly  converted  again  into  ATP  at  the  expense  of  phosphagen  and  no  change  in  the 
ATP  content  of  the  muscle  can  be  detected  ..."  Others  suppose  that  contraction  is 
associated  with  the  formation  of  myosin  —  ATP  and  that  ATP  is  broken  down  in  relaxa- 
tion. By  Sandow^  a  slight  initial  lengthening  (in  a  muscle  under  tension)  after  a  stimulus 
("latency  relaxation")  is  attributed  to  the  formation  of  a  complex  between  activated 
myosin  and  ATP.  Most  of  this  is  pure  speculation,  without  direct  experimental  evi- 
dence. Unlike  Mr.  Stalin  (Historicus^)  I  have  no  general  theory  of  revolutions, 
but  I  did  once  write  an  article  (1932),  which  I  think  is  still  worth  reading,  on  "The 
Revolution  in  Muscle  Physiology"^.  That  was  after  phosphagen  had  deposed  lactic 
acid  from  pride  of  place  as  the  chief  chemical  agent  in  contraction.  At  that  date 
one  could  write:  "On  stimulation,  phosphagen  breaks  down  .  .  .:  this  is  the  primary 
change  by  which  energy  is  set  free".  Only  four  years  earlier  Ritchie^  wrote:  "On 
stimulation  of  a  muscle  fibre  the  wave  of  excitation  passes  down  it;  by  increasing 
the  permeability  of  a  membrane  or  by  some  other  means  it  causes  the  liberation 
of  lactic  acid  from  a  carbohydrate  source.  The  liberated  hydrogen  ions  neutralize  the 
negative  charge  on  a  surface  of  protein,  Meyerhof's  V erkiirzungsort .  .  .  and  thereby 
alter  the  type  of  structure,  the  area  of  surface,  and  the  mechanical  constants.  This  will 
be  the  fundamental  change."  In  the  lactic  acid  era  the  evidence  that  the  formation  of 
lactic  acid  was  the  cause  and  provided  the  eneigy  for  contraction  seemed  pretty  good. 
In  the  phosphagen  era  a  similar  attribution  to  phosphagen  appeared  even  better 
justified.  Now,  in  the  adenosinetriphosphate  era  lactic  acid  and  phosphagen  have  been 
relegated  to  recovery  and  ATP  takes  their  place.  Those  of  us  who  have  lived  through 
two  revolutions  are  wondering  whether  and  when  the  third  is  coming. 

It  may  very  well  be  the  case,  and  none  will  be  happier  than  I  to  be  quit  of  revolu- 
tions, that  the  breakdown  of  ATP  really  is  responsible  for  contraction  or  relaxation: 
but  in  fact  there  is  no  direct  evidence  that  it  is.  Indeed,  no  change  in  the  ATP  has  ever 
been  found  in  living  muscle  except  in  extreme  exhaustion,  verging  on  rigor.  This  is 
explained  by  supposing  that  as  soon  as  ATP  is  broken  down  into  ADP  and  phosphate 
it  is  promptly  restored  in  the  so-called  "Lohmann  reaction"  at  the  expense  of  creatine 
phosphate. 

ADP  +  CP  ->  ATP  +  C 

If  this  happens  after  each  stimulus,  then  the  smallness  of  the  changes  involved  and 
their  quickness  make  it  extremely  difficult  to  gain  any  direct  evidence  on  the  subject. 
In  a  single  twitch,  for  example,  the  heat  set  free  is  about  3  millicaloiies  per  gram, 
which  would  correspond  to  the  liberation  from  ATP  of  2.5-10"'  g  molecule  of  phosphate 
per  giam  of  muscle.  To  measure  so  small  a  change,  reversed  within  the  duration  of  a 
single  twitch,  might  well  seem  an  impossible  task. 

We  should  not,  however,  be  so  satisfied  with  the  explanation  of  why  no  change  in 
ATP  is  ever  found  in  living  muscle  that  we  cease  to  look  for  it :  for  another  possibility 
exists.  The  total  energy  available  from  all  sources  (lactic  acid,  phosphagen  and  ATP) 
for  the  anaerobic  phase  of  contraction  is  about  i  cal/g,  corresponding  to  about  400 
twitches.  The  total  energy  similarly  available  after  poisoning  with  iodoacetate  (from 
phosphagen  and  ATP)  is  about  0.25  cal/g  corresponding  to  about  100  twitches.  From  the 
known  amount  of  ATP  present  is  muscle,  the  total  energy  it  could  provide  by  breaking 
References  p.  11. 


VOL.  4  (1950)  A   CHALLENGE   TO   BIOCHEMISTS  7 

off  one  phosphate  is  about  0.05  cal/g,  corresponding  to  about  20  twitches.  Is  it  not 
possible  that  as  stimulation  proceeds  a  balance  is  reached  at  some  intermediate  level 
between  breakdown  and  restoration  ?  That  is  the  case  with  phosphagen  and  lactic  acid ; 
in  a  muscle  steadily  stimulated  (in  the  presence  of  oxygen)  a  certain  amount  of  phos- 
phagen is  broken  down,  a  certain  amount  of  lactic  is  formed,  and  a  steady  level  is  reached 
between  breakdown  and  recovery.  At  a  still  earlier  stage  one  might  expect  steady 
stimulation  to  provide  at  least  a  temporary  balance  between  ATP  breakdown  and 
restoration. 

In  frogs'  muscles  at  20°  C,  if  ATP  were  the  only  source  of  energy  a  maximal  tetanus 
would  lead  to  its  complete  breakdown  in  about  0.5  sec.  The  suggested  balance,  if  it 
occurred,  would  presumably  be  reached  within  that  time,  and  when  the  stimulus  ended 
restoration  of  the  ATP  might  be  completed  within  another  0.5  sec.  The  times  involved 
are  far  too  short  for  chemical  manipulation:  but  biochemists  need  not  be  disheartened, 
frogs'  and  rabbits'  muscles  are  singularly  ill-suited  to  the  enquiry,  they  are  much  too 
quick,  why  not  use  muscles  which  contract  more  slowly?  The  muscles  of  the  Mediter- 
ranean land  tortoise,  Testudo  graeca,  commonly  imported  before  the  War  into  England 
and  sold  on  barrows  for  i/-  in  London  streets,  take  about  fifteen  times  as  long  to  con- 
tract as  those  of  a  frog  and  their  speed  can  be  further  reduced  about  nine  times  by 
lowering  the  temperature  from  20°  C  to  0°  C,  or  about  five  times  by  lowering  it  to  5°  C. 
This  means  that  the  time  available  for  chemical  manipulation  can  be  reckoned  in  large 
fractions  of  a  minute  instead  of  fractions  of  a  second.  Provided,  therefore,  that  the 
chemical  technique  is  capable  of  determining  a  substantial  part  of  the  total  ATP  with 
reasonable  accuracy,  the  time  involved  can  be  made  so  long  that  sufficient  resolution 
ought  easily  to  be  obtained. 

The  experiment  ought  certainly  to  be  made  and  nobody  could  make  it  better  than 
Otto  Meyerhof  —  for  he  knows  how  to  handle  living  muscles.  The  result  may  not  be 
unequivocal  —  but  it  very  well  may.  If  no  change  in  ATP  is  found,  but  only  a  change 
in  phosphagen,  the  status  quo  remains  and  we  can  all  believe  what  we  like,  provided 
it  is  consistent  with  the  physical  facts  described  below.  But  suppose  it  is  found  that 
ATP  is  broken  down  at  a  rate  decreasing  from  the  start,  reaching  a  steady  concentration 
after  half  a  minute's  stimulation  (corresponding  to  half  a  second  in  a  frog's  muscle  at 
20°  C)  and  is  restored  to  its  original  level  after  (say)  a  further  half  minute  of  rest  and 
recovery.  Then  at  least  we  can  be  assured  that  ATP  is  really  concerned  either  with  the 
contractile  process  itself,  or  with  the  very  early  stages  of  recovery.  There  are  other 
possibilities  and,  without  trying,  it  is  useless  to  speculate  tco  much.  A  German  clinician  is 
said  to  have  remarked :  "Der  Versuch  muss  gemacht  werden  und  sollte  er  hundert  Bauem 
kosten".  A  decision  on  this  important  matter  is  certainly  worth  a  hundred  tortoises. 

But  whatever  may  be  the  outcome  of  this  challenge  to  biochemists,  I  would  invite 
them  also,  in  their  speculations  about  muscle,  to  take  note  of  the  following  facts,  all 
referring  to  contraction  and  relaxation,  as  distinguished  from  recovery. 

1.  There  is  no  sign  of  an  endothermic  process  at  any  stage  of  contraction  or  relaxa- 
tion. If  endothermic  processes  occur  they  are  balanced,  or  overbalanced,  by  exothermic 
ones. 

2.  No  heat  at  all  is  produced  during  relaxation,  apart  from  that  derived  from  the 
degradation  of  work  previously  performed  during  contraction  (in  raising  a  load,  or  in 
stretching  elastic  material  in  series  with  the  muscle).  When  a  muscle  relaxes  without 
load  or  tension,  no  heat  is  produced  after  the  contractile  phase  is  over. 

References  p.  11. 


8  A.  V.  HILL  VOL.  4  (1950) 

3.  It  has  been  found  by  quick  stretches  appHed  to  a  muscle  shortly  after  a  single 
shock  that  the  full  strength  of  the  contraction,  defined  as  the  load  which  a  muscle  can 
just  bear  without  lengthening  (and  equal  to  the  force  of  a  maximal  tetanus)  is  developed 
abruptly  immediately  after  the  end  of  the  latent  period.  It  is  maintained  for  a  time  and 
then  declines  in  "relaxation".  If  stimulation  is  continued,  each  successive  shock  re- 
stores the  strength  of  contraction  to  its  full  height. 

4.  Corresponding  to  (3)  there  is  a  "heat  of  activation"  in  a  twitch,  which  is  inde- 
pendent of  all  other  factors  except  the  fact  of  stimulation.  The  heat  of  activation  starts 
at  its  maximum  rate  before  any  visible  sign  of  contraction  occurs,  declining  to  zero  at 
about  the  moment  when  the  strength  of  contraction  (see  3  above)  begins  to  fall  off,  i.e., 
at  the  end  of  the  contractile  phase. 

5.  The  "heat  of  maintenance"  in  a  prolonged  contraction  is  the  summated  effect 
of  the  heat  of  activation  following  successive  elements  of  the  stimulus.  It  is  greater  at 
first  corresponding  to  the  more  rapid  relaxation  after  a  short  tetanus,  but  after  a  certain 
duration  of  stimulus  it  becomes  constant.  It  is  affected  only  to  a  minor  extent  by  the 
length  of  the  muscle.  It  is  greatly  increased  by  a  rise  of  temperature,  corresponding  to 
the  more  rapid  relaxation. 

6.  In  twitch  and  tetanus  alike,  apart  from  the  heat  of  activation  or  the  heat  of 
maintenance,  energy  is  given  out  in  two  discrete  forms,  (a)  as  mechanical  work  and  b)  as 
heat  of  shortening.  The  heat  of  shortening  is  directly  proportional  to  the  change  of 
length  over  the  whole  range  of  shortening,  and  (for  a  given  change  of  length)  is  inde- 
pendent of  the  work  done. 

7.  Apart  from  heat  of  activation  or  heat  of  maintenance,  the  rate  at  which  total 
energy,  i.e.,  heat  plus  work,  is  given  out,  is  a  linear  function  of  the  load  throughout 

a  contraction :  /  n    ,      \  j   /  7*       z  /  d         t,\ 

[P  +  a)  ax  jilt  =  o{P^  —  P) 

where  x  is  the  amount  of  shortening  up  to  time  t,  P  is  the  load,  dx  is  the  heat  of  short- 
ening, Pq  is  the  maximum  isometric  tension  and  Z)  is  a  constant  related  to  the  maximum 
velocity  of  shortening  under  zero  load. 

8.  The  constant  a  in  (7)  can  be  obtained  either  from  thermal  measurements  or  from 
the  form  of  the  characteristic  relation  between  load  and  velocity  of  shortening.  The 
agreement  is  good. 

9.  Relaxation  is  not  an  active  process.  A  muscle  completely  without  load  or  tension 
does  not  lengthen  again  after  shortening  in  response  to  a  stimulus.  That  its  length  has 
really  changed  and  that  its  fibres  or  fibrils  have  not  gone  into  folds  is  shown  by  the  fact 
that  its  latent  period  is  practically  the  same  at  a  short  length  as  it  is  at  a  greater  one. 
If  a  muscle  had  to  "take  up  the  slack"  in  fibres  or  fibrils  before  its  tension  could  be 
manifested  externally,  the  latent  period  would  be  greatly  prolonged. 

10.  Simultaneous  with  the  earliest  sign  of  mechanical  activity  after  a  shock  is  a 
change  of  opacity.  This  is  due  to  an  alteration  of  light  scattering  (D.  K.  Hill*).  The 
earliest  phase  has  certain  characteristics  which  distinguish  it  from  a  later  phase  which 
continues  into  recovery. 

11.  If  we  can  assume  that  excitation  occurs  at  the  surface  membrane  of  a  muscle 
fibre,  the  propagation  inwards  of  the  change  there  started  cannot  be  due  to  the  diffusion 
inwards  of  some  substance,  e.g.,  Ca  ions  or  acetyl  choline,  initiating  contraction  by  its 
arrival  at  each  point.  Diffusion  is  far  too  slow.  Some  chain-reaction  started  at  the  surface 
is  required. 

References  p.  11. 


VOL.  4  (1950)  A   CHALLENGE   TO   BIOCHEMISTS  9 

Nineteen  years  ago  my  colleagues  and  I  found,  (Hill  and  Kupalov';  Hill  and 
Parkinson^)  in  muscles  stimulated  to  exhaustion  in  nitrogen,  a  lowering  of  vapour 
pressure  considerably  too  large  to  be  accounted  for  by  chemical  changes  known  to  occur, 
if  the  precursors  of  the  chemical  substances  produced  were  themselves  osmotically 
active.  In  normal  muscles  complete  exhaustion  led  to  a  decrease  of  vapour  pressure 
corresponding  to  an  increased  concentration  in  the  free  water  of  a  muscle  of  0.12  M. 
The  production  of  0.35%  lactic  acid  dissolved  in  the  free  water,  (taken  as  0.77  g  per  g) 
of  the  muscle,  would  lead  to  a  concentration  change  of  0.050  M.  The  liberation  of 
creatine  and  phosphate  by  the  complete  breakdown  of  phosphagen  in  amounts  equiva- 
lent to  65  mg.  P/ioo  g  would  give  0.054  M.  The  production  of  phosphate  and  adenylic 
acid  from  ATP  in  amounts  equivalent  to  30  mg  P/ioo  g  would  give  0.012  M.  The  total, 
0.116  M,  is  not  far  from  that  (0.12  M)  calculated  from  the  observed  change  of  vapour 
pressure.  We  have  assumed,  however,  that  the  phosphagen  and  the  ATP  were  not 
themselves  osmotically  active;  if  they  had  been  the  increase  would  have  been  0.031  M 
less,  namely  0.085  M  instead  of  0.12  M.  The  vapour  pressure  measurements  were  cer- 
tainly not  that  much  wrong. 

Again,  in  muscles  poisoned  with  iodoacetale  complete  exhaustion  led  to  a  mean 
decrease  of  vapour  pressure  corresponding  to  an  increased  concentration  of  0.050  M. 
If  phosphagen  and  ATP  breakdown  are  assumed,  as  above,  to  be  the  only  chemical 
reactions  involved,  the  corresponding  change  of  concentration  in  the  free  water  of  the 
muscle  would  be  0.066  M.  It  is  impossible,  however,  in  muscles  adequately  poisoned 
to  ensure  that  some  preliminary  breakdown  of  phosphagen  has  not  occurred :  and  if  the 
poisoning  is  not  quite  sufficient,  there  is  likely  to  be  some  formation  of  lactic  acid.  Either 
cause  would  tend  to  make  the  observed  change  of  vapour  pressure  smaller  than  that 
calculated  from  the  assumed  breakdowns.  Even  so,  had  the  phosphagen  and  ATP 
originally  been  osmotically  active,  the  change  calculated  from  the  constituents  would 
have  been  only  0.035  M,  considerably  less  than  the  0.050  M  observed. 

Unless,  therefore,  some  chemical  reactions  hitherto  unknown  occur  in  a  muscle 
stimulated  to  exhaustion  in  nitrogen,  we  are  forced  to  conclude  that  phosphagen  and 
ATP  are  not  themselves  osmotically  active  in  the  normal  muscle.  This  would  be  the 
case  if  they  were  bound  to  other  molecules  and  their  constituents  only  became  free 
when  they  broke  down.  These  older  experiments  are  worth  recalling  now  because  they 
are  pertinent  to  the  question  of  how  phosphagen  and  ATP  exist  in  the  living  muscle. 
Looking  back  at  them  today  I  see  no  reason  to  question  their  results.  If  those  are  correct, 
ATP  and  phosphagen  exist  in  a  combined  form  in  muscle,  exerting  no  osmotic  pressure 
on  their  own  account  until  they  are  broken  down. 

The  work  which  an  isolated  muscle  of  frog  or  toad  can  perform  under  optimal 
conditions  may  be  as  high  as  40%  of  the  total  energy  given  out  in  the  initial  process,  as 
distinguished  from  recovery  (Hill^).  This  high  efficiency  is  obtained  just  the  same  at 
0°  C  as  at  higher  temperatures,  and  there  are  no  grounds  at  all  for  supposing  that  the 
nature  of  contraction  is  in  any  way  altered,  except  in  speed,  by  a  change  of  temperature. 
The  muscle  twitch  is  rather  stronger  at  0°  C  than  at  25°  C,  and  quite  as  efficient.  If 
theory  predicts  otherwise,  so  much  the  worse  for  the  theory.  The  highest  efficiency  is 
obtained  with  a  comparatively  large  load  and  slow  shortening ;  under  isotonic  conditions, 
with  a  load  about  half  the  maximum  which  the  muscle  can  lift.  In  such  a  contraction 
the  work  done  is  about  twice  the  heat  of  shortening :  two  thirds  of  the  total  energy  set 
free,  in  excess  of  the  heat  of  activation  (or  maintenance),  is  external  mechanical  work. 
References  p.  11. 


10  A.  V.  HILL  VOL.  4  (1950) 

Under  conditions,  therefore,  of  maximum  efficiency,  the  energy  is  liberated  in  about 
the  following  proportions: 

Heat  of  activation  Work  Heat  of  shortening 

or  maintenance 

40  40  20 

At  the  other  extreme,  with  zero  load  and  rapid  shortening,  the  situation  may  be  this : 
Heat  of  activation  Work  Heat  of  shortening 

40  Nil  49 

(The  heat  of  activation  is  the  same  in  both  cases.) 

The  fact  that  the  external  work  may  be  so  large  a  fraction  of  the  whole  energy 
liberated  in  excess  of  the  activation  (or  maintenance)  heat  naturally  makes  one  ask 
whether  the  heat  of  shortening  may  not  itself  really  be  work  degraded  into  heat  in 
overcoming  some  internal  resistance  to  shortening :  in  that  case  energy  would  be  liberated 
in  two  forms  only,  heat  of  activation  (or  maintenance)  and  mechanical  work.  For  two 
reasons,  the  supposed  internal  resistance  cannot  be  of  a  viscous  nature:  (i)  the  heat  of 
shortening  is  independent  of  the  velocity  of  shortening,  and  (2)  the  heat  of  shortening 
per  cm  is  the  same  over  the  whole  range  of  possible  shortening  (if  it  were  due  to  over- 
coming viscous  resistance  it  would  be  inversely  proportional  to  the  length).  The  sup- 
posed resistance  must  be  constant,  and  must  reside  in  lines  or  filaments  parallel  to  the 
axis  of  the  muscle,  it  cannot  be  a  volume  effect.  An  obvious  objection  to  the  theory 
of  a  constant  {e.g.,  frictional)  resistance  a  parallel  to  and  inherent  in  the  contractile 
elements  is  that  there  should  then  be  a  constant  difference  2a  between  the  load  at  which 
a  muscle  just  shortened  and  the  load  at  which  it  just  lengthened:  experiment  "showed 
(Katz^")  that  no  such  difference  exists.  The  objection  would  be  valid  if  a  muscle  were 
a  single  contractile  element,  with  a  parallel  constant  resistance.  In  fact,  however,  a 
muscle  fibre  is  very  long  relative  to  its  thickness,  and  its  diameter  is  by  no  means  con- 
stant throughout  its  length.  There  is  no  reason  to  suppose  that  its  maximum  force  is 
the  same  everywhere.  If  not,  in  an  isometric  contraction  the  stronger  regions  would 
tend  to  shorten  at  the  expense  of  the  weaker  regions,  and  the  constant  resistance  would 
hinder  shortening  at  one  point  and  lengthening  at  another  (possibly  a  very  convenient 
arrangement  in  a  system  of  non-uniform  strength).  With  a  large  number  of  such  elements 
in  series  an  increase  of  load  would  stretch  the  weaker  elements,  a  decrease  of  load  would 
allow  the  stronger  elements  to  shorten:  and  the  difference  of  load  between  observable 
lengthening  and  shortening  would  be  small.  The  objection,  therefore,  is  not  really  valid. 

A  stronger  objection,  raised  in  1938^^,  is  that  there  are  indications  that  the  heat 
of  shortening  changes  sign  when  shortening  becomes  lengthening ;  and  the  heat  generated 
in  overcoming  a  frictional  resistance  does  not  change  sign  when  the  direction  of  motion 
is  reversed.  The  difficulty  is  to  get  muscles  to  lengthen  reversibly  except  at  very  low 
speeds.  Possibly  the  use  of  dogfish  jaw  muscles  (Levin  and  Wyman^^)  which  stand 
stretching  well  would  allow  more  positive  conclusions  to  be  reached.  One  thing  is 
certain,  namely  that  the  work  done  in  making  a  muscle  lengthen  does  not  reappear 
completely  as  heat :  Some  of  it  is  absorbed,  presumably,  in  driving  chemical  reactions 
in  the  endothermic  direction.  The  subject  is  being  investigated  afresh  by  improved 
methods. 
References  p.  11. 


VOL.  4  (1950)  A   CHALLENGE   TO  BIOCHEMISTS  II 

One  final  word  —  to  continue  my  challenge  to  biochemists.  Otto  Meyerhof's  first 
letter  to  me,  as  I  wrote  at  the  beginning,  came  from  Naples:  all  his  life  he  has  been 
ready  to  vary  not  only  his  chemical  technique  but  his  biological  material.  The  proper- 
ties of  animals,  and  of  their  muscular  systems,  vary  over  a  very  wide  range.  There  is 
no  need  to  stick  to  rabbits  and  frogs.  If  a  problem  seems  insoluble  on  one  muscle,  one 
should  try  to  define  it  more  precisely  to  see  where  the  difficulty  lies.  Discussion  with  a 
zoologist,  or  a  visit  to  a  Marine  Laboratory,  may  provide  material  many  times  better 
suited  to  one's  needs.  I  spent  many  years  trying  to  measure  the  heat  production  of  nerve : 
if  I  had  made  the  experiment  on  crabs'  nerves  instead  of  frogs'  the  answer  would  have 
come  in  1912  instead  of  1926.  In  1912  it  was  not  possible  to  define  the  problem  well 
enough  to  get  a  clear  direction  to  non-medullated  nerve,  but  at  least  one  might  have 
taken  a  chance  and  not  persisted  with  the  frog's  sciatic.  If  one's  instruments,  or  methods, 
are  too  slow,  one  can  make  them  relatively  quicker  by  using  slower  material  —  tortoises, 
toads  or  even  sloths.  That  means,  of  course,  that  biochemists,  Hke  biophysicists,  must 
also  be  biologists  (as  Meyerhof  has  always  been  and  as  Hopkins  was)  —  but  why  not? 

REFERENCES 

1  E.  Baldwin,  Dynamic  Aspects  of  Biochemistry.  Cambridge  University  Press  (i947)- 

2  A.  Sandow,  A7in.  N.  Y.  Acad.  Sci.,  47  (1947)  895- 
^  HiSTORicus,  Foreign  Affairs,  27  (i949)  I75- 

*  A.  V.  Hill,  Phvsiol.  Revs,  12  (1932)  56.  . .      xr  ■         ■      ti_       /       Q^ 

5  A.  D.  Ritchie,  The  Comparative  Physiology  of  Muscular  Tissue,  Cambridge  Umversity  Press  (I92«). 

6  D.  K.  Hill,  /.  Physiol.,  107  (1948)  4°  ?• 

'  A.  V.  Hill  and  P.  Kupalov,  Proc.  Roy.  Soc.  B.,  106  (1930)  445- 

8  A.  V.  Hill  and  J.  L.  Parkinson,  Proc.  Roy.  Soc.  B.,  108  (1931)  148- 

9  A.  V.  Hill,  Proc.  Roy.  Soc.  B.,  127  (i939)  434- 
10  B.  Katz,  /.  Physiol.,  96  (i939)  45- 

"  A.  V.  Hill,  Proc.  Roy.  Soc.  B.,  126  (1938)  136- 

12  A.  Levin  and  J.  Wyman,  Proc.  Roy.  Soc.  B.,  loi  (1927)  218. 

Received  March  7th,  1949 


12  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


MUSKELPROTEINE 


HANS  H.  WEBER 

Physiologisches  Instiitif,  Tubingen  (Deutschland) 


Es  ist  wahrscheinlich,  dass  bei  keinem  anderen  Gewebe  Stoffwechsel,  Energetik  und 
kolloidaler  Feinbau  so  gut  bekannt  sind  wie  beim  Skelettmuskel.  Es  ist  sicher,  dass  bei 
keinem  anderen  Gewebe  der  Ztisamnienhang  zwischen  diesen  Eigenschaften  lebender 
Systeme  auch  nur  annahernd  so  weit  geklart  ist,  wie  bei  der  Muskeltatigkeit. 

Bei  den  Muskelproteinen  betraf  sogar  die  erste  fundamentale  Entdeckung  gar  nicht 
die  Proteine  selbst,  sondern  gerade  diesen  Zusammenhang :  1922  stellte  O.  Meyerhof^^ 
fest,  dass  etwa  V3  der  Warmeproduktion  der  Arbeitsphase  des  Muskels  auf  der  Bindung 
der  H-Ionen  der  Milchsaure  durch  die  Muskeleiweisskorper  beruhe  und  dass  in  der 
Erholungsphase  ein  entsprechender  Betrag  der  Verbrennungsenergie  der  Milchsaure 
verbraucht  wiirde,  um  die  H-Ionen  wieder  von  dem  Eiweiss  abzulosen. 

Man  wusste  damals  fast  nichts  iiber  Zahl  und  Art  der  Muskeleiweisskorper. 
Von  FiJRTH^^  hatte  aus  dem  Muskelpressaft  ein  Muskelalbumin  isoliert,  das  Myogen, 
mit  zahlreichen  und  verwickelten  Denaturierungsmechanismen.  Er  hielt  es  ausserdem 
fiir  moglich,  dass  im  Pressaft  auch  noch  ein  besonderes  Protein  vorhanden  sei,  das  er 
fiir  den  Trager  der  Muskelkontraktion  hielt,  und  fiir  das  er  den  Namen  Myosin  vorschlug. 
Er  war  allerdings  nicht  sicher,  dass  dieses  Myosin  ein  Eiweisskorper  sui  getieris  sei  und 
nicht  ein  Denaturierungsprodukt  des  Myogens.  Soweit  diese  Zweifel  die  Anwesenheit 
des  Myosin  im  Muskelpressaft  betrafen,  waren  sie  berechtigt:  denn  das  kontraktile 
Protein  geht  nicht  in  den  Muskelpressaft  iiber^'  ^'^. 

Die  Entdeckung  Meyerhof's  war  trotz  oder  gerade  wegen  dieser  Unsicherheiten 
ausserordentlich  folgenreich.  Denn  Meyerhof  hatte  schon  selbst  gleich  in  seiner  ersten 
Originalarbeit  festgestellt,  dass  die  angefiihrten  Warmetonungen  bei  H'-Bindung  und 
H'-Abgabe  offenbar  bei  alien  Proteinen  in  neutralem  und  alkahschem  Milieu  auftreten. 
Nun  verliiuft  aber  H'-Bindung  und  H'-Abgabe  durch  Carboxylgruppen  in  der  Kegel 
athermisch,  durch  organische  basische  Gruppen  aber  mit  solchen  Warmetonungen,  wie 
sie  Meyerhof  gefunden  hatte.  Das  wiirde  bedeuten,  dass  Eiweisskorper  auf  der  alka- 
lischen  Seite  des  isoelektrischen  Punktes  nicht,  wie  man  bis  dahin  geglaubt  hatte,  mit 
ihren  Carboxylgruppen,  sondern  mit  ihren  basischen  Gruppen  puffern,  oder  mit  anderen 
Worten,  dass  isoelektrische  Eiweissteilchen  nicht  Molekiile  sondern  Zwitterionen  sind. 
Und  so  wurde  die  MEYERHOF'sche  Entdeckung  am  Muskel  zu  einem  fundamentalen 
Argument  der  Zwitterionentheorie  der  Aminosauren  und  Eiweisskorper^^. 

Da  aber  im  iibrigen  grosse  Unterschiede  in  der  Warmetonung  der  H'-Bindung  nicht 
nur  zwischen  Carboxylgruppen  und  basischen  Gruppen  bestehen,  sondern  auch  zwischen 
den  verschiedenen  basischen  Gruppen  unter  sich,  wirkte  die  MEYERHOF-Entdeckung 
noch  weiter.  Jesse  P.  Greenstein^-^  mass  diese  Warmetonungen  an  den  trivalenten 
Literatur  S.  24. 


VOL.  4  (1950)  MUSKELPROTEINE  I3 

Aminosauren  und  ihren  Peptiden.  Er  schuf  damit  die  Unterlagen,  um  die  Warmetonung 
der  Eiweisspufferung  in  verschiedenen  pH-Bereichen  auszuwerten  fiir  die  Beantwortung 
der  Frage,  welche  der  ionogenen  Gruppen  in  dem  jeweiligen  pn-Bereich  Trager  der 
Pufferung  waren^^.  Wenn  wir  heute  am  intakten  Proteinteilchen  Zahl  und  Dissoziations- 
bereich  der  einzelnen  ionogenen  Gruppen  weitgehend  kennen,  so  ist  das  u.a.  eine  Folge 
der  MEYERHOF'schen  Muskelstudien. 

II 

Das  fehlende  systematische  Wissen  um  die  Zahl  und  Art  der  Muskeleiweisskorper 
wurde  in  den  nachsten  15  Jahren  nach  Meyerhof's  Entdeckung  im  Groben  nachgeholt. 
Der  Stand  dieses  Wissens  warde  1934^''  erschopfend  und  1939^  in  den  wesentlichsten 
Ziigen  zusammenfassend  dargestellt.  Das,  was  wir  heute  wissen,  ist  —  unter  Ausschluss 
der  elektrischen  Ladungsverhaltnisse  der  Proteine  und  der  optischen  Resultate*  aus 
Tabelle  I  zu  ersehen. 

Fiir  die  Beurteilung  der  Bedeutung  der  Hauptfraktionen  der  Muskelproteine  gelten 
folgende  Uberlegungen :  die  Myogenfraktion  umfasst  nicht  nur  20%  der  Muskeleiweiss- 
korper, sondern  sie  beansprucht  auch  20%  des  Faservolumens.  80%  des  Faservolumens 
sind  fiir  Myogen  "nichtlosender  Raum"^^.  Das  bedeutet,  dass  Myogen  dort,  wo  es  im 
Muskel  ist,  sich  in  einer  Konzentration  von  20%  vorfindet.  Ebenso  stimmt  der  kolloidos- 
motische  Druck  der  Muskelfaser  recht  gut  mit  dem  osmotischen  Druck  einer  20%igen 
^lyogenlosung  iiberein^'^'  ^'.  Da  Myogen  unter  physiologischen  Bedingungen  >  30% 
loslich  ist,  ist  das  Myogen  also  auch  im  Muskel  selbst  gelost.  Da  diese  Myogenlosung 
im  Muskel  noch  nicht  einmal  den  Raum  des  Sarkoplasmas  vollstandig  beanspruchen 
wiirde,  liegt  es  nahe  wenigstens  den  Hauptteil  der  Fraktion  (Myogen  B)  als  Bestandteil 
des  Sarkoplasma  anzusehen. 

Die  Stromafraktion  umfasst  —  nach  mikroskopischer  Beobachtung  an  der  erschop- 
fend extrahierten  Muskelfaser — bindegewebige  Anteile,  Sarkolemm  und  vielleicht  noch 
einige  weitere  nicht  oder  nicht  wesentlich  doppelbrechende  unlosliche  Strukturanteile. 

tJber  die  Bedeutung  der  Globulin  X-Fraktion  sind  Aussagen  noch  nicht  moglich. 

Die  Stellung  der  Myosinfraktion  in  der  Muskelfaser  wurde  bisher  auf  Grund  fol- 
gender  Tatsachen  beurteilt:  die  Eigendoppelbrechung  der  Faser  betragt  '•^40  (44%) 
der  Eigendoppelbrechung  des  Myosinfadens  gleicher  Eiweisskonzentration^^'  ^^,  wahrend 
die  Stabchendoppelbrechung  sogar  genau  40  %  der  Stabchendoppelbrechung  des  Fadens 
ausmacht^^'  22.  Die  Stabchendoppelbrechung  des  Fadens  ist  dabei  auch  quantitativ  die 
Doppelbrechung  eines  idealen  WiENER'schen  Stabchenmischkorpers.  Da  ferner  auch 
40%  der  Muskeleiweisskorper  der  Myosinfraktion  angehoren,  wurde  gefolgert,  dass  die 
gesamte  Doppelbrechung  des  Muskels  ausschliesslich  auf  der  Doppelbrechung  der 
Myosinfraktion  beruhe  und  dass  auch  im  Muskel  die  Myosin-  (Aktomyosin)  Stabchen 
streng  achsenparallel  angeordnet  sind.  Da  ferner  das  Volumen  der  A-Abschnitte  auf 
ctwa  40%  des  Faservolumens  geschatzt  werden  muss,  ergab  sich  als  zweiter  Schluss, 
dass  wahrscheinlich  alles  Aktomyosin  sich  in  den  doppelbrechenden  Abschnitten  be- 
findet^^.  Daraus  und  aus  der  weiteren  Tatsache,  dass  das  Aktomyosin  der  Trager  der 
rontgenoptischen  Phanomene  des  Muskels  und  ihrer  Veranderung  bei  der  Kontraktion 
ist^'  ^,  ergab  sich  schliesslich,  dass  Aktomyosin  offenbar  das  kontraktile  Protein  sei. 

Nun  fanden  Wolpers*^,  sowie  Schmitt  und  Mitarbeiter^s,  dass  elektronenmikros- 
kopische  Eiweissfaden  von  einer  Dicke  von  50  bis  250  A  in  gleicher  Dichte  den  A-  und 

*  Rontgen-,  Polarisations-  und  Elektronenoptik,  sowie  Streuung  des  sichtbare  Lichtes. 
Literatur  S.  24. 


14 

H.  H.  WEBER 

VOL. 

4  (1950) 

TABELLE 

Protein-Fraktion 

Anteil  am 
Gesamtprotein 

Name  des  einzelnen 
Proteins 

Loslich  zwischen 
Pjj  6  u.  7  bei 

V— I 

c 

(c—  0)  (G*=  400) 

Albumini2 

aoo/o^o 

Myogen  B  (80%  der 
Fraktion) 

0  bis  6//^' 

0.043" 

Myogen  A*  —  Aldolase' 
(20%  der  Fraktion) 

— 

— 

Globulin  X^o 

200/^20 

nicht  bearbeitet 

0.005  A*""  t)is   ? 

0.1437 

40%2» 

L-Myosin^i 

0.05  /J,  bei  ph  6.72",  34 
bis  5.7  fi  bei  pn  5-5® 

2.226 

Myosinioa 

Aktomyosine^^ 

0.3  fj,  bei  Ph  6.726 
bis  3.3 /i  bei  PH  5-5® 

3  bis  4.5'" 

Aktin  (aktiv)32 

0  bis  2  fi^^ 

2.3  bis  3.2^^ 

Aktin  (inaktiv)32 

0  jjfi^ 

O.Ol40 

Stroma 

2oO/o20 

nicht  bearbeitet 

nicht  loslich 

Summe 

1 00% 

Proteine  unbekannter 
Zugehorigkeit 

6%^ 

Tropomyosin^ 

0.1  bis  7  fx^ 

— 

*  G  =  Gefalle 

den  I-Abschnitt  durchziehen.  Infolgedessen  sollte  im  I-Abschnitt  etwa  dieselbe  positive 
Stabchen-Do*  auftreten  wie  im  A-Abschnitt,  d.h.  etwa  70%  der  Gesamt-Do  des  A- 
Abschnittes22.  Diese  Do  der  I-Bande  aber  fehlt!  Der  Widerspruch  wiirde  sich  losen, 
wenn  man  annimmt,  dass  die  elektronenmikroskopischen  Fadenmizellen  des  I-Ab- 
schnittes  eine  negative  Eigen-Do  besitzen,  die  die  positive  Stabchen-Do  ungefahr 
kompensiert.  Tatsachlich  fanden  Szent-Gyorgyi  und  seine  Schiiler^^,  dass  gerade  in 
der  I-Bande  ein  Protein — von  ihnen  N-Protein  genannt — vorhanden  ist  von  betracht- 
licher  negativer  Eigen-Do  und  positiver  Stabchen-Do.  Beim  Brechungsindex  des  Wassers 
wird  die  positive  Stabchen-Do  durch  die  negative  Eigen-Do  vollstandig  aufgehoben. 
Die  Gesamt-Do  der  I-Bande  wird  nach  erschopfender  Extraktion  des  Muskels  sogar 
ganz  schwach  negativ**.,Es  bleibt  zu  priifen,  ob  die  Menge  des  N-Proteinsreicht,  um  aus 

*  Do  :=  Doppelbrechung. 

**  Szent-Gyorgyi^*  nimmt  an,  dass  sich  in  der  I-Bande  die  gleichen  Mengen  und  Strukturen 

an  Aktomyosin  fanden  wie  in  der  A-Bande  und  infolgedessen  die  gleiche  positive  Gesamt-Do  wie 

dort  —  nur  maskiert  durch  eine  entsprechende  hohe  negative  Do  des  N-Protein.  Er  iibersieht  dabei 

aber,  dass  die  von  ihm  angefiihrte  negative  Gesamt-Do  des  N-Protein  nur  bei  Einbettung  in  Medien 

Literatuy  S.  24. 


VOL.  4  (1950) 


MUSKELPROTEINE 


15 


(c=  0) 

(c=o) 

M 

I 

Q 
osmot. 

I 
aus  D20  u.  S20 

6.4 

— 

81000  osmot.3i* 

3.0  aus  M  u.  Sjo 

— 

7.86" 

4.78" 

i.5-io5auss2ou.  D20 

— 

5-5 

— 

— 

— 

— 

• 

-  j26a,  30,  23 

0.926a 

0.533,  40(p) 

0.84-I06(26«) 

I  bis  1.5-  io8(?)33 

128 

100.2  ausMu.  s,o 
i8o(?) 

93  bis  280*0 

0.5** 

14-  lo^ausSjpU.  Djo 

— 

— 

64*"beic=  0.1% 

i 

3.7**"beic=o.2J% 

— 

7600033 

2.5^ 

2.72 

88oooosmot.'^ 
gSoooausSjoU.Dgo^ 

Ill 

56 

Einzelne  Versuchsreihe 

ihm  die  elektronenmikroskopischen  Fadenmizellen  des  I-Abschnittes  aufzubauen.  Die 
Tatsache,  dass  die  Gesamt-Do  der  I-Bande  durch  erschopfende  Extraktion  schwach 
negativ  wird,  deutet  darauf  hin,  dass  sich  in  der  I-Bande  neben  dem  N-Protein  noch  ein 
wenig  extrahierbares  Protein  mit  positiver  Do  findet.  Man  konnte  dabei  an  Tropomyosin^ 
(6%  des  Muskeleiweiss)  oder  auch  an  einen  kleinen  Teil  des  Aktomyosins  denken.  Die 
neuen  Entdeckungen  scheinen  eher  das  Ratsel  der  Stniktur  der  I-Bande  der  Losung 
naher  zu  fiihren  als  zu  neuen  Annahmen  iiber  den  Auf  bau  des  A-Abschnittes  zu  notigen. 
Die  kontraktilen  A-Banden  diirften  zu  ihrem  Auf  bau  den  Hauptteil  des  Aktomyosins 
verbrauchen,  und  das  Aktomyosin  diirfte  also  das  kontraktile  Protein  sein. 


Ill 

1930  hatte  Deuticke^  gefunden,  dass  bei  pn  7  die  Loslichkeit  der  Muskeleiweiss- 

von  hohem  Brecbungsindex  auftritt  (Xylol-Canadabalsam).  Denn  nur  hier  ist  die  hohe  positive 
Stabchen-Do  des  N-Protein  selbst  infolge  des  geringen  Brechungsunterschiedes  zwischen  Eiweiss- 
fadchen  und  Einbettungsmedium  weitgehend  verschwunden. 

Literatur  S.  24. 


l6  H.  H.  WEBER  VOL.  4  (1950) 

korper  als  Folge  excessiven  anaeroben  Stoffwechsels  (Ermudung,  Totenstarre,  langere 
Aufbewahrung  von  Muskelbrei)  deutlich  abnimmt.  1933  fanden  Meyer  und  Weber^", 
dass  bei  24  stiindiger  Aufbewahrung  von  Muskelbrei  die  Loslichkeit  der  Myosinfraktion 
fast  vollig  schwindet.  1938  zeigten  Kamp^^  und  Weber^  am  lebenden  Kaninchen,  dass 
die  Loslichkeitsminderung  durch  Ermiidung  in  Sekunden  und  Minuten  mit  der  Erholung 
wieder  verschwindet.  Sie  zeigten  ferner  am  Frosch,  dass  diese  Loslichkeitsminderung 
ausserordentlich  viel  schneller  auftritt,  wenn  dem  Muskel  durch  Halogenacetat  die 
Milchsaurebildung  unmoglich  gemacht  ist.  Die  Loslichkeitsminderung  beruht  also 
offenbar  auf  einem  Stoffwechselvorgang,  der  durch  die  Bildung  der  Milchsaure  riick- 
gangig  gemacht  wird.  Da  die  L5slichkeitsanderungen  durch  Zusatz  von  Kreatin,  Kreatin- 
phosphat  und  Adenylsaure  nicht  beeinflussbar  waren,  musste  es  sich  um  einen  sehr 
friihen  Stoffwechselprozess  handeln,  der  zeitlich  der  Kontraktion  nahe  steht.  Und 
schliesslich  ergab  sich,  dass  allein  die  kontraktile  Eiweissfraktion,  die  Myosinfraktion, 
durch  diesen  Stoffwechselvorgang  reversibel  in  ihrer  Loslichkeit  geandert  wird. 

1939  entdeckten  Engelhardt  und  Ljubimova^^,  dass  zwischen  Myosinfraktion 
und  Adenosintriphosphat-(ATP)-spaltung  enge  Beziehungen  bestehen:  der  Elastizitats- 
modul  von  Myosinfaden  sinkt  bei  ATP-Gegenwart  ab,  und  das  ATP  wird  gleichzeitig 
vom  Myosin  gespalten.  Diese  Befunde  wurden  1941  von  Needham  und  Mitarbeitern^^ 
erweitert:  auch  die  Viskositat  [r]')  und  die  Stromungsdoppelbrechung  (DRF)  sinken 
unter  ATP  reversibel  ab.  —  1942  gelang  Schramm  und  Weber^^  mit  der  Ultrazentrifuge 
die  Auflosung  der  Myosinfraktion  und  ihre  Trennung  in  mehrere  Komponenten:  eine 
langsam  sedimentierende  Komponente  (L-Myosin)  und  mehrere  schnell  sedimentierende 
Komponenten  (S-Myosin). 

Alle  diese  verschiedenen  Linien  der  Forschung  vereinigten  sich  1942  in  den  sensatio- 
nellen  und  bedeutenden  Ergebnissen  von  Szent-Gyorgyi  und  seinen  Schiilern^^  und  in 
den  Untersuchungen  anderer  Autoren,  die  von  diesen  Ergebnissen  ihren  Ausgang 
nahmen.  Szent-Gyorgyi  bestatigte  die  Befunde  der  NEEDHAM-Gruppe — iibrigens  ohne 
sie  zu  kennen^und  erweiterte  sie  dahin,  dass  durch  ATP  auch  noch  die  Lichtstreuung 
und  die  Loslichkeit  reversibel  beeinflusst  wurden  — •  aber  nicht  der  Myosinfraktion 
sondern  nur  einer  Komponente,  des  Aktomyosin.  Damit  war  auch  der  Befund  Weber 
und  Schramm  bestatigt,  dass  die  Myosinfraktion  aus  mehreren  Komponenten  besteht. 
Einen  gewissen  Abschluss  fand  die  Erklarung  aller  dieser  Phanomene  durch  den  Beweis, 
dass  die  Aktomyosinkomponente  eine  Verbindung  zweier  Fadenproteine,  des  Aktin  und 
des  Myosin,  darstellt,  die  bei  Gegenwart  von  ATP  unter  Anderung  aller  der  Eigen- 
schaften  dissoziiert,  deren  ATP-Abhangigkeit  oben  angefiihrt  wurde.  Schliesslich 
ergaben  die  Untersuchungen  der  Szegeder  Schule  auch  noch,  dass  die  Extrahierbarkeit 
der  Myosinfraktion  aufhort,  sobald  die  gesamte  ATP  des  Muskels  gespalten  ist.  Damit 
war  der  DEUXICKE-KAMP-Effekt  auf  Bildung  des  schwer  loslichen  Aktomyosin  durch 
ATP-Mangel  zuriickgefiihrt. 

IV 

Der  Versuch,  die  Komponenten  der  Myosinfraktion  zu  trennen,  fiihrte  zunachst  nur 
zu  einer  Reindarstellung  des  L-Myosin  (Schramm  und  Weber^^)  bezw.  des  von  Szent- 
Gyorgyi^*  so  genannten  "Myosin"  (kristallisiertes  Myosin).  Es  ist  aber  leicht^^,  L- 
Myosin  und  S-Myosine  sauber  quantitativ  von  einander  zu  trennen :  ein  Muskelextrakt 
von  0.6  ft  (0.3  m  KCl  -f-  0.15  m  Standartphosphat  nach  Szent-Gyorgyi)   wird  auf 

Literatur  S.  24. 


VOL.  4  (1950) 


MUSKELPROTEINE 


17 


0.04  [.I  verdiinnt  und  die  Myosinfraktion  abzentrifugiert.  Aus  der  ATP  freien  Losung  des 
Niederschlages  fallen  dann  bei  pn  6.8  alle  S-Myosine  geschlossen  durch  Verdiinnung  auf 
0.28  bis  0.3  [.I  aus.  Die  iiberstehende  Losung  enthalt  nur  noch  L-Myosin,  das  bei  0.05  /t 
als  Gel  von  0.5  bis  1%  und  bei  0.03  //  als  Gel  von  ^2%  quantitativ  ausfallt.  Der  lockere 
Niederschlag  der  S-Myosine  schliesst  etwas  gelostes  L-Myosin  ein,  dass  durch  weitere 
Umfallungen  entferni  werden  kann*.  Nach  solcher  Trennung  ist  es  leicht  zu  beweisen, 
dass  L-Myosin  mit  dem  Myosin  Szent-Gyorgyi's  und  die  S-Myosine  mit  seinem  Akto- 
myosin  identisch  sind. 

Werden  Extrakte  in  dieser  Weise  aufgeteilt,  so  ordnen  sich  die  Sedimentations- 
konstanten  von  mehr  als  10  Praparaten  des  L-Myosin  in  schwacher  und  geradliniger 
Abhangigkeit  von  der  Eiweisskon-  . 
zentration  zur  Kurve  i  der  Fig.  i. 
Auf  dieser  Kurve  liegen  auch  unsere 
Werte  fiir  "kristallisiertes"  Myosin. 
Bei  c  =  o  betragt  Soq  7-I- 

Die  Sedimentationskonstanten 
der  S-Myosine  sind  viel  grosser, 
hangen  von  der  Eiweisskonzentra- 
tion  viel  starker  und  ausserdem 
nicht  geradlinig  ab  (vergl.  Kurven 
2  und  3  der  Fig.  i).  Bei  den  S- 
Myosinen  ist  vielmehr  i/s  der  Kon- 
zentration  geradlinig  proportional 
nach  der  Formel 


K-c 


5fC  =  o) 


Fig.  I.  Sedimentationskonstante 
Kurve  i :  L-Myosin  O  O  rein  durch  fraktionierte  Um- 
fallung,  -)-+  rein  durch  Kristalhsation,  ^  Ci  aus 
Aktomyosin  der  Kurven  2  und  4  durch  .\TP;  Kurve  2, 
3,  4:  Aktomyosine  ©^3  aus  Muskelextrakt  isoliert 
durch  fraktionierte  Umfallung;  •  5  aus  L-Myosin  der 
Kurve  i  durch  Aktin;  Q  durch  Riickbildung  aus  ^  der 
Kurve  i  bei  Aufspaltung  der  ATP;  Kurve  la:  Denatu- 
riertes  L-Myosin  \/  rein,  ^  zu  50%  gemischt  mit 
undenaturiertem  L-Myosin. 


to     1.11 
cinX 

S20  (c  =  o)   ist   bei    verschiedenen 

Praparaten  der  S-Myosine  sehr  ver- 

schieden,   K  dagegen  weniger:  so 

hat  S20  (c  ==  o)    in   Kurve    2    den 

Wert  93,  in  Kurve  3  den  Wert  280, 

wahrend    K  =  J-/     fiir   die   Kur- 

s/c 

ven  2  und  3  den  Wert  8.8  bezw.  8.2 
besitzt.  Wenn  das  immer  so  ist, 
so  wiirde  es  bedeuten,  dass  die  Wechselwirkung  der  Einzelteilchen  bei  den  verschie- 
denen Aktomyosinen  annahernd  gleich  ist,  wahrend  Gestalt  und  Grosse  der  Teilchen 
von  einem  Aktomyosin  zum  anderen  sehr  verschieden  sein  konnen.  Denn  K  charakte- 
risiert  die  Wechselwirkung,  S20  (c  =  o)  dagegen  das  Einzelteilchen.  Es  wurden  unter 
40  Sedimentationskonstanten  von  S-Myosinen  keine  Werte  gefunden,  die  tiefer  lagen  als 
die  Werte  von  Kurve  2.  In  Abbildung  i  sind  nur  seiche  Konstanten  eingezeichnet,  die 
an  einheitlichen  Praparaten  gefunden  ^vurden.  Reine  Praparate  von  S-Myosinen  ent- 
halten  namlich  haufig  2  S-Myosine  mit  scharf  unterschiedlichen  Sedimentationskon- 
stanten. 

Werden  S-Myosine  mit  einer  geniigenden   Menge  ATP  versetzt,   so  fallen  ihre 
*  Die  Trennung  bei  cinem  0.6  m  KCl-Extrakt  ist  kurz  beschrieben  im  FIAT-Review*". 
Literatur  S.  24. 


i8 


H.  H.  WEBER 


VOL.  4  (1950) 


Sedimentationskonstanten  reversibel  auf  die  Werte  des  L-Myosin  (vergl.  Punkt  3  der 
Kurven  2  und  i  und  Punkt  Q  der  Kurve  4  der  Fig.  i,  siehe  ferner  Fig.  2). 


iiiil!!!!liiilllllli 


Fig.  2.  a)  Aktomyosin  der  Kurve  4  (Fig.  i);  b)  nach  ATP-Zusatz 

Wird  L-Myosin  mit  einer  geniigenden  Menge  Aktin  versetzt,  so  verschwindet  seine 
Sedimentationskonstante  und  es  tritt  dafiir  die  Sedimentationskonstante  eines  S- 
Myosin  auf  (vergl.  Punkt  •  Kurve  i  mit  Punkt  •  5  der  Fig.  i).  Wird  zu  wenig  Aktin 
hinzugesetzt,  so  tritt  ebenfalls  die  Sedimentationskonstante  eines  Aktomyosin  auf,  aber 
es  bleibt  ausserdem  ein  Teil  des  L-Myosin  erhalten. 

Die  Praparate  des  langsam  sedimentierenden  Myosin  und  des  Myosin  nach  Szent- 
Gyorgyi  haben  eine  niedrige,  ATP-unempfindliche  Viskositat,  die  vom  Gefalle  erst  bei 
sehr  niedrigen  Wert  en  starker  abhangt ;  die  Viskositat  der  S-Myosine  ist  f  iir  j  edes  Praparat 
verschieden,  sehr  viel  hoher,  starker  vom  Gefalle  abhangig  und  fallt  auf  ATP-Zusatz 
ungefahr  auf  den  Wert  des  L-Myosin  (vergl.  die  Kurven  i,  2  und  3  der  Fig.  i  und 3)*. 


10 
9 
0 

7 
6 
5 
i 
3 
2 


\ 

y 

\ 

\ 

\, 

\ 

N^ 

V 

^ 

Vw 

\ 

'^^ 

9- 

r*>sg 

2 

V 

">0Q. 

m 

n 

-e 

^S' 

^ 

f- 

0 

0 

1 

(^ 

^ 

^ 

logTl' 
IS 

',* 
1,2 
1,0 
0.0 
0.6 
O.i 
0.2 


1 

2  L— -"'^■^ 


0.2 
0  cm^ 


200 


600 


Gefalle  6  r  _lkl_ 

Fig.  3.  Viskositaten. 
Kurve  i :  L-Myosin,  Kurve  2  und  3 :  Aktomyosine, 
die  Zeichen  fiir  die  einzelnen  Versuchspunkte  haben 
dieselbe  Bedeutung  wie  in  Fig.  i. 


Aktin    12        10         e  5         4  2 

Myosin  0         2  <  6         0         10        12  cm  ^ 

MiichungsyerhSttnii 
Fig.  4.  Viskositaten  kiinstlicher  Akto- 
myosine, Kurve  i  vor  ATP-Zusatz, 
Kurve  2  nach  ATP-Zusatz;  Abszisse 
Mischungsverhaltnis  von  Aktin-  und  L- 
Myosinlosung  in  ml,  Ordinate  log  r]'. 
Aktin  o.385%ig,  L-Myosin  0.701  %ig. 


Genau  genommen  fallt  die  Viskositat  von  Aktomyosinen  durch  ATP  auf  einen  Wert,  der  sich 


Vergleicht  man  nur  die  Aktomyosine  unter  sich,  so  wachsen  Viskositat  und  Sedimentations- 
konstante keineswegs  parallel  (vergl.  Punkt  ^  der  Kurve  4  der  Fig.  i  mit  Punkt  ©  der  Kurve  2 
der  Fig.  3).  Das  ist  nicht  wunderbar:  denn  das  Achsenverhaltnis  wirkt  auf  beide  Phanomene  ent- 
gegengesetzt. 

Literatur  S.  24. 


VOL.  4  (1950) 


MUSKELPROTEINE 


19 


aus  einem  Beitrag  des  freien  L-Myosin  und  freien  Aktin  des  Komplexes  zusammensetzt  —  und  zwar 
so,  dass  sich  unter  ATP  der  log  rj'  der  L-Myosin-  und  der  Aktinkomponente  addiert  und  nicht  etwa 
die  beiden  ?j'  Werte  selbst  (Fig.  4)^^  Da  aber  im  ATP-Versuch  nach  Szent-Gyorgyi  immer  die 
Viskositat  auf  die  Gesamteiweisskonzentration  (L-Myosin  -|-  Aktin)  bezogen  wird  und  da  ferner  r]' 
fiir  Aktin-  und  Myosinlosungen  gleicher  Konzentration  sehr  ahnlich  ist,  fallt  der  Unterschied  nicht 
sehr  auf  (s.  u.). 

Von  der  Konzentration  hangen  die  Viskositaten  aller  Myosinkomponenten  und  des 
aktiven  Aktin  nach  der  ARRHENius-Formel  log  >;'  =  K-c  ab*.  BeiGefalle  1000  betragt 
der  K-Wert  fiir  L-Myosin  0.9  und  streut  fiir  aktives  Aktin  zwischen  0.9  und  1.3. 

Die  haufig  auftretenden^"  Sedimentationskonstanten  der  Kurve  la  der  Fig.  i 
stammen  von  einheitlichem,  denaturierten  L-Myosin:  einheitliche  Praparate  mit  diesen 
Sedimentationskonstanten  und  Mischungen  solcher  Praparate  mit  L-Myosin  geben  die 
niedrige  Viskositat  des  L-Myosin  und  sind  ATP-unempfindlich  (vergl.  Kurve  la  der 
Fig.  I  mit  Kurve  i  der  Fig.  3)^''.  Die 
Komponente  mit  S20  (c  =  o)  =  15  ist 
also  kein  Akto-  oder  S-Myosin.  Und  sie 
entsteht  aus  L-Myosin  im  Laufe  der 
Zeit  und  der  Umf allungen  (vergl.  Fig.  5). 
Die  "Kristallisation"  begiinstigt  durch 
ihre  hohere  Dauer  diesen  Vorgang  mehr 
als  die  oben  beschriebene  Abtrennung 
des  L-Myosin  durch  fraktionierte  Um- 
fallung  (vergl.  Fig.  5b  und  c).  Die 
Denaturierung  vollzieht  sich  offenbar 
in  scharfen  Stufen.  Zwischenwerte 
zwischen  den  Kurven  i  und  la  wurden 
nie  beobachtet.  Mit  fortschreitender 
Denaturierung  wachst  nur  der  Anted 
der  denaturierten  Komponente  (S20 
(c  =  o)  =  15)  auf  Kosten  des  urspriing- 
lichen  L-Myosin  (vergl.  d  und  e  der 
Fig.  5). 


\, 


23   24  25      24  25    26      2i   25  26  2U  25    26  27  28      25   26  27  28  29 


Fig. 5.  Sedimentaiionsgradienien. 
Linker  Gipfel  =  L-Myosin  mit  der  Sedimentations- 
geschwindigkeit  der  Kurve  i  der  Fig.  i,  rechter 
Gipfel  (in  b,  d  und  e)  denaturiertes  L-Myosin  mit 
der  Sedimentationsgeschwindigkeit  der  Kurve  la 
der  Fig.  i.  a)  =  i  X  "kristallisiert"  4  Tage  p.m., 
b)  =  2  X  "kristallisiert"  8  Tage  p.  m.,  c)  =  2  X 
fraktioniert  umgefallt  4  Tage  p.  m.,  d)  =  ebenso, 
aber  20  Tage  p.  m.,  e)  =  2  X  "kristallisiert",  i  X 
umgefallt,  9  Tage  p.m. 


Wahrend  der  letzten  Jahre  wurden  im 
Laboratorium  von  Svedberg  gleichzeitig 
mit  unseren  Untersuchungen  die  Sedimen- 
tationskonstanten der  unfraktionierten  Myo- 
sinlosungen untersucht^".  Die  Ergebnisse 
stimmen  experimentell  mit  den  hier  ange- 
gebenen  Werten  fiir  die  gereinigten  einheitlichen  Komponenten  iiberein.  Dagegen  sind  die  Sedi- 
mentationskonstanten bei  den  schneller  sedimentierenden  Komponenten  etwas  anders  auf  c  =  o 
extrapoliert.  Dies  beruht  darauf,  dass  die  Extrapolationsstrecke  wesentlich  grosser  ist  als  bei  uns 
und  dass  s  und  nicht  i/s  geradlinig  extrapoliert  wurde.  So  werden  die  Sedimentationskonstanten  der 
Kurve  la  (Fig.  i)  fiir  c  ^  o  auf  12  und  der  Kurve  2  auf  50  extrapoliert  statt  auf  15  bezw.  93.  Fiir 
die  langsamste  Komponente  und  ebenso  fiir  das  kristallisierte  Myosin  nach  Szent-Gyorgyi  wird 
SjQ  (c  =  o)  mit  7.2  Svedberg23  angegeben  in  guter  Ubereinstimmung  mit  unserem  Wert  von  7.1 
Svedberg.  Die  angegebenen  Sedimentationsdaten  diirfen  also  als  gesichert  angesehen  werden. 

Fiigen  wir  hinzu,  dass  der  scheinbare  Absorptionskoelhzient  infolge  von  Licht- 
streuung  bei  L-Myosinlosungen  '^^  o.i  cm"^  ist — nach  Abzentrifugieren  sehr  feiner 

*  Dies  gilt  im  Grunde  nur  streng,  wenn  r]'  aus  Messungen  mit  iiblichen  Ostwald-  oder  Ubbe- 
LOHDE-Viskosimetern  ohne  HAGENBACH-Korrektur  berechnet  wird.  Mit  HAGENBACH-Korrektur 
hangt  log  7^' nicht  mehr  ganz  geradlinig  von  der  Konzentration  ab;  die  Abhangigkeit  folgt  dann  der 
Formel  von  G.  V.  Schulz  und  F.  Blaschke^^'  ^s. 


Literatur  S.  24. 


20 


H.  H.  WEBER 


VOL.  4  (1950) 


ungeloster  Partikel  mit  16000  Touren  sogar  nur  0.05  cm~^ — ,  wahrend  er  bei  S-Myosinen 
und  kiinstlichen  Aktomyosinen  rw  i  cm~i,  so  ist  damit  die  Identitat  von  S-Myosinen  mit 
Aktontyosinen  und  von  L-Myosinen  mit  "Myosin"  durch  Ubereinstimmung  in  alien 
Eigenschaften  bewiesen*. 

Zu  Szent-Gyorgyi's  Anschauungen  ergibt  sich  nur  in  einem  wichtigen  Punkt  eine 
Differenz:  Aktomyosine  sedimentieren  mit  verschiedenen  scharf  getrennten  Sedimen- 
tationsgeschwindigkeiten — haufig  sogar  in  derselben  Aktomyosinlosung.  Die  Akto- 
myosinbildung  aus  den  beiden  Komponenten  scheint  also  in  Stufen  stattzufinden  und 
nicht  gleitend  in  beliebiger  Proportion — genau  so  wie  das  L-Myosin  in  scharf  getrennten 
Sedimentationsstufen  denaturiert. 

Die  sparlichen  vorlaufigen  Angaben  liber  Sedimentation  und  Viskositat  des  inak- 
tiven  und  aktiven  Aktin  sind  aus  Tabelle  i  zu  ersehen. 


V 


Werden  die  zahlreichen 


mm  Hg 


1.1 


iO 
0.9 
0.8 
0.7 
0.6 
0.5 
0,4 
0.3 
0.2 
0.1 


/ 

/ 

/ 

'A 

/ 

V. 

/ 

V 

/ 

^ 

^<<^ 

B 

3 

/ 

r 

i- 

X 

K 

0      0.2    OA    0.6    0.8    10     12     U     16 


L-Myosin-Praparationen  der  Sedimentationskurve  i  der 
Fig.  I  auf  ihren  osmotischen  Druck  unter- 
sucht,  so  steigt  der  osmotische  Druck  bis  zur 
Konzentration  2.2%  von  o  auf  1.16  mm  Hg 
(Fig.  6)  *  * .  Aus  der  P/c  Kurve  (Fig.  7)  ergibt 
sich  fur  P/Ciiia  o  der  Wert  2.05 -lo-^,  d.h.  ein 
Teilchengewicht  von  840000  (±  33000). 

Aus  diesem  Teilchengewicht  und  S20 
(c  =  0)  errechnet  sich  D20  (c  =  0)  zu  0.874  *  io~'- 
Der  vorlaufige  Mittelwert  unserer  direkten 
Bestimmung  ergibt  Dgo  =  0.9-10"'***. 

Wird  aus  dem  Teilchengewicht  und  der 
Sedimentationskonstanten  das  Achsenverhalt- 
nis  berechnet,  so  ergibt  es  sich  zu  i/g  =  102. 

Wird  das  Achsenverhaltnis  auf  Grund 
der  Untersuchungen  von  G.  V.  Schulz^" 
liber  Mischungsentropie  und  osmotischen 
Druck  berechnet  nach  der  Formel**** 


I    2.0   2.2 

c  in% 


Fig.  6.  Osmotischer  Druck  von  L-Myosin 


4CB 

JT  A 


10-^ 


so  ergibt  sich  das  Achsenverhaltnis  q  zu  128. 

Da  der  Ausdruck  Myosin  sehr  haufig  fiir  die  Gesamtfraktion  und  ihre  Losungen  gebraucht 
wird,  erscheint  es  als  eine  klare  und  kurze  Bezeichnungsweise,  dies  weiterhin  zu  tun,  das  sogenannte 
"kristallisierte"  Myosin  als  L-Myosin  und  die  Myosin-Aktin-Komplexe  als  Aktomyosin  zu  bezeichnen. 
Die  Ausdriicke  Myosin  A  und  B  fiir  kurz,  bezw.  lang  extrahierte  Gesamtfraktionen  wiirden  sich  gut 
in  diese  Nomenklatur  einfiigen.  Die  allgemeine  Annahme  dieses  Vorschlages  wiirde  die  Verstandigung 
erleichtern. 

**  Die  Methodik  der  "Mcssung  sehr  kleiner  osmotischer  Drttcke"  ist  von  H.  Portzehl  und 
H.  H.  Weber  beschrieben^*. 

***  Der  in  den  FIAT-Reviews  auf  Grund  einer  einzigen  Konzentrationsreihe,  die  von  G.  Bergold 
durchgefiJhrt  wurde,  angefiihrte  Wert  fiir  Djq  (c  =  o)  von  0.45-10—'  hat  sich  bei  Nachpriifung  der 
Unterlagen  als  unzuverlassig  erwiesen  und  muss  fallen  gelassen  werden,  obwohl  er  mit  den  Werten 
iibereinstimmt,  die  Pedersen^^  auf  miindliche  Mitteilung  von  Snellman,  Jenow  und  Erdos  an- 
gegeben  hat. 

****  q  =  Achsenverhaltnis;  g  =  Dichte  des  Eiweiss;  A  =  p/cum  o.  B  =  Jp/c;  c  =  Gramm/Liter. 

Literaiur  S.  24. 


VOL.  4  (1950) 


MUSKELPROTEINE 


21 


Bei  der  ausgezeichneten  experimcntellen  Sicherheit  der  Sedimentationskonstanten 
und  der  Kurve  des  osmotischen  Druckes  erscheint  das  Teilchengewicht  '^^  840000  und 
das  Achsenverhaltnis  --^loo  recht 
zuverlassig.  Die  auf  Grund  von 
S20  und  D20  friiher^'^'  -^'  ^^  ange- 
gebenen  Teilchengewichte  schei- 
nen    dagegen    einer    sorgfaltigen 


p/cW 


0.5 


OA 


0.J 


0.2 


0,1 


q 

0^^ 

.  < 

L  ^ 

0 

\o 

,^ 

&^ 

r^ 

0   \p^ 

yc. 

^-r^ 

^tT 

0 

-""c.^ 

Uberpriifung  von  Djq  zu  bediir- 

fen.  Denn  die  Messungen  von  D.^ 

sind    nicht    nur    bisher    wider- 

spruchsvoll    sondern    auch    sehr 

empfindlich  gegen  Beimengungen 

langsamer  diffundierender  Dena-  c^g/uter 

turierungsformen  des  L-Myosin.  Fig.  7. 

Reine     Praparate     von     L- 
Myosin  scheinen  monodispers  zu  sein.  Fig.  8  zeigt  ein  Sedimentationsdiagramm  einer  L- 
Myosinlosung  nach  Skalenmethode.  Berechnet  man  nach  dem  Verfahren  von  Bergold^ 
den  Betrag,  um  den  sich  die  Gradientenkurve  vom  Zeitpunkt  i  bis  zum  Zeitpunkt  10 

durch  Diffusion  verbreitert  und  addiert  diesen 
Betrag  zur  Breite  der  Kurve  i,  so  erhalt  man 
die  gestrichelte  Glockenkurve,  die  die  Gradien- 
tenkurve 10  einschliesst.  Die  gefundene  Sedi- 
mentation ist  also  einheitlicher,  als  sie  unter 
Beriicksichtigung  des  Diffusionseffektes  hatte 
sein  diirfen.  Der  Grund  liegt  in  der  Zunahme 
der  Sedimentationsgeschwindigkeit  mit  ab- 
nehmender  Konzentration :  die  durch  Diffu- 
sion zuriickgebliebene  Teilchen  sedimentieren 
infolgedessen  schneller  und  die  Gradienten- 
kurve wird  infolgedessen  schmaler  und  in 
ihrem  vorderen  Teil  steiler,  als  sie  es  auf  Grund 
unbeeinflusster  Diffusion  geworden  ware.  Da 
aber  die  Konzentrationsabhangigkeit  beim 
L-Myosin  nicht  gross  ist,  und  da  die  experi- 
mentelle  Gradientenkurve  nicht  unbetracht- 
lich  schmaler  ist,  als  sie  bei  reiner  Diffusion 
sein  miisste,  ist  es  wahrscheinlich,  dass  die 
Sedimentationskonstanten  allcr  cinzelnen 
Myosinteilchen  gleich  sind. 

Dass  auch  die  mechanische  Beweglichkeit 
aller  einzelner  L-Myosinteilchen  anscheinend 
gleich  ist,  d.h.  die  Diffusionskonstante  einheit- 
lich  ist,  geht  aus  Fig.  9  hervor :  Aus  der  Dif- 
fusionsformel  lasst  sich  ableiten,  dass  bei 
einheitlicher  Diffusionskonstante  das  Quadrat  der  Breite  der  Diffusionsgradientenkurve 
geradlinig  vom  log  der  Hohe  abhangt,  in  der  die  Breite  gemessen  ist.  Die  Ungestortheit 
der  Gradientenkurve  folgert  sich  aus  ihrer  Symmetric.  Die  Auswertung  zweier  beliebiger 
Literatur  S.  24. 


«r 

BO 

1 

f 

"3 

70 

1 

'5 

60 

f\ 

\ 

X 

7 

50 

\ 

1 

1 

\ 

A 

;i 

10 

«0 

\ 

' 

I 

1 

A 

30 
20 
10 

\ 

\ 

n 

i 

1  ] 

\ 

7 

'\ 

A 

V 

\ 

k 

/ 

/ 

J 

> 

\ 

s 

N 

\ 

V 

\ 

^ 

-« 

18     19     20    21    22    23     24     25    26    27    23    29     30 

X 

Fig.  8.  Sedinientationsgradienten  von  L-Myosin. 

Sedimentationsgradienten  gefunden,  o-o-o 

berechnet  aus  Kurve  x^  und  Dgo- 


22 


H.  H.  WEBER 


VOL.  4  (1950) 


10 


experimentell  gefundener  Diffusionsgradientenkurven  zeigt  (Fig.  9) ,  dass  die  Quadrate  der 
Breiten  rechts  und  links  der  Symmetrieachse  (x^^  und  x^^)  gleich  oder  fast  gleich  sind,  und 

dass  beide  geradlinig  von  log  H  abhangen.  Es  handelt  sich 
also  um  storungsfreie  Diffusion  mit  einheitlicher  Diffu- 
sionskonstante. 

Einheitliche  Sedimentations-  und  Diffusionskon- 
stante  aber  bedeutet,  einheitliche  Grosse  und  einheitliche 
Gestalt  der  einzelnen  Teilchen  des  L-Myosin. 

Fiir  diese  Grosse  und  Gestalt  ergeben  sich  aus  Sgo 
und  Teilchengewicht  und  unter  der  plausibelen  Annahme 
eines  spezifischen  Volumens  von  0.75  folgende  Masse:  22 
bis  23  A  Dicke  bei  2200  bis  2400  A  Lange  fiir  quadra- 
tischen  bezw.  runden  Querschnitt.  Vorlaufige  friihere 
Angaben*"  sind  durch  diese  Werte  iiberholt. 

Das  /3-Myosin  Dubuissons^  scheint  mit  dem  L-Myosin 
identisch  zu  sein.  SoUte  sich  das  bestatigen,  d.h.  sollten 
die  Spuren  des  y-Myosin  mit  dem  L-Myosin  nichts  zu 
tun  haben,  so  waren  alle  Teilchen  des  L-Myosin  nicht 
nur  in  Grosse  und  Gestalt,  sondern  auch  in  ihrer  elektri- 
schen  Ladung  gleich  (vergl.  auch  Szent-Gyorgyi^^). 

VI 


^.n: 

c 

^' 

<    > 

■  y 

2 

' 

./ 

/ 

/x 

Ofi       Ofi        1,0        1.2 


1A 


1.6        1.0 
logH 

Fig.  9.  x2  =  (Breite  der  Diffu- 

sionsgradientenkurve   von    der 

Symmetrieachse   aus)^. 

nach  rechts  =  x^^. 

X— X  — X  nach  links  =  Xg^ 
fiir  2  verschiedene  Gradienten- 
kurven  (i  und  2). 


An  Meyerhof's  Entdeckung  jenes  Zusammenhanges  zwischen  Kolloidik  und  Stoff- 
wechsel,  der  durch  die  lonisationswarme  der  Proteine  gegeben  ist,  schloss  sich  die  erste 
Periode  systematischer  Erforschung  der  Muskelproteine  an.  Sie  fiihrte  in  der  Feststellung 
der  Wechselwirkungen  zwischen  Adenosintriphosphat  und  Myosin  zu  einem  neuen 
Zusammenhang  von  Stoffwechsel  und  Eiweisszustand —  diesmal  sogar  Zustand  gerade 
des  kontraktilen  Proteins.  Dieser  Zusammenhang  gewann  eine  eindrucksvolle  Aktualitat 
dadurch,  dass  sich  Myosinfaden  auf  ATP-Zusatz  bei  niedriger  lonenstarke  zusammen- 
ziehen  und  bei  hoherer  lonenstarke  wieder  ausdehnen  (Szent-Gyorgyi^^'  ^).  Von  neuem 
folgte  systematische  Proteinforschung  mit  dem  Ziel  einer  verfeinerten  Analyse  gerade 
der  kontraktilen  Eiweissfraktion.  Diese  Analyse  steht  noch  in  ihren  Anfangen.  Infolge- 
dessen  kann  der  Mechanismus  der  ATP-Wirkung  auf  Myosin  nur  mit  Zuriickhaltung 
erortert  werden :  in  Losung  besteht  er  zweifellos  in  einer  reversiblen  Vermin derung  der 
Kohasionskrafte  zwischen  Aktin  und  L-Myosin.  Das  fadenformige  Gel  dagegen  wird 
offenbar  (s.o.)  nicht  von  ATP  sondern  von  der  lonenstarke  reversibel  beeinflusst.  ATP 
scheint  nur  notig  zu  sein,  damit  der  Faden  beim  Ubergang  von  einer  lonenstarke  zur 
anderen  das  neue  Gleichgewicht  wirklich  erreicht  und  nicht  in  einem  falschen  Gleichge- 
wicht  stecken  bleibt.  ATP  macht  offenbar  die  Fadenmolekiile  beweglich,  d.h.  es  setzt 
auch  im  Gel  ihre  Kohasionskrafte  herab.  Es  scheint  somit,  als  genligte  der  ATP-Einfluss 
auf  die  Kohasionskrafte  des  Aktomyosin  zur  Erklarung  der  bisher  vorliegenden  Beob- 
achtungen  am  Gel  wie  am  Sol.  Ob  dieser  Einfluss  allerdings  das  einzige  Prinzip  der 
Wirkung  ist,  muss  solange  off  en  bleiben  wie  man  nicht  weiss,  warum  Pyrophosphat  auf 
Aktomyos'mldsungen  ahnlich  oder  gleich  wirkt  wie  ATP,  wahrend  es  Aktomyos'mf aden 
nicht  beeinflusst^^. 

Die  umgekehrte  Wirkung,  die  Wirkung  des  Myosins  auf  das  ATP  ist  einem  gewissen, 
vorlaufigen  Abschluss  der  Erkenntnis  zugefiihrt  durch  die  Entdeckungen  von  Polis 
Literatur  S.  24. 


VOL.  4  (1950)  MUSKELPROTEINE  23 

UND  Meyerhof^*.  Die  ATPase-Wirkung  des  Myosin  ist  zusammen  mit  einer  kleinen 
Eiweissmenge  abtrennbar,  ohne  dass  die  Wirkung  verloren  geht !  Das  ATPase-Ferment- 
eiweiss  gehort  also  offenbar  nur  in  soweit  zum  Myosin,  als  es  auf  den  Myosinkomplex 
gebunden  ist.  Auf  Grund  der  Monodispersitat  dieses  Komplexes — eben  des  L-Myosin — 
liegt  es  allerdings  nahe  anzunehmen,  dass  es  sich  hier  nicht  um  einen  zufalligen,  sondern 
um  einen  stochiometrischen  Komplex  handelt. 

Und  so  schliesst  heute  die  Betrachtung  der  Muskeleiweisskorper  mit  dem  Namen 
O.  Meyerhof,  mit  dem  sie  vor  einem  guten  Viertelj  ahrhundert  begann. 

ZUSAMMENFASSUNG 

Im  Rahmen  eines  kurzen  zusammenfassenden  Berichtes  iiber  Muskelproteine  werden  einige 
neue  Tatsachen  gebracht : 

1.  Die  Diskrepanz  zwischen  der  parallelfaserigen  Struktur  des  I-Abschnitjtes  und  dem  Fehlen 
von  Stabchendoppelbrechung  der  I-Bande  kann  erklart  werden,  wenn  man  annimmt,  die  Stabchen 
bestanden  aus  dem  stark  negativ  doppelbrechenden  N-Protein  von  Szent-Gyorgyi.  In  der  I-Bande 
wiirden  sich  dann  die  positive  Stabchendoppelbrechung  dieses  Proteins  und  seine  negative  Eigen- 
doppelbrechung  gerade  aufheben. 

2.  Die  L-Myosinkomponente  und  die  Aktomyosinkomponenten  des  Myosin  konnen  sauber 
getrennt  werden. 

3.  Es  werden  fiir  die  einzelnen  isoUerten  Komponenten  Sedimentationskonstanten,  Viskositaten 
und  Werte  fiir  die  Lichtstreuung  angegeben  —  und  ebenso  die  Anderungen  dieser  Werte  bei  Zusatz 
von  ATP  Oder  Aktin. 

4.  Eine  haufig  vorkommende  Komponente  des  Myosin  besteht  aus  einer  scharf  abgegrenzten 
Denaturierungsstufe  des  L-Myosin. 

5.  Aktin  und  L-Myosin  vereinigen  sich  stufenweise  zu  Aktomyosinen  ganz  verschiedener  Sedi- 
mentationskonstanten. 

6.  Das  L-Myosin  sedimentiert  und  diffundiert  monodispers. 

7.  Das  L-Myosinteilchen  ist  ein  Stabchen  von  2200-2400  A  Lange  und  22-23  -^  Dicke. 

8.  Die  beobachteten  ATP-Wirkungen  konnen  vorlaufig  sowohl  im  Sol  wie  auch  im  Gel  als 
eine  reversible  Verminderung  der  Kohasionskrafte  zwischen  L-Myosin  und  Aktin  befriedigend  be- 
handelt  werden. 

SUMMARY 

The  information  available  on  muscle  proteins  is  reviewed  and  in  addition  the  following  new 
facts  are  presented : 

1.  The  discrepancy  of  the  parallel-fibred  structure  of  the  I-band  and  the  lack  of  the  form 
birefringence  might  be  explained  by  supposing  that  the  micelles  consist  of  the  strongly  negative 
birefringent  N-Protein  of  Szent-Gyorgyi.  Thus  the  positive  form  birefringence  of  this  protein  is 
compensated  by  its  own  negative  birefringence. 

2.  It  is  possible  to  separate  completely  both  components:  L-myosin  and  actomyosin. 

3.  The  sedimentation  constants,  viscosities,  and  values  for  light  scattering  of  the  isolated  com- 
pounds are  given.  The  changes  of  these  values  produced  by  addition  of  ATP  or  actin  are  also  indicated. 

4.  It  is  shown  that  one  component  of  the  myosin  which  is  frequently  found  consists  of  a  sharply 
limited  stage  of  denaturated  L-myosin. 

5.  Actin  and  L-myosin  combine  step  by  step  to  actomyosins  of  quite  different  sedimentation 
constants. 

6.  The  sedimentation  and  diffusion  of  L-myosin  is  monodispers. 

7.  The  L-myosin  particle  is  a  micelle  with  a  length  of  2  200-2400  A  and  a  diameter  of  22-23  ■^• 

8.  The  observed  effects  of  ATP  in  sol  as  well  as  in  gel  may  satisfactorily  be  interpreted  as  a 
reversible  weaking  of  the  cohesive  forces  linking  L-myosin  and  actin. 

r£sum£ 

Quelques  faits  nouveaux  sont  d^crits  dans  un  rapport  sur  les  prot^ines  du  muscle. 

I.  En  supposant  que  les  micelles  soient  form^es  par  la  N-prot6ine  de  Szent-Gyorgyi  a  refraction 
double  negative,  il  est  possible  d'interpr^ter  la  discordance  entre  la  structure  fibrillaire  du  segment  I 
du  muscle  et  le  manque  de  la  refraction  double.  Dans  ce  cas,  la  positivite  de  la  refraction  double 
formale  pourrait  etre  compens^e  par  la  negativite  de  la  refraction  double  propre  de  la  meme  proteine. 

Liter atur  S.  24. 


24  H.  H.  WEBER  VOL.  4  (195O) 

2.  On  peut  separer  completement  les  deux  constituants  L-myosine  et  actomyosine. 

3.  Les  constantes  de  sedimentation,  les  viscosites  et  les  valeurs  de  I'absorption  apparente  des 
constituants  isol^s  sont  d^crites.  En  plus,  les  variations  de  ces  valeurs  produites  par  I'addition  d'ATP 
ou  d'actine  sont  donn^es. 

4.  II  est  demontre,  qu'un  constituant  de  la  myosine  frequemment  trouve  est  une  fraction 
exactement  d^limitee  de  L-myosine  denature. 

5.  L-myosine  et  actine  se  combinent  en  plusieurs  etapes  formant  des  actomyosines  avec  des 
constantes  de  sedimentation  completement  differentes. 

6.  La  sedimentation  et  la  diffusion  de  L-myosine  sont  monodisperses. 

7.  Une  particule  de  L-myosine  a  une  longueur  de  2200-2400  A  et  un  diametre  de  22-23  -^• 

8.  A  I'etat  actuel  les  effets  observes  de  I'ATP,  en  solution  ou  en  gel,  peuvent  etre  interpretes 
comme  une  diminution  reversible  des  forces  d'union  entre  L-myosine  et  actine. 

LITERATUR 

1  W.  T.  AsTBURY  UND  S.  DICKINSON,  Nature,  135  (1935)  95,  1765. 

-  K.  Bailey,  Nature,  157  (1946)  36S;  Biochem.  J.,  43  (1948)  271,  279. 

^  G.  Bergold  UND  G.  Schramm,  Naturforschimg,  2b  (1947)  loS. 

*  T.  BARANOWSKi,«Ho^/5e  Seyler's  Z.  physiol.  Chem  ,  260  (1939)  43. 

•^  G.  BoHM  UND  H.  H.  Weber,  Kolloid-Z.,  61  (1932)  269. 

^  F.  BucHTHAL,  Acta  Physiol.  Scand.  13  (1947)  i^?- 

'  G.  T.  CoRi  [nach  brief licher  Mitteilung). 

^  H.  J.  Deuticke,  Pfliigers  Arch.  ges.  Physiol.,  224  (1930)  i,  44. 

^  M.  DuBuissoN,  Experientia,  2/10  (1946)  i;  3/11  (1947)  i. 
1"  R.  E.  Duff,  Proc.  Soc.  Exptl  Biol.  Med.,  29  (1932)  508. 

i"**  J.  T.  Edsall  und  J.  T.  Edsall  und  A.  v.  Muralt,  /.  Biol.  Chem.,  89  (1930)  289a,  315. 
11  W.  A.  Engelhardt  und  Ljubimova,  Nature,  145  (1939)  668;  Biokhimiya,  4  {1939)  716. 
1^  V.  FiJRTH,  Arch,  exptl.  Path.  Pharmakol.,  36  (1895)  231. 

1^  M.  G^rendas  und  a.  G.  Matoltsy,  Hung.  Acta  Physiol.,  i  (1948)  116,  121,  128. 
"  N.  Gral;6n,  Biochem.  J.,  33  (1939)  1342. 
1^  J.  P.  Greenstein,  /.  Biol.  Chem.,  loi  (1933)  602. 
1^  W.  Haumann  und  H.  H.  Weber,  Biochem.  Z.,  283  (1935)  146. 
^^   F.  Kamp,  Biochem.  Z.,  307  (1941)  226. 

1^  H.  Kaumanns  und  H.  H.  Weber,  M akromolekulare  Chemie  (erscheint  demnachst). 
1*  O.  Meyerhof,  Pfliigers.  Arch.  ges.  Physiol.,  195  (1925)  22;  204  (1924)  295. 
-"  K.  Meyer  und  H.  H.  Weber,  Biochem.  Z.,  266  (1933)  i37- 

21  D.  M.  Needham,  J.  Needham,  S.  C.  Shen   und  A.  S.  C.  Lawrence,  Nature,  147  (1941)  766. 
-2  D.  Noll  und  H.  H.  Weber,  Pfliigers  Arch.  ges.  Physiol.,  233  (1934)  -34- 
'^•^  K.  O.  Pedersen,  Ann.  Rev.  Biochem.  (1948)  169. 
-*  D.  B.  PoLis  UND  O.  Meyerhof,  /.  biol.  Chem.,  169  (1947)  389,  401. 
'^^  H.  PoRTZEHL  UND  H.  H.  Weber,  Mukromolckulare  Chemie  (im  Erscheinen). 
-^  H.  Portzehl,  G.  Schramm  und  H.  H.  Weber  (1943)  unveroffentlicht. 
-""H.  Portzehl  und  H.  H.  Weber  (erscheint  demnachst). 
2'  E.  Roth,  Biochem.  Z.,  318  (1946)  74. 

-^  F.  O.  Schmitt,  C.  E.  Hall  und  Jakus,  Biol.  Bull.,  90  (1946)  32. 
28"  G.  V.  Schulz,  Z.  Naturforsch.,  2a  (1947)  348. 
28   G.  V.  Schulz  und  F.  Blaschke,  /.  prakt.  Chem.  158  (1941)  130. 
^"  O.  Smellmann  und  M.  Tenow,  Bicchim.  Biophys.  Acta,  2  (1948)  384. 
^^  G.  Schramm  und  H.  H.  Weber,  Kolloid  Z.,  100  (1942)  242. 
•'i«R.  Stover  und  H.  H.  Weber,  Biochem.  Z.,  259  (1933)  269. 
*2  Szent-Gyorgyi  und  Mitarbeiter,  Studies  Inst.  med.  Chem.  Univ.  Szeged   (G.  Karger,  Basel,   New 

York)  I  (1942);  2  (1942);  3  (1943);  Hung.  Acta  Physiol.,  i  (1948)  2,  3  (1948);  4,  5  (1948). 
^^  Szent-Gyorgyi,  Nature  of  Life,  Academic  Press  Inc.  New  York. 
^*  Szent-Gyorgyi,  Chemistry  of  Muscular  Contraction,  Academic  Press  Inc.  Xcw  York. 
'■'^  H.  H.  Weber,  Biochem.  Z.,  218  (1930)  i. 
^^  H.  H.  Weber,  Pfliigers  Arch.  ges.  Physiol.  235  (1934)  205. 
^^   H.  H.  Weber,  Ergeb.  Physiol.,  36  (1934)  109. 
■**  H.  H.  Weber,  Naturwissenschaften    27  (1939)  33- 
**  H.  H.  Weber,  Eiweisskorpcr  als  Riesenionen,  Schriften  Kunigsberg  Gelehrten-Ges.,  Naturw.  Klasse 

H.  4  (1942). 
*°  H.  H.  Weber,  FIAT-Review,  Band  III,  Physiologic  Abschnitt  Muskel  (im  Erscheinen). 
^^  C.  Wolpers,  Deut.  med.  Wochsch.  29/30  (1944)  495;  Sitzbcr.  Berlin  Med.  Ges.  von  24.5.45 

Eingegangen  den  4.  April  1949 


VOL.  4  (1950)  BIOCHIMICA   ET   BIOPHYSICA   ACTA  25 


MODIFICATIONS  DANS  LA 

STRUCTURE  PHYSICO-CHIMIOUE  DE  L'EDIFICE  CONTRACTILE  AU 

COURS  DU  CYCLE  DE  LA  CONTRACTION  MUSCULAIRE 

par 

M.  DUBUISSON 

Laboratoirc  de  Biologic  generale,  Faculte  des  Sciences,  Universite  de  Liege  [Belgique) 


INTRODUCTION 

Pendant  fort  longtemps,  les  recherches  effectuees  sur  le  muscle,  et  qui  ressortis- 
saient  de  trois  disciplines  differentes :  la  morphologic,  la  physiologic  et  la  biochimie,  sont 
restees  sans  connections;  les  techniques  auxquelles  ces  domaines  devaient  faire  appel 
etaient  de  nature  trop  differente  et  les  resultats  obtenus  par  les  divers  chercheurs 
offraient  peu  de  recoupements.  Nul  n'ignore  encore  le  role  de  pionnier  que  notre  Maitre 
O.  Meyerhof,  que  nous  fetons  ici,  a  joue  dans  ce  rapprochement,  si  extraordinairement 
fecond,  entre  la  physiologic  et  la  biochimie  du  muscle.  Ses  travaux  sont  si  classiques,  si 
nombreux,  constituent  un  exemple  si  merveilleux  de  logique,  de  profondeur  et  de  pers- 
picacite,  qu'ils  forment  une  gerbe  modele  dont  nous  sommes  loin  d'avoir  cueilli  aujour- 
d'hui  tous  les  epis.  Je  ne  puis  evoquer  sans  une  certaine  emotion  des  notions  —  comme 
celles  qui  etablissent  les  relations  quantitatives  entre  le  travail  du  muscle  et  son  meta- 
bolisme  — •  qui  nous  sont  devenues  maintenant  si  familieres  que  nous  avons  presque 
oublie  qu'elles  ne  furent  pas  tout  de  suite  evidentes  et  qu'il  a  fallu  bien  du  genie  et  du 
talent  pour  les  etablir;  je  ne  puis  contempler  sans  emerveillement  la  liste  des  enzymes 
qui  interviennent  dans  le  cycle  des  generateurs  d'energie  du  muscle  et  dont  un  si  grand 
nombre  ont  ete  decou verts  par  ce  Maitre. 

Transformations  moleculaires  d'une  part,  travail  musculaire  de  I'autre:  qu'avons- 
nous  entre  les  deux  ? 

Que  sait-on  aujourd'hui  du  mecanisme  grace  auquel  I'energic  chimique  est  trans- 
formee  en  travail  mecanique  ? 

Helas,  la  route  est  difficile.  Les  deux  domaines  se  recoupent  au  niveau  de  la  machine 
musculaire,  formee  de  proteines  de  structure  qui  sont  d'autant  plus  difficiles  a  etudier 
qu'elles  existent,  in  vivo  et  in  situ,  non  pas  comme  la  plupart  des  proteines-enzymes : 
librement  dissoutes  dans  le  sue  musculaire  et  par  consequent  aisement  extractibles  sans 
trop  de  risques  de  modifications,  mais  sous  une  forme  d'association  tres  particuliere 
qui  assure  precisement  cette  structure.  Les  precedes  d'extraction  nous  forcent  a  briser 
celle-ci  pour  ne  retrouver,  dans  nos  extraits,  que  des  morceaux  dont  le  degre  de  disper- 
sion, I'orientation  spatiale,  la  structure  et  les  groupements  prosthetiques  eventuels  sont 
indiciblement  bouscules.  Si  nous  commen^ons  aujourd'hui  a  connaitre  un  certain  nombre 
de  proprietes  de  ces  proteines  de  structures,  considerees  in  vitro,  disons  le  tout  de  suite : 
Bibliograpkie  p.  36(37. 


26  M.  DUBUISSON  VOL.  4  (1950) 

nous  sommes  fort  loin  de  pouvoir  nous  representer  I'edifice  contractile  en  place  dans  le 
muscle  et  toute  tentative  consistant  a  expliquer  comment,  par  I'intervention  de  cet 
edifice,  les  generateurs  d'energie  produisent  un  travail,  ne  peut  etre  par  consequent  que 
fort  speculative  et  tout  au  plus  une  source  plus  ou  moins  suggestive  d'hypotheses  de 
travail,  ce  qui  n'est  d'ailleurs  pas  un  faible  merite. 

Le  nombre  de  proteines  de  structure  qui  ont  ete  Fob  jet  de  recherches  est  deja 
considerable:  citons  la  myosine  (elle-meme  vraisemblablement  complexe:  myosines 
^p  /Sg  (DuBUissoN^),  y  (DuBuissoN^),  I'actine  (Straub^'  *>  ^)  (sous  la  forme  monomere: 
G-actine  et  polymere:  F-actine),  la  combinaison  des  actines  aux  myosines  (F-acto- 
myosine,  G-actomyosine^),  la  tropomyosine  de  Bailey',  la  N  proteine  de  Gerendas  et 
Matoltsy^.  On  possede  les  methodes  pour  extraire  ces  proteines  et  les  separer  les  unes 
des  autres  et  les  resultats  obtenus  sont  deja  qualitativement  et  quantitativement  tres 
reproductibles. 

Ces  techniques  ont  toutes  en  commun  d'attaquer  la  pulpe  musculaire,  prealable- 
ment  finement  divisee  par  des  procedes  mecaniques,  par  des  solutions  dont  les  carac- 
teristiques  principales  ne  sont  pas  tant  de  posseder  une  action  specifique  sur  la 
solubilite  de  ces  molecules  que  d'avoir  une  influence  specifique  sur  leur  extractihilite, 
c'est-a-dire  une  action  disruptive  sur  les  forces  qui  maintiennent  en  place  ces  proteines  de 
structure^. 

Tons  ceux  qui  ont  extrait  ces  proteines  savent  cela  et  je  n'enfonce  qu'une  porte 
ouverte  en  le  repetant.  Mais  peut-etre  n'a-t-on  pas  suffisamment  songe  au  parti  que 
Ton  peut  en  tirer,  sur  un  terrain  ou  la  physiologic  rencontre  cette  biochimie  particuliere. 
Personne  ne  contestera  que  V edifice  contractile  doit  posseder,  a  Vetat  raccourci,  une  structure 
bien  differente  de  celle  quHl  possede  a  Vetat  reldche.  Cette  difference:  c'est  le  noeud  du 
probleme.  Elle  implique  un  remaniement  des  elements  constitutifs,  des  modifications 
des  relations  spatiales,  physico-chimiques,  des  changements  dans  les  modes  de  liaison. 
On  peut  ainsi,  a  priori,  prevoir  que  V extractihilite  des  proteines  de  structure  ne  peut  etre 
la  meme  si  Von  part  de  pulpe  de  muscle  contracts  ou  de  muscle  au  repos.  Et  Ton  saisit  aussi 
tout  de  suite  que,  dans  la  mesure  ou  il  est  possible : 

a)  de  preparer  des  pulpes  musculaires  repondant  a  ces  deux  etats  extremes  du  cycle 
contractile:  I'etat  de  reldchement  et  I'etat  de  contracture; 

b)  d'analyser  qualitativement  et  quantitativement  le  composition  protidique  de  ces 
extraits ; 

c)  d'etablir  I'existence  de  changements  d' extractihilite  de  I'une  ou  I'autre  de  ces 
proteines  de  structure : 

Ton  se  trouve  a  meme  d'aborder  le  probleme  de  la  contraction  musculaire  par  un 
nouvel  angle,  a  la  fois  physiologique  et  biochimique  et,  par  consequent,  de  nature  a 
apporter  des  renseignements  inedits  au  probleme  general  de  la  connaissance  du  meca- 
nisme  de  la  fonction^'  ^". 

C'est  dans  ce  domaine  que  mes  collaborateurs  et  moi  travaillons  depuis  un  certain 
nombre  d'annees. 

Je  voudrais  ici  offrir  a  mon  Maitre  O.  Meyerhof,  sous  la  forme  d'un  aper^u  general 
de  nos  resultats*,  les  fruits  de  notre  modeste  contribution  a  I'etude  du  probleme  de  la 
contraction  musculaire,  dont  11  fut  I'un  des  plus  intenses  animateurs. 


*  Les  travaux  effectu6s  dans  notre  laboratoire,  et  dont  il  sera  question  dans  cet  article,  sont 
cit6s  dans  les  r6f6rences  sous  les  numeros:  i,  2,  9,  10.  11,  12,  13,  14,  15,  16,  17,  28,  29,  34,  36,  37. 

Bibliographic  p.  36l3y. 


VOL.  4  (1950)  CONTRACTION    MUSCULAIRE  27 

I.   PREPARATION  D'EXTRAITS  PROTIDIQUES  DE  PULPES  DE  MUSCLES  DE  LAPIN  SE 
TROUVANT   DANS    UN    INSTANT   DEFINI    DU    CYCLE    DE    LA   CONTRACTION 

La  preparation  d'extraits  musculaires  quelconques  necessite  toujours  a)  la  division 
mecanique  du  tissu,  b)  I'extraction  a  basse  temperature  pour  eviter  les  denaturations, 
autolyses,  etc. 

I,  Lorsqu'il  s'agit  de  muscles  normaux  et  au  repos,  il  faut  que  ni  le  hachage,  ni 
I'abaissement  de  temperature  n'entrainent  une  stimulation  des  fibres  musculaires.  De 
nombreux  tatonnements  ont  montre  que  le  procede  le  plus  sur  consiste  tout  d'abord  a  re- 
froidir  le  muscle,  non  pas  brutalement  en  le  plongeant  dans  I'eau  glacee  ou  I'air  liquide,  ce 
qui  conduit  a  coup  sur  a  une  certaine  stimulation  ou  meme  une  contracture,  au  moins  des 
fibres  peripheriques^^,  mais  graduellement,  en  plagant  les  muscles  non  encore  excises 
dans  une  chambre  froide  (i  a  2°  C)  pendant  au  moins  une  heure.  On  pent  ensuite  hacher 
le  tissu  au  moyen  d'un  broyeur  a  viande  du  genre  Latapie,  ou  placer  le  muscle  refroidi 
dans  une  enceinte  a  — 20  a  — 30°  C,  dans  laquelle  le  muscle  se  congelera  et  pourra  etre 
ensuite  coupe  au  microtome  a  congelation*,  en  tranches  de  20  a  40  //  d'epaisseur.  Ce 
dernier  procede  fournit  des  extraits  plus  riches  et  de  composition  plus  constante  que 
I'autre^^. 

S'il  s'agit  d'obtenir  des  muscles  se  trouvant  a  Vetat  de  raccourcissement  maximum, 
provoque,  par  exemple,  par  la  stimulation  electrique,  le  seul  moyen  connu  d'immobiliser 
le  tissu  en  cet  etat  consiste  a  le  plonger  dans  Pair  liquide.  Nous  avons  montre  que  le 
procede  est  moins  sur  que  Ton  pouvait  a  priori  le  supposer :  le  refroidissement  brusque 
paralyse,  dans  une  certaine  mesure,  les  processus  d'excitation  au  niveau  de  certaines 
fibres  avant  que  celles-ci  aient  pu  etre  saisies  par  la  congelation.  Aussi  observe-t-on 
frequemment,  au  moment  de  I'immersion  —  bien  que  le  tetanos  electrique  soit  main- 
tenu  — ,  un  relachement  musculaire,  plus  ou  moins  considerable^'  ^°.  L'obtention,  par 
cette  methode,  de  fibres  musculaires  contractees  est  done  souvent  un  effet  du  hasard 
et  necessite  un  certain  tatonnement. 

Plus  sur  a  obtenir  est  I'etat  de  raccourcissement  maximum  que  fournissent  certains 
agents  ou  facteurs  contracturants  tels  le  monoiodoacetate^^,  la  str3^chnine^^,  le  rigor 
mortis^^. 

2.  Quel  que  soit  le  procede  utilise  pour  la  dilaceration  du  tissu,  la  mise  en  solution 
de  certaines  proteines  demands  la  presence  de  solutions  salines,  dont  Taction  doit  etre 
plus  ou  moins  prolongee  et  facilitee  par  une  agitation  appropriee,  sufiisamment  douce 
cependant  pour  eviter  I'apparition  de  mousse  dont  on  connait  I'influence,  par  action  de 
surface,  sur  la  denaturation  des  proteines.  Comme  on  le  salt,  Taction  dissolvante  des 
solutions  salines  sur  les  globulines  est,  grosso  modo,  proportionnelle  k  fi  ^  ^  Vz  ^^^,  ou 
C  est  la  concentration  des  ions  et  V  leur  valence,  a  condition  de  rester  en  dega  de  la 
limite  du  salting  out.  Mais  la  nature  des  ions  n'est  pas  sans  influence;  elle  depend  de  leur 
degre  d'hydratation  et  de  leur  pouvoir  de  s'associer  aux  proteines.  II  en  resulte  que  Tutili- 
sation  de  solutions  de  composition  diverse  conduit  a  l'obtention  d'extraits  qui  peuvent 
reveler  des  richesses  dissemblables  en  proteines.  Les  differences  constatees  peuvent  porter 
sur  la  "qualite"  comme  sur  la  "quantite"  de  proteines  extraites.  (C'est  ainsi  que  Tacto- 
myosine  est  plus  aisement  mise  en  solution  dans  les  solutions  de  KCl  que  dans  les  solu- 
tions de  MgCla,  de  meme  force  ionique,  et  que  (NH4)2S04  extrait  peu  de  myosine  /3, 

Nous  utilisons  pour  cela  un  microtome  a  congelation  dont  le  mouvement  est  entraine  par  un 
moteur  electrique,  ce  qui  permet  de  d6biter  100  g  de  muscle  en  dix  minutes. 

Bibliographie  p.  36137. 


28  M.  DUBUISSON  VOL.  4  (1950) 

qui  parait  denaturee  in  situ  sous  I'influence  de  ce  seP).  Ce  qui  nous  parait  essentiel, 
c'est  d'eviter,  autant  que  possible,  dii  moins  au  cours  iVune  premiere  Hape  de  ce  genre 
d'etudes,  rutilisation  d'electrotytes  dont  on  sait  par  avance  I'influence  nuisible  sur  le 
degre  de  polymerisation  de  certaines  proteines  (KI),  sur  la  grandeur  des  particules 
(uree),  sur  leur  solubilite  (Ca,  metaux  lourds).  Le  plus  prudent  est  d'employer  des  ions 
"naturels"  tels  que  K+,  Na+,  Cl~,  HCOg",  HP04~~,  H2P04~,  aux  p^  les  plus  physiolo- 
giques  possible. 

3.  Jusqu'a  quel  point  ces  methodes  d'extraction  permettent  I'obtention  de  solutions 
de  proteines  inalterees  est  un  probleme  qu'il  convient  tout  d'abord  de  bien  poser.  II 
est  evident  que  les  solutions  de  proteines  obtenues  a  la  suite  d'une  extraction  aussi 
prudente  que  possible  ne  pen  vent  jamais  etre  considerees  comme  des  solutions  au  sein 
desquelles  les  molecules  sont  dispersees  sous  une  forme  identique  a  celle  qui  existe  a 
I'interieur  du  myone.  Les  proteines  peuvent  etre  associees,  in  vivo,  de  fa^on  bien  plus 
complexe  at  avoir  ete  dissociees  d'un  support  insoluble,  ou  separees  de  groupements 
prosthetiques  qui  y  sont  naturellement  attaches,  par  Taction  meme  des  solutions  salines 
d'extraction.  Elles  peuvent  aussi  se  trouver,  in  vivo,  sous  une  forme  orientee,  peu 
soluble,  passer  dans  les  solutions  d'extraction  dans  un  etat  beaucoup  plus  disperse, 
plus  chaotique,  et  y  presenter  une  structure  secondaire  (enroulements  ou  deplissements 
des  chaines  principales)  totalement  differente.  Lorsque  nous  envisageons  des  conditions 
d'extraction  qui  fournissent  un  minimum  d'alteration  des  constituants  protidiques, 
ceci  veut  signifier,  par  consequent,  que  ces  conditions  seront  celles  dans  lesquelles  s'ob- 
servera  un  minimum  de  denaturation,  c'est-a-dire  de  formation  de  produits  insolubles, 
sans  prejuger  des  autres  modifications  que  nous  venous  d'envisager.  Nous  trouvons,  en 
effet,  dans  le  muscle,  un  exemple  curieux  fourni  par  les  myosines  /3.  Ces  proteines,  une 
fois  isolees,  sont  solubles  au  p^  7-2  et  a  //  0.20.  Cependant  une  solution  d'extraction  de 
cette  composition  n'extrait  que  tres  peu  de  myosine  ^  d'une  pulpe  musculaire  finejnent 
divisee:  il  faut  utiliser  des  solutions  de  force  ionique  comprises  entre  0.5  et  i  pour 
extraire  au  maximum  ces  myosines;  mais  une  fois  dispersees,  on  pent  garder  ces  pro- 
teines en  solution  a  [.i  0.20^.  Nous  sommes  ici  en  presence  d'un  cas  typique  d'extraction 
d'une  molecule  qui  n'existe  surement  pas,  in  vivo  et  in  situ,  dans  I'etat  ou  nous  la  trou- 
vons dans  I'extrait;  mais,  sans  autres  recoupements,  il  n'est  pas  possible  de  dire  si  ce 
fait  est  du  a  une  moindre  solubilite,  in  vivo,  parce  que  la  molecule  presenterait  une 
orientation  pseudo-cristalline  de  ses  molecules  ou  parce  qu'elle  y  serait  combinee  avec 
d'autres  substances,  sous  la  forme  d'un  complexe  insoluble,  mais  que  les  solutions  salines 
dissocient.  Nous  trouverons  encore  plus  loin  d'autres  exemples  analogues. 

Mais  c'est  la  precisement  une  situation  des  plus  precieuses  pouvant  contribuer 
a  eclaircir  le  probleme  des  transformations  physiologiques  in  vivo  et  in  situ,  des  proteines 
musculaires.  A  egalite  de  conditions  d'extraction,  si  deux  muscles,  consideres  a  des 
etats  fonctionnels  diffcrents,  fournissent  systematiquement  des  extraits  de  composition 
dissemblable,  c'est  precisement  parce  que  les  forces  de  liaison  sont  plus  solides  dans 
I'un  des  deux  cas,  forces  que  la  solution  d'extraction  n'est  pas  capable  de  briser.  Et  ceci 
montre  combien  il  est  important,  dans  la  poursuite  de  ce  genre  d'etudes,  d'utiliser  des 
solutions  dont  Taction  sur  les  proteines  et  les  forces  de  liaison  qui  les  unissent  soit  aussi 
temperce  que  possible,  par  la  nature  et  la  concentration  des  ions  qu'elles  contiennent 
comme  par  leur  pj^.  On  sait,  a  propos  de  ce  dernier  facteur,  que  le  pfj  des  solutions 
d'extraction  a  une  influence  considerable  sur  la  stabilite  des  extraits  soumis  a  la  dialyse. 
Jacob^*'  ^^  a  montre,  dans  mon  laboratoire,  par  Tetude  systematique  d'extraits  dialyses 
Bibliographie  p.  36  j 3/. 


VOL.  4  (1950)  CONTRACTION    MUSCULAIRE  29 

48  heures  a  toute  une  serie  de  p^,  la  formation  de  complexes  d'agregation  denatures 
dans  les  zones  acides  et  etabli  que  la  zone  de  securite  est  relativement  etroite  et  se  con- 
fond  avec  les  p^  biologiques:  6.5-7.6. 

II.  TECHNIQUE   RENDANT  POSSIBLE   L'ANALYSE   QUANTITATIVE   ET  QUALIT.\TIVE   DES 
EXTRAITS   AVEC    UN    MINIMUM    D'ALTERATION 

La  methode  la  meilleure  sera  evidemment  celle  qui  permettra  une  analyse  des 
extraits  avec  un  minimum  de  manipulations:  il  faut  tacher  de  ne  point  modifier  le  p^j, 
la  force  ionique,  la  concentration  relative  de  chaque  constituant,  etc.  On  salt  que  deux 
splendides  techniques  nous  permettent,  aujourd'hui,  d'analyser  des  extraits  dans  de 
semblables  conditions:  Tultracentrifugation  et  I'electrophorese.  La  derniere,  due  surtout 
aux  recherches  de  Tiselius,  a  ete,  de  beaucoup,  la  plus  utilisee;  elle  est  generalement 
plus  facilement  accessible  aux  laboratoires  de  biochimie  et  fournit  des  resultats  plus 
selectifs  que  I'ultracentriiugation ;  nous  I'employons  intensivement  pour  I'etude  des 
proteines  musculaires  depuis  1942.  Elle  permet  de  determiner,  par  la  mesure  des  de- 
placements  de  frontieres  protidiques  (gradients),  sous  I'infiuence  d'un  champ  electrique : 
le  nombre  de  constituants  presents,  la  vitesse  de  chacun  d'eux  au  p^  choisi  et,  par  con- 
sequent, le  p.i.  (vitesse  nulle),  les  proportions  de  chacun  des  constituants  par  la  mesure 
des  surfaces  occupees  par  chaque  gradient  sur  les  cliches  et,  dans  une  certaine  mesure, 
leur  degre  d'homogeneite,  c'est-a-dire  la  tendance  a  r"etalement"  de  ces  gradients  dans 
le  temps,  qui  resulte  a  la  fois  de  cette  heterogeneite  et  des  phenomenes  de  diffusion 
moleculaires.  La  seule  manipulation  a  faire  subir  aux  extraits  consiste  a  les  dialyser 
pendant  au  moins  40  heures,  a  0°  C,  contre  une  solution  de  p^  et  de  force  ionique  choisie. 

II  convient  de  preciser  que  les  diagrammes  electrophoretiques  correspondant  a  des 
extraits  tissulaires  ne  peuvent  reveler  toutes  les  proteines  presentes  dans  cet  extrait : 
on  ne  pent  pratiquement  deceler  une  composante  que  si  sa  concentration  dans  I'extrait 
depasse,  en  valeur  absolue,  0.02%,  par  la  methode  de  Tiselius-Longsworth^^' ^'. 
Comme  la  concentration  totale  en  proteines  des  extraits  dialyses  est  rarement  superieure 
a  3%,  seuls  sont  decelables  les  constituants  dont  le  taux,  dans  I'extrait,  est  superieur 
a  2%.  Encore  faut-il  que  les  substances  presentes  seulement  en  faibles  quantites  posse- 
dent,  au  Ph  considere,  une  vitesse  qui  ne  soit  pas  trop  voisine  de  celle  d'autres  consti- 
tuants. Dans  le  cas  du  muscle,  le  nombre  important  de  gradients  de  proteines  et  leur 
heterogeneite  moleculaire  font  que  ces  gradients  se  separent  en  general  incompletement ; 
les  conditions  sont  done  defavorables  pour  mettre  en  evidence  la  presence  de  proteines 
dont  la  concentration  est  peu  importante.  Or,  beaucoup  de  proteines  (surtout  les  pro- 
teines-enzymes)  existent  dans  le  muscle  a  des  concentrations  faibles;  ainsi  s'explique 
le  nombre  relativement  restreint  de  gradients  differents  dans  les  traces  d'electrophorese, 
alors  que  les  travaux  enzymologiques  nous  laissent  prevoir  la  .presence,  dans  le  muscle, 
d'un  nombre  beaucoup  plus  considerable  de  proteines  solubles  dans  les  solutions  salines 
(une  cinquantaine  peut-etre?). 

III.  existence  de  modifications  d'extractibilite  de  certaines  proteines 

MUSCULAIRES  DU  LAPIN  SELON  LE  MOMENT  DU  CYCLE  DE  LA  CONTRACTION 

Aucune  description  ne  pent  remplacer  I'examen  et  le  commentaire  des  deux  figures 
ci-dessous  qui  representent,  chez  un  meme  Lapin  (muscles  homolateraux),  d'une  part, 
Bibliographie  p.  36I3J. 


30 


M.  DUBUISSON 


VOL.  4  (1950) 


le  cliche  electrophoretique  d'un  muscle  nonnal  et  an  repos,  hache  et  extrait  pendant 
une  heure,  au  moyen  de  1.5  volumes  de  Na2HP04:  0.048  m  —  Na2HP04:  0.006  m  — 
NaCl:  0.20  m  (24  heures  de  dialyse  contre  la  meme  solution)  et,  d'autre  part,  celui 
d'un  muscle  contracts,  immobilise  en  cet  etat  dans  Pair  liquide  et  traite  ensuite  de  la 
meme  fagon  que  le  muscle  temoin*. 

Ces  deux  cliches  montrent  que  ces  muscles  fournissent  des  extraits  differents  en 
plusieurs  points. 

I.  En  ce  qui  concerne  le  groupe  des  myogenes,  nous  sommes  ici  en  presence  en  realite 
d'une  collection  de  proteines,  de  vitesses  electrocinetiques  fort  semblables,  qui  presque 
toutes  apparaissent  deja  dans  les  extraits  aqueux  de  muscles  et  qui  doivent  etre  con- 
siderees  comme  des  proteines  existant,  in  vivo  et  in  situ,  sous  une  forme  soluble^.  Ces  pro- 


A 

Adomyosme  — 

-  MyosineP 

Mr  Myogenes 

A 

Myoalbumine       |H^|^ 

Myoalbumine      ^^^^|^^r^ 

Actomyosine  -^M  '^^V 

1  Pi^Myogenes 

Myosineft,—! 

Actomyosine  ^■\^y°^''"^l^ 

t^ogenes 


1    Ci 


Myoalbumine[ 


Myoalbumine  \' 
Actomyosine  ^jI 


MyosineP, 


Myogenes 


Fig.  I.  Proteinogrammes  electrophoretiques  (methode  de  Tiselius-Longsworth)  d'extraits  muscu- 
laires  du  Lapin,  /x:  0.35,  pn:  7-4o,  ' — -  50000  secondes  d'electrophorese.  En  A,  muscles  nortnaux  et 
au  repos,  refroidis  lentement.  En  B,  muscles  contractes  par  stimulation  et  immobilisation  dans  cet 
etat  par  congelation  instantanee.  En  traits  interrompus:  les  gradients  de  I'actomyosine  et  des 
myosines  jS^  et  ^2  du  muscle  normal  au  repos. 


teines  comprennent  le  myogene  de  Weber^^,  les  myogenes  A  et  B  de  Baranowski^^^^, 
I'aldolase^^^  (qui  est  une  partie  du  myogene  A  de  Baranowski  (Engelhardt^^,  Meyer- 
HOF  et  Beck^*),  la  glyceraldehyde  deshydrogenase  (Cori,  Stein  et  Cori^^),  la  phos- 
phoglucomutase  (Najjar^^)  et  probablement  bien  d'autres  proteines-enzymes  dont  la 
Vitesse  electrocinetique  ne  nous  est  pas  encore  connue^'  ">  ^'^. 

Rien  ne  permet  de  distinguer  le  groupe  d'ensemble  de  ces  myogenes  dans  les  extraits 
de  muscles  au  repos  ou  de  muscles  contractes ;  il  semble  bien  que  la  distribution  quan- 
titative et  qualitative  de  ces  proteines  dans  les  extraits  ne  subisse  pas  de  modification 
au  cours  du  cycle  de  la  contraction^*'  ^'  '^^. 


*  Nous  n'envisagerons  pas,  dans  cet  article,  le  cas  du  muscle  epuise  par  stimulations  r^petees 
dont  le  cas  s'apparente  plus  a  I'etude  du  m^tabolisme  musculaire  qu'a  celle  du  mode  de  fonctionne- 
ment  de  I'^difice  contractile  (Dubuisson^'). 

Bibliographie  p.  jOjjy. 


VOL.  4  (1950)  CONTRACTION    MUSCULAIRE  3I 

2.  Aucune  difference  ne  s'observe  non  plus  au  niveau  du  gradient  h  (Jacob^*)  qui 
represente  la  myoalbumine  de  Bate-Smith^". 

3.  En  dehors  du  cas  des  myogenes  et  de  la  myoalbumine,  la  distribution  de  tous  les 
autres  constituants  est  modifiee  dans  Vetat  de  contraction. 

Ces  autres  constituants  sont : 

a.  les  myosines.  Electrophoretiquement,  la  myosine  classique  de  Weber-Edsall^I'^^ 
preparee  selon  Greenstein  et  Edsall^^,  a  partir  de  muscles  au  repos,  est  caracterisee 
par  trois  gradients  que  nous  avions  appele  a  I'epoque  de  ces  recherches:  myosines  a, 
^  et  y^'  ^*'  ^.  Banga  et  Szent-Gyorgyi^^  ont  montre  que  ces  preparations  de  myosine, 
selon  Greenstein  et  Edsall,  contiennent  deux  constituants:  la  myosine  proprement 
dite  et  une  combinaison  de  cette  myosine  (actomyosine)  a  une  proteine  du  stroma, 
I'actine,  plus  tard  isolee  par  Straub^"^,  et  que  Ton  pent  obtenir  des  echantillons  conte- 
nant  des  taux  variables  de  ces  deux  constituants  en  faisant  varier  le  temps  d'extrac- 
tion:  plus  celui-ci  est  prolonge,  plus  il  y  a  de  I'actomyosine  en  solution. 

Ayant  reussi  plus  tard  a  separer  deux  myosines,  a  et  j8^^,  des  trois  constituants 
electrophoretiques  de  la  myosine,  nous  avons  pu  montrer  que  le  gradient  a  correspond 
reellement  a  I'actomyosine  de  Szent-Gyorgyi  et  le  gradient  j3  a  la  myosine  proprement 
dite^.  La  myosine  y,  d'ailleurs  tres  faiblement  representee  dans  ces  extraits,  n'a  pas  encore 
pu  etre  isolee. 

L'aspect  des  gradients  actomyosine  et  myosine  des  extraits  totaux  de  muscles 
normaux  est  caracteristique^'  ^*.  Ces  deux  gradients,  de  vitesse  voisine,  ne  se  separent 
que  lorsque  les  electrophoreses  sont  suffisamment  prolongees.  Le  premier  (actomyosine) 
est  tou jours  beaucoup  plus  aigu  que  le  gradient  de  la  myosine  dans  le  compartiment 
ascendant  de  la  cellule  d'electrophorese.  La  forte  viscosite  des  solutions  d'actomyosine 
freine  considerablement  les  phenomenes  de  diffusion  qui  sont  la  cause  principale  de 
I'etalement  des  gradients;  en  outre,  le  gradient  d'actomyosine  separe  nettement  la 
colonne  de  proteines  en  deux  regions:  I'une  turbide  et  une  autre  non  turbide  (les  solu- 
tions d'actomyosine  possedent  une  turbidite  elevee). 

Dans  le  compartiment  descendant,  le  gradient  actomyosine  est,  au  contraire,  forte- 
ment  etale  dans  les  extraits  totaux.  Cet  aspect  dissymetrique  existe  aussi  pour  le  gra- 
dient de  la  myosine  qui  parait  unique  du  cote  ascendant,  mais  nettement  bifide  du 
cote  descendant  (^^  et  ^2^).  Les  raisons  de  ces  asymetries  sont  encore  pen  evidentes; 
elles  resultent  sans  doute  d'interactions  entre  I'actomyosine  et  la  myosine,  car  si  I'on 
etudie  electrophoretiquement  des  solutions  pures  d'actomyosine  ou  de  myosine,  les 
figures  ascendantes  et  descendantes  sont  symetriques  pour  chacune  de  ces  proteines^^ 
(compte  tenu  de  la  dissymetrie  classique  due  au  principe  meme  de  la  methode  elec- 
trophoretique). 

Les  caracteristiques  electrocinetiques  (en  io~^  cm/volt/sec)  de  ces  deux  gradients 
sont  {/u:  0.40  (Na2HP04:  0.048  m  —  NaH2P04:  0.006  m  —  NaCl:  0.25  m,  pg:  7.3  a  7.4^) : 

asc.  desc. 

actomyosine —  3.1  — 

myosine —  2.9  —  2.4  (jSj)  —  2.6  (/Sg) 

Si  Ton  se  reporte  maintenant  aux  extraits  de  muscles  contractes,  les  differences 
sont  extremement  grandes.  II  n'y  a  plus  ici  qu'une  tres  faible  quantite  d'actomyosine 
visible  cette  fois,  a  I'anode  comme  a  la  cathode,  tandis  que  le  gradient  des  myosines  j3 
Bibliographie  p.  36I37. 


32  M.  DUBUISSON  VOL.  4  {1950) 

a  completement  disparu^.  (Ce  qui  explique  precisement  la  visibilite  des  gradients  d'ac- 
tomyosine  et  a  I'anode  et  a  la  cathode). 

Mais  il  existe  encore  d'autres  differences  entre  le  muscle  normal  et  le  muscle  con- 
tracte.  Dans  ce  dernier  cas,  les  extraits  contiennent  une  composante  nouvelle,  que  nous 
avons  appele  provisoirement  " contractine'"  et  qui  est  toujours  absente  ou  faiblement 
representee  dans  les  extraits  de  muscles  normaux  (dont  les  fibres  ne  sont  d'ailleurs  pas 
toujours  exemptes  d'un  certain  degre  de  contracture^").  Les  caracteristiques  electro- 
cinetiques  de  cette  nouvelle  composante  sont,  dans  les  memes  conditions  que  celles 
mentionnees  ci-dessus^: 

asc.  desc. 

—  2.35        —2.05 

Enfin,  dans  les  extraits  de  muscles  contractus,  entre  les  gradients  formes  par 
Tactomyosine  et  la  myoalbumine,  on  voit  accumule  une  certaine  quantite  de  materiel 
protidique  tres  heterogene,  sp,  visible  aussi  bien  du  cote  descendant  que  du  cote  ascen- 
dant et  qui  represente  une  augmentation  notable  du  materiel  sp  toujours  present,  mais 
en  faibles  quantites,  dans  les  extraits  de  muscles  normaux. 

Ajoutons  que  les  constatations  decrites  ci-dessus  sont  valables,  quelles  que  soient 
les  causes  de  la  contracture  (ac.  monoiodoacetique,  strychnine,  rigor  mortis)  et  identiques 
aux  cas  de  contraction  par  stimulation  et  immobilisation  par  Pair  liquide^^. 

DISCUSSION 

Inextractibilite  totale  des  myosines  ^  par  KCl,  apparition  de  contractine  et  de 
certaines  proteines  du  groupe  sp,  voila  des  faits  essentiels  qui  caracterisent  la  contraction 
ou  la  contracture,  quelle  que  soit  la  cause  de  celle-ci.  Et  le  parallelisme  entre  le  degre  de 
raccourcissement  et  ces  modifications  protidiques  est  si  etroit  qu'en  cas  de  contracture 
incomplete  {rigor  mortis  en  voie  de  formation),  on  pent  observer  des  ctats  intermediaires 
caracteristiques^^. 

Or,  si  au  lieu  par  exemple  d'immobiliser  le  muscle,  amene  en  contraction  par  un 
bref  tetanos,  dans  Fair  liquide,  on  interrompt  I'excitation  pour  le  laisser  se  relacher,  il 
fournira  le  meme  extrait  protidique  que  le  muscle  normal.  Les  modifications  d'extrac- 
tibilite  du  muscle  contractc  doivent  done  etre  reversibles;  elles  ne  se  constatent  que  si 
Ton  saisit  la  machine  "sur  le  vif". 

Examinons  tout  d'abord  le  cas  des  myosines  j8.  Le  passage  en  solution  de  ces  myo- 
sines ne  pent  etre  une  simple  dissolution.  Tout  d'abord,  les  quantites  de  cette  substance 
que  Ton  peut  extraire  d'un  muscle  dependent  du  degre  de  division  du  tissu,  ce  qui 
n'est  point  le  cas  pour  les  proteines  appartenant  au  groupe  des  myogenes^^.  Les  muscles, 
finement  divises  au  moulin  a  viande  genre  Latapie  fournissent  —  toutes  autres  condi- 
tions etant  egales  —  moins  de  myosine  que  les  muscles  coupes  finement  au  microtome 
a  congelation  en  tranches  de  0.02  mm^^.  Les  myosines  appartiennent  done  a  des  struc- 
tures spatialement  peu  accessibles  aux  solutions  salines,  sans  doute  parce  qu'elles  sont 
protegees  par  des  structures  morphologiques.  Rappelons  ensuite  (voir  p.  28)  que  de 
nombreux  dosages  nous  ont  montre  que  I'extraction  des  myosines  j3  necessite  des  solu- 
tions plus  concentrees  que  celles  qui  permettent  de  garder  simplement  en  solution  ces 
memes  myosines.  Ces  substances  isolees  sont  en  effet  tres  solubles  a  une  force  ionique 
de  0.20  a  0.25  (KCl  0.25  m,  de  pn  7.00) ;  mais  si  Ton  fait  agir  semblable  solution  sur  la 
Bibliographie  p.  36  J 37. 


VOL.  4  (1950)  CONTRACTION    MUSCULAIRE  33 

pulpe  musculaire,  on  extrait  seulement  ^/g  des  myosines  que  Ton  peut  obtenir  si  I'on 
traite  la  pulpe  musculaire  avec  une  solution  de  KCl  0.6  m^.  Ceci  indique  qu'entre  les 
myosines  j8  isolees  et  les  myosines  jS  telles  qu'elles  existent  in  situ  dans  le  muscle,  il 
existe  de  profondes  dissemblances  que  Ton  peut  sans  doute  rapporter  au  fait  que,  dans 
ce  dernier  cas,  ces  myosines  font  partie  de  structures  complexes  dont  elles  se  dissocient 
d'autant  plus  aisement  qu'on  les  attaque  par  des  solutions  salines  concentrees. 

A  la  lumiere  des  travaux  de  I'ecole  de  Szent-Gyorgyi^'  ^^,  qui  montrent  I'affinite 
de  la  myosine  pour  cette  proteine  du  stroma:  I'actine,  on  pourrait  penser  que  les  struc- 
tures complexes  auxquelles  nous  venons  de  faire  allusion  sont  constituees  par  de  I'acto- 
myosine.  Mais  si  tel  etait  le  cas,  il  faudrait  admettre  que  la  solution  d'extraction  brise 
les  forces  de  liaison  entre  I'actine  et  les  myosines  ^  (ces  forces  paraissent  devoir  etre  des 
ponts  SH^^'  ^)  et  permette  la  dispersion  de  cette  derniere  dans  I'extrait,  tandis  que 
I'actine  resterait  insoluble  dans  les  conditions  de  nos  extractions.  Malheureusement,  les 
solutions  d'actomyosine  ne  sont  jamais  scindees  en  actine  et  en  myosine  sous  I'infiuence 
de  sels  (KCl:  0.6  m) ;  s'il  en  etait  autrement,  il  ne  pourrait  jamais  y  avoir  d'actomyosine 
dissoute  dans  une  solution  saline. 

Quoi  qu'il  en  soit  de  la  nature  du  complexe  auquel  sont  normalement  associees 
les  myosines  /3,  nous  devons  admettre,  puisque  ces  myosines  sont  devenues  inextrac- 
tibles  dans  la  pulpe  de  muscles  contractes  ou  contractures,  que  le  raccourcissement  a 
modifie  leurs  forces  de  liaison:  elles  sont  desormais  inaccessibles  aux  solutions  salines 
utilisees.  II  est  sans  doute  assez  pertinent  de  penser  que  c'est  I'etablissement  de  ces 
forces  de  liaison  meme  qui  entraine  la  mise  en  tension  (contraction  isometrique)  ou  le 
raccourcissement  (contraction  isotonique)  de  la  machine  contractile  et  leur  disparition 
qui  assure  son  relachement. 

En  ce  qui  concerne  la  contractine^,  on  peut  envisager  plusieurs  causes  a  son  appari- 
tion dans  les  extraits  de  muscles  contractes.  II  est  tout  d'abord  possible  que  la  contractine, 
dont  I'apparition  accompagne  la  disparition  des  myosines  ^,  soit  en  realite  une  partie 
des  proteines  j8  transformee,  par  exemple,  par  le  gain  ou  la  perte  de  quelque  groupement 
prosthetique  qui  en  modifierait  les  proprietes  electrocinetiques.  Signalons  cependant 
qu'il  ne  semble  exister  aucune  relation  quantitative  entre  la  disparition  des  myosines 
/3  et  I'apparition  de  contractine  dans  le  cas  des  contractures  non  maximales.  II  est 
possible  d'admettre  aussi  que  I'on  a  affaire  a  une  proteine  qui  devient  extractible  lorsque 
la  machine  est  a  I'etat  raccourci,  parce  qu'elle  est  liberee  a  ce  moment  de  complexes, 
ordinairement  indissociables  par  les  solutions  salines.  On  en  arriverait  en  somme,  dans 
cette  derniere  eventualite,  a  constater,  pour  la  contractine,  I'inverse  de  ce  qui  se  presente 
pour  les  constituants  de  la  myosine  ^,  qui  ne  sont  plus  liberables  par  KCl,  lorsque  le 
muscle  est  a  I'etat  contracted. 

Quant  a  la  nature  de  la  contractine,  nous  savons  seulement  ceci:  cette  proteine 
precipite  mal  dans  les  conditions  011  precipite  le  myosine  de  Weber-Edsall  (acto- 
myosine  +  myosines  j8,  y),  soit  a  /x :  0.05  et  au  pjj  6.3.  Elle  ne  peut  etre  extraite  du  muscle 
contracte  a  une  force  ionique  inferieure  a  0.15-0.20.  Elle  ne  peut  non  plus  correspondre 
a  la  phosphorylase  b  de  Cori^^'  "*",  qui  apparait  dans  les  muscles  fatigues  par  suite 
de  la  transformation  de  phosphorylase  a,  car  elle  n'est  jamais  presente  dans  les  muscles 
fatigues  par  stimulations  et  relaches ;  de  plus,  le  taux  de  contractine  est  bien  superieur 
a  celui  des  phosphorylases^.  On  ne  peut  exclure,  a  priori,  cette  possibilite  que  la  contrac- 
tine corresponde  a  cette  proteine  dont  nous  avons  trouve  des  traces  dans  la  plupart  des 
preparations  de  myosine  de  Weber-Edsall  du  Lapin  et  que  nous  avons,  a  cette  epoque, 
Bibliographie  p.  J6/J7. 
3 


34  M.  DUBUISSON  VOL.  4  (1950) 

appelee  myosine  ■■/■.  Nous  I'avions  trouvee  beaucoup  plus  abondante  dans  les  prepara- 
tions de  myosine  faites  a  partir  de  muscles  de  Mollusques  (muscles  pedieux),  quisont 
d'ailleurs  des  muscles  tres  excitables  et  qu'on  ne  peut  reduire  en  pulpe  sans  en  provoquer 
la  contracture.  Au  p^  7.3  a  7.4  et  ^i  0.35  a  0.40,  la  vitesse  de  la  contractine  est  de  —  2.35 
(asc.)  et  de  —  2.05  (desc.) ;  celle  de  la  myosine  y  est,  dans  les  memes  conditions,  pratique- 
ment  la  meme,  peut-etre  un  peu  plus  faible  (—2.25)  (asc).  II  y  a  lieu  cependant  de  noter 
que,  contrairement  a  la  myosine  y,  la  contractine  ne  precipite  pas  dans  les  conditions 
oil  precipite  la  myosine  de  Weber-Edsall  dans  laquelle  on  reconnait  la  presence  de 
myosine  y,  bien  qu'en  faibles  quantites.  Enfin,  de  recentes  analyses  electrophoretiques 
effectuees  sur  des  echantillons  de  G-actine,  de  F-actine  et  de  tropomyosine*  montrent 
que  la  contractine  ne  peut  etre  aucune  de  ces  proteines-la.  Par  centre,  il  semble  qu'existe 
certaines  analogies,  qui  font  I'objet  de  recherches  actuelles,  entre  la  contractine  et  la 
N-proteine  de  GerendAs  et  Matoltsy^  qui  entre  dans  la  constitution  des  portions 
isotropes  des  myofibrilles.  L'emplacement  meme  de  ce  nucleoproteide  dans  la  fibre 
musculaire  donnerait  un  interet  particulier  a  ce  rapprochement. 

On  peut  se  demander  maintenant  s'il  n'est  pas  possible  de  trouver  des  solutions 
d'extraction  qui  possedent  la  propriete  a)  ou  bien  de  briser  les  forces  de  liaison  qui 
maintiennent  si  solidement  les  myosines  /S  a  d'autres  substances  au  moment  de  la  con- 
traction et  qui  seraient  en  consequence  susceptibles  d'extraire  ces  proteines  d'un  muscle 
contracte  ou  contracture;  b)  ou  bien  de  briser  les  forces  de  liaison  qui  rendent  inex- 
tractible  la  contractine  des  muscles  normaux.  C'est  egalement  ce  qui  fait  I'objet  de 
nos  recherches  actuelles,  dont  les  resultats  preliminaires,  fort  encourageants,  seront 
publics  sous  peu  et  semblent  devoir  etre  de  nature  a  eclairer  grandement  la  connaissance 
de  la  structure  de  I'edifice  contractile. 

CONCLUSIONS 

Seules  les  proteines  extraites  par  les  solutions  de  force  ionique  elevee  doivent  etre 
considerees  comme  des  constituants  engages  iyi  vivo  et  in  situ,  dans  des  complexes  qui 
sont  par  eux-memes  insolubles.  Or,  il  se  trouve  precisement  que  ce  sont  ces  proteines 
la  dont  I'extraction  est  la  plus  modifiee  au  cours  du  cycle  de  la  contraction.  II  est  ainsi 
tout  naturel  de  penser  que  le  fonctionnement  de  la  machine  contractile  est  essentielle- 
ment  caracterise  par  la  formation  ou  la  dissociation  de  ces  complexes.  Cette  conclusion 
est  en  harmonic  avec  les  theories  suggerees  par  Szent-Gyorgyi^'  ^^,  selon  lesquelles  le 
mecanisme  de  la  contraction  resulterait  de  la  transformation  de  I'actomyosine  sous 
I'infiaence  de  sels  et  d'A.T.P. ;  mais  ceci  n'est  qu'une  solution  approchee,  comme  le 
reconnait  d'ailleurs  lui-meme  ce  chercheur.  Tout  d'abord,  la  myosine  elle-meme  est  une 
substance  compliquee.  EUe  est  constituee  d'au  moins  deux  composantes  electrophore- 
tiques: j3,  et  ^2^  slle  contient  I'A.T.Pase*^,  qui  est  un  enzyme  n'etant  vraisemblablement 
qu'accroche  a  la  myosine;  elle  contient  encore  d'autres  enzymes:  une  desaminase*^, 
l^apoferment  d'un  enzyme  susceptible  de  transformer  I'arginine  et  I'histidine  en 
creatine*^.  Le  cycle  de  la  contraction  affecte  aussi  une  autre  myosine :  la  composante  y, 
electrocinetiquement  distincte  de  jS^.  Le  substrat  auquel  les  myosnies  peuvent  se  Her 
contient  siirement  I'actine  de  Straub  (sous  la  forme  de  F-actine  vraisemblablement, 
etant  donnee  la  force  ionique  du  muscle)  et  peut-etre  meme  la  tropomyosine  de  Bailey 

*  Recherches  inedites. 
Bibliographie  p.  jOjjy. 


VOL.  4  (1950)  CONTRACTION    MUSCULAIRE  35 

et  la  nuclcoproteine  de  GerendAs  et  Matoltsy.  Enfin,  la  liaison  des  myosines  a 
I'edifice  contractile  au  moment  de  la  contraction  est  concomitante  de  la  liberation  de  la 
contractine,  nettement  distincte  de  I'actomyosine  et  des  myosines  /S^  et  jSg.  Ce  soat 
la  des  faits  qui  permettent  de  penser  que  la  machine  contractile  est  beaucoup  plus  com- 
pliquee  que  Ton  serait  tente  de  le  croire.  Deja,  les  travaux  de  I'ecole  de  Szent-Gyorgyi 
ont  montre  par  la  decouveite  de  I'actomyosine,  que  les  myofibrilles  ne  sont  pas  unique- 
ment  constituees  de  myosine,  comme  on  I'avait  cru  jusqu'alors;  mais  il  serait  dangereux 
de  penser  que  le  schema  de  la  contraction  musculaire  construit  sur  la  base  actomyosine 
—  ATP  —  KCl  —  MgClg  est  satisfaisant,  malgre  ce  que  certaines  experiences  faites  avec 
des  fils  prepares  au  moyen  de  cette  substance  peuvent  avoir  de  spectaculaire  (super- 
precipitation  ou  forte  deshydratation  (cynaerese)  sous  I'influence  de  selsoud'A.T.P.**"*^). 
On  ne  fera  certes  jamais  trop  d'experiences  dans  le  genre  de  celles  qui  furent  faites  par 
Needham  et  collaborateurs^"'  °^,  ainsi  que  par  I'ecole  de  Szent-Gyorgyi,  sur  les  pro- 
prietes  des  myosines  sous  Paction  de  telle  ou  telle  substance;  mais  on  n'en  fera  jamais 
assez  pour  poser  tout  d'abord,  dans  toute  son  ampleur,  le  probleme  "physiologique"  qui 
consiste  a  determiner  combien  de  -proteines  appartiennent  reellement  aux  structures  dont 
les  modifications  assurent  le  mecanisme  de  la  contraction  et  du  relachement  musculaires 
et  comment  se  modifient  leurs  modes  de  liaison  au  cours  du  cycle  de  la  contraction. 
C'est  une  premiere  contribution  a  ce  genre  d'investigation  dont  les  resultats  ont  ete 
resumes  ici.  lis  montrent  qu'en  s'efforgant  de  dissocier  les  complexes  protidiques,  avec 
le  moins  de  brutalite  possible,  en  attaquant  leurs  forces  de  liaison  par  des  solutions 
d'extraction  de  composition  appropriee,  afin  de  liberer  progressivement  les  elements 
detachables,  on  pent,  par  des  comparaisons  faites  sur  des  muscles  se  trouvant  en  divers 
etats  fonctionnels  (relaches,  contractes)  se  rendre  compte  par  la  methode  electropho- 
retique,  de  I'etablissement  ou  d'^  la  rupture  de  liaisons  qui  unissent  les  elements  qui 
participent  a  la  contraction.  Les  resultats  obtenus  jusqu'ici  sont  encore  fort  difficiles  a 
interpreter  et  ne  peuvent  pas  encore,  pas  plus  d'ailleurs  que  ceux  obtenus  par  d'autres 
voies,  servir  a  construire  une  theorie  de  la  contraction  et  du  relachement  musculaires. 
Si  certains  elements  permettent  de  penser  que  le  cycle  de  la  contraction  est  du  a  la 
formation  et  a  la  dissociation  de  complexes  constitues  d'actine,  de  myosines  jS^  et  ^^y 
de  contractine,  etc.,  il  nous  faut  encore  mieux  connaitre  la  structure  de  ces  complexes 
et  leurs  modifications  au  cours  du  cycle  de  la  contraction.  Et  ceci  est  un  chemin  dont 
le  parcours  est  encore  long  et  difficile. 

RliSUMfi 

L'edifice  contractile  doit  poss^der,  a  I'etat  raccourci,  une  structure  bien  differente  de  celle  qu'il 
possede  a  I'etat  relache.  Cet  edifice  etant  essentiellem^nt  constitue  de  proteines,  on  doit  s'attendre 
a  ce  que  I'extractibilite  de  ces  substances,  au  moyen  de  solutions  salines  ayant  une  action  plus  ou 
moins  disruptive  sur  les  forces  de  liaison  qui  maintiennent  les  proteines  en  place  dans  l'edifice,  doit 
etre  differente  selon  que  Ton  considere  le  muscle  a  I'etat  contracte  —  ou  contracture  —  ou  relach^. 

C'est  effectivement  ce  que  nous  avons  pu  constater.  Pour  ne  citer  que  les  faits  les  plus  saillants: 
tandis  que  les  myosines  fi  deviennent  inextractibles  par  les  solutions  salines  utilisees,  lorsque  la 
machine  musculaire  se  trouve  a  I'etat  contracte,  une  nouvelle  proteins:  la  contractine  apparait  dans 
les  extraits.  Ces  observations  sont  discutees.  II  apparait  que  la  methode  d'investigation  employee, 
qui  fait  appel  simultanement  a  des  techniques  physiologiques,  physico-chimiques  et  biochimiques, 
est  loin  d'avoir  fourni  tons  les  renseignements  qu'elle  est  susceptible  de  nous  apporter  dans  la  con- 
naissance  du  probleme  du  mecanisme  general  de  la  contraction  musculaire. 

SUMMARY 

The  contractile  apparatus  must  possess,  in  the  shortened  state,  a  structure  which  differs  from 
that  in  the  relaxed  state.  As  it  is  essentially  composed  of  proteins,  one  must  expect  the  extractabilit  v 

Bibliographic  p.  36JJJ. 


36  M.  DUBUISSON  VOL.  4  (1950) 

of  these  substances  —  as  efifected  by  salt  solution,  possessing  a  more  or  less  disruptive  action  on  the 
forces  which  keep  the  proteins  in  their  place  in  the  structure  —  to  differ  when  the  muscle  is  in  state 
of  contraction  or  relaxation. 

This  we  have  been  able  to  observe.  The  most  remarkable  facts  are:  When  the  muscle  is  in  state 
of  contraction  the  myosins  /3  cannot  longer  be  extracted  by  the  salt  solutions  employed,  but  then  a 
new  protein,  the  contractine,  appears  in  the  extracts.  These  observations  are  discussed.  The  method  of 
investigation  employed,  requiring  at  one  time  physiological,  physico-chemical  and  biochemical 
techniques,  does  not  yet  appear  to  have  revealed  all  information  it  is  expected  to  yield  in  contribu- 
tion to  the  understanding  of  the  mechanism  of  muscle  contraction. 


ZUSAMMENFASSUNG 

Der  Kontraktionsapparat  muss  im  verkiirzten  Zustand  eine  andere  Struktur  haben,  als  im 
Ruhezustand.  Da  er  grosstenteils  aus  Proteinen  besteht,  so  ist  zu  erwarten,  dass  die  Extrahierbarkeit 
dieser  Substanzen  mit  Salzlosungen  aus  kontrahiertem  und  ruhendem  Muskel  verschieden  sein  wird, 
denn  die  Salzlosungen  wirken  mehr  oder  weniger  spaltend  auf  die  Bindungen  welche  die  Proteine 
in  der  Struktur  zusammenhalten. 

Wir  konnten  dies  in  der  Tat  beobachten.  Nennen  wir  nur  die  hervorragendsten  Falle:  Wenn 
der  Muskel  kontrahiert  ist,  konnen  die  ^-Myosine  nicht  mehr  durch  Salzlosungen  extrahiert  werden, 
aber  ein  neuer  Eiweisstoff ,  das  Kontraktin,  tritt  in  den  Extrakten  auf.  Die  verwendete  Methode,  die 
gleichzeitig  von  physiologischen,  physiokochemischen  und  biochemischen  Arbeitsweisen  Gebrauch 
macht,  scheint  noch  lange  nicht  alle  Aufklarungen  zum  Verstandnis  des  Mechanismus  der  Muskel- 
kontraktion  gegeben  zu  haben,  die  sie  verschaffen  konnte. 


BIBLIOGRAPHIE 

1  M.  DuBUissoN,  Experientia,  4  (1948)  437. 

2  M.  DuBUissoN,  Experientia,  2  (1946)  258. 

3  F.  B.  Str.\ub,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  2  (1942)  3- 

4  F.  B.  Straub,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  3  (i943)  23. 

5  F.  B.  Straub,  Hung.  Acta  Physiol.,  i  (1948)  150- 

6  A.  Szent-Gyorgyi,  Chemistry  of  muscular  contraction,  Acad.  Press,  New- York  1947. 
■^  K.  Bailey,  Nature,  157  (1946)  368. 

8  M.  GerendAs  et  a.  G.  Matoltsy,  Hung.  Acta  Physiol.,  i  (1948)  124. 
^  M.  DuBUissoN,  Biolog.  Revs  (sous  presse). 

10  M.  DUBUISSON,  Arch,  intern,  physiol.,  61  (1948)  93- 

11  M.  DUBUISSON,  Experientia,  3  (1947)  372- 

12  J.  Jacob,  Experientia,  3  (1947)  241. 

13  p.  Crepax,  J.  Jacob  et  J.  Seldeslachts,  Biochim.  Biophys.  Acta  (sous  presse). 
1*  J.  Jacob,  Biochem.  J.,  41  (1946)  808. 

15  J.  Jacob,  Biochem.  J.,  42  (1948)  71. 

1"  M.  DUBUISSON  ET  J.  Jacob,  Bull.  soc.  roy.  sci.,  Liege,  3  (i945)  i33- 

1'  M.  DuBUissoN,  Les  Prolines  musculaires.  Exposes  annuels  de  Biochimie  Medicale,  S^rie  IX,  Paris, 

Masson  1948. 
18   H.  H.  Weber,  Ergeb.  Physiol.,  36  (1934)  io9- 
1^  L.  Chrobak  et  T.  Baranowski,  Compi.  rend.  acad.  sci.  U.R.S.S.,  28  (194°)  724- 

20  T.  Baranowski,  Compt.  Rend.  soc.  biol.,  130  (1939)  1182. 

21  T.  Baranowski,  Compt.  Rend.  acad.  sci.  U.R.S.S.,  28  (1940)  722. 

22  T.  Baranowski,  Biochimica  U.R.S.S.,  5  (194°)  i74- 
22bE.  C.  Bate-Smith,  Biochem.  J.,  34  (1940)  1122. 

23  V.  A.  Engelhardt,  Yale  J.  Biol,  and  Med.,  15  (1942)  21. 

2*  O.  Meyerhof  et  L.  V.  Beck,  J.  Biol.  Chem.,  156  (1944)  ^°9- 

25  G.  T.  Cori,  M.  W.  Slein  et  C.  F.  Cori,  /.  Biol.  Chem.,  159  (i945)  565- 

2«  V.  A.  Najjar,  /.  Biol.  Chem.,  175  (1948)  281. 

2'  A.  DisT:fecHE,  Biochim.  Biophys.  Acta,  2  (1948)  265. 

28  J.  Jacob,  Bull.  soc.  roy.  sci.  Liege,  4  (i945)  231. 

29  J.  Jacob,  Experientia,  2  (1946)  no. 

30  E.  C.  Bate-Smith,  Proc.  Roy.  Soc,  B,  124  (i937)  136. 

31  J.  T.  Edsall,  /.  Biol.  Chem.,  89  (1930)  289. 

32  A.  V.  Muralt  et  J.  T.  Edsall,  Trans.  Faraday  Soc,  26  (1930)  837. 

33  J.  P.  Greenstein  et  J.  T.  Edsall,  /.  Biol.  Chem.,  133  (1940)  397- 


VOL.  4  (1950)  CONTRACTION   MUSCULAIRE  37 

34  M.  DuBUissoN,  Differenciation  electrophoretique  et  separation  de  diverses  composantes  dans  les 
myosines  de  muscles  au  repos  et  fatigues,  de  Mammiferes  et  de  Mollusques.  Un  symposium  sur  les 
Proteines,  Paris  1947. 

35  I.  Banga  et  a.  Szent-Gyorgyi,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  i  (1941-1942)  5. 

36  M.  DuBuissoN,  Experientia,  2  (1946)  412. 

"  J.  GoDEAUX,  Bull.  soc.  roy.  sci.  Liege  (1944)  217. 

38  K.  Bailey  et  S.  V.  Perry,  Biochim.  Biophys.  Acta,  i  (1947)  506. 

39  G.  T.  CoRi  ET  C.  F.  CoRi,  /.  Biol.  Chem.,  158  (1945)  321. 
*«  G.  T.  CoRi,  /.  Biol.  Chem.,  158  (1945)  333. 

*i  V.  A.  Engelhardt,  Advances  in  Enzymol.,  6  (1946)  147. 

42  Ferdman,  Biochem.  J.  (Ukraine),  18  (1946)  no. 

43  F.  Menne,  Z.  physiol.  Chem.,  279  (1943)  105. 

4*  T.  Erdos,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  1  (1941-42)  59. 

45  T.  Erdos,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  3  (1943)  57. 

46  M.  GerendAs,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  i  (1941-42)  47. 
*'  M.  Gerendas  et  Szent-Gyorgyi,  Enzymologia,  9  (1941)  117. 

**  A.  Szent-Gyorgyi,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  1  (1941)  17. 

49  A.  Szent-Gyorgyi,  Stud.  Inst.  Med.  Chem.  Univ.  Szeged,  2  (1942)  25. 

50  J.  Needham,  S.  C.  Shen,  D.  M.  Needham  et  A.  S.  C.  Lawrence,  Nature,  147  (1941)  766. 

51  M.  Daity,  A.  Kleinzeller,  A.  S.  C.  Lawrence,  M.  Miall,  J.  Needham,  D.  Needham  et  Shih- 
Schang  Shen,  /.  Gen.  Physiol.,  27  (1944)  355. 

Regu  le  22  mars  1949 


38  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


ACTOMYOSIN  AND  MUSCULAR  CONTRACTION 

by 

A.  SZENT-GYORGYI* 

Marine  Biological  Laboratory  Woods  Hole,  Massachusetts  and 
Laboratory  of  Physical  Biology,  Experimental  Biology  and  Medicine  Institute,  Bethesda  14, 

Maryland  (U.S.A.) 


It  has  been  shown  in  the  author's  laboratory^"  that  two  structural  proteins  can  be 
extracted  from  the  muscle  fibril,  actin  (F.  B.  Straub)  and  myosin.  The  two,  if  mixed 
at  a  proper  ionic  concentration,  unite  to  a  complex,  actomyosin,  which  has  the  remark- 
able property  of  contractility.  Actomyosin  threads  contract  under  influence  of  ATP. 
This  contraction,  though  imitating  in  many  ways  contraction  of  muscle,  differs  from 
it  also  in  several  respects.  Two  of  these  differences  are  rather  striking  and  led  Buchthal, 
Deutsch,  Knappeis,  and  Petersen,  as  well  as  Astbury,  Perry,  Reed,  and  Spark 
to  the  conclusion  that  "contraction"  of  actomyosin  has  little  to  do  with  muscular 
contraction.  According  to  Astbury,  "contraction"  of  actomyosin  is  simply  a  colloidal 
synaeresis  while  muscular  contraction  is  an  entirely  different  phenomenon.  The  two 
observations,  on  which  this  conclusion  was  based,  were  the  following:  muscle  contracts 
anisodiametrically,  becoming  shorter  and  thicker  without  changing  volume,  while 
"contracting"  actomyosin  threads  become  shorter  and  proportionately  thinner,  thus 
simply  shrinking.  The  second  objection  is  based  on  Buchthal's  observation:  while  an 
unloaded  actomyosin  thread  shortens  in  ATP,  a  loaded  thread  lengthens  in  the  same 
solution,  thus  behaving  contrary  to  muscle  which  shortens  whether  loaded  or  unloaded. 

In  this  paper,  the  author,  after  pointing  out  certain  analogies  between  the  con- 
traction of  muscle  and  actomyosin,  hopes  to  show  that  the  objections  raised  by  Buch- 
thal and  Astbury  can  easily  be  explained  and  do  not  plead  for  a  basic  dissimilaritj^ 
of  the  two  processes. 

If  a  washed  fibre  bundle  of  the  musculus  psoas  of  the  rabbit  is  suspended  in  a 
Ringer  solution,  containing  o.ooi  M  Mg  and  0.2%  ATP,  it  contracts  and  develops  the 
same  tension  as  the  muscle  developed  maximally  i}i  vivo,  showing  that  it  was  the  normal 
mechanism  of  contraction. which  has  been  put  into  motion  by  ATP.  This  reaction  is 
very  specific,  and  all  attempts  to  produce  it  with  any  other  substance  than  ATP  have 
hitherto  failed.  The  same  muscle  fibre  can  be  made  to  shorten  also  by  other  means,  as 
for  instance,  by  heat.  At  70°  shortening  may  be  extensive,  but  no  appreciable  tension 
will  be  produced. 

If  the  same  washed  psoas  muscle  is  suspended  in  water  and  decomposed  in  the 
Waring  blender  into  a  suspension,  on  addition  of  the  salts  of  the  Ringer  solution,  a 
moderate  fiocculation  will  be  observed.  On  addition  of  ATP  an  excessive  precipitation 
occurs  which  has  been  termed  "superprecipitation".  Evidently,  this  superprecipitation 

*  Special  Fellow,  U.  S.  Public  Health  Service. 
References  p.  41. 


VOL.  4  (1950)  ACTOMYOSIN    AND    MUSCULAR   CONTRACTION  39 

is,  in  its  essence,  identical  with  muscular  contraction,  having  been  elicited  by  the  same 
specific  substance  under  a  similar  condition,  the  only  difference  being  the  destruction 
of  the  fibrillary  architecture. 

We  can  go  one  step  further  and  dissolve  out  of  the  freshly  minced  psoas  the  con- 
tractile matter,  actomyosin,  by  prolonged  extraction  by  means  of  Weber's  alkaline 
0.6  M  KCl.  This  actomyosin  behaves  like  the  suspended  muscle  giving  flocculation  in 
presence  of  salts  and  superprecipitation  in  presence  of  ATP.  The  last  step  of  degradation 
of  the  muscle  may  be  the  isolated  extraction  of  actin  and  myosin.  The  two  proteins, 
if  mixed,  unite  to  actomyosin  which  gives  the  same  reactions  as  actomyosin  extracted 
or  the  suspended  psoas.  This  stepwise  decomposition  of  the  psoas  thus  gives  identical 
results  all  the  way,  and  the  reactions,  elicited  by  the  h'ghly  specific  ATP,  are,  in  all 
phases,  so  similar  that  there  can  be  little  doubt  about  the  essential  identity  of  these 
reactions.  Naturally,  we  must  bear  in  mind  that  the  fibril  has  its  specific  architecture 
which  is  present  no  more  in  suspensions.  . 

Instead  of  making  a  suspension  out  of  our  actomyosin,  we  can  also  bring  it  into 
the  form  of  a  gel  and  make  of  this  gel,  by  the  method  of  Weber,  a  fibre  again.  Suspended 
in  pure  water,  the  thread  will  swell.  Addition  of  salts  will  make  this  swelling  regress, 
a  reaction  which  evidently  corresponds  to  the  flocculation  of  our  actomyosin  or  muscle 
suspensions.  On  addition  of  ATP  the  thread,  if  thin  enough,  will  shorten  rapidly,  a 
reaction  which  evidently  corresponds  to  the  superprecipitation  of  our  suspensions  and 
corresponds  thus,  also,  in  its  essence,  to  contraction  in  muscle. 

After  having  pointed  out  these  analogies  of  actomyosin  and  muscle,  let  us  consider 
the  dissimilarities,  quoted  above. 

Muscle  shortens;  actomyosin  shrinks.  This  is  certainly  true,  and  our  problem  is 
whether  this  difference  is  due  to  a  difference  in  the  very  essence  of  the  reaction  or  whether 
it  is  due  merely  to  the  rough  structural  difference  between  fibril  and  actomyosin  thread. 
In  the  former,  as  shown  by  the  electron  microscopic  studies  of  Hall,  Jacus,  and 
ScHMiTT*,  the  contractile  filaments  run  all  along  the  muscle  fibril  continuously,  parallel 
to  the  axis.  On  extraction  these  filaments  are  broken  up  into  fragments  which  are 
distributed  at  random  in  the  actomyosin  thread.  If,  in  contracting  muscle  the  filaments 
become  shorter  and  wider,  the  muscle  will  have  to  do  the  same  —  become  shorter  and 
wider  without  changing  volume.  If  the  same  shortening  of  filaments  occur  in  the  acto- 
myosin thread  which  contains  the  fragments  unoriented,  at  random  distribution,  the 
shortening  of  the  very  same  filaments  has  to  make  the  thread  contract  equally  in  all 
directions,  that  is  make  it  shrink. 

That  this  difference  is  actually  due  only  to  this  difference  in  orientation  can  easily 
be  shown.  If  the  thread  is  gently  stretched,  as  shown  by  Gerendas,  the  filaments 
become  oriented  parallel  to  the  axis  similarly  to  muscle.  If  ATP  is  made  to  act  on  such* 
an  oriented  thread,  this  thread  will  shorten  and  become  wider,  thus  contract  without 
changing  volume,  similarly  to  muscle.  The  same  is  true,  as  shown  by  Buchthal  and 
his  associates  after  drying  which  acts  as  stretching. 

Perry,  Reed,  Astbury,  and  Spark  explain  the  "synaeresis"  of  actomyosin  bj^ 
a  lateral  association  of  particles.  That  this  explanation  cannot  be  correct  is  shown  by 
the  anisodiameteral  contraction  of  the  oriented  actomyosin  threads.  In  this  structure 
the  filaments  are  oriented  parallel  to  the  axis.  Their  lateral  association  could  only  make 
the  thread  thinner  and  never  shorter,  while  the  experiment  shows  that  actually  the 
opposite  happens  and  the  thread  becomes  shorter  and  wider. 
References  p.  41. 


40  A.  SZENT-GYORGYI  VOL.  4  (1950) 

In  order  to  be  able  to  discuss  the  stretching  of  the  loaded  actomyosin  thread  in 
ATP,  we  have  to  give  our  attention  for  an  instant  to  another  effect  of  ATP,  independent 
of  contraction.  Fresh  muscle  is  elastic.  Post  mortem  the  ATP  is  disintegrated  and, 
parallel  to  its  disappearance,  the  muscle  becomes  inelastic,  as  shown  by  Th.  Erdos, 
Bate-Smith,  and  Bendall.  It  is  possible  to  show  that  it  was  actually  the  disappearance 
of  ATP  which  induced  this  difference.  A  washed  psoas  fibre  is  inelastic.  If  suspended  in 
Ringer,  containing  ATP,  it  becomes  elastic  again.  This  shows  that  in  absence  of  ATP, 
links  are  developed  between  neighbouring  micells  which  make  the  system  rigid, 
making  slipping  and  relative  motion  impossible.  These  are  abolished  by  ATP.  This 
effect  of  ATP  is  independent  of  its  second  effect,  contraction.  If  ATP  did  not  have  the 
first  effect,  it  could  not  induce  contraction  at  all  because  the  system  would  be  too  rigid. 
This  effect  of  ATP  was,  in  fact,  the  very  first  specific  effect  discovered  of  ATP  on 
"myosin"  by  Engelhardt,  Ljubimowa,  and  Meitina  who  found  that  ATP  makes 
"myosin"  threads  more  extensible.  The  decrease  of  dynamic  softness  of  actomyosin 
induced  by  ATP  has  also  been  studied  extensively  by  Buchthal  and  his  associates. 

After  this  short  discussion  we  can  consider  now  the  extension  of  loaded  actomyosin 
threads.  If  an  actomyosin  thread  is  loaded,  it  will  not  stretch  because  it  is  rigid,  its 
particles  being  held  together  by  the  links  or  cohesive  forces  described  before.  If  ATP  is 
added  these  forces  will  be  abolished  and,  under  action  of  the  load,  the  short  fragments 
of  filaments  of  which  the  thread  is  composed,  will  begin  to  slip  under  influence  of  the 
load,  and  the  thread  will  lengthen,  even  if  at  the  same  time  these  fragments  shorten. 
The  situation  will  be  different  in  an  unloaded  thread.  There  will  be  no  force  present  to 
cause  slipping,  and  the  shortening  micells  will  make  the  thread  contract  or  "shrink" 
according  to  its  co-axial  or  random  distribution.  In  the  muscle  fibre  there  can  be  no 
slipping  because  the  filaments  run  continuously  through  the  fibrils,  and  so  the  muscle 
can  shorten  only  if  its  filaments  contract,  whether  loaded  or  unloaded. 

Perry,  Reed,  Astbury,  and  Spark  stress  one  more  difference  between  muscular 
contraction  and  the  contraction  in  actomyosin  threads:  the  time  factor.  Muscle  may 
contract  several  hundred  times  per  second,  while  even  thin  threads  need  seconds  for 
their  contraction.  Here  again  the  difference  lies  in  steric  relations  and  not  in  principle. 
If  diffusion  and  friction  are  eliminated,  the  ATP  contraction  is  instantaneous.  This 
can  be  shown  in  washed  psoas-fibres  suspended  0°  C  in  a  solution.  At  this  temperature 
the  fibres  develop  only  a  very  weak  tension.  If  they  are  transferred  into  a  Ringer  of,  say 
25°  C,  the  development  of  a  high  tension  is  instantaneous.  Rapid  reaction  can  also  be 
demonstrated  in  thin  actomyosin  threads,  to  which  ATP  is  added  in  such  a  way  as  to 
reach  the  thread  from  one  side.  On  this  side  the  actomyosin  contracts  and  makes  the 
thread  bend  or  curl  up  rapidly. 

The  differences  in  behaviour  of  muscle  and  actomyosin  can  thus,  in  the  instances 
discussed,  be  explained  satisfactorily  by  the  rough  structural  differences  of  both  for- 
mations and  need  not  be  ascribed  to  the  difference  in  underlying  reactions. 

SUMMARY 

It  is  shown  that  the  contraction  of  muscle,  superprecipitation  of  its  suspensions,  superprecipita- 
tion  of  actomyosin  and  contraction  of  actomyosin,  ehcited  by  ATP,  are  related  phenomena. 

Differences  in  behaviour,  as  for  instance  anisodiametry  of  shrinking  in  muscle  and  isodiametry 
of  shrinking  in  unoriented  actomyosin  gels,  can  be  explained  by  the  differences  in  structure.  The 
same  is  true  for  the  difference  of  muscle  and  loaded  actomyosin  threads,  the  latter  of  which,  contrary 
to  muscle,  lengthen  under  influence  of  ATP. 

References  p.  41. 


VOL.  4  (1950)  ACTOMYOSIN   AND    MUSCULAR   CONTRACTION  4I 

RfiSUMfi 

II  a  et6  montr^  que  la  contraction  du  muscle,  la  superprecipitation  de  ses  suspensions,  la  super- 
precipitation  de  I'actomyosine  et  la  contraction  de  I'actomyosine,  provoqu^es  par  I'ATP,  sont  des 
phenomenes  connexes. 

Des  differences  de  comportement,  comme  par  exemple  I'anisodiametrie  de  retr6cissement  du 
muscle  et  I'isodiametrie  de  retrecissement  de  gels  d'actomyosine  non  orientee,  peuvent  etre  expliqu6es 
par  les  differences  de  structure.  Le  cas  est  le  meme  en  ce  qui  concerne  la  difference  entre  les  fibres 
musculaires  et  les  filaments  d'actomyosine  charges,  ces  derniers  s'allongeant,  contrarrement  au 
muscle,  sous  I'influence  de  I'ATP. 

ZUSAMMENFASSUNG 

Es  wird  gezeigt,  dass  die  Zusammenziehung  des  Muskels,  die  Super-Fallung  seiner  Suspensionen, 
die  Super-Fallung  von  Aktomyosin  und  die  Zusammenziehung  von  Aktomyosin,  hervorgerufen  durch 
ATP,  mit  einander  zusammenhangende  Erscheinungen  sind. 

Verschiedenheiten  des  Verhaltens,  wie  z.B.  die  Anisodiametrie  des  schrumpfenden  Muskels  und 
die  Isodiametrie  der  Schrumpfung  in  unorientierten  Aktomyosin-Gelen,  konnen  durch  die  Struktur- 
verschiedenheiten  erklart  werden.  Dasselbe  gilt  fiir  die  Unterschiede  zwischen  Muskel  und  belasteten 
Aktomyosin-Faden,  welch  letztere  sich  im  Gegensatz  zum  Muskel  unter  der  Einwirkung  von  ATP 
dehnen. 

REFERENCES 

1  W.  T.  AsTBURY,  Proc.  Roy.  Soc.  Ser.  B.,  134  (1947)  303. 

2  S.  V.  Perry,  R.  Reed,  W.  T.  Astbury,  and  L.  C.  Spark,  Biochim.  Biophys.  Acta,  2  (1948)  674. 

3  E.  C.  Bate-Smith  and  J.  R.  Bendall,  /.  Physiol.,  106  (1947)  177. 

*  Fr.  Buchthal,  a.  Deutsch,  C.  G.  Knappeis,  and  A.  Petersen,  Acta  Physiol.  Scand.,  13  (1947) 
167. 

^  V.  A.  Engelhardt,  M.  H.  Ljubimowa,   and   R.  A.  Meitina,   Compt.  rend.  acad.  sci.  U.R.S.S., 
30  (1941)  644. 

®  Th.  Erdos,  Studies  Inst.  Med.  Chem.  Univ.  Szeged.,  3  (1943)  51  (see  also  Ref.  10). 

^  M.  Gerendas,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  i  (1941-42)  47. 

8  C.  E.  Hall,  M.  A.  Jakus,  and  F.  O.  Schmitt,  Biol.  Bull.,  90  (1946)  32. 

^  F.  B.  Straub,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  2  (1942)  3. 

^o  A.  Szent-Gyorgyi,  Chemistry  of  Muscular  Contraction,  Academic  Press,  New  York  1947. 
"  H.  H.  Weber,  Arch.  ges.  Physiol.,  235  (1934)  193. 

It  is  a  great  pleasure  and  privilege  to  offer  these  lines  to  one  of  the  most  distin- 
guished pioneers  of  muscle  research;  I  wish  him  long  years  of  undisturbed  scientific 
activity. 

Received  April  13th,  1949 


42  BIOCHIMICA   ET   BIOPHYSICA   ACTA  VOL.  4  (1950) 


^lYOSIN  AND  ADENOSINETRIPHOSPHATE  IN  RELATION  TO  MUSCLE 

CONTRACTION 

by 

D.  M.  NEEDHAM 
Biochemical  Laboratory,  Cambridge  [England) 


The  conception  of  energy  provision  by  the  spHtting  off  of  the  terminal  phosphate 
group  of  ATP,  under  the  influence  of  myosin  or  actomyosin  acting  as  ATPase,  is  central 
in  current  hypotheses  of  muscle  contraction.  Indeed,  in  many  aspects  of  metabolism 
we  find  evidence  that  ATP  serves  as  a  readily  expended  store  of  energy  and  that  much 
of  the  free  energy  of  oxidation  and  glycolysis  goes  to  its  resynthesis.  In  these  circum- 
stances, it  is  strange  to  reflect  that  we  are  still  without  accurate  knowledge  of  the 
amount  of  free  energy  available  in  this  reaction ;  we  do  know,  however,  that  it  is  sur- 
prisingly small,  only  of  the  order  of  12000  g  cals  per  g/mol  H3PO4  set  free.  Still  more 
surprising  is  the  small  difference  in  free  energy  content  (only  about  6000-8000  g  cals) 
which  separates  the  "energy-rich"  phosphate  bonds  from  the  "energy-poor"  phosphate 
bonds.  It  is  probably  because  of  its  ability  to  deal  in  these  small  stages  of  energy  transfer 
that  the  living  cell  achieves  its  high  efficiency.  Thus  even  normal  aerobic  contraction  is 
about  20%  efficient  when  tension  production  or  work  performance  is  compared  with 
heat  production;  and  anaerobic  contraction  about  40%  efficient.  The  anaerobic  recovery 
phase  (when  creatinephosphate  formation  is  going  on  at  the  expense  of  carbohydrate 
breakdown  to  lactic  acid)  is  over  90%  efficient:  there  is  little  heat  production  during 
this  period  and  the  formation  of  the  energy-rich  phosphate  bonds  goes  on  with  scarcely 
any  waste  in  the  form  of  heat.  We  shall  return  to  this  point  later. 

The  fact  that  no  breakdown  of  ATP  has  been  demonstrated  in  normal  contraction, 
but  only  becomes  observable  in  fargoing  fatigue,  has  recently  been  emphasized  by 
A.  V.  HiLL^.  By  the  use  of  the  new  micro-methods,  for  example  those  of  Kalckar^, 
it  should  now  be  possible  to  estimate  ADP  in  amounts  of  the  order  to  be  expected  during 
a  single  twitch  or  a  very  short  series  of  twitches.  Although  rephosphorylation  by  means 
of  creatine  phosphate  probably  follows  with  great  rapidity,  by  using  slow-moving  muscle 
at  low  temperature  it  might  thus  be  possible  to  detect  a  period  of  ATP  breakdown 
unobscured  or  only  partly  obscured  by  resynthesis. 

ATPase  activity  in  vivo  and  in  vitro 

The  close  connection  of  ATP  breakdown  with  energy  provision  for  contraction  once 
conceded,  two  very  important  questions  arise  —  the  exact  conditions  of  the  ATPase 
activity  and  its  timing. 

That  myosin  can  act  as  ATPase  is  wellknown^  but,  as  Bailey  has  shown*,  the 
optimal  conditions  for  the  activity  of  myosin  prepared  in  the  classical  manner  and 
containing  little  actomyosin,  are  not  those  to  be  expected  within  the  muscle  fibre.  The 
References  p.  4g. 


VOL.  4  (1950)  MYOSIN,  ATP,  AND    MUSCLE    CONTRACTION  43 

activity  is  very  low  around  pn  74,  the  activity  of  fresh  preparations  increasing  pro- 
gressively up  to  and  beyond  p^  10;  Ca++  is  an  essential  activator  and  Mg++  exercises 
a  strong  antagonism  to  Ca++^.  These  facts  have  led  Mommaerts  and  Seraidarian*, 
to  repudiate  the  possibility  that  ATP  can  break  down  in  the  fibre  at  more  than  a  small 
fraction  of  the  rate  required  to  produce  the  increase  in  free  phosphate  observed  on 
contraction.  But  here  some  recent  experiments  of  Keilley  and  Meyerhof''  seem  likely 
to  throw  important  light  on  a  dark  place.  In  a  study  of  the  ATPase  activity  of  various 
protein  fractions  from  muscle,  they  found  with  myosin  alone  the  high  pn  optimum  and 
the  Ca++-Mg++  antagonism  already  mentioned;  but  with  actomyosin  (made  from 
■"crystalline  myosin"  and  purified  actin)  they  observed  in  presence  of  Ca++  an  optimum 
activity  around  Ph  T-T ,  almost  unaltered  by  addition  of  Mg++.  Szent-Gyorgyi^  had 
already  remarked  on  Mg++  activation  of  the  ATPase  activity  of  "impure  natural 
actomyosin"  but  this  effect  may  have  been  due  to  presence  of  myokinase.  Mommaerts 
and  Seraidarian^  report  experiments  on  ATPase  activity  of  actomyosin  at  p^  7.0 
and  Ph  9.0  where  Mg++  showed  its  antagonistic  effect  to  Ca++.  It  certainly  seems  that 
further  enzymic  examination  of  actin,  myosin  and  their  combinations  might  lead  to 
illuminating  results. 

Keilley  and  Meyerhof'  describe  also  the  preparation  from  muscle  of  a  second 
Mg++-activated  ATPase,  p^  optimum  6.8,  containing  no  myosin  or  actin,  but  possibly 
■associated  with  mitochondrial  particles;  this  may  correspond  to  the  ATPase  found  in 
the  mitochondria  of  other  tissues  (Schneider^)  but  not  yet  so  thoroughly  investigated. 

It  is  clear  from  this  study  that  it  would  be  a  difficult  matter  to  specify  at  present 
the  optimal  conditions  for  tne  muscle  ATPase  activity.  Further  it  has  to  be  remembered 
that  there  is  considerable  evidence  (to  be  discussed  later)  for  localization  of  materials 
in  the  muscle  fibre.  This  applies  to  the  adenylic  compounds  and  to  inorganic  salts,  so 
th^t  we  cannot  assume  that  the  ionic  concentrations  where  the  enzyme  is  acting  in  vivo 
are  the  same  as  the  overall  ionic  concentrations.  Nor  have  we  data  from  which  to  gauge 
the  extent  of  p^  variation  within  the  fibre. 

the  timing  of  ATPase  activity  in  vivo  and  the  effect  of  ATP  on  myosin 

We  come  now  to  the  timing  of  the  ATPase  activity:  does  it  occur  simultaneously 
with  contraction  or  with  relaxation?  With  this  is  bound  up  the  whole  question  of  the 
details  of  interaction  between  myosin  and  ATP.  Does  ATP  enter  into  combination  with 
myosin  as  a  result  of  the  stimulus  or  is  it  always  in  some  kind  of  combination  with  some 
part  of  the  myosin  chain  ?  Does  the  ATP  in  combining  with  the  myosin  act  as  a  trigger 
to  set  off  the  energy  liberation  and  the  shortening  of  the  myosin  ?  Do  tension  develop- 
ment and  work  performance  depend  on  simultaneous  ATP  breakdown?  Or  does  the 
energy  liberated  in  contraction  come  in  the  first  place  from  energy  stored  in  the  myosin 
chains,  the  energy  from  ATP  dephosphorylation  being  used  during  relaxation  to  recon- 
stitute the  chains  in  their  initial  state? 

None  of  these  questions  can  be  answered  with  assurance.  We  shall  consider  briefly 
the  results  obtained  from  experiments  in  vitro  on  the  effect  of  ATP  on  myosin  and 
actomyosin  since  it  is  from  further  pursuit  of  such  analytical  procedures  that  we  can  best 
hope  to  get  a  clue  to  the  intimate  mechanism  of  contraction.  But  at  the  present  time 
perhaps  the  best  indication  of  an  answer  to  any  of  these  questions  comes,  not  from  any 
results  ifi  vitro  but  from  the  fact  that,  in  the  living  muscle,  relaxation  gives  the  impression 
References  p.  49. 


44  D.  M.  NEEDHAM  VOL.  4  (1950) 

of  being  an  active  process.  For  example,  during  onset  of  fatigue,  it  is  the  relaxation 
phase  which  becomes  slowed  rather  than  the  contraction  phase.  This  would  suggest  an 
answer  in  the  affirmative  to  the  last  question. 

THE    EFFECT   OF   ATP   ON    MYOSIN    SOLS 

The  experiments,  during  1941  and  1942  of  J.  Needham  and  his  collaborators  in 
Cambridge^"  and  of  Szent-Gyorgyi  and  his  collaborators  in  Szeged^^  showed  the  highly 
specific  reversible  effect  of  ATP  in  diminishing  the  double  refraction  of  flow  and  the  vis- 
cosity of  solutions  of  myosin  (prepared  in  the  classical  way)  in  0.5  M  KCl.  The  decrease 
in  the  length  to  breadth  ratio  of  the  micelles  thus  indicated  was  traced  by  the  Szent- 
Gyorgyi  school  to  the  splitting  of  actomyosin;  and  the  isolation  of  the  new  muscle 
protein,  actin,  by  Straub^^  followed. 

In  a  recent  publication,  Jordan  and  Oster^^  have  described  experiments  on  the 
light-scattering  properties  of  solutions  of  classical  myosin  in  0.5  M  KCl  before  and  after 
addition  of  ATP ;  they  interpret  their  data  on  the  change  in  ratio  of  forward  to  backward 
scattering  as  showing  an  increase  in  coiling  of  the  protein  particles,  these  being  present, 
before  ATP  addition,  in  the  form  of  slightly  coiled  rods. 

The  validity  of  this  interpretation  depends  upon  the  presence  of  the  actomyosin 
in  the  solution  in  the  form  of  discrete  rods  and  not  in  the  form  of  the  branching  network 
to  be  seen  in  electron  micrographs.  It  is  very  possible  that  the  dilute  solution  used  did 
contain  rod-shaped  particles  especially  as  it  had  been  subjected  to  ultra  centrifugation, 
which  might  be  expected  to  carry  down  the  network. 

An  increased  coiling  of  such  actomyosin  particles  (or  of  myosin  particles  formed 
from  them)  under  the  influence  of  ATP  would  obviously  be  of  importance  in  consider- 
ations of  muscle  contraction  and  further  work  along  these  lines,  including  observations 
on  pure  myosin  (myosin  A),  will  be  of  much  interest. 

It  is  a  matter  too,  for  future  experiment  to  decide  whether  evidence  for  the  in- 
creased coiling  of  the  rods  (after  they  are  set  free  from  the  network)  can  be  obtained 
from  electron  micrographs.  So  far  attention  has  been  concentrated  on  the  behaviour 
of  the  network  with  ATP  and  the  appearance  of  the  resulting  debris  has  not  been  closely 
studied. 

Another  step  forward  in  our  knowledge  of  the  interaction  of  myosin,  actin  and  ATP 
was  gained  by  the  observations  of  Bailey  and  Perry^^  on  the  effect  of  -SH  reagents. 
They  showed  a  close  correlation  between  the  effect  of  reagents  which  oxidize  or  combine 
with  -SH  groups  in  inhibiting  ATPase  activity  of  myosin  on  the  one  hand  and  its  power 
to  combine  with  altin  on  the  other.  Thus  certain  -SH  groups  of  myosin  are  necessary 
for  its  combination  with  ATP  (and  this  is  in  line  with  much  other  information  about 
enzymes  concerned  with  ATP).  These  same  -SH  groups  are  necessary  for  combination 
of  myosin  with  actin,  and  if  ATP  is  added  to  actomyosin  it  displaces  actin  from  these 
groups  and  itself  combines.  These  results  are  important,  not  only  in  throwing  light  on 
the  mechanism  of  the  dissociation  effect.  The  earlier  experiments  of  the  Needham  and 
Szent-Gyorgyi  groups  had  indicated  an  interaction  between  ATP  and  the  protein 
responsible  for  the  double  refraction  of  flow  and  the  high  viscosity ;  that  is  to  say,  they 
made  it  unlikely  that  ATPase  activity  of  the  myosin  preparations  was  to  be  put  down 
to  presence  of  small  amounts  of  some  other  protein.  This  line  of  argument  is  strongly 
re-inforced  by  the  work  of  Bailey  and  Perry  which  forms  the  best  evidence  so  far 
References  p.  4g. 


VOL.  4   (1950)  MYOSIN,  ATP,  AND    MUSCLE    CONTRACTION  45 

for  the  ATPase  activity  of  myosin  itself.  The  knowledge  gained  from  the  study  of  inter- 
action between  ATP  and  myosin  sols  must  clearly  play  a  useful  part  in  our  progress 
towards  understanding  of  muscle  contraction.  But  it  does  not  seem  that  any  deductions 
having  a  direct  bearing  on  the  question  we  have  raised  can  be  drawn  from  it  at  the 
moment.  Certainly  a  deduction  recently  made  from  the  results  of  Dainty  et  al.'^^  by 
MoRALES^^  that  "the  catalytic  activity  of  ATPase,  that  is  of  acto-myosin,  rises  exponen- 
tially with  disorientation  of  the  protein"  is  not  justified. 

THE    EFFECT   OF   ATP    ON    MYOSIN   THREADS 

The  similarity  in  rod  and  intrinsic  birefringence  and  in  the  X-ray  diagram  between 
artifical  myosin  threads  and  muscle  fibres  led  to  hope  that  important  progress  might 
be  made  by  study  of  the  effect  of  ATP  upon  such  threads ;  especially  since  it  was  found 
that  they  still  retain  ATPase  activity  and  could  withstand  a  certain  amount  of  tension 
(Engelhardt^'^)  without  breaking. 

Engelhardt  used  threads  made  from  classical  myosin  and  containing  about  2% 
protein.  Subjected  to  loads  of  about  200  mg  such  threads  show  a  reversible  extension. 
If  the  threads  are  tested,  immersed  not  in  KCl  solution  but  in  0.005  ^I  ATP,  this  exten- 
sibility is  increased  by  50-100%. 

This  effect  of  rise  in  extensibility  with  loaded  threads  is  in  contrast  to  the  striking 
shortening  effect  obtained  by  Szent-Gyorgyi^^  with  unloaded  actomyosin  threads 
(myosin  B),  suspended  in  dilute  (0.05  M)  KCl.  Addition  of  ATP  (0.002  M)  led  to  isodi- 
mensional  contraction,  with  shortening  up  to  66%.  This  shrinkage  of  the  actomyosin 
thread  is  accompanied  by  great  loss  of  water,  the  percentage  falling  from  about  97  to  50. 

The  observations  of  Buchthal  et  al}^  form  a  link  between  these  two  sets  of  obser- 
vations. Using  actomyosin  threads  (which  had  been  dried  to  a  N  content  of  16.15%  and 
then  allowed  to  imbibe  water  for  30  minutes  from  0.9%  NaCl  solution)  they  found  that 
addition  of  0.002  M  ATP  caused  a  30%  shortening  of  the  unloaded  thread;  while  with 
a  load  of  no  mg  there  was  an  increase  in  length  of  30%.  Even  so  small  a  load  as  5  mg 
caused  a  slight  lengthening. 

Perry  et  alP  have  contributed  an  instructive  electron  microscope  and  X-ray  study 
of  the  synaeretic  effect  of  ATP  on  actomyosin  gel  in  0.05%  KCl.  The  photographs  of 
the  control  gel  show  a  dense  tangled  network.  After  ATP  addition,  the  network  has 
opened  out;  it  would  appear  that  small  linear  fibres  are  first  formed  (as  might  be  ex- 
pected on  a  splitting  to  actin  and  myosin)  and  that  these  aggregate  side  by  side  to  form 
denser  fibres.  The  X-ray  diagrams  from  the  same  material  show  no  ^fundamental  differ- 
ence between  actomyosin  before  and  after  synaeresis.  These  observEftions  were  taken  to 
indicate  that  there  is  no  increased  intramolecular  folding  with  intramolecular  synaeresis, 
but  rather  that  the  water  loss  is  intermolecular  accompanied  by  lateral  aggregation. 

When  all  these  facts  are  considered  together  it  seems  that  the  discrepancy  originally 
felt  between  the  results  of  Engelhardt  and  of  Szent-Gyorgyi  disappears.  The  effect 
of  the  x\TP  in  both  cases  is  to  cause  breakdown  of  the  actomyosin  network  followed  by 
aggregation  of  the  particles  and  squeezing  out  of  water.  When  the  thread  is  loaded,  the 
fall  in  elasticity  consequent  upon  the  disappearance  of  the  network  is  the  obvious 
aspect;  when  the  thread  is  unloaded,  this  aspect  is  not  noticeable  but  the  shortening 
due  to  S5maeresis  can  manifest  itself. 

Buchthal  et  al}^  have  reported  that  treatment  of  fresh  actomyosin  threads  (3% 
References  p.  4g. 


46  D.  M.  NEEDHAM  VOL.  4  (1950) 

protein)  with  the  sulphydryl  reagents  iodoacetate  and  porphyrexid  causes  decreased 
shortening  when  ATP  is  subsequently  added.  The  interpretation  of  these  results  is  not 
immediately  obvious  for,  when  sulphydryl  reagents  are  added  to  actomyosin  sols  in 
0.5  M  KCl,  there  is  a  decrease  in  viscosity  as  would  be  expected  if  the  reagent,  like  ATP, 
broke  the  link  between  the  actin  and  the  myosin.  The  effect  is  slower  than  with  ATP 
itself,  probably  because  the  reagents  react  also  with  other  -SH  groups  (for  example,  in 
the  actin)  while  the  ATP  reacts  specificially  with  the  connecting  groups.  In  the  case  of 
the  threads,  there  seems  no  reason  why  -SH  reagents  should  inhibit  the  splitting  of  the 
myosin  from  the  actin;  possibly  the  attachment  of  ATP  to  the  myosin,  as  well  as  the 
presence  of  free  -SH  groups,  is  involved  in  the  further  changes  in  state  of  aggregation 
and  it  is  these  wh'ch  become  impossible. 

It  seems  doubtful  whether  these  phenomena  of  synaeresis  are  connected  with  the 
mechanism  of  contraction.  If  the  removal  of  water  is  intermoiecular,  this  would  lead 
in  vivo  to  a  narrowing  rather  than  a  shortening  of  the  fibres,  since  both  myosin  and  actin 
are  known  to  be  arranged  with  their  long  axes  parallel  to  the  fibre  axis.  Perry  et  al}^ 
have  remarked  on  this  and  also  pointed  out  that  though  the  loss  of  water  associated 
with  volume  contraction  is  very  rapid,  the  reverse  process  (which  might  be  analogous 
to  relaxation)  is  slow  and  there  is  little  information  as  to  its  degree. 

the  localization  of  substances  in  the  striated  fibre 

The  anisotropic  (A)  band  seems  to  have  had,  from  the  early  days  of  work  on  muscle 
fibres,  a  particular  interest  for  observers.  In  spite  of  the  many  variations  and  discrepan- 
cies of  description  of  the  h'stological  appearance  of  contracted  muscle  (depending  on 
the  different  sources  of  the  muscle ;  differences  in  preparation,  whether  fresh  or  fixed  and 
stained;  differences  in  optical  set-up;  and  differences  in  degree  of  contraction)  there  has 
been  a  widespread  if  by  no  means  unan'mous  opinion  that  it  is  the  A-band  which  be- 
comes shorter  in  appea^-ance  on  contraction  of  the  muscle  while  the  I-band  may  show 
little  change  or  even  become  longer. 

This  conclusion  was  probably  based  partly  on  the  formation  of  contraction  bands 
(see  below)  in  strong  contraction ;  a  condition  where  the  position  of  the  staining  mate- 
rial has  actually  become  reversed  with  respect  to  the  A-  and  I-bands;  but  studies 
like  those  of  Buchthal  et  al.^^  on  single  living  fibres  do  show  a  decrease  in  the  A/I 
ratio  in  early  conti  action.  The  work  of  Buchthal  et  al.  was  quantitative  and  showed, 
in  short  isometric  tetani,  a  decrease  in  length  of  the  A-band  of  18%,  an  increase  in 
the  I-band  of  28%. 

The  visible  changes  in  length  of  the  A-  and  I-bands  have  often  been  taken  as  indi- 
cating that  the  actual  contractile  process  was  limited  to  the  A-bands;  the  I-bands, 
thorgh  not  necessarily  considered  as  passive,  being  the  seat  of  less  important  changes. 
The  conception  of  mo^e  recent  yea^-s  took  the  form  that  in  the  A-bands  the  protein 
micelles  undergo  folding  while  this  process  is  much  less  or  gives  place  to  unfolding  in 
the  I-bands. 

The  idea  that  the  protein  of  the  A-band  actually  differed  in  kind  from  that  of  the 
I-band  was  given  up  as  more  accurate  estimates  of  the  myosin  content  of  the  muscle 
became  available.  For  several  years  the  view  was  then  prevalent  that  the  fibril  consists 
of  collections  of  myosin  chains,  a^'ranged  in  the  anisotropic  bands  with  their  long  axes 
parallel  to  the  fibril  axis,  but  in  the  isotropic  bands  having  much  less  orderly  arrange- 
References  p.  4g. 


VOL.  4  (1950)  MYOSIN,  ATP,  AND    MUSCLE    CONTRACTION  47 

merit.  However  the  recent  production  of  electron  micrographs  of  muscle  fibres  (Hall, 
Jakus,  AND  ScHMiTT^^),  showing  continuous  micelles  passing  stra-ght  through  A- and 
I-bands,  brought  the  realization  that  the  lack  of  orderly  arrangement  in  the  I-bands 
(if  it  exists)  must  be  at  a  level  of  dimension  below  the  resolving  power  of  the  electron 
microscope.  The  work  of  Dempsey  et  al}~  about  the  same  time,  demonstrated  the  pre- 
sence in  I-bands  of  lipoids  with  negative  double  refraction  and  the  possibility  of  con- 
verting striated  fibrils,  by  thorough  extraction  with  fat  solvent,  into  fibrils  of  uniform 
positive  double  refraction.  Matoltsy  and  Gerendas^?  also  report  experiments  indi- 
cating the  presence  of  a  substance  of  negative  double  refraction  in  the  I-band. 

The  present-day  conception  is  therefore  rather  that  the  fibrils  consist  uniformly 
through  their  length  of  bundles  of  myosin  (or  actomyosin)  chains  pursuing  an  apparently 
straight  course,  and  as  far  as  our  knowledge  of  these  chains  goes,  there  is  no  obvious 
reason  for  a  localization  of  the  contraction  process  in  the  A-bands. 

We  have  some  further  knowledge  indicating  a  high  degree  of  localization  of  other 
substances  within  the  fibre,  and  also  (a  matter  of  particular  interest)  in  some  cases 
suggesting  a  change  in  location  during  contraction.  Since  the  possibility  arises  that 
changes  in  position  of  non-myosin  material  may  affect  the  visible  length  of  the  A-  and 
I-bands,  this  subject  may  be  pursued  a  little  further.  Thus  there  is  good  evidence 
(Caspersson  and  Thorell^^)  that  material  with  selective  absorption  at  a  wave-length 
of  265  m/i  is  concentrated  in  the  I-bands  in  resting  muscle;  in  muscle  after  vigorous 
contraction  there  is  spread  of  the  material  into  the  A-bands.  The  adenylic  compounds 
are  the  most  likely  to  be  responsible  for  this  effect ;  it  has  also  been  suggested  that  they 
may  contribute  to  the  negative  double  refraction  of  the  I-band  (Barer^^). 

Then  we  have  the  more  recent  work  of  Scott  and  Packer^^  (using  a  rapid  freeze- 
drying  method  and  careful  avoidance  of  water  to  prevent  movement  of  soluble  salts) 
confirming  a  good  deal  of  earlier  work  in  finding  the  greater  part  of  the  ash  in  the  A- 
band.  There  were  indications  that  this  localization  applied  to  calcium  and  magnesium. 
Finally  it  has  long  been  known  that  the  A-bands  contain  material  wh'ch  stains  deeply 
with  basophilic  dyes.  This  material  seems  to  contribute  to  the  dark  colour  of  the  A-band 
in  fresh  fibres  in  ordinary  I'ght,  but  does  not  seem  to  be  concerned  with  the  double 
refraction  of  the  A-band.  Histological  literature  abounds  with  detailed  descriptions  of 
the  movement  of  this  material  (the  A  substance)  during  contraction.  Such  descriptions 
are  usually  concerned  with  fixed  and  stained  material  but  as  of  more  interest  we  may 
take  the  example  of  the  m^re  recent  work  of  Speidel"  on  living  muscle  of  vertebrates 
and  invertebrates.  He  describes,  as  the  fibre  shortens,  first  a  shortening  of  both  A  and  I ; 
secondly  a  blurring  of  cross  striae  when  the  sarcomeres  have  shortened  by  about  one 
third,  as  if  the  daT-k  refracting  material  were  undergoing  profound  redistribution  or 
chemical  change ;  thirdly,  concentration  of  the  da^k  refractive  material  (the  contraction 
band)  about  each  Z  disc  (crossing  the  centre  of  what  was,  during  rest,  the  I-band). 

It  is  interesting  that  the  electron  microg  -aphs  of  Hall,  Jakus,  and  Schmitt^^  show 
material  (of  wh'ch  we  know  only  that  it  has  h'gh  electron-scattering  power  and  h'gh 
affinity  for  phosphotungstic  acid)  concentrated  in  the  resting  fibril  in  the  A-band.  When 
fibres  a'-e  stained  with  phosphotungstic  ac'd  and  fixed  in  different  stages  of  contraction, 
stages  can  be  made  out  in  the  electron  microg-aphs  indicating  the  spreading  of  stainable 
material  from  the  A-bands,  until  at  about  40%  shortening  a  state  is  reached  with 
a  narrow  dense  band  in  the  position  of  the  Z-membrane,  the  rest  of  the  fibre, 
including  the  A-band,  being  uniform  with  comparatively  faint  staining.  The  close 
References  p.  4g. 


48 


D.  M.  NEEDHAM 


VOL.  4  (1950) 


correspondence  with  the  behaviour  of  the  "A"  substance  described  above  is  striking. 
These  observations  may  be  summarized  as  follows : 


TABLE  I 

I-Band 

Movement  during  contraction 

A-Band 

Lipoids 

Adenylic  compounds 

Salts,  perhaps  especially  Ca  and  Mg 

Basophilic  A  substance 
Electron  scattering  substances 

It  seems  that  there  must  be  some  intimate  connection  between  the  three  classes 
of  substance  mentioned  in  the  3rd.  column;  whether  the  same  substances  are  actually 
responsible  for  the  staining  and  the  electron-scattering  phenomena  we  do  not  know. 

Besides  these  localizations  which  have  been  recognized,  and  which  must  have 
significance  for  contraction  it  seems  likely  that  there  may  be  much  localization  still 
unknown.  In  particular  it  is  to  be  expected  that  the  soluble  protein  fractions,  myogen 
and  globulin  X,  including  most  of  the  enzyme  equipment  of  the  muscle,  instead  of  being 
merely  dissolved  in  the  sarcoplasm,  will  show  pattern. 


CONCLUSION 

If  one  is  to  make  any  sort  of  tentative  picture  of  the  mechanism  of  contraction, 
one  must,  under  present  conditions,  be  allowed  a  bias  towards  one  side  or  the  other 
in  answering  the  question  "Is  relaxation  of  the  fibril  an  active  process,  requiring  pro- 
vision of  free  energy?"  The  writer  would  like  to  take  the  standpoint  that  an  affirmative 
answer  best  fits  the  observed  physiological  behaviour  during  relaxation  and  that  obser- 
vations on  the  relations  of  heat  production  and  on  the  effect  of  work  on  heat  production 
are  not  at  variance  with  this  view. 

One  can  make  the  basic  assumptions  that,  in  the  stimulated  muscle,  chemical 
reaction  becomes  possible  between  groups  situated  along  the  protein  chain;  that  this 
reaction  goes  on  with  production  of  free  energy  and  that  in  the  resting  muscle  there 
is  some  configurational  hindrance  to  its  taking  place.  Further,  one  can  assume  that 
the  number  of  these  groups  which  can  react  together  will  depend  upon  the  length  which 
the  muscle  is  made  to  assume,  being  fewer  at  greater  lengths  and  increasing  in  number 
as  the  muscle  shortens.  It  is  known  that  during  a  twitch  the  amount  of  "shortening 
heat"  production  is  proportional  to  the  degree  of  shortening  of  the  muscle,  while  the 
rate  of  "shortening  heat"  production  is  dependent  on  the  speed  with  which  the  muscle 
shortens,  (A.  V.  Hill^^).  Thus  for  shortening  a  given  distance,  the  "shortening  heat" 
production  is  the  same,  whether  the  shortening  is  slow  or  fast.  But  the  rate  of  shortening 
depends  on  the  load,  being  slower  the  greater  the  load;  thus  at  slower  rates  of  shortening 
between  two  given  lengths,  more  work  is  done  and  more  energy  must  be  produced, 
since  the  heat  remains  the  same.  If  this  energy  production  is  the  result  of  the  interaction 
of  the  same  groups  at  different  rates  of  shortening,  we  must  suppose  that,  at  the  slower 
rates,  repeated  interaction  takes  place.  When  speculations  are  made  as  to  the  timing 
of 'ATP  breakdown,  it  is  usually  supposed  that  this  is  confined  either  to  the  contraction 
phase  or  to  the  relaxation  phase  (in  the  latter  case  its  energy  being  used  to  restore 
energy-rich  protein  linkages).  If  we  suppose  that,  when  work  is  done,  before  a  pair 
References  p.  4g. 


VOL.  4  (1950)  MYOSIN,  ATP,  AND    MUSCLE    CONTRACTION  49 

of  groups  can  react  together  a  second  time,  they  must  have  been  put  back  into  their 
original  state  by  means  of  free  energy  provided  by  reaction  with  ATP,  we  see  that  ATP 
breakdown  could  begin  within  the  contraction  phase,  even  though  it  were  associated 
with  restoration  of  the  chains. 

A.  V.  HiLL^s  has  shown  that  the  relaxation  phase  of  a  twitch  is  free  from  heat,  if 
the  work  done  is  not  allowed  to  degenerate  into  heat.  During  this  period,  on  the  view 
under  discussion,  ATP  breakdown  would  be  continuing  the  process  of  separating  the 
reactive  groups,  a  process  leading  now  to  the  lengthening  of  the  fibril.  Since  no  heat 
is  associated  with  the  relaxation  phase,  this  process  would  seem  to  be  100%  efficient, 
and  the  waste  heat  associated  with  the  contraction  phase  would  appear  to  be  due  to 
the  primary  reaction  along  the  protein  chains.  As  we  have  seen,  the  anaerobic  recovery 
process  (immediately  following  an  anaerobic  contraction)  is  knov/n  to  go  on  with  very 
little  heat  wastage;  it  is  not  unhkely  that  there  is  a  similar  efficiency  in  the  relaxation 
process.  A  mechanism  suggested  for  the  transfer  of  energy  (Kalckar^*^;  Dainty  et  al.)  is 
the  transfer  of  phosphate  from  ATP  to  the  protein  chains ;  this  still  remains  a  possibility, 
(see  F.  BuCHTHAL  et  al.^^). 

The  Veykiirzungsort  still  retains  its  mystery  but  we  begin  perhaps  to  see  in  what 
direction  solution  lies. 

I  am  indebted  to  Dr  K.  Bailey  and  Professor  W.  T.  Astbury  for  the  benefit  of 
discussion  with  them. 

REFERENCES 

1  A.  V.  Hill,  Nature,  163  (1949)  320. 

2  H.  Kalckar,  /.  Biol.  Chem.,  167  (1947)  445. 

3  M.  N.  LiuBiMOVA  AND  W.  A.  E.  Engelhardt,  Biochimia,  4  (1939)  716. 
■*  K.  Bailey,  Biochem.  J.,  36  (1942)  121. 

*  G.  D.  Greville  and  H.  Lehmann,  Nature,  152  (1943)  81. 

^  W.  F.  H.  M.  Mommaerts  and  K.  Seraidarian,  /.  Gen.  Physiol.,  30  (1947)  401. 
^  W.  W.  Keilley  and  O.  Meierhof,  /.  Biol.  Chem.,  176  (1948)  591. 

*  A.  Szent-Gyorgyi,  Muscle  Contraction,  New  York  1947. 

*  W.  C.  Schneider,  /.  Biol.  Chem.,  165  (1946)  585. 

1°  J.  Needham,  S.-C.  Shen,  D.  M.  Needham,  and  A.  S.  C.  Lawrence,  Nature,  147  (1941)  766- 

"  i.  Banga,  T.  Erdos,  M.  Gerendas,  W.  F.  H.  M.  Mommaerts,  F.  B.  Straub,  and  A.  Szent- 

Gyorgyi,  Studies  Inst.  Med.  Chem.  Szeged,  i  (1941)  42. 
12  F.  B.  Straub,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  2  (1942)  3. 
1^  W.  J.  Jordan  and  G.  Oster,  Science,  108  (1948)  188. 
1^  K.  Bailey  and  S.  V.  Perry,  Biochim.  Biophys.  Acta,  i  (1947)  506. 
15  M.  Dainty,  A.  Kleinzeller,  A.  S.  C.  Lawrence,  M.  Miall,  J.  Needham,  D.  M.  Needham,  and 

S.  Shen,  /.  Gen.  Physiol.,  27  (1944)  355- 
1"  M.  F.  Morales,  Biochim.  Biophys.  Acta,  2  (1948)  618. 
•^  W.  A.  Engelhardt,  Advances  in  Enzymology,  6  (1946)  147. 
'**  F.  Buchthal,  a.  Deutsch,  G.  G.  Knappeis,  and  A.Petersen,  Acta  Physiol.  Scand.,  13  (1947)  167. 

19  S.  V.  Perry,  R.  Reed,  W.  T.  Astbury,  and  L.  C.  Spark,  Biochim.  Biophys.  Acta,  2  (1948)  674. 

20  F.  Buchthal,  G.  G.  Knappeis,  and  J.  Lindhard,  Skand.  Arch.  Physiol.,  73  (1936)  162. 

21  C.  E.  Hall,  M.  A.  Jakus,  and  F.  O.  Schmitt,  Biol.  Bull.  Woods  Hole,  90  (1946)  32. 

22  E.  W.  Dempsey,  G.  B.  Wislocki,  and  M.  Singer,  Anat.  Record,  96  (1946)  221. 
2^  A.  G.  Matoltsy  and  M.  Gerendas,  Hung.  Acta.  Physiol.,  1  (1947)  116. 

2*  R.  Caspersson  and  B.  Thorell,  Acta  Physiol.  Skand.,  4  (1943)  97- 

25  R.  Barer,  Biol.  Revs,  23  (1948)  159. 

-*  G.  H.  Scott  and  D.  M.  Packer,  Anat.  Record,  74  (1939)  31. 

"  C.  C.  Speidel,  Am.  J.  Anat.,  65  (1939)  471. 

28  A.  V.  Hill,  Proc.  Roy.  Soc.  Lond.  Ser.  B.,  136  (1949)  I95- 

29  A.  V.  Hill,  /.  Physiol.,  107  (1948)  29  P. 

^°  F.  Buchthal,  A.  Deutsch,  G.  G.  Knappeis,  and  A.  Munch-Petersen,  Acta  Physiol.  Skand. 
16  (1948)  326. 

Received  April  12th,  1949 


50  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


A  CONSIDERATION  OF  EXPERIMENTAL  FACTS  PERTAINING  TO  THE 
PRIMARY  REACTION  IN  MUSCULAR  ACTIVITY 

by 

W.  F.  H.  M.  MOMMAERTS* 
Department  of  Biochemistry,  Duke  University  School  of  Medicine  Durham,  N.  C.  (U.S.A.) 


One  of  the  most  significant  results  of  the  investigations  of  Meyerhof  and  his 
associates  was  the  demonstration  that,  of  all  known  metabolic  processes  the  splitting 
of  adenosine  triphosphate  (ATP)  is  most  directly  connected  with  the  fundamental 
mechanical  event  in  contracting  muscle  (Lohmann^'';  Meyerhof^O;  see^'.  Chapter  II). 
Notwithstanding  its  importance  this  result  is  subject  to  two  limitations.  For  one  thing, 
the  nature  of  the  breakdown  of  ATP  is  still  not  yet  satisfactorily  settled;  the  assumption 
now  popular  that  it  is  due  to  a  straightforward  hydrolysis  by  the  enzyme  myosin- 
ATPase  leads,  at  the  present  state  of  knowledge,  to  difficulties.  On  the  other  hand,  the 
introductory  statement  as  well  as  Lohmann's  original  conclusion  contained  the  res- 
triction "of  all  known  metabolic  processes".  It  is  possible  that  ATP,  before  becoming 
decomposed,  engages  in  other  more  intimate  reactions  with  the  contractile  structure, 
as  will  be  emphasized  in  this  paper.  These  restrictions  do  not  diminish,  they  rather 
enhance  the  emphasis  on  ATP,  and  it  is  exactly  here  that  the  most  direct  link  between 
the  study  of  muscular  metabolism  and  the  modern  analysis  of  its  function  exists. 

An  essential  contribution  to  this  latter  category  has  been  made  by  Szent-Gyorgyi^^ 
by  his  discovery  of  the  contractility  of  actomyosin,  his  biochemical  analysis  of  the  com- 
ponents of  this  complex  substance,  and  by  the  study  of  various  aspects  of  its  behaviour. 
This  work  has  repeatedly  been  summarized  in  greater  or  lesser  detail  (/.c.^^.  37, 38-  27)_ 
There  are.however,  a  few  points  which  may  be  discussed  as  a  suitable  introduction  to 
the  problem  of  this  essay. 

If  ATP  is  indeed  the  ultimate  action  substance  of  muscle,  as  Szent-Gyorgyi  in 
logical  continuation  of  Meyerhof's  worJ<  assumes,  it  is  to  be  expected  that  addition  of 
this  compound  to  a  muscle  will  evoke  contractions.  This  has  been  achieved.  Contractions 
were  obtained  by  Buchthal  et  al.^'  ^'  ^  by  close  arterial  injection  of  ATP,  and  by  its 
application  to  isolated  muscle  fibers.  The  latter  effect  was  also  studied  in  a  quantitative 
manner  by  Rozsa^^,  using  a  different  method.  Since  Buchthal  finds  the  effect  to  persist 
after  curarization,  it  may  appear  difficult  to  assume  an  indirect  stimulation.  Never- 
theless, the  possibility  that  ATP  in  such  experiments  activates  the  excitatory  process  of 
the  muscle,  rather  than  the  contractile  structure  directly,  has  to  be  kept  in  mind. 
.Rozsa's  results  indeed  suggest  this  to  be  the  case.  Since  the  excitatory  process  in  its 
turn  activates  or  liberates  the  ATP  present,  this  Buchthal-Rozsa  effect  may  play  an 
essential  role  in  the  conduction  of  the  contraction  wave. 

A  simpler  and  more  convincing  system  is  what  the  writer  proposes  to  call  the  fibril 
preparation,  which  has  been  introduced  by  Szent-Gyorgyi^^,  i,  page  24.  Its  great 

*  Established  investigator  of  the  American  Heart  Association. 
References  p.  56I3J. 


VOL.  4  (1950)  PRIMARY   REACTION    IN    MUSCULAR   ACTIVITY  5I 

signijEicance  has  been  underlined  by  Meyerhof^i.  If  a  muscle  with  parallel  fibre  arrange- 
ment is  kept  in  distilled  water  for  a  prolonged  time,  and  is  frozen  and  thawed,  one 
obtains  a  preparation  which  consists  essentially  of  the  original  undisturbed  fibrils,  and 
from  which  the  soluble  constituents  of  the  sarcoplasma,  including  all  factors  which  have 
to  do  with  irritability,  have  been  removed.  No  stimulation  will  cause  contraction  of 
these  fibrils.  They  shorten,  however,  promptly  if  ATP  in  a  proper  electrolytic  medium 
is  added.  In  this  case  there  appears  to  be  little  doubt  that  ATP  has  directly  acted  upon 
the  contractile  structure  itself. 

The  analysis  has  gone  further.  One  can  extract  and  fractionate  the  muscle,  and 
obtain  a  crystalline  protein,  myosin  (Szent-Gyorgyi,  I.e.)  and  supposedly  pure  actin 
(Straub^^'  34).  Combined  with  each  other,  they  form  the  complex  actomyosin  which  can 
also  be  extracted  directly  (Szent-gyorgyi,  I.e.)  and  from  which  threads  may  be  spun. 
These  threads,  suspended  in  the  same  solution  of  KCl  and  MgClo  as  is  used  with  the  fibril 
preparation,  will  contract  in  response  to  the  addition  of  ATP  (Szent-Gyorgyi  I.e.). 
It  is  true  that  these  threads,  unhke  fibrils,  become  shorter  and  thinner  instead  of  thicker. 
This  is  however  merely  a  consequence  of  the  fact  that  the  actomyosin  particles  in  such 
a  thread  are  very  imperfectly  orientated.  After  initial  difficulties  (Gerendas13),Buchthal 
et  al.^  have  succeeded  in  preparing  well  orientated  threads,  and  these  behave  in  accor- 
dance with  the  rule  by  becoming  thicker  during  contraction.  Two  objections  have  been 
made.  Buchthal  et  al.,  at  the  International  Congress  of  Physiology  in  Oxford  (1947)^ 
(repeated  by  Perry  et  al.^^)  raised  the  difficulty  that  such  threads,  when  loaded,  do  not 
contract  but  become  stretched  upon  addition  of  ATP.  This  may  be  due  to  the  circum- 
stance that  in  the  formation  of  the  threads  very  few  and  weak  points  of  intermicellar 
attachment  are  formed,  which  are  not  able  to  carry  any  strain.  Since  the  action  of  ATP 
upon  actomyosin  includes  a  disaggregative  effect  as  well,  the  plasticity  of  the  threads 
is  actually  increased  by  ATP.  In  the  fibrils  on  the  other  hand,  very  strong  intermicellar 
bonds  exist  in  the  densely  packed  system.  The  second  objection,  made  by  Astbury  at 
the  Experimental  Cytology  Congress  in  Stockholm  (1947)  (Perry  et  al.^^),  was  that  upon 
electron-microscopical  investigation  actomyosin,  after  treatment  with  ATP  at  0.05  M 
KCl,  0.005  M  MgClo,  showed  a  dispersion  of  the  original  aggregates,  with  no  indication 
of  a  true  contraction.  Since  however  after  the  addition  of  ATP,  during  the  drying  of  the 
preparation,  the  salt  concentration  had  to  increase  and  pass  the  limit  above  which  the 
actomyosin  dissolves  and  disaggregates,  this  experiment  has  no  bearing  upon  the, 
mechanism  of  contraction.  Finally,  the  same  authors^^,  (p.  677)  object  that,  even  if  the 
shortening  of  actomyosin  threads  may  imitate  the  contraction  of  muscle,  these  threads 
show  no  relaxation.  According  to  all  we  know  about  muscle,  however,  relaxation  would 
seem  to  be  the  more  comphcated  phenomenon.  That  this  has  not  yet  been  reproduced 
in  vitro  is  no  objection  against  a  contribution  relevant  to  contraction.  The  objection  is 
invalid  the  more  so,  since  the  contraction  process  in  threads  takes  place  to  an  extreme 
extent.  Such  extreme  shortenings  are  irreversible  even  in  vivo  (Ramsey's  deltastate^i). 
It  seems  thus  that  Szent-gyorgyi's  observations  on  the  effect  of  ATP  upon  actomyosin 
are  not  subject  to  any  serious  inconsistency  at  this  moment. 

A  further  simplification  may  be  achieved  by  working  not  with  carefully  prepared 
actomyosin  threads,  but  with  a  suspension  of  finely  precipitated  actomyosin  flocks. 
Addition  of  ATP  will  cause  their  contraction  as  well.  Since  they  are  perfectly  disoriented, 
their  contraction  will  take  place  in  all  dimensions  equally.  It  is  manifested  by  an 
increased  tendency  of  the  flocks  to  settle  (Szent-Gyorgyi's  "superprecipitation"),  and 
References  p.  sSj^j. 


52 


W.  F.  H.  M.  MOMMAERTS 


VOL.  4  (1950) 


its  extent  can  be  quantitatively  established  by  determining  the  volume  of  the  gel  pellet 
after  centrifugation  (Mommaerts^^).  One  can  thus  study  contraction  at  various  levels  of 
subcellular  and  supermolecular  organization. 

A  still  simpler  system  is  a  solution  of  actomyosin  in  0.5  M  KCl.  As  Szent-Gyorgyi 
has  discribed^^'  ^^,  the  high  viscosity  of  such  a  solution  is  greatly  decreased  by  ATP. 
The  analysis  of  this  effect  has  shown  that  it  is  not  due  to  a  contraction  of  dissolved 
actomyosin  micells^-'  '^'  ^^'  ^^.  The  true  reason,  as  is  well  established  now,  is  a  disaggre- 
gation of  the  actomyosin  into  its  components,  myosin  and  actin.  Although  the  immediate 
connection  between  this  disaggregation  and  the  contraction  at  lower  ionic  strengths  is 
not  clear,  it  may  be  presumed  that  the  first  effect  of  ATP  is  identical  in  both  cases.  One 
of  the  aspects  of  this  first  effect  apparently  is  an  elimination  of  certain  intermolecular 
bonds.  In  the  case  of  dissolved  actomyosin,  which  is  on  the  verge  of  disaggregation,  the 
complex  falls  apart.  At  low  salt  concentration,  where  more  or  other  bonds  may  exist, 
this  dissociation  cannot  reveal  itself,  but  the  contraction  can.  It  appears  unlikely  that 
in  solutions  of  actomyosin  contraction  takes  place  side  by  side  with  the  disaggregation. 
For  it  is  an  empirical  fact  (Szent-Gyorgyi,  I.e. ;  Erdos^^)  that  without  actin  myosin 
cannot  contract ;  in  0.5  M  KCl  solution,  ATP  separates  the  actin  and  myosin  so  that  no 
contractile  complex  then  exists.  Although  the  relation  between  the  two  effects  is  not 
understood,  the  study  of  the  disaggregation  in  solution  is  highly  useful,  for  it  enables 
a  great  variety  of  experiments  to  be  performed  which  would  not  be  possible  in  strongly 
heterogeneous  systems.  As  a  result  of  the  study  of  this  viscosity  effect,  mainly  three 
h  conclusions  seem  possible: 

3.0i 1 1 — r 1 1 i 1  First,  the  effect  is  fast.  It  appa- 

rently takes  a  fraction  of  a  second 
to  reach  completion.  Methods  for 
the  exact  study  of  its  time  course 
have  not  yet  been  available. 

2.o\ 1 1 i \ 1  \  I  The  second  conclusion   needs 

more  elaborate  explanation^^.  Fig.  i 
shows  a  few  examples  of  the  visco- 
simetric  measurement  of  the  effect 
of  ATP  upon  an  actomyosin  solu- 

i.o\ 1 -/ — \     jf     I     y^    I  I  I  i         tion.  It  will  be  seen  that,  after  the 

initial  viscosity  response  a  recovery 
effect  sets  in,  which  takes  more  time 
the  more  ATP  had  been  added.  It 
is  inhibited  by  Mg+  and  activated 
by  Ca+-ions,  and  is  to  be  identified 
with  the  removal  of  the  ATP  by 
the  ATPase  associated  (Polis  and 
Meyerhof^")  with  the  myosin.  The 
viscosity  response  itself  is  not  inhi- 
bited by  Mg+  and  activated  by  Ca+ 
(rather  the  opposite)  and  can  also 
take  place  if  no  hydrolysis  occurs. 
Hence  the  second  conclusion:  the 
effect  of  ATP  upon  the  aggregation  of  actomyosin  is  not  caused  by  any  known  breakdown 
References  p.  sOj^y. 


A.T.P  added 

1 

®/" 

©/ 

®y 

^ 

> 

/ 

/ 

/ 

/ 

J^ 

/ 

^- 

(5) 

1 

20'  30 

Time 

Fig.  I.  Effect  of  ATP  upon  the  viscosity  of  an  acto- 
myosin solution.  At  zero  time,  ATP  is  added.  In  all 
experiments,  2.5  mg  actomyosin  were  present  per  ml, 
dissolved  in  0.5  molar  KCl  at  neutral  reaction.  Curve  i 
(/\)  refers  to  an  experiment  in  the  presence  of  o.ooi 
molar  CaClg,  curve  5  (n)  to  an  experiment  with  0.00 1 
molar  MgClj.  The  amount  of  ATP  added  was  25-10-* 
moles  in  the  experiments  i,  2  and  5;  50-10-*  in  3; 
200 •  10—*  in  4  (see  text). 


VOL.  4  (1950) 


PRIMARY    REACTION    IN    MUSCULAR   ACTIVITY 


53 


50 


1 

1 

^ 

.— -e 

' 

1 

'/ 

1/ 

A 

/ 

! 

J 

/ 

T 

/ 

1 

1 

/ 

\    1    i 

1    ! 

1        1        i 

1 

1 

5  ?0- 


10  ■  10-^ 


of  the  ATP.  In  an  attempt  to  specify  the  nature  of  this  primary  reaction  between  ATP 
and  actomyosin,  the  quantitative  relation  between  the  amount  of  xA.TP  added  and  the 
magnitude  of  the  physical  effect 
has  been  studied^^'  ^^.  Because  of* 
particular  experimental  dithcul-| 
ties,  the  results  have  not  yet  been^ 
satisfactory,  but  an  example  as 
that  of  Fig.  2  shows  that  one  has 
to  assume  the  formation  of  a  spa- 
ringly dissociated  compound  be- 
tween ATP  and  (acto)- myosin. 
Further  quantitative  researches 
are  in  progress.  The  relationships 
depend  on  whether  Mg+  or  Ca+  are 
present  and  the  best  result  in  the 
presence  of  the  promoting  Mg+ 
showed  that  i  mole  of  ATP  causes 
the  maximal  change  in  as  mxuch  as 
300000  grams  of  myosin.  The  dot- 
ted line  (Fig.  2)  represents  what 
would  be  expected  if  the  ATP- 
actomyosin  complex  would  be 
completely  undissociated;  the  de- 
viation of  this  from  the  actual  curve 
is  possibly  still  less  than  is  indi- 
cated by  the  results,  which  are 
obtained  by  difficult  measurements 
in  a  rapidly  changing  system.  The 
third  conclusion  reads  therefore: 
the  effect  of  ATP  upon  a  measured 

physical  property  of  actomyosin  is  due  to  the  formation  of  a  sparingly  dissociated 
ATP-(acto-)  myosin  complex.  One  is  led  to  a  similar  conclusion  by  studies  of  the  same 
combination  in  heterogeneous,  contractile  actomyosinsystems,  but  I  had  insufficient 
opportunity  to  study  this  in  full  detail.  In  solution,  the  measured  effect  was  maximal 
when  I  mole  of  ATP  was  present  for  300000  gram  myosin.  It  is  possible  that  upon 
addition  of  more  ATP,  more  is  bound  and  stronger  physical  changes  are  induced.  This 
cannot  be  measured  in  solution,  but  may  possibly  be  found  in  further  studies  with 
different  methods.  Not  more  can  thus  be  stated  than  that  300000  gram  myosin  combine 
with  at  least  i  mole  of  ATP,  or  roughly  that  100  mg  myosin,  present  in  one  gram  of 
muscle,  combine  with  3-  io"~'  mole  ATP  or  more. 

Naturally,  the  mere  demonstration  that  ATP,  when  interacting  with  actomyosin, 
actually  combines  with  it  (most  probably  with  the  myosin  component  only),  is  yet  no 
explanation  of  the  mechanism  of  its  action.  In  this  connection,  the  question  arises 
whether  ATP  is  only  bound  to  myosin,  or  whether  any  further  reaction  takes  place 
between  them.  More  precisely  the  question  may  be  asked  whether  myosin  is  phos- 
phorylated  by  ATP.  The  author  spent  a  summer  trying  to  demonstrate  such  a  phos- 
phorylation. Actomyosin  and  ATP  were  allowed  to  react  in  a  proper  medium,  and  were 
References  p.  56J5J. 


15-10-' 

Amciini  of  A  TF"  added 

Fig.  2.  Dissociation  curve  of  the  ATP-myosin  complex. 
The  effect  of  varying  quantities  of  ATP  upon  the  magni- 
tude of  the  viscosity  drop  (at  0° ;  extrapolated  to  zero- 
time)  of  actomyosin  was  studied.  System:  20  mg 
actomyosin  in  10  ml  0.5  molar  KCl,  0.02  molar  MgClj. 
Abscissa:  amounts  of  ATP  added  to  this  system. 
Ordinate:  viscosity  drop,  expressed  as  percentage  of 
the  effect  obtained  with  a  large  excess  of  ATP.  The 
dotted  line,  tentatively  drawn -as  representing  complete 
absence  of  dissociation,  indicates  that  the  maximal 
effect  is  reached  when  5.7-10-^  moles  ATP  combine 
with  20  mg  actomyosin,  corresponding  to  i  mol  ATP 
per  300000  g  myosin.  The  difference  between  the  dotted 
and  the  experimental  line  indicates  that  at  half-equi- 
librium the  concentration  of  free  ATP  is  much  less  than 
the  total  ATP  concentration  of  3-io— ®  moles  per  liter 
(see  text) 


54  W.  F.  H.  M.  MOMMAERTS  VOL.  4  (1950) 

then  separated  by  centrifugation.  Considerable  quantities  of  P  were  found  in  the  pre- 
cipitate. Eventually  it  was  found  out,  however,  that  the  apparent  phosphorylation  was 
proportional  to  the  amount  of  calcium  in  the  system,  and  what  appeared  to  be  a  phos- 
phorylation turned  out  to  be  nothing  else  than  a  coprecipitation  of  actomyosin,  Ca+  and 
inorganic  phosphate,  the  latter  being  formed  by  enzymatic  splitting  of  the  ATP*.  It  is 
true  that  without  Ca+  very  small  amounts  of  P  were  found  in  the  sediment,  but  those 
were  neglected  at  that  time. 

Meanwhile,  however,  Buchthal,  Deutsch  et  al.^  conducted  their  study  of  just  this 
small  effect.  They  find  amounts  of  about  or  above  15  fi  gram  P  per  100  mg  myosin 
(Professor  Buchthal  kindly  provided  me  with  additional  data  not  given  in  the  prelimi- 
nary paper),  which  would  correspond  to  5-10"''  mole  or  more  of  P  transferred  to  100  mg 
myosin  (i  gram  muscle).  This  is  the  same  order  of  magnitude  as  that  of  the  combination 
between  ATP  and  myosin.  Indeed,  Buchthal,  Deutsch  et  al.  also  measured  an  uptake 
of  nucleotide.  It  thus  seems  likely  that  the  primary  reaction  between  ATP  and  myosin 
does  not  remain  restricted  to  a  mere  combination,  but  is  followed  by  more  intricate 
interactions  as  well. 

In  spite  of  the  insulBcient  information  available,  some  further  quantitative  aspects 
of  the  (acto-) myosin- ATP  dissociation  curve  just  referred  to  may  be  discussed.  We 
indicate  the  molar  concentrations  of  the  myosin  (taking  the  relative  weight  of  the  unit 
combining  with  one  ATP),  the  complex, and  the  ATP  with  0^,  Cma  and  c^.  From  viscosity 
measurements,  as  described  above,  it  would  be  possible  to  determine  the  value  of  K, 
most  easily  by  measuring  the  c^  at  which  half  the  maximal  viscosity  response  is  obtained 
(for  Cma  =  ^u>  K  =  c^~^).  This  problem  is  now  under  investigation,  but  previously  no 
values  for  c^  have  been  obtained  due  to  experimental  difficulties.  Naturally,  only  the 
concentration  of  free  ATP  is  relevant  here;  Engelhardt^°  (page  i8g),  who  attempted 
to  calculate  an  equilibrium  constant  from  my  earlier  measurements^^  erroneously  took 
the  total  ATP  amount  present  in  the  system.  If  the  total  ATP  concentration  is  below 
io~^,  (see  Fig.  2)  c^  is  very  much  smaller,  perhaps  around  io~^.  Thus,  K  will  be  of  the 
order  of  10''  or  more,  and  the  value  of  RT  In  K  will  be  in  the  range  of  10  000  calories, 
a  very  considerable  free  energy  effect. 

There  is  an  independent  way  of  estimating  the  quantitative  relationships  between 
ATP  and  myosin  in  a  single  elementary  contractile  event.  As  is  well  known  (comp. 
Lundsgaard,  I.e.),  in  iodoacetate  poisoning,  where  the  muscle  uses  up  its  stores  of 
'^  P,  some  seventy  contractions  are  possible.  Such  a  muscle,  before  beginning  its  activity, 
contains  some  2.5-10"^  moles  of  . — -  P  per  gram,  counting  only  the  terminal  P  of  the 
ATP.  One  can  look  upon  every  twitch  as  one  elementary  event  involving  a  fraction  of 
this  --^^  P  in  the  form  of  ATP,  which  first  combines  with  myosin,  and  is  thereupon  decom- 
posed. For  simplicity  of  argument,  it  will  be  assumed  that  the  poisoned  muscle  performs 
some  50  full,  rather  than  70  decreasing  twitches.  Since  2.5-10"^  moles  z^-  P  enable  to 
50  full  twitches,  one  elementary  event  involves  the  reaction  of  5-io~'  moles  of  • — '  P 
with  the  contractile  structure,  followed  by  direct  or  indirect  degradation  into  inorganic 
phosphate.  Since  this  same  amount  of  muscle  contains  nearly  100  mg  myosin,  it  is  found 
that  in  every  complete  elementary  process  i  mole  of  '--'  P  reacts  with  200000  gram 
myosin.  This  value  is  so  close  to  the  proportion  of  i  ATP  to  1-3  hundred  thousands 
myosin  which  I  regularly  found  in  vitro  that  it  would  be  hard  to  consider  it  as  a  mere 
coincidence. 


*  The  critical  attitude  of  Dr.  Gerhard  Schmidt  is  gratefully  acknowledged. 
References  p.  56J57. 


VOL.  4  (1950)  PRIMARY   REACTION    IN    MUSCULAR   ACTIVITY  55 

It  is  still  difficult  to  judge  the  exact  physiological  meaning  of  the  described  reaction, 
but  it  is  of  obvious  interest  to  see  whether  a  theory  ascribing  to  it  the  significance  of  the 
primary  event  in  muscular  activity  would  meet  the  standards  set  by  Hill's  thermal 
measurements.  As  is  well  known,  a  single  anaerobic  twitch,  in  which  the  primary  event 
would  take  place  only  once,  is  accompanied  by  an  appearance  of  about  3-10"^  calories 
per  gram  muscle^*.  In  the  given  picture,  this  primary  event  would  involve  the  combi- 
nation of  3-10"'  or  more  moles  of  ATP  with  the  structure  protein.  Thus  the  heat  effect 
of  this  combination  per  mole  ATP  would  have  to  be  10  000  calories  or  less.  This  has  not 
yet  been  measured,  but  the  requirement  seems  to  be  quite  in  line  with  what  could  be 
expected.  It  seems  a  permissible  hypothesis  therefore  to  identify  the  primary  event  of 
contraction  with  a  combination  and  further  reaction  between  ATP  and  (acto-)  myosin. 

We  shall  now  turn  to  a  discussion  of  the  chemical  basis  of  relaxation,  and  will  have 
to  correlate  this  event,  by  exclusion,  with  the  enzymatic  breakdown  of  ATP  or  its 
myosincomplex.  In  connection  with  the  close  association  between  ATPase  and  myosin, 
the  current  assumption  is  that  it  is  the  myosin- ATPase  itself  which  hydrolyses  the  ATP, 
and  thus  makes  the  energy  of  this  reaction  available  to  the  contractile  structure.  After 
an  extensive  study  of  the  activity  of  myosin-ATPase  it  has  been  estimated^^  that  in 
muscle  the  overall  speed  of  hydrolysis  by  this  enzyme  can  amount  to  only  about  3  •  io~3 
mg  P  per  mg  myosin  per  minute.  The  actual  speed  of  ATP  breakdown  in  active  mam- 
malian muscle  is  much  higher.  From  Lundsgaard's^^  results  with  frog  muscles  the 
writer  estimated  the  speed  of  this  process  to  be  around  2-  io~^  mg  P  per  mg  myosin  per 
minute,  and  a  reinvestigation  of  all  relevant  data  ("Chapter  III)  gave  rise  to  the  same 
or  even  higher  values.  Likewise,  Braverman  and  Morgulis^  essentially  confirmed 
these  results  and  reported  the  same  disproportion.  To  reformulate  the  difficulty:  the 
actual  speed  of  breakdown  of  ATP  in  active  muscle  proceeds  a  hundred  times  faster  than 
the  myosin-ATPase  under  the  given  circumstances  can  account  for.  Several  explanations 
of  this  discrepancy  seem  possible.  Either,  intact  muscle  contains  unknown  potent 
activators  of  the  myosin-ATPase.  Or,  the  true  reaction  is  not  at  all  a  hydrolysis  of  ATP, 
but  a  phosphorus  transfer  to  some  acceptor ;  in  fact  there  are  indications  (Lundsgaard^^  ; 
Cori  and  Cori'')  that  a  P-transfer  of  ATP  to  fructose-6-phosphate  under  formation  of 
hexose-diphosphate  is  a  significant  reaction.  Further,  it  is  not  yet  possible  to  judge 
which  role  the  new  ATPase  described  by  Kielley  and  Meyerhof^^  has.  Several 
possibilities  for  a  solution  of  the  dilemma  thus  seem  to  exist,  and  the  identification  of 
the  exact  course  of  ATP  breakdown  may  throw  a  significant  light  upon  the  question  of 
relaxation.  At  this  moment  however,  no  suggestions  seem  to  be  indicated. 

The  above  considerations  have  been  developed  on  the  basis  of  in  vitro  experiments 
only,  and  the  task  remains  of  identifying  the  sequence  of  events  in  the  contraction  cycle 
of  a  living  muscle.  In  this  field,  we  owe  most  direct  and  illuminating  experiments  to 
Dubuisson^'  ^,  who  studied  the  rapid  pn  changes  which  accompany  a  contraction.  It 
was  found  that  first  an  acidification  occurs  which  in  favourable  specimens  was  preceded 
by  a  small  reaction  change  in  the  opposite  direction.  Then  there  is  an  alkalinization, 
followed  in  turn  again  by  an  acidification.  The  last  two  changes  could  be  identified 
convincingly:  they  are  due  to  the  dephosphorylation  of  phosphocreatine,  and  to  the 
formation  of  lactic  acid.  The  latter  process  takes  place  only  after  the  mechanical  events, 
the  former  is  coincident  with  the  relaxation.  The  initial  acidification  is  correlated  with 
the  initiation  of  the  contraction  process,  and  is  therefore  of  great  interest.  Dubuisson 
assumes  it  to  be  due  to  hydrolysis  of  ATP,  but  this  conclusion  is  tentative ;  acidification 
References  p.  sOJsy. 


56  W.  F.  H.  M.  MOMMAERTS  VOL.  4  (1950) 

might  likewise  be  caused  by  the  binding  of  ATP  by  myosin  followed  by  phosphorylation 
of  the  latter.  On  this  point  therefore,  no  decision  seems  possible  as  yet. 

With  respect  to  the  moment  at  which  the  energy  of  metabolism  is  made  available 
to  the  contractile  apparatus,  it  is  now  customary  (see-'')  to  distinguish  two  possible 
mechanisms.  In  the  first  of  these,  chemical  energy  may  be  transferred  at  the  very  mo- 
ment of  contraction,  when  it  is  necessary.  The  alternative  possibility  is  that  the  primary 
event  merely  releases,  by  a  trigger  action,  a  spontaneous  contractile  process  (often 
paralleled  with  the  shortening  of  stretched  rubber),  and  that  it  is  the  event  of  relaxation 
which  is  linked  with  exergonic  m.etabolic  reactions  in  order  to  restore  the  active  state. 
The  latter  category,  the  so  called  postenergization  mechanisms,  seems  difficult  to  recon- 
cile with  the  results  of  Fenn  and  Hill^^,  is  indicating  rather  the  existence  of  contraction- 
coupling.  Nevertheless,  postenergization  hypotheses  are  rather  in  demand  at  present, 
and  the  opinion  seems  to  prevail  that  Szent-Gyorgyi's  work  may  lead  to  this  type  of 
inter  probation,  a  viewpoint  taken,  e.g.,  in  the  speculations  of  Morales^^.  As  the  present 
communication  shows,  the  analysis  of  the  effects  discovered  by  Szent-Gyorgyi  gives, 
on  the  contrary,  rise  to  a  preenergizationtheory. 

It  has  been  the  purpose  of  this  discussion  to  show  where  the  actual  experimental 
analysis  of  the  contractile  event,  in  terms  of  ATP-actomyosin  interaction,  at  present 
stands.  No  detailed  theory  seems  warranted,  or,  as  Meyerhof  said  in  1930^^  (p.  280) : 
"Es  soil  daher  hier  weniger  eine  bestimmte  Theorie  ausgearbeitet  werden  als  die  fest- 
gestellten  Tatsachen  und  die  sich  daraus  ergebenden  mehr  oder  weniger  wahrschein- 
lichen  Folgerungen  zusammengefasst  sowie  Missverstandnisse  gegeniiber  der  Auslegung 
dieses  Tatbestandes  beseitigt  werden".  But  neither  should  the  impression  prevail 
that  "...  (man)  auch  heute  eigentlich  noch  gar  nichts  weiss". 


REFERENCES 

1  T.  Braverman  and  S.  Morgulis,  /.  Gen.  Physiol.,  31  (1948)  411. 

2  F.  BucHTHAL,  A.  Deutsch,  and  G.  G.  Knappeis,  Acta  Physiol.  Scand.,  8  (1944)  271. 

^  F.  BucHTHAL,  A.  Deutsch,  G.  G.  Knappeis  and  A.  Munch-Petersen,  Acta  Physiol.  Scand.,  13 
(1947)  167. 

*  F.  BucHTHAL,  A.  Deutsch,  G.  G.  Knappeis,  and  A.  Munch-Petersen,  Nature,  162  (1948)  965. 
'  F.  BucHTHAL  and  G.  Kahlson,  Acta  Physiol.  Scand.,  8  (1944)  317. 

^  F.  BucHTHAL  and  G.  Kahlson,  Acta  Physiol.  Scand.,  11  (1946)  284. 

■^  G.  T.  CoRi  and  C.  F.  Cori,  /.  Biol.  Chem.,  126  (1936)  119. 

^  M.  Dubuisson,  /.  Physiol.,  90  (1937)  6. 

®  M.  Dubuisson,  Experientia,  3  (1947)  213. 

"  V.  A.  Engelhardt,  Advances  in  Enzymol.,  6  (1926)  147. 

^  T.  Erdos,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  3  (1943)  57- 

2  W.  O.  Fenn,  /.  Physiol.  58  (1923)  175. 

*  M.  Gerendas,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  i  (1942)  47. 

*  A.  V.  Hill,  Muscular  Activity,  Williams  and  Wilkins  Comp.,  1926. 
5  A.  V.  Hill,  Proc.  Roy.  Soc,  B  126  (1938)  136. 

W.  W.  KiELLEY  AND  O.  Meyerhof,  /.  Biol.  Chem.,  176  (1948)  591. 

K.  Lohmann,  Biocheni..  J.,  271  (1934)  264. 

E.  Lundsgaard,  Biochem.  J.,  227  (1930)  51. 

O.  Meyerhof,  Die  chemischen  Vorgange  im  Muskcl,  Springer,  Berlin,  1930. 
2"  O.  Meyerhof,  Ann.  N.  Y.  Acad.  Set.,  45  (1944)  377. 
2^  O.  Meyerhof,  Arch.  Biochem.,  15  (1947)  166. 

22  W.  F.  H.  M.  Mommaerts,  Studies  Inst.  Med.  Chem.  Szeged,  1  (1942)  37. 
-3  W.  F.  H.  M.  Mommaerts,  Arkiv  Kemi,  Mineral.  Geol.,  19  A  (1945)  No.  17. 
2*  W.  F.  H.  M.  Mommaerts,  Arkiv  Kemi,  Mineral.  Geol.,  19  A  (1945)  No.  18. 
25  W.  F.  H.  M.  Mommaerts,  /.  Gen.  Physiol.,  31  (1948)  361. 
2'  W.  F.  H.  M.  Mommaerts  and  K.  Seraidarian,  /.  Gen.  Physiol.,  30  (1947)  201. 


VOL.  4  (1950)  PRIMARY    REACTION    IN    MUSCULAR   ACTIVITY  57 

^^  W.  F.  H.  M.  MoMMAERTS,  Mitsculur  Activity,  Interscience  Inc.,  New  York,  1949. 

-^  M.  F.  Morales,  Biochini.  Biophys.  Acta,  2  (194S)  618. 

-*  S.  V.  Perry,  R.  Reed,  W.  F.  Astbury,  and  L.  C.  Spark,  Biochim.  Biophys.  Acta,  2  (1948)  874. 

^°  Pons  and  O.  Meyerhof,  /.  Biol.  Chem.,  169  (1927)  389. 

31  R.  W.  Ramsey  and  S.  F.  Street,  /.  Cellular  Comp.  Physiol.,  25  (1940)  n. 

*2  F.  Rozsa,  Hung.  Acta  Physiol.,  i  (1946)  16. 

^■^  F.  B.  Straub,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  2  (1942)  3. 

■'*  F.  B.  Straub,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  3  (1943)  23. 

■'^  A.  Szent-Gyorgyi,  Studies  Inst.  Med.   Chem.   Univ.  Szeged,  vols.   1-3   (containing  38  separate 

papers),  S.  Karger,  Basle  (1942-1943). 
•'^  A.  Szent-Gyorgyi,  Acta  Physiol.  Scand.,  9  (1945)  suppl.  25. 

•"  A.  Szent-Gyorgyi,  Chemistry  of  Muscular  Contraction,  Academic  Press,  New  York,  1947. 
^^  A.  Szent-Gyorgyi,  Nature  of  Life,  Academic  Press,  New  York,  1948. 

Received  March  loth,  1949 


58  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


SOME  FACTORS  INFLUENCING  THE  CONTRACTILITY  OF  A 
NON-CONDUCTING  FIBER  PREPARATION* 

by 

SAUL  KOREY** 

Department  of  Neurology,  College  of  Physicians  and  Surgeons, 
Columbia  University  New  York,  N.Y.  (U.S.A.) 

INTRODUCTION 

One  of  the  most  important  contributions  of  Otto  Meyerhof  was  the  discovery 
of  the  high  energy  which  may  be  contained  in  phosphorylated  compounds.  Following 
the  description  of  phosphocreatine  (phosphagen)  by  Fiske  and  Subbarow^  and  Eggle- 
TON  AND  Eggleton^,  Meyerhof  found,  in  1927,  that  the  enzymatic  decomposition  of 
this  compound  is  connected  with  the  liberation  of  a  large  amount  of  heat^.  The  energy 
released  is  about  10  000  to  12000  g  calories  as  compared  with  2000  to  3000  of  other 
phosphorylated  compounds,  e.g.,  hexose  mono-  and  diphosphate,  pentose  and  triose 
phosphate  and  other  esters,  i.e.,  all  those  compounds  where  the  phosphate  is  linked  to 
an  alcoholic  hydroxyl.  Meyerhof  found  a  similar  high  energy  in  argininephosphate 
which  in  many  invertebrates  takes  the  place  of  creatinephosphate*.  A  few  years  later, 
when  in  his  laboratory,  Lohmann  had  isolated  adenosinetriphosphate  (ATP)  from 
muscle,  Meyerhof^  showed  that  about  24000  g  calories  are  released  by  the  breakdown 
of  ATP  to  adenosinemonophosphate  (AMP).  This  is  about  the  same  amount  of  energy 
for  each  of  the  two  P  as  that  derived  from  the  P  of  phosphocreatine.  Soon  afterwards, 
two  more  phosphorylated  compounds,  intermediates  in  glycolysis,  were  found  to  be  rich 
in  energy:  phosphoenol  pyruvic  acid^  and  1.3-diphosphoglyceric  acid,  isolated  by  Nege- 
LEIN  AND  Bromel  in  Warburg's  laboratory''.  The  great  significance  of  Meyerhof's 
discoveries  of  energy-rich  phosphates  for  the  understanding  of  intermediate  metabolism 
and  the  far  reaching  implications  have  been  reviewed  in  this  country  by  Lipmann^ 
and  Kalckar^. 

Among  all  the  energy  rich  phosphorylated  compounds,  ATP  plays  a  special  role. 
Originally  the  study  of  this  compound  was  limited  to  the  glycolytic  cycle.  More  recent 
studies,  however,  have  shown  that  ATP  has  a  more  general  importance,  as  the  source  of 
energy  in  intermediate  cellular  reactions,  as  e.g.,  acetylation  (Nachmansohn^*^),  urea  for- 
mation (RatnerII)  and  many  others.  Although  the  essential  role  of  ATP  in  intermediate 
metabolism  becomes  continuously  more  evident,  its  function  in  the  muscle  cell  in  which 
it  was  first  discovered  and  studied  is  still  one  of  the  most  challenging  problems  to  biolo- 
gists. From  the  work  of  Meyerhof  and  his  associates,  it  appeared  likely  that  ATP  was 
involved  in  the  primary  changes  of  the  protein  during  muscular  contraction.  No  other 


*  This  investigation  was  supported  by  a  research  grant  from  the  Division  of  Research  Grants 
and  Fellowships  of  the  National  Institutes  of  Health,  U.S.  Public  Health  Service. 
**  Senior  Fellow  in  Neurology,  U.  S.  Public  Health  Service. 

References  p.  6y. 


VOL.  4  (1950)  CONTRACTILITY   OF   A   NON-CONDUCTING   FIBER  59 

chemical  reaction  is  known  to  be  more  closely  associated  with  the  contractile  mechanism. 
A  new  development  was  initiated  in  1939  by  Engelhardt  and  Lyubimova^^'  ^^  when 
they  tested  this  idea  by  studying  the  interaction  between  ATP  and  myosin,  which  at 
that  time,  was  the  main  protein  considered  to  be  associated  with  contraction.  Under 
the  stimulus  of  their  observations,  the  reaction  between  ATP  and  the  muscle  proteins  has 
been  extensively  studied  and  considerable  progress  has  been  achieved  essentially  by 
the  work  of  the  Needhams  and  Szent-Gyorgyi  and  their  associates^*'  ^^'  ^^.  The 
demonstration  by  Straub  of  a  second  protein,  actin,  which  combines  with  myosin  to 
actomyosin,  was  an  important  advance  in  the  study  of  the  primary  reactions  which 
may  underly  the  contractile  process^'^. 

However,  if  the  interaction  of  ATP  and  actomyosin  is  studied  in  solution,  the  ele- 
ment of  organization  of  the  protein  is  not  included.  Recently,  Szent-Gyorgyi*  has 
described  a  muscle  fiber  preparation  which  contracts  in  the  presence  of  ATP.  The  usual 
electric  stimulus  is  ineffective.  This  indicates  that  the  conductive  membrane  is  inactive. 
In  a  normal  muscle  fiber,  whether  stimulated  directly  or  indirectly,  the  activation  of 
the  conductive  membrane  which  envelopes  the  muscle  cell  intervenes  between  stimulus 
and  contraction.  It  is  only  through  the  activity  of  thi^  membrane  that  the  contractile 
process  is  initiated.  Since  in  the  preparation  of  Szent-Gyorgyi  the  conductive  mecha- 
nism is  excluded  but  the  contractile  units  are  still  functioning,  as  demonstrated  by 
the  ATP  induced  contraction,  this  fiber  offers  a  most  suitable  material  for  the  study  of 
factors  influencing  contraction  independent  of  the  action  of  the  conductive  membrane. 
Such  a  differentiation  is  of  considerable  interest  for  the  understanding  of  some  muscular 
disorders,  especially  myotonia  and  familial  periodic  paralysis.  It  is  with  this  problem 
in  mind  that  the  present  study  has  been  initiated. 


material;' AND    METHODS 

The  psoas  major  of  a  rabbit  was  isolated  by  dissection  and  then  tied  at  either  end  to  an  appli- 
cator stick.  This  preserved  the  resting  length.  The  muscle  was  removed  in  toto  by  severing  its  connec- 
tion at  origin  and  insertion.  It  was  placed  in  50%  glycerol,  kept  in  the  icebox  overnight  and  then 
stored  in  50%  glycerol  at — 10°  C.  The  fibers  of  the  psoas  muscle  of  the  rabbit  pass  throughout  the 
length  of  the  muscle  in  a  parallel  fashion. For  the  present  study  this  muscle  appeared  suitable  but 
other  striated  muscles  may  be  used  in  a  similar  way. 

The  main  features  of  the  glycerol  preserved  fibers  are :  the  ease  with  which  a  small  numbei  of  fibers 
(about  3-10)  can  be  stripped  from  the  main  bulk  of  the  muscle;  the  retention  of  the  structural  organi- 
zation of  the  fibrils;  the  modification  of  the  cell  membrane  to  an  unexcitable  state;  and  finally  the 
fiber's  ability  to  contract  on  the  addition  of  ATP. 

By  grasping  the  desired  amount  of  muscle  fibers  in  a  forceps,  they  can  be  peeled  from  the  muscle 
belly  by  exerting  a  slight  tension.  Forceful  pulling  on  the  fibers  being  detached  causes  partial  inter- 
ruptions in  their  continuity  which  can  be  noted  by  holding  the  fibers  to  the  light  and  observing  regions 
of  increased  transparency.  Fiber  groups  0.5  to  i  mm  in  diameter  were  separated  from  the  muscle 
for  study. 

The  microscopic  appearance  of  the  unstained  preserved  fibers  was  similar  to  the  normal  un- 
treated fibers  from  the  same  animal.  However,  the  volume  of  sarcoplasm  was  diminished  and  the 
diameters  of  the  fibers  were  decreased  appreciably. 

The  contractions  of  the  unloaded  fibers  were  studied  in  various  experiments.  After  a  number 
of  preliminary  observations,  the  experiments  were  carried  out  in  the  following  way.  The  fibers  were 
suspended  in  a  constant  volume  of  mammalian  Ringer's  solution  according  to  Krebs.  The  con- 
tractions were  recorded  by  an  isotonic  system  on  a  kymograph  moving  at  3  cm  per  minute.  The 
suspended  fibers  were  kept  in  a  bath  of  constant  temperature  which  could  be  varied  according  to 


*  I  am  greatly  indebted  to  Professor  Szent-Gyorgyi  for  the  demonstration  of  this  preparation 
which  made  this  study  possible. 

References  p.  6y. 


60  S.  KOREV  VOL.  4  (1950) 

the  requirements  of  the  experiment.  The  standard  ATP  solution  or  others  tested  were  added  at  a 
rapid  and  fairly  constant  rate  reaching  the  suspended  fiber  almost  instantaneously. 

Electrical  stimulation  applied  directly  to  the  fibers  did  not  cause  contraction.  The  fibers  were 
inert  to  supramaximal  single  and  tetanic  shocks.  On  the  addition  of  ATP  to  the  environment  of  the 
fibers,  a  definite  and  easily  recorded  contraction  developed.  As  the  fibers  shortened,  their  diameters 
increased.  In  this  respect  the  contractions  resembled  isotonic  contractions  of  normal  muscle.  However, 
the  fibers  did  not  readily  relax  following  the  contraction  induced  by  ATP.  It  was,  therefore,  necessary 
to  use  new  fibers  for  each  determination.  The  stability  and  constancy  of  fiber  groups  became  all  the 
more  important  for  this  reason.  During  the  first  two  weeks  of  preservation  the  libers  were  found  to 
be  unstable  and  variable.  On  exposure  to  isotonic  solutions  of  salts,  e.g.,  contained  in  mammalian 
Ringer's  or  saline,  pseudo-contractile  movements  were  occasionally  observed.  After  the  second  week 
of  preservation,  more  dependence  could  be  placed  on  the  stability  of  the  fibers. 

The  ATPase  activity  of  the  homogenates  of  the  fibers  was  determined  by  the  method  described 
by  Du  Bois  AND  PotterI^. 

RliSULTS 

Addition  of  ATP 

Of  the  compounds  tested,  ATP  and  ADP  alone  elicited  contraction  of  the  libers*. 
A  particularly  significant  group  of  substances  are  listed  in  Table  I.  The  fiber  apparently 

TABLE  I 

COMPOUNDS     TESTED     TO     INDUCE     CONTRACTION     OF     UNLOADED     NON-CONDUCTIVE     MUSCLE     FIBERS 
(rabbit),    the    ATP    AND    ADP   FIGURES    INDICATE    THE    LOWEST    CONCENTRATION    WITH    WHICH    CON- 
TRACTION   WAS    OBSERVED.    THE    FIGURES    OF    THE    OTHER    COMPOUNDS    INDICATE    THE    HIGHEST    CON- 
CENTRATION   TESTED 


Compound 

Concentration  (mg/ml) 

Contraction 

Adenosinetriphosphate        (ATP) 
Adenosinediphosphate         (ADP) 
.\denosinemonophosphate  (AMP) 
Inorganic  Pyrophosphate 
Acetylcholine 
Adrenaline 

0.04 

0-5 

100. 0 

44.0 

2.0 

2.0 

+ 
+ 
0 
0 
0 
0 

reacted  in  a  selective  manner  to  ATP  and  ADP.  The  threshold  concentration  of  ATP 
requisite  for  contraction  was  less  than  that  of  ADP.  Moreover,  with  equimolar  solutions 
of  ATP  and  ADP,  the  degree  of  shortening  was  greater  in  the  case  of  the  former.  The 
amount  of  shortening  of  fibers  was  found  to  depend  on  the  concentration  of  the  ATP 
solution  employed,  approaching  a  maximum  asymptotically  (Figs  i,  2). 

Unlike  myosin  threads,  the  loaded  fibers  contracted  rather  than  extended  in  the 
presence  of  ATP.  Moreover,  if  the  fibers  were  incapable  of  shortening  because  the  load 
was  excessive,  extension  did  not  occur  on  the  addition  of  ATP. 

Effect  of  temperature 

When  the  suspended  fibers  and  the  added  solutions  of  standard  ATP  were  main- 
tained at  37°  C,  the  extent  of  shortening  was  5.4  times  as  great  as  observed  under  similar 
conditions  at  10°  C  (Fig.  3).  Calculated  on  this  basis  there  was  an  increase  in  the  amount 
of  contraction  by  a  factor  of  1.9  for  each  lo*^  C  rise  in  temperature  between  10°  and  37°  C. 


*  I  am  greatly  obliged  to  Dr  Harry  G.  Albaum,  Brooklyn  College,  for  supplying  adenosine- 
diphosphate  and  adenosinemonophosphate.  The  ADP  was  tested  enzymatically  b}"  Dr  Albaum  and 
found  by  his  method  to  be  free  of  ATP. 

References  p.  6y. 


VOL.  4  (1950) 


CONTRACTILITY    OF    A    NON-CONDUCTING    FIBER 


61 


Fig.  I.  The  series  of  tracings  represent  the  ATP  induced  isotonic  contraction  of  fibers  8.5  cm  in  length 

recorded  on  a  kymograph  moving  3.0  cm  per  minute.  Concentrations  of  ATP  decreasing  from  0.04  M 

in  the  first  to  o.ooi  M  in  the  last  tracing.  Magnification  6  X  . 


g>75 


10 


0        0.05       .01  .02  .03  Oh 

Molarity  ATP 

Fig.  2.  Degree  of  shortening  of  fibers  suspended 
in  an  isotonic  recording  system  as  function  of 
increasing  concentrations  of  ATP.  Ordinates: 
Degree  of  shortening  at  a  given  time  in  arbi- 
trary units.  Abscissae:  M  ATP  concentration. 
All  fibers  were  of  equal  length  (8.5  cm). 


Fig.  3.  Effect  of  temperature  on  the  ATP 

induced  isotonic  contraction.  Lower  curve 

contraction  at  10°  C,  upper  curve  at  37°  C. 

ATP  concentration  0.002  M. 


Effect  of  pH 

The  present  experiments  were  carried  out  at  p^  7.4-7.6.  It  was  observed  that  the 
fibers  deteriorated  rapidly  in  solutions  beyond  the  limits  of  p^  6.8  and  7.8. 

Effect  of  ions 

Sodium  ATP  caused  contraction  of  the  libers  in  the  absence  of  other  ions.  However, 
magnesium  ion  activated  the  reaction  of  ATP  with  the  contractile  proteins  of  the  fibers 
as  shown  by  the  increased  extent  of  shortening  in  equimolar  solutions  of  ATP  (Fig.  4). 
The  optimal  concentration  of  magnesium  ion  was  i  •  io~^  M.  Potassium  in  similar 
concentrations  did  not  manifest  the  activating  effect  of  magnesium.  In  the  presence  of 
References  p.  6y. 


62 


S.  KOREY 


VOL.  4  (1950) 


^00 


t  150 


100 


calcium  ion  at  i  •  io~2  M  there  was  precipita- 
tion of  the  nucleotide  and  therefore  the  effect 
cannot  be  evaluated.  On  the  basis  of  these 
observations  a  solution  of  ATP  in  i  •  lO"^  M 
MgClg  was  used  as  standard  to  produce  con- 
traction. 

A  TPase  activity 

The  role  of  ATPase  in  the  interaction 
between  muscle  protein  and  ATP  has  been 
repeatedly  investigated.  It  is  still  a  matter 
of  discussion^^'  ^^  at  which  phase  of  muscle 
activity  the  enzyme  is  required.  It  appeared 
therefore  of  great  interest  to  determine  to 
what  extent  the  ATPase  activity  is  preserved 
in  the  preparation  used.  Table  II  shows  the 
rate  of  decrease  of  the  enzyme  activity.  The 
determinations  revealed  a  gradual  decline  of 

activity  to  about  20%  of  the  initial  value,  at  which  level  the  activity  appeared  to  remain 

stable. 

On  the  addition  of  ATP  enzymatically  inactive  fibers  when  loaded  remained  at 

resting  length  and  no  extension  was  noted. 

TABLE  II 
ATPase   activity   of   muscle   fibers   of   rabbit 
preserved  in  glycerol  at  io°  c,  tested  at37°c 


Mg(-logM) 

Fig.  4.  The  effect  of  magnesium  ion  in 
varying  concentrations  on  the  extent  of 
isotonic  shortening  of  fibers  exposed  to 
0.002  M  sodium  ATP.  The  isotonic  short- 
ening caused  by  sodium  ATP  alone  is 
arbitrarily  assigned  as  100%. 


Time  of  preservation 
(days) 

/xg  P/mg/15  min 

0  (fresh 

25-30 

4 

15-17 

16 

10-12 

20 

6-8 

23 

6-8 

30 

6-8 

Inhibitors  of  contraction 

Since  it  is  known  that  ATPase  has  -SH  groups^^,  the  effect  of  -SH  inhibitors  were 
studied  to  find  reversible  inhibitors  of  the  contractile  process.  Fiber  bundles  of  a  dia- 
meter of  0.5  mm  or  less  were  soaked  in  solutions  of  various  compounds  and  then  immer- 
sed in  I  •  io~2  M  ATP  standard.  In  suitable  cases,  the  fibers  after  soaking  were  set  up 
in  the  isotonic  system  and  quantitative  measurements  made. 

It  was  apparent  that  compounds  which  combined  with  sulfhydryl  groups  effectively 
inhibited  contraction  of  the  fibers  (Table  III).  Of  these  compounds  sodium  o-iodoso- 
benzoate  and  mapharsen  (we/a-amino-/)flra-hydroxyphenylarsinoxide)  proved  to  be 
reversible  inhibitors.  The  inhibitory  effect  of  mapharsen  was  reversed  by  washing  the 
fibers  in  saline  whereas  addition  of  cysteine  to  saline  was  required  to  remove  the  inhibi- 
tion produced  by  o-iodosobenzoate.  HgClg  in  i  •  io~*  M  concentration  caused  irreversible 
References  p.  6y. 


VOL.  4  (1950) 


CONTRACTILITY   OF   A   NON-CONDUCTING   FIBER 


63 


TABLE  III 

INHIBITION  OF  ATP  INDUCED  CONTRACTION  IN  NON-CONDUCTING  MUSCLE  FIBERS  (rABBIT)  BY  SOME 
COMPOUNDS  REACTING  WITH  -SH  GROUPS.  AFTER  EXPOSURE  THE  FIBERS  WERE  SOAKED  IN  SALINE 
CONTAINING  O.OI   M  CYSTEINE  EXCEPT  IN  THE  CASE  OF  MAPHARSEN  IN  WHICH  SALINE  ALONE  PROVED 

TO   BE    EFFECTIVE 


Compound 

Concentration 
(M) 

Exposure 
(min) 

Time  of  washing 
(min) 

Reversibility 

o-Iodosobenzoate 
o-;Iodosobenzoate 
Mapharsen 
Mercuric  chloride 
Mercuric  chloride 

I- 10-3 
5-IO-* 

I-IO-* 

I- 10-^ 
I • 10-3 
3"  10-^ 

20-25 

30-40 

20 

20 

9 

7 

120 
90 
10 

>  120 

>  120 

>  120 

+ 
+ 
+ 

inhibition  of  contraction.  At  a  concentration  of  i-io"^  M,  however,  the  inhibitory 
effect  of  this  compound  appeared  negHbible. 

Other  compounds  tested  and  found  without  an  inhibitory  effect  on  the  contractile 
process  were  sodium  monoiodoacetate  i-io"^  M,  sodium  pyrophosphate  4.4- lo"^  M, 
sodium  arsenate  3-io~^  M,  sodium  arsenite  i-io"^  M  and  antimony,  tartrate  and 
chloride,  8- 10-2  M. 

Since  o-iodosobenzoate  is  a  reversible  inhibitor  of  contraction,  the  following  ex- 
periments were  carried  out.  Thirty  fiber  units  6-8  cm  in  length  were  placed  in  a  solution 
of  o-iodosobenzoate  5  •  io~*  M  in  saline.  At  various  intervals  2  cm  were  cut  from  seme 
of  these  fibers,  and  the  sections  tested  for  contractility  in  a  standard  ATP  solution 
I-  io~2  M.  After  40  minutes,  none  of  the  parts  of  the  fibers  so  tested  contracted  on  ex- 
posure to  the  ATP  standard.  The  fibers  were  then  removed  from  the  inhibiting  solution. 
Ten  were  placed  in  saline  containing  cysteine  in  i  •  lO"^  M,  the  remainder  in  ATP 
i-io~2  M  for  either  2  or  10  minutes.  The  experimental  groups  which  did  not  contract 
during  exposure  to  ATP  were  removed  from  the  ATP  and  washed  in  saline  for  10  minutes 
and  then  soaked  in  saline  with  i  •  lO"^  M  cysteine  for  12  hours.  The  fibers  placed  directh^ 
in  the  cysteine  saline  solution  were  tested  by  removing  a  unit  and  exposing  it  to  ATP 

1  •  io~2  M.  Contractility  had  returned  in  go  minutes.  The  fibers  which  were  soaked  either 

2  or  10  minutes  in  ATP  prior  to  their  transfer  into  the  cysteine  saline  were  tested  for 
return  of  contractility  in  a  manner  similar  to  the  former  group.  During  the  12  hours 
of  observation,  measurable  shortening  responses  did  not  appear. 

When  fibers  soaked  in  o-iodosobenzoate  i  •  lO"^  M  ceased  to  contract,  they  were 
washed  in  sahne  for  10  minutes  and  homogenized.  At  that  period,  the  ATPase  activity 
of  their  homogenates  ranged  from  3-5  //g  P/mg/15  min.  A  part  of  the  saline  washed 
fibers  were  then  regenerated  in  a  solution  of  cysteine  i  •  lO""^  M.  At  the  earliest  moment 
when  contractility  returned,  the  homogenate  revealed  an  ATPase  activity  of  5.5-8 
iWg  P/mg/15  min. 

Fibers  preserved  in  glycerol  and  then  soaked  in  cold  saline  for  10  days  retained  their 
ability  to  contract  when  their  ATPase  activity  was  6  //g  P/mg/15  min  or  above. 
Below  this  level  contraction  was  absent. 

Effect  of  biologically  active  compounds 

Fibers  were  exposed  for  30  to  60  minutes  to  a  number  of  substances  known  to 
have  an  effect  on  the  contraction  of  normal  muscle.  In  Table  IV  are  listed  the  compounds 

References  p.  6y. 


64 


S.  KOREY 


VOL.  4  (1950) 


and  the  concentrations  used.  The  degree  of  shortening  on  addition  of  standard  ATP  was 
compared  with  control  fibers.  To  determine  the  possibiHty  of  simultaneous  activation 
of  the  contractile  process,  test  solutions  of  adrenaline,  acetylcholine  and  histamine  were 
prepared  in  ATP  standard.  These  were  added  to  fibers  which  had  been  previously 
soaked  in  corresponding  solutions  without  ATP.  None  of  the  compounds  enumerated 
affected  the  ATP  induced  contraction,  whether  the  fibers  were  exposed  to  them  prior 
to  the  contact  with  ATP  or  simultaneously. 

TABLE  IV 

COMPOUNDS    WHICH     H.^D     NO     EFFECT    IN     THE      CONTRACTIONS 

INDICATED    ON    THE    NON-CONDUCTING    MUSCLE    FIBER    (RABBIT) 

NOR     CHANGED     THE     ATP     INDUCED      ISOTONIC     CONTRACTION. 

TIME    OF    EXPOSURE :    30-60    MIN    24°  C 


Compound 

Concentration 
mg/mm 

Compound 

Concentration 
mgm/mm 

Adrenaline 

0.002-0.09 

Strychnine 

0-5 

Acetylcholine 

i.o     -2.0 

Veratrine 

0.5 

E  serine 

0.05  -2.0 

Rvanodin 

1.0 

Prostigmine 

0.5     -1.0 

Digitoxin 

0.2 

Caffeine 

0.5 

Histamine 

1.0 

DFP 

1.0 

Quinine 

0.4-0.6 

Cocaine 

1.0 

DISCUSSION 

The  Szent-Gyorgyi  preparation  may  be  considered  a  prototype  of  the  contractile 
elements  of  normal  muscle.  For  the  study  of  contraction,  it  is  intermediate  between 
the  intact  cell  and  isolated  systems  (and  proteins)  in  solution.  Since  the  structure  of 
the  preserved  iibers  appears  similar  to  the  normal,  they  probably  retain  a  considerable 
degree  of  the  orientation  and  organization  of  the  contractile  proteins  originally  present. 
Partly  for  this  reason,  contraction  rather  than  extension,  as  seen  in  the  randomly 
constituted  myosin  threads,  occurs  after  the  addition  of  ATP  to  the  loaded  iibers.  Also 
the  supportive  action  of  the  sarcolemma  mechanically  prevents  separation  of  the 
fibrils'  contractile  units  while  they  are  undergoing  spatial  rearrangement  associated 
with  the  process  of  contraction. 

ATP  and  ADP  but  not  adenylic  acid  cause  contraction  of  the  fibers.  Quantitative 
relationships  between  concentrations  of  ATP  and  ADP  and  the  degree  of  shortening  of 
the  fibers  require  further  investigations.  It  is  apparent,  however,  that  ATP  is  at  least 
10  times  more  effective  in  causing  shortening  than  an  equivalent  amount  of  ADP. 
Since  no  enzyme  is  known  to  exist  in  muscle  which  splits  ADP,  the  effect  obtained  with 
ADP  may  appear  surprising.  In  previous  observations  reported  ADP  preparations  were 
not  entirely  free  of  significant  amounts  of  ATP  and  the  action  of  such  preparations  could 
be  attributed  to  ATP.  The  preparation  of  ADP  used  in  these  experiments  was  free  of 
ATP,  as  tested  enzymatically.  However,  it  is  possible  that  the  ADP  was  converted  by 
myokinase  to  ATP  prior  to  its  action.  The  fibers  have  not  been  examined  for  the  pre- 
sence of  this  enzyme. 

Under  the  conditions  of  the  present  experiments,  contraction  of  the  fibers  was  not 
followed  by  comparable  relaxation  despite  washings  in  solution  containing  NaCl,  KCl, 
References  p.  6y. 


VOL.  4  (1950)  CONTRACTILITY   OF   A   NON-CONDUCTING   FIBER  65 

CaClg  or  MgClg  in  various  concentrations.  Fibers  which  contracted  as  little  as  20%  of 
initial  length  were  not  restored  to  their  original  length.  Relaxation  may  be  a  more 
complicated  process  than  contraction  depending  on  the  integration  of  several  reactions 
performed  poorly,  if  at  all,  in  this  preparation.  That  ATP  induces  contraction  and  not 
relaxation  of  the  fibers  does  not  indicate  at  which  phase  of  contraction  dephosphoryla- 
tion  of  ATP  occurs^^. 

It  has  been  observed  that  fibers  inhibited  from  contraction  by  o-iodosobenzoate 
and  then  exposed  for  2  minutes  to  ATP  did  not  regain  their  contractility  after  prolonged 
washing  in  cysteine  saline.  This  may  indicate  a  reaction  of  ATP  with  proteins  of  the 
fiber  possibly  independent  of  that  initiating  contraction.  This  observation  may  offer 
an  explanation  for  the  inability  of  the  fibers  to  relax,  since  in  the  usual  experiments 
performed  to  measure  isotonic  contraction,  the  fibers  were  exposed  to  ATP  for  Deriods 
longer  than  2  minutes. 

It  is  noteworthy  that  the  contraction  of  the  fibers  produced  by  ATP  is  enhanced 
by  the  addition  of  magnesium  ions.  This  effect  finds  its  analogy  in  the  action  of  this 
ion  in  increasing  the  adsorption  of  ATP  by  actomyosin^^.  The  magnitude  of  the  effect 
and  the  optimal  concentration  of  magnesium  ion  at  which  it  occurs  are  in  harmony 
with  similar  observations  in  isolated  ATP-actomyosin  systems. 

Activation  of  the  fiber  contraction  by  magnesium  contrasts  to  its  depressing  effect 
on  the  intact  muscle^".  Further  observations  are  necessary  to  decide  whether  this  may 
indicate  that  the  magnesium  effect  in  the  intact  fiber  is  due  to  an  action  on  the  conduc- 
tive membrane. 

Compounds  like  mapharsen  and  o-iodosobenzoate  which  reversibly  inhibited  contrac- 
tion of  the  fibers  inactivate  ATPase  activity  of  myosin^i.  The  inhibitors  are  not  specific 
for  ATPase  but  rather  oxidize  or  combine  with  thiol  groups  in  general.  By  measuring 
the  ATPase  activity  of  the  homogenates  of  the  fibers,  one  may  secure  an  index  of  their 
efficacy  in  affecting  available  -SH  groupings.  However,  the  inactivation  of  ATPase  may 
not  be  directly  correlated  with  the  ability  of  these  inhibitors  to  prevent  contraction. 
The  sulfhydryl  groups  binding  actin  to  myosin,  e.g.,  are  susceptible  to  effects  of  these 
inhibitors-^.  The  loss  of  fiber  contractility  may  be  related  to  a  stabilization  or  blocking 
of  sulfhydryl  linkages  of  the  contractile  proteins  themselves. 

By  means  of  the  elemental  contractile  system  under  study,  the  action  of  the  bio- 
logically important  compounds  listed  in  Table  IV  can  be  further  differentiated.  All  the 
substances  enumerated  are  known  to  affect  the  process  of  contraction  of  intact  muscle 
fibers.  Since  they  are  ineffective  in  influencing  fiber  contraction  produced  by  ATP,  their 
site  of  action  may  be  assumed  to  be  elsewhere.  From  data  available  it  is  probable  that 
they  affect  contraction  of  intact  fibeis  through  their  action  on  the  conductive  membrane 
of  the  muscle  either  at  the  neuromuscular  junction  or  along  the  fibers.  Of  particular 
interest  in  this  connection  is  the  absence  of  any  effect  of  the  cholinesterase  inhibiting 
compounds,  such  as  diz'sopropylfluorophosphate  and  eserine,  on  the  contractile  process. 
This  does  not  support  the  assumption  of  a  general  toxic  effect  of  these  compounds  as 
proposed  by  some  investigators,  but  is  consistent  with  the  view  which  attributes  their 
effect  to  blocking  conductions^. 

The  observations  presented  show  the  usefulness  of  the  non-conductive  contractile 
preparation  of  muscle  described  by  Szent-Gyorgyi.  The  system  simplifies  the  study 
of  the  contractile  process  and  offers  an  opportunity  to  study  chemical  and  pharmaco- 
logical factors  affecting  contraction  as  distinct  from  conduction. 
References  p.  6y. 


66  S.  KOREY  VOL.  4  (1950) 

I  am  grateful  to  Dr  David  Nachmansohn  for  his  suggestions  and  advice  in  the 
conduct  of  this  research. 

SUMMARY 

A  preparation  of  muscle  fibers  preserved  in  glycerol  has  been  described  by  Szent-Gyorgyi,  in 
which  the  contractile  elements  remain  intact  whereas  the  conductive  membrane  is  not  functioning. 
Properties  of  such  fibers  and  factors  influencing  the  contractile  mechanism  independent  of  conduction 
have  been  studied.  The  following  essential  results  have  been  obtained. 

1.  Of  a  great  number  of  compounds  tested,  only  ATP  and  ADP  induced  contraction.  The  con- 
centration of  ADP  required  was  more  than  ten  times  higher  than  that  of  ATP.  Adenylic  acid  and 
inorganic  pyrophosphate  had  no  effect  in  high  concentrations.  The  same  is  true  for  a  great  number 
of  compounds  like  acetylcholine,  adrenaline,  DFP,  eserine  and  many  others  which  are  known  to 
affect  the  normal  muscle  fiber  preparation. 

2.  Quantitative  evaluations  have  shown  that  4-  lo-^  M  of  ATP  is  close  to  the  optimum  to  induce 
the  contraction  of  the  non-conducting  fiber  but  concentrations  as  low  as  i  •  10-^  M  had  a  measurable 
effect. 

3.  The  extent  of  shortening  increased  strongly  with  temperature,  for  each  10"  C  rise  between 
10°  and  37°  C  by  a  factor  of  1.9. 

4.  The  Ph  optimum  was  found  to  be  between  7.4  and  7.6.  The  fibers  deteriorated  rapidly  in 
solutions  beyond  the  limits  of  6.8  and  7.8. 

5.  Magnesium  ions  activate  the  reaction  of  ATP  with  the  contractile  proteins.  The  optimal 
concentration  was  i  •  10--  M. 

6.  The  ATPase  activity  in  the  fiber  preparation  declined  greatly  during  the  first  three  weeks 
to  about  20%  of  the  initial  value  at  which  level  the  activity  appears  to  remain  stable. 

7.  The  effect  of  -SH  inhibitors  has  been  studied.  Two  of  these  compounds,  o-iodosobenzoate 
and  mapharsen,  proved  to  be  reversible  inhibitors  of  the  contractile  process. 

RfiSUMfi 

Szent-Gyorgyi  a  decrit  une  preparation  de  fibres  musculaires  preservees  dans  le  glycerol  dans 
laquelle  les  elements  contractiles  restent  intacts  tandis  que  la  membrane  conductive  ne  fonctionne 
pas.  Les  proprietes  de  telles  fibres  et  les  facteurs  qui  influencent  le  mecanisme  contractile  independant 
de  conduction  ont  ete  etudies.  Voici  les  principaux  resultats  obtenus. 

1.  Sur  un  grand  nombre  de  composes  etudies  seuls  I'ATP  et  I'ADP  induisaient  une  contraction. 
La  concentration  d'ADP  requise  etait  plus  de  dix  fois  superieure  a  celle  d'ATP.  L'acide  adenylique 
et  le  pyrophospate  inorganique  n'avaient  pas  d'effect  a  des  concentrations  elevees.  II  en  etait  de 
meme  pour  un  grand  nombre  de  composes  tels  que  I'acetylcholine,  I'adrenaline,  le  FDP,  I'eserine  et 
beaucoup  d'autres  dont  nous  savons  qu'ils  affectent  une  preparation  normale  de  fibres  musculaires. 

2.  Des  evaluations  quantitatives  nous  ont  montre  qu'une  concentration  de4- 10-2  M  d'ATP  est 
pres  de  I'optimum  qui  induit  la  contraction  d'une  fibre  non-conductive;  cependant  des  concentrations 
aussi  faibles  que  i-io-^  M  produisaient  un  effet  mesurable. 

3.  Le  raccourcissement  devenait  plus  fort  lorsque  la  temperature  augmentait;  le  facteur  etait 
de  1.9  pour  toute  augmentation  de  10°  C,  dans  I'intervalle  de  10  et  37°  C. 

4.  Le  Ph  optimum  se  trouvait  entre  7.4  et  7.6.  Le  fibres  se  gataient  rapidement  dans  des  solutions 
ayant  un  pn  inferieur  a  6.8  ou  superieur  a  7.8. 

5.  Les  ions  de  magnesium  activaient  la  reaction  de  lATP  avec  les  proteines  contractiles.  La 
concentration  optima  etait  de  i  •  10--  M. 

6.  L'activit^  adenosine  triphosphatasique  diminuait  rapidement  dans  la  preparation  de  fibres 
jusqu'a  environ  20%  de  sa  valeur  initiale  puis,  a  ce  niveau,  elle  semblait  rester  stable. 

7.  Nous  avons  etudie  egalement  I'effet  des  inhibiteurs  d'  -SH;  deux  de  ces  composes,  I'o-iodoso- 
benzoate  et  le  mapharsene  sont  des  inhibiteurs  reversibles  du  processus  contractile. 

ZUSAMMENFASSUNG 

Szent-Gyorgyi  hat  ein  in  Glycerin  konserviertes  Muskelpraparat  beschrieben,  in  dem  die  kon- 
traktilen  Elemcnte  intakt  bleiben,  wahrend  die  Icitende  Membrane  nicht  funktioniert. 

Die  Eigenschaften  solcher  Fasern  und  die  Faktoren,  welche  den  von  Konduktion  unabhangigen 
Kontraktionsmechanismus  beeinflussen,  wurden  untersucht.  Dies  sind  die  wichtigsten  Ergebnisse. 

I.  Von  der  grossen  Anzahl  der  untersuchten  Verbindungen  bewirkten  nur  ATP  und  ADP  eine 
Kontraktion.  Die  notige  Konzentration  war  fiir  ADP  zehnmal  grosser  als  fur  ATP.  Adenylsaure  und 
anorganisches  Pyrophosphat  hatten  in  hohen  Konzentrationen  keine  Wirkung.  Das  Gleiche  gilt  fiir 

References  p.  f>7 ■ 


VOL.  4  (1950)  CONTRACTILITY   OF   A   NON-CONDUCTING   FIBER  67 

eine  grosse  Anzahl  von  Verbindungen,  wie  Acetylcholin,  Adrenalin,  DFP,  Eserin  und  viele  andere, 
deren  Wirkung  auf  normale  Muskelfaserpraparate  bekannt  ist. 

2.  Quantitative  Schatzungen  haben  ergeben,  dass  das  Optimum  fiir  die  Kontraktion  einer  nicht 
leitenden  Faser  nahe  bei  4-10-2  ^  ATP  liegt,  aber  schon  Konzentrationen  von  i  •  10-^  M  batten  eine 
messbare  Wirkung. 

3.  Die  Verkiirzung  wird  bei  steigender  Temperatur  grosser;  fiir  eine  Steigerung  von  je  10°  C 
zwischen  10  und  37°  C  betragt  der  Faktor  1.9. 

4.  Wir  fanden  ein  pn-Optimum  zwischen  7.4  und  7.6.  Die  Fasern  verderben  rasch  in  Losungen 
deren  pn  unter  6.8  oder  ijber  7.8  liegt. 

5.  Magnesiumionen  aktivieren  die  Reaktion  von  ATP  mit  Kontraktions-Proteinen.  Die  optimale 
Konzentration  betrug  i-io-^  M. 

6.  Die  ATPase-Aktivitat  des  Faserpraparates  nimmt  wahrend  der  ersten  drei  Wochen  stark 
ab  und  scheint  dann  bei  ungefahr  20%  des  Anfangwertes  konstant  zu  bleiben. 

7.  Die  Wirkung  von  -SH-Hemmstoffen  wurde  untersucht  und  gefunden,  dass  zwei  von  Ihnen, 
Jodosobenzoat  und  Mapharsen  reversible  Inhibitoren  des  Kontraktionsprozesses  darstellen. 

REFERENXES 

1  C.  H.  FisKE  AND  Y.  SuBBAROw,  /.  Btol.  Ckcm.,  81  (1929)  629. 

-  p.  Eggleton  and  G.  p.  Eggleton,  Biochem.  J.,  21  (1927)  190. 

^  O.  Meyerhof  and  J.  SuRANYi,  Biochem.  Z.,  191  (1927)  106. 

*  O.  Meyerhof  and  K.  Lohmann,  Biochem.  Z.,  196  (1928)  49. 

5  O.  Meyerhof  and  L.  Lohmann,  Biochem.  Z.,  253  (1932)  431. 

^  K.  Lohmann  and  O.  Meyerhof,  Biochem.  Z.,  273  (1934)  6°- 

'  E.  Negelein  and  H.  Bromel,  Biochem.  Z.,  303  (1939)  132. 

^  F.  LiPMANN,  Advances  in  Enzymol.,  i  (1941)  99. 

^  H.  M.  Kalckar,  Chem.  Revs,  28  (1941)  71. 

^°  D.  Nachmansohn  AND  A.  L.  Machado,  /.  Neurophysiol.,  6  (1943)  397. 
"  S.  Ratner,  /.  Biol.  Chem.,  170  (1947)  761. 

12  V.  A.  Engelhardt  AND  M.  N.  Lyubimova,  Nature,  144  (1939)  668. 
"  V.  A.  Engelhardt,  Advances  in  Enzymol.,  6  (1946)  147. 
"  D.  M.  Needham,  Biochem.  J.,  36  (1942)  113. 

1*  A.  Szent-Gyorgyi,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  i,  2,  3  (1941-1943). 
^'  A.  Szent-Gyorgyi,  Muscular  Contraction,  Academic  Press,  1947. 
"  F.  B.  Straub,  Studies  Inst.  Med.  Chem.  Univ.  Szeged,  2,  i  (1942). 
^*  K.  P.  DuBois  AND  V.  R.  Potter,  /.  Biol.  Chem.,  150  (1943)  185. 
"  O.  Meyerhof,  Ann.  N.Y.  Acad.  Sci.,  17  (1947)  815. 
2°  C.  A.  Naaske  AND  B.  Gibson,  Am.  J.  Physiol.,  127  (1939)  486. 
21  T.  P.  Singer  and  E.  S.  G.  Barron,  Proc.  Soc.  Exptl  Biol.  Med.,  56  (1944)  120. 
"  K.  Bailey  and  S.  V.  Perry,  Biochim.  Biophys.  Acta,  i  (1947)  506. 
23  D.  Nachmansohn,  Bull.  John  Hopkins  Hasp.,  83  (1948)  463. 

Received  April  gth,  1949 


PART  II 
NERVE 


MORPHOLOGY  IN  MUSCLE  AND  NERVE  PHYSIOLOGY 

by 

FRANCIS  O.  SCHMITT 

Department  of  Biology,  Massachusetts  Institute  of  Technology, 
Cambridge,  Mass.  (U.S.A.) 


As  applied  to  biology,  morphology  embraces  the  study  of  the  structure  of  cell  and 
tissue  constituents  from  gross  and  microscopic  anatomy  through  the  colloidal  range  and 
even  to  the  molecular  and  atomic  levels.  With  the  introduction  of  electron  microscopy 
it  is  now  possible  to  visualize  directly  the  structure  of  objects  throughout  the  colloidal 
range.  It  is  not  unrealistic  to  expect  that  technical  development  will  make  possible 
direct  visualization  of  such  biologically  important  objects  as  the  smaller  protein  mole- 
cules and  possibly  even  the  polypeptide  chains.  Simultaneously  the  theory  and  tech- 
niques of  X-ray  diffraction  are  also  progressing.  This  method  is  already  able  to  deal 
effectively  with  the  analysis  of  the  internal  architecture  of  certain  crystalline  proteins  ; 
a  major  hurdle  appears  to  be  the  development  of  suitable  computing  methods — a 
matter  chiefly  of  technology  and  patience.  Progress  is  also  being  made  in  the  analysis 
of  the  less  regularly  constructed,  but  no  less  important  biologically,  fibrous  proteins  and 
complexes  of  proteins  with  lipids,  nucleic  acids  and  polysaccharides.  This,  too,  is  a 
matter  of  painstaking,  patient  development  of  techniques,  experimental  and  theoretical. 

Morphology  is  a  science  in  its  own  right.  The  analysis  of  the  detailed  structure  of 
the  molecules  and  complexes  which  occur  in  tissues  is  largely  the  task  of  the  physicist 
who,  in  turn,  must  depend  upon  the  chemist  to  identify,  isolate,  purify  and  characterize 
the  individual  constituents.  In  the  normal  course,  as  physicists  and  chemists  become 
interested  in  such  substances,  one  may  expect  knowledge  in  this  branch  of  crystallo- 
graphy slowly  to  unfold.  Slowly  because  such  complex,  frequently  imperfectly  structured 
materials  are  not  attractive  to  most  crystallographers  who  are  likely  to  regard  them  as 
"sick  crystals",  as  one  colleague  expresses  it.  Actually,  some  of  the  most  important 
protein  crystals  are  far  from  "sick"  structurally;  upon  the  regularity  of  the  internal 
structure  of  their  molecules  depend  such  fundamental  vital  properties  as  are  manifested 
in  the  phenomena  of  immunology,  genetics,  and  the  ordered  processes  of  growth  and 
development.  Relatively  minute  changes  in  the  structure  of  certain  protein  molecules 
may  make  the  organism  sick  (Pauling  et  al.^,  recently  referred  to  sickle  cell  anemia  as 
a  "molecular  disease" !).  The  great  biological  significance  of  structural  studies  has  stimu- 
lated many  physicists  and  chemists  to  devote  their  efforts  to  the  problem.  Hopefully 
their  numbers  will  grow. 

The  detailed  analysis  of  biomolecular  structure  is  a  long  term  task.  The  analysis 
starts  with  a  rough  characterization  of  the  main  structural  features  of  a  particular  tissue 
entity.  With  the  aid  of  the  electron  microscope  the  biologist  relatively  untrained  in  the 
discipline  of  crystallography  can  and  must  take  an  active  in  this  phase.  As  the  analysis 
References  p.  76}yy.  68 


VOL.  4  (1950)  MORPHOLOGY   IN    MUSCLE   AND    NERVE    PHYSIOLOGY  69 

becomes  more  detailed,  eventually  leading  to  the  localization  of  the  constituent  atoms, 
the  task  becomes  more  that  of  the  crystallographer.  The  physiologist  and  biochemist 
must  make  use  of  the  information  available  at  the  moment  in  attempting  to  account 
for  biological  phenomena. 

To  what  extent  has  structure  analj^sis  been  of  assistance  in  solving  major  physiolo- 
gical problems  and  what  is  the  outlook  for  further  advance  in  this  field?  In  seeking  a 
perspective  regarding  such  a  question  a  consideration  of  muscle  contraction  and  nerve 
conduction  may  be  instructive  because  of  the  contrast  which  these  problems  present  in 
respect  of  inherent  susceptibility  to  morphological  investigation  and  to  progress  already 
accomplished.  The  following  account  is  necessarily  brief  and  attempts  merely  to  indicate 

the  trend  of  research  in  this  field. 

* 

MUSCLE    CONTRACTION 

Contractility  is  particularly  favourable  for  morphological  study  because  it  involves 
structural  changes  at  all  levels  of  observation.  The  voluminous  literature  of  muscle 
histology,  devoted  largely  to  striated  muscle,  led  to  few  important  physiological  clues. 
Perhaps  the  "reversal  of  striation"^  on  contraction  was  among  the  most  suggestive. 
Even  observations  in  polarized  light  were  difficult  to  interpret.  The  positive  form  bire- 
fringence indicated  that  the  fibrous  proteins  have  widths  small  with  respect  to  the 
wavelength  of  light.  The  relative  isotropy  of  the  /  bands  was  long  misinterpreted  as 
indicating  disorientation  in  these  regions.  Muralt  and  Edsall's  demonstration  of  the 
positive  birefringence  of  myosin  focused  attention  on  this  protein  as  the  contractile 
substance  of  muscle.  Astbury's  identification  of  myosin  as  the  source  of  the  wide- 
angle  X-ray  pattern  of  muscle,  together  with  his  hypothesis  of  intramolecular  folding 
during  contraction,  helped  to  seek  in  myosin  the  substratum  of  contraction^. 

In  the  short  time  since  electron  microscopy  has  been  applied  to  the  problem,  im- 
portant advances  have  been  made.  The  view  that  myosin  is  localized  in  the  A  bands, 
already  discredited  by  quantitative  considerations,  was  disproven  by  electron  micro- 
scopy, which  showed  that  the  protein  filaments  extend  as  parallel  bundles  continuously 
through  both  A  and  /  bands*.  The  relative  isotropy  of  the  /  bands  is  therefore  not  due 
to  disorientation.  Recently  the  view  has  been  taken  that  the  isotropy  results  from  the 
presence  of  negatively  birefringent  substances  in  the  /  bands  which  compensate  the 
positive  birefringence  of  the  myosin;  this  material  has  been  variously  reported  as 
nucleotides^'  ^,  lipids'  and  phosphoproteins  (A''  material)^. 

In  contraction  the  protein  filaments  remain  relatively  straight  and  parallel,  indi- 
cating that  the  contractile  unit  is  thinner  than  the  filaments  (ca  150  A).  The  distribution 
of  the  dense  material  in  the  A  bands  and  on  the  Z  membrane  changes  in  agreement  with 
the  histological  picture  of  reversal  of  striation. 

Morphological  studies  were  greatly  stimulated  by  advances  in  our  concepts  of 
mechano-chemical  coupling  mediated  by  high-energy  phosphate  bonds  and  by  the 
discovery  by  the  Szeged  group  that  myosin  is  composed  of  two  proteins,  a  water-soluble 
myosin  and  actin,  the  actomyosin  complex  being  sensitive  to  the  action  of  adenosine- 
triphosphate  (ATP).  The  general  morphological  features  of  the  water-soluble  myosin 
and  the  globular  and  fibrous  actin  were  soon  demonstrated  with  the  electron  microscope®, 
together  with  the  dissociating  effect  of  ATP  on  the  actomyosin  threads^". 

Of  great  significance  in  the  morphological  approach  to  the  contractile  mechanism 
References  p.  76lyy. 


70  F.  O.  SCHMITT  VOL.  4  (1950) 

is  the  axial  periodicity  demonstrated  both  by  small-angle  X-ray  dil^raction^^  and  by 
electron  microscopy*.  This  period  has  a  value  of  about  400  A  in  uncontracted  fibres  and 
appears  to  be  characteristic  of  muscle  generally,  for  Bear  has  observed  it  not  only  in 
vertebrate  striated  muscle  but  also  in  various  invertebrate  muscles  which  are  generally 
regarded  as  being  of  the  smooth  type.  In  electron  micrographs  the  filaments  have  a 
beaded  appearance  which  gives  rise  to  a  fine  banding  of  the  myofibril,  the  distance 
between  bands  being  about  400  A.  Draper  and  Hodge^^  have  shown  the  period  very 
strikingly  in  electron  micrographs  of  platinum-shadowed  preparations.  In  their  prelimi- 
nary note  they  state  that  the  axial  period  varies  inversely  with  the  degree  of  shortening 
of  the  muscle.  Variations  in  the  400  A  period  with  fibre  length  were  also  noted  by 
Bennett^^  who  believes  to  haye  shown  that  the  filaments  have  a  helical  structure.  If 
these  points  are  satisfactorily  documented  and  confirmed  we  shall  have  visual  evidence 
of  the  contractile  phenomenon  at  the  near-molecular  level. 

Actually  the  relation  between  the  400  A  axial  period  demonstrated  by  X-ray 
diffraction  and  the  pseudo-period  of  about  the  same  value  seen  in  electron  micrographs 
is  not  clear.  The  largest  meridional  spacing  observed  in  the  X-ray  patterns  is  about 
27  A  which  is  an  order  of  the  larger  period.  If  the  situation  is  similar  to  that  of  para- 
myosin^*'  ^^  one  might  expect  that  the  period  which  might  be  observable  as  cross  bands 
in  the  electron  microscope,  would  have  a  value  of  about  27  A ;  the  larger  period  of  about 
400  A  would  be  manifested  as  a  geometric  pattern  of  discontinuities  within  the  bands. 
However,  depending  on  the  type  of  geometry  of  the  intraperiod  structure,  discon- 
tinuities at  a  spacing  larger  than  27  A  may  appear  in  electron  micrographs.  The  solution 
of  this  problem  will  have  to  await  a  more  detailed  X-ray  analysis  and  attainment  of 
very  considerably  increased  electron  microscope  resolution  of  the  structure  of  the 
filaments. 

AsTBURY,  Perry,  Reed,  and  Spark^^  have  observed  a  spacing  of  54  A  in  fibrous 
actin.  At  large  angles  the  pattern  is  not  that  of  an  alpha  protein.  This  led  the  authors 
to  the  conclusion  that  the  large-angle  pattern  of  muscle  is  due  to  myosin  while  the  small- 
angle  pattern  is  due  to  actin;  the  full  muscle  pattern  derives  from  actomyosin  which 
exists  as  a  complex  in  muscle.  While  this  may  prove  to  be  the  case,  the  diffraction  evi- 
dence is  not  yet  sufficiently  detailed  to  require  this  conclusion. 

The  electron  microscope  investigation  of  contractility  might  be  facilitated  by 
examination  of  in  vitro  models  such  as  the  actomyosin-ATP  system  described  by  Szent- 
Gyorgyi^'.  This  would  be  true  if  such  systems  permitted  higher  resolution  than  could 
be  achieved  in  the  myofibril  and,  particularly,  if  the  essential  properties  of  such  a  system 
faithfully  portray  those  of  muscle.  Recently  Szent-Gyorgyi^^  has  found  that  muscles 
thoroughly  extracted  with  50%  glycerol  at  low  temperatures  are  capable  of  contraction 
when  treated  with  ATP  and  produce  the  same  tension  as  the  intact  muscle  when  maxi- 
mally excited.  Differences  in  the  behaviour  of  this  model  as  compared  with  intact  muscle 
are  attributed  to  the  fact  that  the  model  may  lack  some  of  the  proteins,  lipids  and  other 
substances  with  which  the  actomyosin  is  normally  associated  in  muscle.  From  studies 
of  this  model,  as  from  the  previous  one  of  Varg.\^^,  the  conclusion  was  reached  that 
contractile  substance  is  composed  of  functional  units,  "autones",  and  that  contraction 
represents  an  all-or-none  equilibrium  reaction  of  these  units ;  contraction  and  relaxation 
are  two  distinct  allotropic  states  of  the  autones. 

Unfortunately,  as  admitted  by  Szent-Gyorgyi^^  and  as  amplified  by  Sandovv^" 
none  of  the  partial  systems  and  models  thus  far  proposed  fully  displays  the  essential 
References  p.  76I77. 


VOL.  4  (1950)  MORPHOLOGY    IN    MUSCLE    AND    NERVE    PHYSIOLOGY  7I 

properties  of  muscle.  So  far  as  the  morphological  evidence  is  concerned,  Perry,  Reed, 
AsTBURY,  AND  Spark^^  have  shown  by  X-ray  and  electron  microscope  studies  that  the 
changes  which  occur  when  ATP  is  added  to  actomyosin  is  an  intermolecular  syneresis, 
the  contraction  occurring  in  a  direction  normal  to  that  which  characterizes  muscle  con- 
traction. Moreover,  there  is  no  evidence  from  X-ray  results  for  the  existence  of  two 
distinct  states  of  the  "auxones".  Upon  contraction  the  large-angle  pattern  indicates 
a  change  from  an  alpha  to  a  poorly  defined,  disoriented  beta  configuration.  Efforts  to 
obtain  a  characteristic  small-angle  pattern  from  contracted  muscle  have  thus  far  met 
with  failure.  What  httle  electron  microscope  evidence  bears  on  this  point  suggests  that 
the  400  A  axial  period  shows  a  continuous  change  in  value  with  change  in  fibre  length 
rather  than  two  distinct  states. 

However  valuable  partial  systems  and  models  may  be  from  the  biochemical  view- 
point, it  is  evident  that,  in  the  investigation  of  structural  mechanism  which  is  charac- 
teristic of  muscle,  final  answers  will  be  obtained  by  observation  of  nothing  less  complex 
than  the  muscle  fibre  itself. 

There  is  no  reason  to  doubt  that  the  combination  of  X-ray  diffraction  and  electron 
microscopy  will  be  equal  to  the  task  of  disclosing  the  molecular  changes  which  occur  in 
contraction.  The  small-angle  X-ray  analysis  is  particularly  promising  and  may  be 
expected  in  the  near  future  to  portray  the  main  features  of  the  lattice  of  Bear's  Tjq^e  II 
protein.  The  more  detailed  structure  at  smaller  separations,  involving  the  configurations 
of  polypeptide  chains  in  relaxed  and  contracted  muscle  seems  more  difficult  of  unique 
solution  unless  more  diffraction  data  can  be  obtained  at  large  angles. 

An  electron  microscope  investigation  of  the  extra-filamentous  structures  of  the 
striated  myofibril,  including  the  materials  concerned  in  the  "reversal  of  striation",  the 
Z  membranes  and  the  binding  material  which  connects  filaments  to  each  other  and  to 
the  sarcolemma  laterally,  offers  much  promise.  However,  primary  interest  attaches  to 
the  detailed  structure  within  the  filament  and  the  changes  of  this  structure  with  con- 
traction. As  compared  with  paramyosin  the  task  of  the  electron  microscopist  will  be 
considerably  more  exacting  because  of  the  smaller  spacings  involved.  Obviousty,  at  this 
level  of  size  the  most  critical  judgement  of  image  quality  and  of  optical  artifacts  will 
be  required. 

NERVE    CONDUCTION 

The  problem  of  nerve  conduction  contrasts  strikingly  with  that  of  muscle  con- 
traction as  regards  the  contributions  of  morphology.  This  is  due  to  the  fact  that  the 
changes  whicht  occur  in  a  nerve  fibre  when  an  impulse  is  conducted  are  far  more  subtle 
than  those  occurring  during  contraction  and  also  to  the  fact  that  chemical  characteri- 
zation of  nerve  fibre  constituents,  particularly  the  proteins,  is  almost  completely  lacking. 
Until  about  the  turn  of  the  century  the  extensive  histological  literature  emphasized 
primarily  the  neurofibrils  which  were  regarded  by  many  as  the  substratum  of  impulse 
conduction.  In  its  most  stimulating  form  this  hypothesis  visualized  the  interface  be- 
tween axoplasm  and  neurofibril  as  the  locus  of  the  electro-chemical  changes  which 
underlie  impulse  propagation^^.  Bethe's^^  demonstration  of  a  difference  of  stainability 
of  neurofibrils  under  the  anode  and  cathode  of  a  polarizing  current,  due  to  the  presence 
in  axoplasm  of  a  hypothetical  "fibrillary  acid",  attracted  little  attention  though  the 
phenomenon  seems  quite  genuine  and  has  some  renewed  interest  in  the  light  of  recent 
References  p.  76JJJ. 


72  F.  O.  SCHMITT  VOL.  4  (195O) 

polarization  experiments^*.  The  ascendency  of  the  membrane  theory  together  with  a 
growing  distrust  of  structures  which  can  be  demonstrated  only  after  fixation  caused 
physiologists  to  lose  interest  in  morphology  as  an  immediate  aid  in  studying  the  mecha- 
nism of  impulse  propagation.  To  many  physiologists  the  nerve  fibre  became  essentially 
a  tube  limited  by  a  metastable  interfacial  film  and  containing  a  salt  solution  plus 
certain  metabolizing  substances  capable,  in  some  way,  of  maintaining  the  structural 
integrity  of  the  fibre  and  of  furnishing  the  energy  needed  for  impulse  propagation. 

The  conservative  nature  of  the  processes  involved  in  the  generation  and  propa- 
gation of  the  spike  wave  was  demonstrated  by  studies  of  the  thermal  and  oxidative 
changes.  The  excess  oxygen  consumption  per  impulse  may  be  very  small  at  low  rates 
of  stimulation^^  and,  after  treatment  with  azide,  nerve  is  capable  of  conducting  action 
waves  of  undiminished  amplitude  with  no  accompanying  increase  in  oxygen  consump- 
tion's. 

Currently  there  is  renewed  interest  in  the  coupling  of  reactions  of  chemical  metabo- 
lism with  bioelectric  processes.  In  addition  to  the  much  debated  question  of  the  role  of 
acetylcholine^^"^^  and  of  other  "Erregungsstoffe"^",  suggestions  have  been  offered  linking 
particular  chemical  reactions  with  the  polarization  potentiaP^.  ATP-ase  has  also  been 
invoked^-"^*.  However,  there  is  as  yet  no  general  agreement  as  to  the  role  of  such  sub- 
stances. 

In  the  field  of  electrophysiology  much  progress  has  been  made  in  the  more  accurate 
description  of  the  electrical  properties  of  the  nerve  fibre  at  rest  and  during  activity. 
However,  the  present  period  is  characterized  by  fundamental  disagreement  among  the 
most  competent  investigators  about  the  nature,  origin  and  significance  of  the  polari- 
zation and  action  potentials^^^^^  Characteristic  also  is  the  failure  of  the  electrical  studies 
to  provide  definitive  clues  as  to  the  structure  and  chemical  composition  of  the  reacting 
system. 

The  appalling  ignorance  about  the  chemical  composition,  particularly  of  the 
proteins,  of  peripheral  nerve  may  in  part  be  due  to  the  unattractiveness  of  investigating 
a  tissue  in  which  the  structure  of  interest  is  presumably  a  paucimolecular  layer  of 
uncertain  location.  Amino  acid  analyses  have  been  made  on  the  socalled  "neurokeratin" 
but  the  location  of  this  protein  is  uncertain.  Originally  the  term  was  applied  to  the  pro- 
tein of  the  myelin  sheath.  However,  Block^  concluded  that  it  is  more  probably/  located 
in  the  axis  cylinder  and  may  be  the  protein  of  which  the  neurofibrils  are  composed. 
A  pseudo-nucleoprotein  was  isolated  from  the  axons  of  the  giant  fibres  of  the  squid  and 
from  lobster  nerves^^.  Since  this  complex  seems  to  occur  in  the  central  nervous  system 
as  well  as  in  peripheral  nerve  it  was  considered  characteristic  of  nerve  and  was  termed 
"neuronin".  Its  possible  relation  to  neurofibrils  is  not  known.  The  chemical  characteri- 
zation of  this  entity  is  at  best  very  sketchy,  but  it  can  at  least  be  definitely  localized 
in  the  axon.  Chemical  investigations  are  now  being  carried  on  by  J.  Folch  and  his 
collaborators  on  the  proteins  and  lipids  of  the  brain.  Already  a  liponucleoprotein  and 
several  other  proteins  have  been  isolated  and  partially  characterized'*".  Though  it  is 
impossible  at  present  to  say  whether  these  proteins  are  located  in  the  perikarion,  the 
axon  or  in  extrafibrillar  material,  it  may  be  possible,  once  the  pure  constituents  are 
thoroughly  characterized,  to  devise  methods  by  which  their  presence  in  the  components 
of  peripheral  nerve  may  be  demonstrated. 

In  view  of  the  situation  as  outlined  above,  it  is  perhaps  not  surprising  that  mor- 
phological studies  have  thus  far  contributed  relatively  little  to  an  understanding  of 
References  p.  76lyy. 


VOL.  4  {1950)  MORPHOLOGY   IN    MUSCLE   AND    NERVE    PHYSIOLOGY  73 

impulse  propagation  in  nerve.  To  gain  a  perspective  as  to  the  promise  of  further  inves- 
tigations at  or  near  the  molecular  level  it  will  be  useful  to  consider  what  information  of 
this  sort  is  now  at  hand.  The  discussion  will  be  centered  around  the  two  chief  components 
of  the  fibre,  the  axon  (myelin)  sheath  and  the  axon  (axis  cylinder). 

THE   AXON    SHEATH 

The  general  features  of  the  molecular  architecture  of  the  myelin  sheath  have  been 
deduced  from  polarized  light  and  X-ray  diffraction  studies*^  Essentially  the  sheath 
consists  of  lipid-protein  layers  about  180  A  thick  wrapped  concentrically  about  the  axon. 
The  lipid  phases  exist  as  smectic  mesomorphic  double  layers  of  mixed  lipids,  the  paraffin 
chains  being  oriented  normal  to  the  planes  of  the  layers,  i.e.,  radially  in  the  sheath.  The 
protein  component  is  intercalated  between  double  layers  of  lipids  in  thin  sheets  esti- 
mated to  have  an  over-all  thickness  of  25-30  A  per  period.  This  is  presumably  the  protein 
which,  on  fixation,  gives  rise  to  the  neurokeratin  network.  In  view  of  our  ignorance  of 
the  properties  of  this  protein  it  is  impossible  to  say  anything  about  its  configuration  in 
the  very  thin  layers  in  the  sheath.  When  nerve  is  dried  the  thickness  of  the  layers  is 
reduced  by  about  25  A  and  a  considerable  fraction  of  the  sheath  lipids  is  extravasated 
from  the  organized  structure  to  form  separate  lipid  phases.  In  the  skeleton  of  the  original 
structure  which  remains  it  seems  probable  that  a  fraction  of  the  lipid  molecules  is  firmly 
bonded  to  the  thin  protein  layers  and  that  this  linkage  maintains  the  structure  in  the 
dried  sheath.  The  nature  of  this  linkage  can  only  be  surmised  though  one  may  suspect 
that  the  acid  groups  of  the  cephalin  molecules  may  be  involved. 

Thus  far  electron  microscopy  has  contributed  little  to  our  knowledge  of  sheath  struc- 
ture though  advances  in  this  direction  may  be  expected  when  sectioning  methods  are 
applied.  From  osmic  acid  fixed  nerves  disintegrated  with  sonic  oscillations,  Sjostrand 
[unpublished)  has  observed  fragments  of  very  thin  layers  which  may  have  been  derived 
from  the  myelin  sheath.  He  had  previously  demonstrated  with  the  electron  microscope 
that  the  outer  limbs  of  the  retinal  rods  consist  of  stacks  of  thin  discs*^'  ^^.  This  is  in 
agreement  with  the  polarized  light  analysis  which  indicated  that,  like  those  of  the  myelin 
sheath,  the  thin  layers  contain  lipid  and  protein  components  oriented  perpendicular  and 
parallel,  respectively,  to  the  planes  of  the  layers.  It  has  been  suggested**  on  very 
inadequate  grounds,  that  the  protein  of  the  rod  outer  limbs  may  be  a  type  of  "neu- 
rokeratin". De  Robertis  and  the  writer  have  also  observed  thin  layers  in  preparations 
from  fragmented  myelinated  nerves.  Curiously  the  fragmented  layers  frequently  show 
characteristic  angular  cleavage.  If  the  layers  actually  derive  from  the  sheath  this  type 
of  cleavage  is  unexpected  since  the  sheath  has  thus  far  been  considered  to  be  uniaxial 
with  optic  axes  normal  to  the  layers.  Measurements  of  the  thickness  of  the  layered 
fragments  may  help  determine  their  origin  since  the  over-all  thickness  of  the  sheath 
layers  is  known  from  X-ray  data. 

The  X-ray  and  polarized  light  results  concern  only  the  highly  organized  lipid- 
protein  substance  of  the  sheath.  Determination  of  the  detailed  structure  of  the  various 
other  sheath  components  which  have  been  observed  histologically  must  await  electron 
microscope  study  in  thin  sections.  Among  these  structures  are  the  boundaries  of  the 
sheath  at  the  incisures,  the  Golgi  funnels  and  spirals  of  Rezzonico,  the  axolemma  mem- 
brane, the  Schwann  cell  and  the  outer  fibrous  investiments.  The  structure  at  the  node 
will  be  particularly  interesting  because  the  limiting  envelope  of  the  fibre  at  this  point 
References  p.  76I77. 


74  F-  O.  SCHMITT  VOL.  4  (1950) 

has  especial  physiological  significance.  Technical  difficulties  make  it  hard  to  study  this 
surface  structure  with  polarized  light. 

From  polarized  light  studies  it  has  been  suggested  that  all  nerve  fibres  may  possess 
a  lipid-protein  sheath  having  the  same  type  of  architecture  as  that  of  the  myelin  sheath*^. 
Such  a  sheath  has  been  demonstrated  in  several  types  of  invertebrate  fibres  though  the 
investigation  has  not  yet  been  extended  to  the  so-called  naked  fibres  such  as  the  Remak 
fibres.  In  the  limiting  case  the  naked  fibre  may  possess  a  surface  structure  no  more 
complex  than  the  plasma  membrane  itself.  The  polarized  light  method  is  probably 
sufficiently  sensitive  to  detect  molecular  orientation  in  such  paucimolecular  layers. 
However,  the  bearing  of  such  data  on  the  problem  of  impulse  propagation  would  still 
remain  to  be  shown. 

No  direct  connection  between  sheath  ultrastructure  and  physiological  properties 
has  been  demonstrated,  although  a  correlation  has  been  pointed  out  between  sheath 
birefringence,  e.g.,  essentially  lipid  concentration,  and  velocity  of  impulse  propagation*^. 
This  correlation  is  at  best  only  rough  when  applied  to  the  fibres  of  a  particular  type  of 
nerve  but  seem  more  suggestive  when  fibres  of  widely  different  types  of  nerves  are  con- 
sidered. For  several  types  of  vertebrate  and  invertebrate  fibres  having  approximately 
equal  conduction  velocities,  Taylor*^  found  that  the  product  of  fibre  diameter  and 
sheath  birefringence  is  roughly  constant. 

THE    .\XON 

The  most  interesting  structures  in  the  axon  are,  of  course,  the  neurofibrils.  Only  in 
exceptional  cases  can  these  objects  be  observed  in  the  fresh  fibres,  the  chief  lore  of  the 
literature  being  concerned  with  fixed  and  stained  preparations.  The  neurofibrils  may 
approach  the  limit  of  microscopic  resolution  in  fixed  and  stained  preparations.  Hence 
it  is  readily  understandable  that,  if  they  pre-exist  in  the  fresh  axon,  they  may  not  be 
visible,  particularly  if  refractive  index  relations  are  unfavourable.  In  the  dark  field 
microscope  Ettisch  and  Jochims*^  observed  no  structure  in  the  fresh  axon  though  very 
fine  collagen  fibrils  of  the  connective  tissue  were  clearly  visible,  indicating  a  fundamental 
difference  in  the  two  types  of  fibres.  After  treatment  with  reagents  such  as  CaClg  or 
fixatives,  neurofibrils  immediately  appear.  Apparently  only  slight  colloidal  alterations 
suffice  to  make  them  visible.  It  was  concluded  by  Peterfi^^  that  the  fresh  axon  is  a 
rodlet  sol  capable,  under  very  slight  chemical  provocation,  of  forming  a  fibrous  system. 
He  suggested  that  the  mutual  interaction  of  the  elongated  micelles  may  be  intimately 
associated  with  impulse  propagation. 

Electrical  studies  have  failed  to  indicate  any  direct  role  of  axoplasm  except  as  a 
passive  conductor  of  current.  An  electrode  may  be  inserted  into  the  axon  of  the  squid 
giant  fibre  without  blocking  conduction.  But  if  the  inner  surface  of  the  cell  membrane 
is  injured  conduction  ceases^'  *^.  However,  Curtis  and  Cole's*^  statement  that  "This 
makes  it  seem  rather  unlikely  that  there  is  an  internal  structure  in  the  axon  which 
takes  a  prominent  part  in  the  active  mechanism  of  propagation"  must  be  accepted  with 
caution  since  there  is  no  evidence  that  the  manipulation  mentioned  disrupted  any 
axonic  structures  which  might  be  present  as  it  did  the  membrane  structure. 

Changes  in  the  colloidal  organization  of  the  axon  with  activity  have  been  sought, 
but  thus  far  the  experimental  techniques  have  been  very  crude.  It  has  been  claimed 
that  the  fibre  exhibits  changes  in  contour  with  electrical  polarization,  swelling  at  the 
References  p.  76lyy. 


VOL.  4  (1950)  MORPHOLOGY   IN    MUSCLE    AND    NERVE    PHYSIOLOGY  75 

anode  and  flattening  at  the  cathode-^.  More  recently  Flaig^"  believed  to  have  shown 
that  the  viscosit}^  and  turgor  of  the  axoplasm  of  the  squid  giant  fibre  is  considerably- 
increased  during  activity.  He  suggested  that  excitation  increases  the  viscosity  by  shifting 
the  sol-gel  equilibrium.  If  Flaig's  results  are  confirmed,  careful  investigation  of  the 
light  scattering  by  the  axon  might  be  warranted.  The  existence  of  elongate  particles 
in  the  fresh  axon  is  demonstrated  by  the  positive  birefringence  which,  though  weak,  is 
measureable  in  large  axons  such  as  in  the  squid  giant  fibre.  Semi-quantitative  analysis 
of  the  positive  form  birefringence  indicated  that  though  the  oriented  fibrous  structures 
occupy  a  small  portion  of  the  axon  volume,  they  must  have  a  considerable  degree  of 
regularity  of  internal  structure,  for  their  intrinsic  birefringence  is  comparable  with  that 
of  myosin  or  collagen  fibres^^. 

No  change  in  molecular  orientation  in  the  axoplasm  of  squid  giant  fibres  during 
activity  could  be  detected  by  polarization  optical  means^^.  Using  a  sensitive  photo- 
electric method  capable  of  recording  small  changes  in  birefringence  without  appreciable 
time  lag,  it  was  concluded  that  if  any  change  occurred  it  was  less  than  0.2%  of  the  initial 
birefringence  for  the  spike  process  and  less  than  o.oS°o  for  the  slow  recovery  processes. 
Unless  more  sensitive  methods  yield  positive  results  it  may  be  concluded  that  impulse 
propagation  is  associated  with  little  if  any  change  in  orientation  of  the  elongate  par- 
ticles of  the  axon. 

From  electron  microscope  studies,  Richards,  Steinbach,  and  Anderson"^ 
described  contorted  fibrils  composed  of  kinked  elongate  particles  in  a.xoplasm  extruded 
from  squid  giant  fibres.  They  suggested  that  these  structures  may  form  the  basis  of 
neurofibrils.  De  Robertis  and  Schmitt^*  observed  characteristically  double-edged 
fibrils  in  electron  micrographs  of  material  obtained  by  sonic  fragmentation  of  frozen 
sections  of  formalin  fixed  nerves  of  various  types.  Such  structures  had  never  before  been 
observed.  For  descriptive  purpose  the  fibrils  were  tentatively  called  "neurotubules". 
The  dense  material  at  the  edges  is  for  the  most  part  removed  by  washing  with  water. 
It  is  not  yet  clear  to  what  extent  this  dense  material  is  associated  with  the  fibrils  in  the 
natural  state  and  to  what  extent  it  may  have  been  deposited  upon  them  during  the 
preparative  procedure. 

After  staining  with  phosphotungstic  acid  or  shadowing  with  heavy  metal  the  fibrils 
have  a  cross-striated  appearance.  The  axial  period  averages  about  650  A  and  detailed 
intraperiod  structure  has  been  observed.  Since  this  period  is  similar  to  that  of  collagen^^ 
and  since  nerve  fibres  are  closely  invested  with  connective  tissue  the  possibility  that 
neurotubules  may  be  collagen  fibres  invested  with  dense  materials  of  undetermined 
origin  was  carefully  considered.  The  fragmentation  technique  employed  makes  it 
difi&cult  to  determine  the  location  of  the  neurotubules  in  the  nerve  fibre.  All  the  evidence 
was  consistent  with  the  view  that  they  are  of  axonic  origin.  Important  in  the  reasoning 
was  the  fact  that  typical  double-edged  fibrils  were  not  observed  in  preparations  of  nerves 
which  had  been  allowed  to  undergo  degeneration  in  vivo  (Wallerian)  or  in  vitro^^. 
However,  in  recent  experiments  on  late  degeneration,  results  at  variance  with  those 
previously  described  were  obtained.  Preparations  from  nerves  degenerated  for  as  long 
as  three  weeks  were  not  devoid  of  double-edged  fibrils  but  contained  them  in  considerable 
abundance.  The  reason  for  this  discrepancy  is  not  clear.  However,  in  view  of  the  impor- 
tance of  the  degeneration  changes  to  the  argument  that  fibrils  are  of  axonic  origin,  the 
entire  matter  is  being  reinvestigated.  Speculation  as  to  the  possible  role  of  the  neuro- 
tubules in  nerve  function  would  be  premature  at  this  time. 
References  p.  yOjyy. 


76  F.  O.  SCHMITT  VOL.  4  (1950) 

Recent  experiments  suggesting  that  axoplasm  may  be  continuously  moving  peri- 
pherally from  the  cell  body  in  the  normal  neuron^''  ^  have  stimulated  renewed  interest 
in  the  colloidal  properties  of  the  axon  as  they  concern  trophic  phenomena.  It  seems 
probable  that  application  of  the  thin  sectioning  technique  may  prove  valuable  in 
studying  axon  structure  with  the  electron  microscope  and  that  such  studies  may  throw 
light  on  the  physical  basis  of  trophic  processes. 

The  axons  of  fresh  fibres  offer  little  promise  for  X-ray  diffraction  studies  because 
of  their  high  water  content.  It  was  estimated  that  the  axon  proteins  of  the  squid  giant 
fibre  account  for  only  3  or  4%  of  the  wet  weight  of  the  fibre^^.  Dried  frog,  lobster  and 
crab  nerves  show  equatorial  diffractions  at  about  11  A.  It  is  probable  that  these  diffrac- 
tions arise  from  connective  tissue  because  alcohol-dehydrated  axons  isolated  from  squid 
giant  fibres  showed  only  two  disoriented  rings  at  about  4.7  and  10  A,  characteristic  of 
denatured  protein^^.  These  patterns  are  similar  to  those  obtained  from  fibres  spun  from 
axis  cylinder  protein.  These  X-ray  investigations  of  axon  structure  were  not  exhaustive 
and,  in  view  of  current  electron  microscope  results,  warrant  further  careful  study. 

From  the  above  account  it  is  clear  that  the  problem  of  structure  analysis  in  nerve 
is  a  formidable  one.  It  is  particularly  challenging  because  of  the  high  sensitivity  of  the 
colloidal  organization  to  physical  or  chemical  manipulation  and  because  the  chemical 
reactions  underlying  the  physiological  process  are  completely  unknown. 

There  can  be  little  doubt  that  X-ray  and  electron  microscope  techniques,  if  suffi- 
ciently acutely  applied,  are  capable  of  penetrating  to  or  near  the  molecular  level  in 
nerve  as  has  already  been  accomplished  in  the  case  of  contractile  tissue.  Hardly  more 
than  a  beginning  has  been  made  thus  far.  Progress  with  the  morphological  problem 
would  be  greatly  accelerated  if  the  chemical  properties  of  the  nerve  proteins  were  known. 
The  biochemical  problem  is  itself  quite  formidable  bur  there  is  no  reason  to  doubt  that 
it  would  yield  if  subjected  to  a  concerted  attack  by  modern  methods  of  isolation  and 
characterization.  The  bioelectric  aspects  have  attracted  the  best  efforts  of  many  com- 
petent investigators  and  their  analysis  is  still  proceeding.  The  time  has  come  for  an 
equally  concentrated  attack  upon  the  morphological,  biochemical  and  enzymological 
aspects.  Only  thus  may  we  expect  to  make  significant  progress  with  a  problem  as  com- 
plex as  that  of  nerve  function. 

REFERENCES 

^  L.  Pauling,  H.  A.  Itano,  S.  J.  Singer,  and  I.  C.  Wells,  Science,  109  (1949)  443. 

2  H.  E.  Jordan,  Physiol.  Rev.,  13  (1933)  301. 

^  W.  T.  AsTBURY,  Proc.  Roy.  Soc.  (London)  B,  134  (1947)  303. 

*  C,  E.  Hall,  M.  A.  Jakus,  and  F.  O.  Schmitt,  Biol.  Bull.,  90  (1946)  32. 

^  R.  Caspersson  and  B.  Thorell,  Acta  Physiol.  Scand.,  4  (1942)  97. 

®  C.  L.  Hoagland,  Currents  in  Biochemical  Research,  D.  Green,  Ed,  Interscience,  New  York  1946, 

413- 

'  E.  W.  Dempsey,  G.  B.  Wislocki,  and  M.  Singer,  Anat.  Record.,  96  (1946)  221. 

^  A.  G.  Malotsy  and  M.  GerendAs,  Nature,  159  (1947)  502. 

®  M.  A.  Jakus  and  C.  E.  Hall,  /.  Biol.  Chem.,  167  (1947)  705. 

^°  S.  V.  Perry,  R.  Reed,  W.  T.  Astbury,  and  L.  C.  Spark,  Biochem.  Biophys.  Acta,  2  (1948)  674. 
"  R.  S.  Bear,  /.  Am.  Chem.  Soc,  67  (1945)  1625. 
^2  M.  H.  Draper  and  A.  J.  Hodge,  Nature,  163  (1949)  576. 
^^  H.  S.  Bennett,  Anat.  Record,  103  (1949)  7. 
"  R.  S.  Bear,  /.  Am.  Chem.  Soc,  66  (1944)  2043. 

^5  C.  E.  Hall,  M.  A.  Jakus,  and  F.  O.  Schmitt,  /.  Applied  Phys.,  6  (1945)  459- 
"  W.  T.  Astbury,  S.  V.  Perry,  R.  Reed,  and  L.  C.  Spark,  Biochim.  Biophys.  Acta,  i  (i947)  379- 
"  A.  Szent-Gyorgyi,  Muscular  Contraction,  Academic  Press,  New  York  1947. 


VOL.  4  (1950)  MORPHOLOGY   IN    MUSCLE    AND    NERVE    PHYSIOLOGY  77 

18  A.  Szent-Gyorgyi,  Biol.  Bull.,  96  (1949)  140. 

1*  L.  Varga,  Hung.  Acta  Physiol.,  i  (1946)  i,  138. 

20  A.  Sandow,  Ann.  Rev.  Physiol.,  11  (1949)  297. 

21  S.  V.  Perry,  R.  Reed,  W.  T.  Astbury,  and  L.  C.  Spark,  Biochim.  Biophys.  Acta,  2  (1948)  674. 

22  T.  Peterfi,  Handb.  d.  norm.  u.  path.  Physiol.,  9  (1929)  81. 

23  A.  Bethe,  Allgemeine  Anatomic  und  Physiologic  des  Nervensy stems,  Thieme,  Leipzig  1903. 

24  R.  LoRENTE  DE  N6,  Studics  Rockefeller  Inst.  Med.  Res.,  Nos.  131  and  132  (1947). 
2^  F.  O.  ScHMiTT,  Cold  Spring  Harbor  Symposia  Quant.  Biol.,  4  (1936)  188. 

26  R.  W.  Doty  and  R.  W.  Gerard,  Federation  Proc,  8  (1949)  35. 
2'  D.  Nachmansohn,  Ann.  N.  Y.  Acad.  Sci.,  47  (1946)  395. 

28  D.  Nachmansohn,  Federation  Proc,  8  (1949)  116. 

29  R.  W.  Gerard,  B.  Libet,  and  D.  Cavanaugh,  Federation  Proc,  8  (1949)  55. 
3°  A.  V.  MuRALT,  Die  Signaliibermittlung  ini  N erven,  Birkhauser,  Basel  1946. 

31  A.  M.  Shanes  and  D.  E.  S.  Brown,  /.  Cellular  Comp.  Physiol.,  19  (1942)  i. 

32  B.  Libet,  Federation  Proc,  7  (1948)  72. 

33  W.  Sharples,  H.  Grundfest,  and  D.  Nachmansohn,  Federation  Proc,  7  (1948)  113. 
3*  W.  Feldberg  and  C.  Hebb,  J.  Physiol.,  107  (1948)  210. 

35  H.  Grundfest,  Ann.  Rev.  Physiol.,  9  (1947)  477. 

36  J.  C.  EccLES,  Ann.  Rev.  Physiol.,  10  (1948)  93. 

3'  D.  P.  C.  Lloyd  and  A.  K.  McIntyre,  Ann.  Rev.  Physiol.,  11  (1949)  173. 

^8  R.  J.  Block,  /.  Biol.  Chem.,  119  (1937)  ^I- 

39  R.  S.  Bear,  F.  O.  Schmitt,  and  J.  Z.  Young,  Proc.  Roy.  Soc,  London)  B,  123  (1937)  49^- 

4"  J.  FoLCH-Pi  AND  L.  L.  UzMAN,  Federation  Proc,  7  (1948)  155. 

■*!  F.  O.  ScHMiTT,  R.  S.  Bear,  and  K.  J.  Palmer,  /.  Cellular  Comp.  Physiol.,  18  (1941)  31. 

42  F.  Sjostrand, /.  Applied  Phys.,  19  (1948)  1188. 

*3  F.  Sjostrand,  /.  Cellular  Comp.  Physiol.,  in  press. 

4*  W.  J.  Schmidt,  Z.  Zellforsch.  u.  niikrokop.  Anat.,  22  (1935)  504. 

45  F.  O.  Schmitt  and  R.  S.  Bear,  Biol.  Rev.,  14  (1939)  27. 

^6  G.  W.  Taylor,  /.  Cellular  Comp.  Physiol.,  20  (1942)  359. 

"  G.  Ettisch  and  J.  JocHiMS,  Pfliiger's  Arch.,  215  (1927)  525. 

48  A.  L.  Hodgkin  and  A.  F.  Huxley,  Nature,  144  (1939)  710. 

49  H.  J.  Curtis  and  K.  S.  Cole,  /.  Cellular  Comp.  Physiol.,  15  (1940)  147. 
5°  J.  V.  Flaig,  J.  Neurophysiol.,  10  (1947)  211. 

*i  R.  S.  Bear,  F.  O.  Schmitt,  and  J.  Z.  Young,  Proc.  Roy.  Soc.  [London),  B  123  (1937)  505. 

52  F.  O.  Schmitt  and  O.  H.  Schmitt,  /.  Physiol.,  98  (1940)  26. 

53  A.  G.  Richards,  H.  B.  Steinbach,  and  T.  F.  Anderson,  /.  Cellular  Comp.  Physiol.,  21  (1943)  129. 

54  E.  De  Roberts  and  F.  O.  Schmitt,  /.  Cellular  Comp.  Physiol.,  31  (1948)  i. 

55  F.  O.  Schmitt,  C.  E.  Hall,  and  M.  A.  Jakus,  /.  Cellular  Comp.  Physiol.,  20  (1942)  11. 

56  E.  De  Roberts  and  F.  O.  Schmitt,  /.  Cellular  Comp.  Physiol.,  32  (1948)  45. 

5^   J.  Z.  Young,  In  Growth  and  Form.  Essays  presented  to  d'Arcy  Thompson,  Clarendon  Press,  Oxford 

(1945)  41- 

58  P.  Weiss  and  H.  B.  Hiscoe,  /.  Exptl  Zool.,  107  (1948)  315. 

59  F.  O.  Schmitt,  R.  S.  Bear,  and  G.  L.  Clark,  Biodynamica  (1939)  No.  50. 

Received  May  19th,  1949 


78  BIOCHIMICA   ET   BIOPHYSICA   ACTA  VOL.  4  (l950)' 


STUDIES  ON  PERMEABILITY  IN  RELATION 
TO  NERVE  FUNCTION 

I.  AXONAL  CONDUCTION  AND  SYNAPTIC  TRANSMISSION 

by 

DAVID  NACHMAXSOHN 

Department  of  Neurology,  College  of  Physicians  and  Surgeons,  Columbia  University, 

New  York,  N.Y.  (U.S.A.) 

INTRODUCTION 

Cellular  boundaries  are  endowed  with  the  ability  either  to  permit  or  to  prevent  the 
entrance  and  leakage  of  various  compounds  and  metabolites.  This  makes  possible  the 
elimination  of  waste  products  and  the  supply  of  substances  important  for  ionic  equi- 
librium, energy  requirements,  and  other  vital  functions  of  the  cell.  There  are  many 
indirect  indications  for  the  selective  permeability  of  the  membranes  covering  the  cell. 
The  g'eat  importance  of  this  property  for  the  understanding  of  cellular  mechanisms  and 
of  the  action  of  compounds  applied  externally,  which  includes  most  pharmacolog'cal 
effects,  has  long  been  recognized.  Nevertheless,  surprisingly  little  is  known  in  regard  to 
the  factors  wh'ch  determine  and  affect  permeability  of  cellular  boundaries.  Direct 
measurements  are  extremely  difficult.  The  introduction  of  isotopes  as  research  tool  in 
biology,  mainly  due  to  the  work  of  Hevesy^  and  Schoenheimer  and  Rittenberg^, 
has  opened  a  new  pathway  to  the  approach  of  the  problem,  but  the  obstacles  to  be 
overcome  are  still  tremendous.  The  lucid  appraisal  of  the  field  by  Krogh^  in  his  Croonian 
lecture  shows  that  in  spite  of  some  progress  in  recent  years  this  aspect  of  cellular  function 
is  in  its  initial  phase. 

The  permeability  of  the  surface  membranes  of  the  nerve  cell  is  of  particular  interest. 
Physiologists  of  the  last  century  have  already  postulated  that  changes  in  permeability 
must  be  intimately  assoc'ated  with  the  function  of  the  neuron,  i.e.,  with  the  propagation 
of  the  nerve  impulse.  Du  Bois-Reymond  who  first  established  conclusively  that  nerve 
activity  is  associated  with  flow  of  current  devoted  much  time  to  testing  the  possibility 
that  the  source  of  the  electromotive  force  for  the  electrical  manifestations  observed  may 
be  ionic  concentration  g-adients  between  the  interior  of  the  cell  and  its  outer  environ- 
ment*. When,  in  the  later  part  of  the  nineteenth  century,  physico-chemical  invest'gations 
revealed  the  marked  potential  differences  whxh  may  be  produced  by  semipermeable 
membranes,  the  existence  of  such  membranes  was  postulated  as  a  basis  for  the  electrical 
manifestations  during  the  passage  of  the  nerve  impulse.  Ostwald^  wrote  in  iSgo:  "An 
den  halbdurchlassigen  Membranen  kommen  weit  grossere  Potentialdifferenzen  zustande 
als  in  gewChnlichen  Flussig'ceitsketten.  Es  ist  vielleicht  nicht  zu  gewagt  schon  hier  die 
Vermutung  auszusprechen ,  dass  nicht  nur  die  Strome  in  Muskeln  und  Nerven  sondern 
auch  namentlich  die  ratselhaften  Wirkungen  der  elektrischen  Fische  durch  die  hier 
References  p.  93I95. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  I  79 

erorterten  Eigenschaften  der  halbdurchlassigen  Membranen  ihre  Erklarung  linden 
werden".  From  the  discussions  of  Du  Bois-Reymond,  Hermann,  Ostwald  and  others 
concerning  the  mechanism  underlying  the  generation  of  the  electric  currents  during 
nerve  activity  there  finally  emerged  the  membrane  theory  formulated  by  Bernstein 
early  in  this  century^.  This  theory  forms  the  basis  of  all  modern  concepts  of  conduction 
and  has  been  an  extremely  useful  working  hypothesis.  Essentially  the  theory  assumes 
that  the  nerve  fibre  in  resting  condition  is  surrounded  by  a  polarized  membrane,  selec- 
tively permeable  to  potassium  ions.  The  concentration  of  these  ions  inside  the  nerve 
fibre  is  high  compared  with  that  outside.  There  is,  therefore,  a  tendency  for  the  potassium 
ions  to  move  to  the  outside,  but  they  are  kept  back  by  the  negative  ion  for  which  the 
membrane  is  impervious  at  rest.  Thsre  thus  develops  a  positive  charge  on  the  outside 
surface  of  the  membrane  and  a  negative  cha-ge  on  the  inside.  When  a  stimulus  reaches 
the  surface,  a  breakdown  of  resistance  occurs ;  the  permeability  for  the  negative  ion  is 
increased,  resulting  in  a  depolarization.  The  depolarized  point  of  the  membrane  is 
negative  to  the  adjacent  region;  whereby  a  small  electric  current,  the  "Stromchen"  of 
Hermann,  is  generated.  This  current  in  its  turn  stimulates  the  adjacent  region,  leading 
there  to  a  depolarization.  The  same  process  is  repeated  in  successive  parts  of  the  nerve 
fibre  and  in  this  way  the  impulse  is  propagated  along  the  axon. 

Recent  developments  have  made  necessary  a  modification  of  the  membrane  theory 
in  its  original  form.  It  has  been  shown  by  Curtis  and  Cole'^  and  by  Hodgkin  and 
Huxley^  that  during  the  passage  of  the  impulse  there  occurs  not  only  a  depolarization 
but  an  actual  reverse  of  the  charge.  This  result  was  obtained  in  experiments  on  the  giant 
axon  of  Squid  by  the  introduction  of  an  electrode  into  the  interior  of  the  axon  and  by 
direct  determination  of  the  potential  across  the  membrane.  The  spike  potential  was 
found  to  be  markedly  greater  than  the  potential  difference  in  rest,  in  some  cases  it  was 
nearly  twice  as  great.  There  are  some  technical  difficulties  which  make  the  exactness  of 
the  absolute  values  uncertain,  but  the  fact  that  the  charge  is  reversed  during  activity 
appears  to  be  unquestionable  and  well  established.  It  follows  that  the  assumption  of 
a  simple  depolarization  cannot  be  maintained.  The  process  responsible  for  the  gsneration 
of  the  flow  of  current  is  complex  and  is  not  merely  an  abolition  of  the  resting  potential. 

The  availability  of  radioactive  ions  made  possible  the  study  of  the  movement  of 
ions  across  the  neuronal  surface  membrane.  Such  investigations  were  initiated  during 
the  last  two  years  by  Hodgkin  and  Huxley^  and  Keynes^"  in  England  and  by  Rothen- 
berg  in  this  laboratory^^.  The  results  will  be  fully  discussed  in  the  following  paper. 
They  show  that  sodium  and  potassium  ions  are  being  constantly  exchanged,  the  latter 
at  least  to  some  degree  between  the  inside  of  the  axon  and  its  outer  environment.  The 
ionic  equilibrium  is  a  dynamic  and  not  a  static  condition.  The  conclusion  is  similar 
to  that  encountered  in  many  other  fields  where  radioactive  or  stable  isotopes  were  used 
(Schoenheimer12) 

During  activity  the  outflow  of  potassium  and  the  influx  of  sodium  are  greatly 
increased.  The  data  of  the  two  laboratories  are  in  good  agreement  and  supplement  each 
other.  According  to  the  Cambridge  group  about  2  •  lo"^^  mole  of  potassium  leaks  per  cm^ 
surface  per  impulse;  Rothenberg's  experiments  indicate  that  the  influx  of  sodium  is 
about  4-io~i2  mole  per  cm^  per  impulse.  The  question  how  this  movement  of  the  two 
species  of  ions  in  opposite  direction  may  account  for  the  reverse  of  the  cha-ge  is  still 
open.  No  satisfactory  hypothesis  has  been  advanced  so  far.  It  is  obvious,  however,  that 
events  must  take  place  in  the  active  membrane,  the  site  of  the  electrical  manifestations. 
References  p.  93195- 


80  D.  NACHMANSOHN  VOL.  4  (iQSO) 

which  make  this  accelerated  ionic  flow  possible,  and  others  which  restore  the  resting 
condition.  Experimental  evidence  that  such  events  actually  take  place  during  the  passage 
of  the  impulse  has  been  obtained  by  observations  of  Cole  and  Curtis^^  carried  out 
with  the  giant  axon  of  Squid.  These  investigators  measured  the  impedance  changes 
with  alternating  current  of  varying  frequency  applied  across  the  nerve  fibre.  The 
impedance  was  always  reduced  during  the  passage  of  the  impulse.  Analysing  their 
results,  they  concluded  that  the  membrane  resistance  breaks  down  during  activity  from 
about  1000  ohms  per  square  centimeter  to  about  40  ohms  per  square  centimeter. 

The  assumption  of  a  process  in  the  membrane  responsible  for  the  electrical  mani- 
festations is  not  in  contrast  but  in  full  agreement  with  all  classical  views.  As  was  stated 
by  Keith  Lucas  and  Adrian^*  more  than  30  years  ago,  all  facts  indicate  that  the 
energy  for  the  propagation  of  the  nerve  impulse  cannot  be  derived  from  the  stimulus 
itself  as  in  the  case  of  a  sound  wave.  According  to  the  English  investigators  the  energy 
must  be  supplied  locally  by  a  "propagated  disturbance".  The  most  likely  assumption 
as  to  the  nature  of  the  "propagated  disturbance"  is  that  of  a  series  of  chemical  reactions 
producing  a  change  of  the  proteins  or  lipoproteins  of  the  membrane  and  resulting  in  an 
increased  permeability.  Some  kind  of  trigger  mechanism  must  be  responsible  for  the 
change  by  which  the  ionic  concentration  gradient,  inactive  in  rest,  becomes  effective. 
This  concentration  gradient  appears  to  be  the  most  probable  source  of  the  electromotive 
force.  The  change  in  the  membrane  required  for  this  process  must  be,  from  the  thermo- 
dynamic point  of  view,  associated  with  an  irreversible  loss  of  energy.  The  reversal  will 
require  energy  supply  which  can  be  conceivably  derived  from  chemical  reactions  only. 
It  is  remarkable  that  Keith  Lucas  {I.e.)  in  logical  conclusion  of  his  views  postulated 
that  conduction  must  be  associated  with  heat  production,  although  at  that  time  all 
attempts  to  demonstrate  it  had  failed.  In  1926,  however,  A.  V.  Hill  and  his  associates 
were  able  to  demonstrate  heat  production  associated  with  nerve  activity  after  they  had 
developed  the  recording  instruments  to  an  amazingly  h'gh  degree  of  perfections^.  In  the 
same  year  evidence  was  obtained  by  Gerard  and  Meyerhof  that  conduction  is  accom- 
panied by  extra  oxygen  uptake^^. 

These  investigations  have  established  the  experimental  basis  for  the  assumption 
that  conduction  is  associated  with  chemical  reactions.  The  finer  mechanism,  however, 
remained  unknown.  A.  V.  Hill's  Liversidge  lecture:  Chemical  Wave  Transmission  in 
Nerve,  delivered  in  1932,  was  a  challenge  to  biochemists  to  approach  this  central  problem 
of  neurophysiologyS^^.  Without  a  satisfactory  answer  as  to  the  nature  of  the  chemical 
changes  generating  the  flow  of  current,  no  decisive  progress  in  the  understanding  of  the 
mechanism  of  nerve  function  will  be  achieved.  The  difficulty  of  finding  this  answer  is 
easily  understood  if  we  consider  the  information  obtained  by  the  physical  recordings. 
The  initial  heat  per  gram  nerve  per  impulse  in  a  frog  sciatic  nerve  is  of  the  order  of 
magnitude  of  io~^  gcal.  The  chemical  reactions  involved  in  the  primary  event  must  take 
place  within  one-tenth  of  a  millisecond  or  less.  Reactants  in  a  process  of  such  a  high  speed, 
metabolized  in  amounts  of  such  a  small  order  of  magnitude,  cannot  be  measured  directly. 

Otto  Meyerhof's  pioneer  work  on  muscular  contraction  has  shown  how  much 
information  as  to  the  mechanism  of  cellulan function  may  be  obtained  by  the  study  of 
enzymic  reactions  and  by  correlating  them  with  events  recorded  with  physical  methods  on 
the  living  cell.  By  the  successful  linking  of  cellular  metabolism  and  function  Meyerhof's 
work  opened  new  pathways  and  was  perhaps  still  more  revolutionary  than  in  other  fields. 

It  was  under  the  inspiration  obtained  in  Professor  Meyerhof's  laboratory  that 
References  p.  93I95. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  I  81 

the  writer  has  tried  to  approach  the  problem  of  nerve  metaboKsm  in  relation  to  function 
in  a  way  similar  in  principle  to  that  which  had  proved  so  satisfactory  and  valuable  in 
the  study  of  muscular  contraction.  It  is  a  particular  pleasure  and  privilege  to  pay  tribute 
to  Professor  Meyerhof  at  the  occasion  to  which  this  volume  is  dedicated  by  reviewing 
some  aspects  of  this  work. 

Role  of  Acetylcholine  in  Conduction 

Since  the  discovery  of  the  powerful  pharmacological  effects  of  acetylcholine  by 
Reid  Hunt  and  Taveau^'  early  in  this  century,  the  compound  has  attracted  the 
attention  of  physiologists.  Observations  of  Magnus,  Dale,  Loewi,  Cannon  and  many 
others  suggested  that  acetylcholine  may  be  released  from  nerve  endings  and  act  as  a 
"mediator"  of  nerve  impulse  to  the  effector  organ.  There  were  many  difficulties  and 
contradictions  and  the  theory  of  chemical  mediation  encountered  increasing  opposition 
(Fulton^^,  Eccles^^). 

During  the  last  14  years  the  writer  and  his  associates  have  offered  evidence  indi- 
cating that  the  theory  in  its  original  form  has  to  be  modified.  Based  on  the  approach 
outlined  above,  a  great  variety  of  facts  have  accumulated  suggesting  that  the  release 
and  removal  of  acetylcholine  are  intracellular  processes^^^s^  They  seem  to  be  closely 
associated  with  the  alterations  in  the  active  membrane  which  occur  during  the  passage 
of  the  impulse.  The  transmitting  agent  is  the  flow  of  current  but  in  the  chain  of  events 
which  generate  the  "Stromchen"  the  acetylcholine-esterase  system  appears  to  play  an 
essential  role. 

The  important  data  have  recently  been  summarized  at  a  Symposium  on  the  physio- 
logical role  of  acetylcholine^^.  A  more  detailed  and  comprehensive  presentation  may  be 
found  in  the  textbook  on  Hormones^*.  It  may  suf&ce  to  mention  here  briefly  a  few 
essential  facts,  supporting  the  assumption  of  the  necessity  of  acetylcholine  in  conduction. 
Studies  on  the  enzyme  which  hydrolyses  acetylcholine,  acetylcholine-esterase,  have 
revealed  the  following  features:  i.  The  reaction  occurs  at  an  extremely  high  rate,  the 
"turnover  number"  is  20000000  per  minute  or  even  higher,  indicating  that  one  molecule 
of  ester  may  be  hydrolysed  in  3-4  miUionth  of  a  second^^  or  possibly  even  faster  (un- 
published data) .  This  high  speed  is  pertinent  for  any  assumption  correlating  a  chemical 
reaction  directly  with  the  electrical  manifestations  of  conduction.  2.  Acetylcholine- 
esterase  is  present  in  all  conducting  tissues  throughout  the  whole  animal  kingdom^^.  27_ 
3.  The  enzyme  is  localized  exclusively  in  the  surface  where  the  bioelectrical  phenomena 
occur.  This  is  in  contrast  to  many  other  enzymes  required  for  conduction,  as  for  instance 
the  respiratory  enzymes^.  4.  The  concentrations  of  the  enzyme  are  adequate  to  account 
for  an  amount  of  acetylcholine  metabolized  which  is  compatible  with  the  assumption  of 
an  essential  role  in  conduction.  5.  The  enzyme  in  conducting  tissues  has  a  number  of 
properties  by  which  it  may  be  easily  distinguished  from  other  esterases  occurring  in 
the  organism^^'  ^9.  Only  in  erythrocytes  the  same  type  of  esterase  is  found.  Since  the 
physiological  substrate  is  known  to  be  acetylcholine,  the  use  of  the  term  acetylchohne- 
esterase  for  this  enzyme  has  been  recently  proposed^". 

All  these  features  of  acetylcholine-esterase,  however  suggestive,  would  not  yet 
permit  the  assumption  of  its  essentiality  for  conduction.  The  enzyme  activity  has, 
however,  been  correlated  in  many  ways  with  the  electrical  events  of  conduction.  In 
experiments  on  the  electric  organ  of  Electrophorus  electricns  a  direct  proportionality  has 
been  established  between  the  voltage  of  the  action  potential  and  the  concentration  of 
References  p.  93I95. 
6 


82  D.  NACHMANSOHN  VOL.  4  (1950) 

acetylcholine-esterase  over  a  wide  range,  varying  from  0.5  to  22  volts  per  cm^i.  No  other 
enzyme  tested  shows  any  parallelism.  The  result  supports  the  assumption  of  a  close 
relation  and  interdependence  between  these  electrical  and  chemical  processes. 

Using  the  same  material,  it  has  been  shown  that  the  energy  released  by  the  break- 
down of  phosphocreatine  is  adequate  to  account  for  the  total  electrical  energy  released 
by  the  action  potential.  It  appears  probable  that  phosphocreatine  acts,  as  in  muscle, 
only  as  a  reserve  for  energy  rich  phosphate  and  that  the  breakdown  of  adenosine  tri- 
phosphate (ATP)  precedes  that  of  phosphocreatine.  In  contrast  to  muscular  contraction, 
however,  it  appears  for  many  reasons  unlikely  that  ATP  may  be  the  primary  reaction 
associated  with  conduction  ^3. 24  jf  ^^q  postulate  that  acetylcholine  may  be  directly 
associated  with  conduction  is  correct,  the  hydrolysis  of  the  ester  should  precede  the 
breakdown  of  ATP  and  the  energy  released  by  the  latter  used  for  the  synthesis  of  acetyl- 
choline. In  accordance  with  this  postulate,  an  enzyme,  choline  acetylase,  was  extracted 
from  brain  which  in  cell  free  solution  synthesizes  acetylcholine  using  the  energy  of 
^jp32,  33  It  Y^ras  the  first  demonstration  that  acetylation,  occurring  so  frequently  in 
intermediate  metabolism,  requires  ATP  energy  and,  more  generally,  that  ATP  energy 
may  be  used  outside  the  glycolytic  cycle,  in  which  its  crucial  role  had  been  shown,  first 
by  Meyerhof  and  his  associates  and  later  extended  by  the  work  of  Parnas,  the  Coris, 
Needham,  Szent-Gyorgyi  and  many  others. 

Finally  it  has  been  shown  with  a  great  variety  of  conducting  tissues,  nerve  and 
muscle,  that  inactivation  of  acetylcholine-esterase  by  specific  inhibitors  results  in  an 
abolition  of  conduction^''  ^4  jj^jg  effect  is  easily  reversible  with  compounds  which 
inhibit  the  enzyme  reversibly.  With  DFP,  an  inhibitor  which  inactivates  the  enzyme 
irreversibly,  the  abolition  of  conduction  becomes  irreversible.  However,  the  irreversible 
inactivation  of  the  enzyme  is  a  relatively  slow  process.  Its  rate  depends  on  a  great 
number  of  factors^^.  Therefore,  this  compound  was  particularly  suitable  for  testing  the 
essentiality  of  acetylcholine  in  conduction.  A  striking  parallelism  has  been  established 
in  nerves  exposed  to  DFP  between  the  progressive  inactivation  of  acetylcholine-esterase 
and  the  abolition  of  conduction  as  a  function  of  time  and  temperature.  In  no  way  is  it 
possible  to  dissociate  conduction  from  acetylcholine-esterase  activity^^'  ".  Claims  to  the 
contrary  were  shown  to  be  due  to  the  use  of  inadequate  techniques.  The  minimum 
amount  of  enzyme  required  for  unimpaired  conduction  is  relatively  small,  about  10% 
of  the  total  activity  present.  Considering  the  smallness  of  the  initial  heat,  the  remaining 
activity  is,  however,  still  adequate^.  The  excess  is  not  unusual  and  is  in*  accordance 
with  the  experience  with  other  enzymes,  but  it  led  to  some  misinterpretations  in  the 
early  phase  of  the  investigations. 

The  view  that  the  acetylcholine-esterase  system  is  essential  in  conduction  appears  to 
be  well  established.  The  precise  function  of  the  ester  is,  however,  unknown.  It  is  possible 
that,  during  activity,  a  higher  rate  of  collision  of  sodium  or  potassium  ions  with  the  ace- 
tylcholine-protein  or  lipoprotein  complex  leads  to  a  release  of  the  ester.  This  process  may 
be  an  essential  factor  in  the  alterations  of  the  membrane  proteins  leading  to  an  increased 
permeability.  The  possibility  of  a  rapid  removal  of  the  active  ester  by  acetylcholine- 
esterase  which  would  restore  the  resting  condition  permits  such  an  assumption.  No  other 
process  is  known  to  have  the  necessary  speed.  An  electrogen'c  action  of  the  ester  may  be 
demonstrated  in  electric  tissue,  as  will  be  discussed  later.  In  connection  w'th  the  great 
number  of  other  electrical  and  chemical  observations  the  hypothesis  appeals  worthy  of 
consideration.  In  this  connection,  the  experiments  reported  in  the  following  paper  on 
References  p.  93 195 ■ 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  I  83 

the  effect  of  inhibitors  of  acetylcholine-esterase  on  the  ion  permeability  are  also  of 
interest  although  still  far  from  conclusive. 

It  was  mentioned  above  that  the  esterase  in  the  red  blood  cell  has  the  same  charac- 
teristic features  as  the  esterase  in  conductive  tissue.  There,  too,  the  enzyme  is  localized 
exclusively  in  the  surface  membrane^^.  It  is  therefore  of  interest  that  Greig  and 
Holland*"  have  described  observations  suggesting  that  inhibitors  of  choline  ester 
splitting  enzymes  may  affect  the  permeability  of  red  blood  cells.  If  this  hypothesis  be 
confirmed,  it  will  be  another  support  for  the  assumption  of  a  similar  function  of  acetyl- 
choline in  the  neuronal  surface  membrane.  Analogies  as  to  the  permeability  of  these 
two  types  of  cells  have  long  been  known  to  physiologists. 

Difference  between  conduction  and  synaptic  transmission 

In  view  of  the  evidence  that  acetylcholine  has  an  essential  function  in  conduction 
it  appears  necessary  to  reconsider  the  role  of  the  ester  in  synaptic  transmission.  It  is 
the  purpose  of  this  article  to  analyse  the  question  how  the  earlier  observations,  suggesting 
the  theory  of  chemical  mediation,  may  be  integrated  into  the  picture  resulting  (I)  from 
the  enzyme  studies  and  (II)  from  the  attempt  to  correlate  the  chemical  and  physical 
events  of  nerve  activity. 

The  theory  of  chemical  mediation  was  based  essentially  on  two  facts:  i.  the  stimu- 
lating effect  of  acetylcholine  in  relatively  small  amounts  (a  few  //g)  upon  synaptic 
junctions,  and  2.  the  appearance  of  acetylcholine  in  the  perfusion  fluid  of  such  foci 
following  nerve  stimulation.  The  complete  inertness  of  the  fibre  to  acetylcholine  even 
if  applied  in  high  concentrations  (up  to  20  g  per  liter)  was  considered  as  definite  proof 
that  the  physiological  function  of  the  ester  is  limited  to  the  synapse. 

a)  Impermeability  of  the  axonal  surface  membranes  to  acetylcholine.  Studies  on  the 
permeability  of  the  axonal  surface  membranes  have  thrown  new  light  on  this  problem 
and  have  provided  a  satisfactory  explanation  for  the  discrepancy  between  the  earlier 
observations  and  the  conclusions  necessitated  by  the  enzymatic  studies.  The  investi- 
gations were  carried  out  on  the  giant  axon  of  Squid.  This  material  is  unusually  favourable 
in  view  of  the  large  diameter  (0.5  to  0.7  mm)  of  the  axon.  It  is  possible  to  extrude  the 
axoplasm  from  the  cell  interior  of  this  preparation  without  contamination  by  substances 
attached  to  the  outside  surface.  The  axoplasm  thus  obtained  may  be  analysed  for  com- 
pounds to  which  the  axon  has  been  exposed  for  various  periods  of  time.  In  this  way  the 
inside  concentration  of  these  compounds  and  if  desired  the  rate  of  penetration  may 
be  determined. 

It  was  found  that  those  inhibitors  of  acetylcholine-esterase  which  alter  and  abolish 
conduction,  like  eserine  and  DFP,  penetrate  into  the  axoplasm,  although  the  rates  of 
penetration  of  the  different  compounds  may  var^'  considerably^".  In  striking  contrast 
to  the  compounds  mentioned  prostigmine,  an  extremely  potent  inhibitor  of  acetylcholine- 
esterase,  does  not  affect  conduction  even  in  h^'gh  concentrations  (lO"^  M)^*.  This  com- 
pound was  not  found  in  the  axoplasm,  although  the  methods  used  were  highly  sensitive 
and  adequate  to  detect  an  extremely  small  fraction  of  the  concentration  of  the  com- 
pound present  on  the  outside.  The  experiments  show  that  the  axonal  surface  membranes 
are  impervious  to  prosfigTiine  and,  moreover,  that  the  site  of  the  acetylchoHne-esterase 
associated  with  conduction  must  be  inside  a  structural  barrier  which  makes  the  enzyme 
inaccessible  to  the  inhibitor.  Eserine  is  a  tertiary  amine  and  lipid  soluble,  prostigmine 
is  a  quaternary  ammonium  salt  and  lipid  insoluble.  It  appears  likely  that  the  difference 
References  p.  93/95. 


84 


D.  NACHMANSOHN 


VOL.  4  (1950) 


100 


50 


in  chemical  structure  and  properties  is  responsible  for  the  difference  in  permeability  of 
these  two  types  of  compounds.  Possibly  the  lipid  membrane,  known  to  surround  all 
axons,  whether  myelinated  or  not,  may  be  the  structural  barrier. 

Acetylcholine  like  prostigmine  is  a  methylated  quaternary  ammonium  salt.  The 
failure  of  acetylcholine  to  affect  conduction  was  explained  by  the  assumption  that  the 
axonal  surface  membrane  may  be  impervious  to  the  choline  ester.  This  assumption  has 
been  tested  directly  in  the  following  way.  The  axons  were  exposed  to  acetylcholine 
labelled  with  N^^.  High  concentrations  (20  gram  per  liter)  were  used.  When  the  axoplasm 
was  tested  for  the  presence  of  N^^,  only  insignificant  traces  were  present.  These  traces, 
moreover,  were  largely  accounted  for  by  the  contamination  of  the  acetylchoHne  used 
with  tertiary  amine  containing  N^^.  Tertiary  amine  labelled  similarly  with  isotopic  N 
penetrated  rapidly  and  an  equilibrium  between  the  inside  and  outside  concentration 
was  obtained  within  60  minutes*^.  Fig.  i  demonstrates  the  results  obtained. 

The  experiments  show  conclusively  that 
the  axonal  surface  membranes  are  im- 
pervious to  acetylcholine.  They  explain  why 
the  fibre  remains  inert  when  the  ester  is 
applied  externally,  even  in  high  concentra- 
tions. The  fact  that  the  action  of  the  ester  is 
limited  to  the  synaptic  junction  indicates 
that  the  active  membrane  may  be  reached 
at  these  foci  even  by  those  compounds  which 
do  not  penetrate  into  the  interior  of  the 
axon  or  the  muscle  fibre.  The  peculiar  abil- 
ity of  the  synapse  to  react  to  compounds 
which  do  not  affect  axonal  conduction  ap- 
pears thus  to  be  due  to  a  difference  in  ana- 
tomical structure.  This  applies  also  to  curare 
which,    as  recent  observations  have  shown 

(KlNG*2,    WiNTERSTEINER    AND    DuTCHER*^), 

has  as  active  principle  a  methylated  quater- 
nary ammonium  salt.  The  observation  of 
Claude  Bern.\rd  that  this  compound  acts 
exclusively  on  the  neuromuscular  junction 
and  does  not  affect  nerve  or  muscle  fibres 
was  for  a  century  the  basis  underlying  the  assumption  that  the  neuromuscular  junction 
has  special  properties.  It  seemed  to  support  the  view  that  the  fundamental  mechanism 
of  transmission  may  differ  from  that  of  conduction. 

On  the  basis  of  the  investigations  described,  the  schematic  presentation  of  the 
neuromuscular  junction  in  Fig.  2  may  serve  as  illustration  of  the  situation.  Only  the 
compounds  on  the  left  side  are  capable  of  acting  everywhere,  because  they  may  penetrate 
through  the  structural  barriers.  In  contrast,  the  compounds  on  the  right  side  act  only 
upon  the  post-synaptic  membrane  which  appears  to  be  either  less  or  not  at  all  protected. 
The  nerve  ending  itself,  although  not  surrounded  by  myelin,  appears  also  to  be  protected 
by  a  structural  barrier  since,  according  to  Bronk^*,  it  is  inexcitable  even  by  relatively 
high  concentrations  of  acetylcholine  in  the  perfusion  fluid. 

Recently  it  was  found  that  tetraeth}  1  pyrophosphate  (TEPP)  does  not  affect  con- 

References  p.  93l95- 


y 
y 
y 

y 
y 

^^^' 

/ 

/ 
/ 

y 

y 

15 


25 


60 
Min.  of  exposure 

Fig.  I.  Rate  of  penetration  of  trimethylamine 
and  acetylcholine  labelled  with  N^*  into  the 
interior  of  the  giant  axon  of  Squid.  The  ratio 
of  the  concentration  of  the  N  of  these  com- 
pounds inside  (Ci)  to  th9,t  outside  (Co)  is 
plotted  against  the  time  of  exposure  in  mi- 
nutes. The  dotted  line  indicates  the  rate  of 
penetration  of  N  on  exposure  to  trimethyl- 
amine (286  fig  N  per  ml),  the  straight  line, 
that  of  the  N  found  on  exposure  to  acetylchol- 
ine (1430  /ig  N  per  ml  of  which  55  /ig  were 
non-quaternary  N)*^. 


VOL.  4  (1950) 


PERMEABILITY   AND    NERVE    FUNCTION,  I 


85 


Mefhylafed  quaiernary 

ammonium  salfs 

Carer  ine 

Aceiylcholine 

Prosfigmine 


Active  membrane 


Posfsynapiic  membrane 


Structural  barrier 


Fig.  2.  Scheme  of  the  neuromuscular  junction.  A 
structural  barrier  protects  nerve  and  muscle  fibre 
against  the  action  of  methylated  quaternary  am- 
monium salts.  These  compounds  act  only  on  the 
postsynaptic  membrane,  which  apparently  is  either 
less  or  not  at  all  protected.  Other  compounds,  like 
eserine,  DFP,  strychnine,  and  procaine,  being  able 
to  penetrate  through  the  structural  barrier,  act  upon 
the  active  membrane  of  nerve   and    muscle  fibre^^. 


duction*'^.  TEPP  is  an  extremely  potent  inhibitor  of  acetylcholine-esterase,  much  more 
powerful  than  eserine,  prostigmine  and  DFP.  TEPP  inactivates  the  enzyme  irreversibly 
like  DFP  but  this  effect  is  immediate,  in  contrast  to  the  slowly  progressive  action  of 
]3pp46_  Nevertheless,  in  a  frog  sciatic 
nerve  exposed  to  TEPP  in  concentrations 
(2  mg  per  ml)  several  thousand  times  as 
high  as  those  required  to  inactivate 
completely  and  irreveisibly  the  enzyme 
in  solution,  conduction  remains  intact. 
This  suggests  that  the  acetylcholine- 
esterase  retains  its  activity.  Under  the 
same  conditions  DFP  which  penetrates 
into  the  interior  abolishes  conduction 
and  enzyme  irreversibly,  although  it  is 
thousand  times  less  potent  as  inhibitor. 
The  only  apparent  explanation  for  the 
failure  of  TEPP  to  penetrate  into  the 
axon  is  its  insolubility  in  lipid.  Since 
this  property  applies  also  to  methylated 
quaternary  ammonium  salts,  the  as- 
sumption gains  further  support  that 
the  structural  barrier  may  be  a  lipid 
membrane  surrounding  nerve  and 
muscle  fibre  but  absent  at  the  post-synaptic  membrane  of  synaptic  junctions.  But 
whatever  the  anatomical  location  and  the  chemical  nature  of  the  barrier  may  finally 
turn  out  to  be,  it  is  of  decisive  importance  to  recognize  its  existence.  The  barrier  has 
not  been  identified  morphologically  but  has  to  be  postulated  on  the  basis  of  the 
physico-chemical  and  enzyme  studies  described. 

It  has  been  reported  that  intact  nerves  may  split  at  least  25%  or  more  of  the  acetyl- 
choline which  may  be  hydrolyzed  during  the  same  period  by  the  ground  nerve*'.  On 
the  basis  of  this  result,  it  was  concluded  that  acetylcholine  may  penetrate  into  the 
interior.  Since  it  has  been  shown  that  acetylcholine  does  not  penetrate  into  the  axon, 
even  if  applied  in  high  concentrations,  the  more  likely  conclusion  from  this  observation 
is  the  location  of  part  of  the  enzyme  outside  the  barrier.  It  has  never  been  claimed  that 
all  the  esterase  present  is  inside  and  necessary  for  conduction.  The  experiments  reported*' 
were  carried  out  with  the  manometric  technique  in  which  the  CO2  output  is  measured. 
There  has  recently  been  introduced  by  Hestrin  a  new  simple  and  rapid  chemical  method 
which  makes  possible  a  direct  determination  of  the  acetylcholine  removed  by  hydroly- 
sis**. This  method  is  based  upon  the  reaction  of  0-acyl  groups  with  hydroxylamine  in 
alkaline  medium.  It  is  more  specific  than  the  manometric  method,  especially  when 
large  amounts  of  tissue  are  necessary  and  simultaneous  chemical  reactions  cannot  be 
excluded.  Using  this  method  it  has  been  found  that  the  acetylcholine-esterase  activity 
of  the  ground  nerve  is  about  twice  as  high  as  the  manometric  method  indicates.  The 
intact  nerve  splits  acetylcholine  at  a  rate  which  is  only  a  small  fraction  (about  5  to  7%) 
of  the  total  activity*^.  This  activity  is  suppressed  by  prostigmine  which  like  acetyl- 
choline does  not  penetrate  into  the  interior.  Complete  inhibition  of  this  enzyme  activity 
does  not  affect  conduction.  The  meaning  of  the  small  amount  of  esterase  on  the  outside 
References  p.  93I95. 


86  D.  NACHMANSOHN  VOL.  4  (1950) 

of  the  barrier  is  not  clear.  The  activity  may  be  due  to  an  unspecified  esterase  other  than 
acetylcholine-esterase  or  to  the  presence  of  small  blood  vessels,  microscopic  muscle 
fibrils  or  cut  nerve  fibres  where  the  surface  may  be  reached  by  the  ester.  This  is,  how- 
ever, entirely  irrelevant  for  the  major  problem  involved. 

The  elucidation  of  the  situation  became  possible  by  the  fortunate  circumstance  that 
so  many  different  kinds  of  extremely  potent  inhibitors  of  acetylcholine-esterase  were 
available:  reversible  and  irreversible  types  of  inhibitors  and  in  each  of  the  two  groups 
compounds  which  penetrate  and  others  which  do  not  penetrate.  This  combination  made 
it  possible  to  find  a  satisfactory  answer  to  some  of  the  most  pertinent  questions  involved: 
I.  the  necessity  of  acetylcholine-esterase  for  conduction;  2.  the  existence  of  a  barrier 
for  methylated  quaternary  ammonium  salts,  and  3.  the  localization  of  the  enzyme  in 
respect  to  the  barrier. 

Even  if  a  compound  affects  both  axon  and  synapse,  there  may  still  be  a  great 
difference  as  to  the  concentration  required.  Chemical  substances  may  act  upon  the 
apparently  unprotected  active  surface  of  the  post-synaptic  membrane  in  concentrations 
much  smaller  than  those  necessary  for  affecting  the  nerve  or  muscle  fibre.  An  interesting 
illustration  is  provided  by  the  experiments  of  Roeder  and  his  associates*®,  who  found 
that  DFP  abolishes  synaptic  transmission  in  much  lower  concentrations  than  those 
which  affect  conduction.  DFP  is  very  lipid  soluble  and  may  therefore  accumulate  in 
the  myeline  sheath  to  a  certain  concentration  before  penetrating  into  the  aqueous 
interior  of  the  fibre  in  concentrations  sufficiently  high  to  inactivate  the  enzyme  and, 
consequently,  to  abolish  conduction.  At  the  time  when  conduction  disappears,  the 
concentration  of  DFP  is  small  in  the  axoplasm  compared  with  that  in  the  outside 
fluid^^.  This  finding  supports  the  assumption  that  the  concentration  of  DFP  at  the  site 
of  action  may  be  small  and  is  consistent  with  the  potency  of  the  compound  as  inhibitor 
of  acetylcholine.  The  necessity  of  a  high  outside  concentration  may  be  attributed  to 
the  relatively  slow  rate  of  penetration.  In  the  case  of  eserine,  the  distribution  between 
inside  and  outside  at  the  same  period,  i.e.,  at  the  time  when  the  action  potential  has 
disappeared,  is  very  different.  The  rate  of  penetration  will  be  determined  by  the  pro- 
perties of  the  various  chemical  compounds  on  the  one  hand  and  by  the  properties  of 
the  various  surface  membranes.  Additional  factors  may  be  of  importance,  such  as  the 
affinity  of  the  compound  to  the  enzyme,  its  potency  as  inhibitor  and  the  kinetics  of  the 
inhibition.  In  view  of  the  complexity  of  the  process,  it  is  not  surprising  that  in  applying 
potent  inhibitors  of  acetylcholine-esterase,  the  phenomena  observed  may  differ  sharply 
in  so  many  respects,  although  the  underlying  cause  is  the  same  chemical  reaction. 

The  action  of  procaine,  one  of  the  compounds  marked  on  Fig.  2,  requires  comment. 
The  blocking  of  conduction  by  this  and  other  similar  anaesthetics  cannot  be  explained 
in  terms  of  acetylcholine-esterase  inhibition.  These  compounds  are  weak  inhibitors  of 
acetylcholine-esterase,  although  other  esterases  may  be  affected  more  strongly^". 
Thimann^^  has  pointed  out  that  these  compounds  have  some  resemblance  in  structure 
to  acetylcholine,  but  are  tertiary  amines.  They  will,  therefore,  easily  penetrate  into 
the  interior  and  they  may  act  competitively  with  the  ester  on  some  proteins  or  lipo- 
proteins of  the  membrane.  Since  apparently  they  do  not  depolarize  the  membrane^^^ 
it  is  possible  to  assume  that  they  form  a  complex  but,  in  contrast  to  acetylcholine,  they 
do  not  change  the  condition  of  the  protein.  However,  they  may  prevent  the  action 
of  the  ester  released  and  thereby  block  conduction,  whereas  otherwise  the  resting 
condition  may  remain  unchanged.  This  is  consistent  with  the  apparent  failure  of  cocaine, 
References  p.  93I95. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  I  87 

described  in  the  following  paper,  to  produce  a  significant  change  in  permeability. 

b)  Release  of  acetylcholine  during  activity.  In  view  of  the  permeability  studies 
described,  the  limitation  of  the  action  of  acetylcholine  to  the  synapse,  if  the  ester  is 
applied  externally,  cannot  be  used  as  an  indication  for  a  special  role  at  this  junction, 
as  was  proposed  by  the  theory  of  chemical  transmission.  For  the  same  reason,  the  second 
fact  on  which  the  hypothesis  was  built  has  to  be  reconsidered.  The  appearance  of  acetyl- 
choline in  the  perfusion  fluid  of  the  synapse  following  nerve  stimulation  must  be  attrib- 
uted to  the  absence  of  an  insulating  membrane.  If  acetylcholine  cannot  pass  through 
the  structural  barrier  into  the  interior,  it  will  not  be  able  to  leak  from  the  inside  to  the 
outside  in  stimulated  nerve  and  muscle  fibres.  The  only  site  where  such  leakage  will 
be  possible  is  the  postsynaptic  membrane.  However,  even  at  the  synaptic  junction  the 
ester  does  not  appear  under  physiological  conditions.  Dale  and  his  associates  have 
repeatedly  emphasized  that  the  ester  appears  in  their  experiments  only  if  the  normal 
mechanism  responsible  for  the  rapid  removal  of  the  ester,  viz.,  acetylcholine-esterase,  is 
largely  inactivated  by  the  presence  of  eserine.  Even  in  presence  of  the  drug,  the  amounts 
leaking  out  were  extremely  small,  about  one  hundred-thousandth  of  that  required  to 
set  up  a  stimulus.  On  the  basis  of  more  recent  experiments,  in  which  acetylcholine  was 
applied  directly  to  the  motor  end  plate,  the  difference  was  of  the  same  order  of  magni- 
tude. Such  a  difference  is  not  easily  explained  in  terms  of  chemical  mediation.  It  is 
true  that  in  Loewi's  original  observations  on  the  frog  heart,  no  eserine  was  present. 
However,  considerable  difficulties  were  encountered  by  him  as  well  as  other  investigators 
when  they  tried  to  reproduce  the  appearance  of  the  ester.  For  this  reason,  Loewi's 
theory  was  repeatedly  criticized^^'  ^*.  When  a  heart  preparation  has  been  perfused  for 
a  certain  period  of  time  with  Ringer's  solution,  the  post-synaptic  membrane  may  not 
be  in  a  completely  normal  condition  and  may  therefore  permit  leakage  of  the  compound, 
which  under  physiological  conditions  may  be  rapidly  inactivated.  The  condition  of  the 
membrane  may  depend  upon  a  variety  of  factors,  such  as  the  length  of  the  perfusion 
period,  the  composition  of  the  perfusion  fluid,  the  condition  and  the  species  of  the  frog 
used,  etc.  Variations  of  these  factors  may  explain  the  difficulties  encountered  by  a 
number  of  investigators  who  tried  to  reproduce  this  observation.  The  same  consideration 
may  be  applied  to  the  finding  of  Kibjakow^^,  who  in  1932  described  the  appearance  of 
acetylcholine  in  the  perfusion  fluid  of  the  synaptic  ganglion  in  absence  of  eserine.  His 
observations  were  questioned  by  Dale's  school,  but  it  is  conceivable  that  with  the  less 
perfect  perfusion  technique  in  Kibjakow's  experiments,  the  active  membrane  suffered 
more  damage  and  thus  permitted  the  leakage  of  traces  just  in  the  measurable  range. 
So  far  there  is  no  conclusive  evidence  that  the  appearance  of  the  ester  outside  the  cell 
is  a  physiological  event. 

It  is  an  interesting  psychological  phenomenon,  encountered  frequently  in  the  pro- 
gress of  science  as  well  as  in  the  work  of  individual  investigators,  that  certain  observa- 
tions are  neglected  or  even  discarded  because  they  are  inconvenient,  puzzling  and  do 
not  fit  into  preconceived  ideas.  Later,  when  the  views  have  changed,  the  facts  may 
suddenly  gain  significance  and  it  becomes  possible  to  integrate  them  into  the  general 
picture.  The  release  of  acetylcholine  at  the  synapse  assumes  a  new  aspect  if  considered 
in  connection  with  other  pertinent  observations  which  at  the  time  of  their  presentation 
did  not  find  sufficient  attention. 

In  1933,  simultaneously  with  or  even  prior  to  the  finding  of  Dale  that  acetylcholine 
appears  in  the  perfusion  fluid  of  the  sympathetic  ganglion  or  of  the  neurc muscular  junc- 
References  p.  93I95. 


88  D.  NACHMANSOHN  VOL.  4  (1950) 

tion,  Calabro^^  had  shown  that,  following  prolonged  stimulation  of  the  rabbit  vagus, 
an  acetylcholine-like  substance  is  released  from  the  cut  end  into  the  surrounding  fluid. 
BiNET  AND  MiNz"  found,  in  1934,  that  from  the  transsected  surface  of  nerves  a  compound 
is  released  which  increases  the  sensitivity  of  the  leech  muscle  to  acetylcholine.  Calabro's 
findings  were  confirmed  and  extended  by  Bergami^^  and  by  Babski  and  Kisljuk^*'  ^^. 
In  1937  VON  Muralt^^  described  a  diffeience  of  the  acetylcholine  content  between 
stimulated  and  unstimulated  nerves.  In  view  of  the  possibility  of  a  very  rapid  disap- 
pearance of  the  active  ester,  he  developed  a  special  technique  by  which  he  "shot"  the 
nerves  into  liquid  air.  Tested  by  bioassay  after  a  short  period  of  extraction  the  amount 
of  acetylcholine  was  0.2  jug  per  gram  in  the  stimulated  as  compared  with  0.12  //g  per 
gram  in  the  control  nerve.  In  a  large  series  of  experiments  the  difference  between 
stimulated  and  control  nerve  was  later  found  to  be  0.09  //g  per  gram^-.  However,  the 
difference  between  the  two  nerves  disappears  if  extraction  is  continued  for  a  longer 
period  of  time.  There  is,  therefore,  some  uncertainty  as  to  the  interpretation.  It  is 
conceivable  that  the  acetylcholine  released  from  its  complex  is  present  in  a  free  form 
and  therefore  diffuses  from  the  frozen  tissue  during  extraction  more  rapidly  than  that 
part  of  the  acetylcholine  which  is  bound  to  protein  or  lipoprotein. 

Even  in  sensory  nerves  release  of  acetylcholine  has  been  demonstrated  by  Brecht 
AND  Corsten^^  from  the  cut  end  after  stimulation.  These  investigators  used  a  remarkably 
sensitive  method,  the  contraction  of  the  frog  lung  in  presence  of  eserine,  and  hereby 
succeeded  in  detecting  the  ester  released.  The  amounts  are  still  smaller  than  those 
released  from  motor  nerves,  but  this  difference  appears  consistent  with  the  smaller  rate 
of  metabolism  indicated  by  the  lower  concentrations  of  acetylcholine-esterase  and 
choline  acetylase".  It  is  significant  that  the  release  of  acetylchoHne  has  been  demon- 
strated in  parasympathetic,  motor  and  sensory  nerve  fibres.  The  situation  is  pertinent 
in  connection  with  the  finding  that  the  enzymes  which  form  and  hydrolyse  acetylcholine 
are  present  in  all  types  of  nerves  and  that  the  inactivation  of  acetylcholine-esterase 
invariably  leads  to  abolition  of  conduction. 

The  facts  described  support  the  assumption  that  there  is  no  difference  in  principle 
between  the  release  of  acetylcholine  at  the  synapse  and  in  the  axon,  except  that  in  the 
latter  case  the  ester  cannot  pass  through  the  structural  barrier.  They  make  it  appear 
still  more  probable  that  this  release  is  an  intercellular  process  and  that  the  appearance 
outside  the  cell  at  the  synapse  must  be  attributed  either  to  the  poisoning  of  the  enzymic 
mechanism,  normally  preventing  the  leakage  or  to  some  other  damage  of  the  active 
surface  where  it  is  least  protected  and  most  vulnerable.  At  the  time  when  these  findings 
were  described,  acetylcholine  was  considered  to  be  a  chemical  mediator  and  since 
chemical  transmission  in  the  axon  is  inconceivable,  it  was  difficult  to  integrate  them 
into  the  general  picture.  Little  or  no  attention  was  consequently  paid  to  these  findings. 
Von  Muralt  has  been  very  cautious  in  his  statements  as  to  the  possible  significance 
of  the  release  of  acetylcholine  in  the  nerve  fibre.  He  called  the  ester  an  "Aktions- 
substanz",  meaning  that  it  may  be  important  like  many  other  substances  for  nerve 
activity  in  the  axon  as  well  as  at  the  synapse.  This  caution  was  well  justified  at  a  time 
when  nothing  was  known  about  the  high  speed  of  the  reaction,  the  effects  of  acetyl- 
choline-esterase inhibitors  on  conduction  and  the  great  variety  of  other  factors 
known  today.  These  facts  had  to  be  established  before  it  became  possible  to  assume 
a  direct  association  of  the  ester  with  the  generation  of  the  electric  currents  which 
propagate  the  impulse.  In  the  light  of  recent  developments,  however,  the  situation 

References  p.  93/9 5. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  I  89 

has  changed.  The  demonstration  of  the  release  of  acetylchoHne  in  the  axon  appears 
as  relevant  as  that  at  the  synaptic  junction  and  requires  a  modification  of  the  original 
interpretation. 

The  structural  barrier  for  acetylcholine  present  in  the  fibre  and  its  absence  in  the 
post-synaptic  membrane  may  be  considered  as  the  main  reason  that  the  attention  of 
many  physiologists  was  focused  for  such  a  long  time  on  the  synapse  only.  Very  little 
is  known  concerning  the  properties  of  the  barrier  and  the  factors  affecting  it.  The 
observations  on  the  permeability  of  neuronal  surface  membranes  described  in  this  and 
the  following  paper  are  only  an  initial  phase  in  the  attempt  of  analysing  the  problem. 
Its  importance  can  hardly  be  overemphasized,  not  only  for  the  understanding  of  the 
cellular  mechanism  but  of  the  pharmacology  and  pathology  of  the  nervous  system  as 
well.  The  development  of  new  drugs  may  be  greatly  facilitated  if  the  structural  factors 
determining  the  permeability  and  the  rate  of  penetration  are  known.  In  many  cases  an 
action  may  be  desirable,  preferably  or  exclusively,  on  the  synapse,  in  others,  upon  both 
axon  and  synapse. 

The  existence  of  structural  barriers  and  the  great  variations  of  their  properties 
may  account  for  the  many  obstacles  encountered  and  the  many  contradictory  reports 
when  the  two  criteria  of  chemical  mediation  were  applied  to  different  types  of  synapses. 
The  unnumerable  differences  of  anatomical  structure,  the  biochemical  composition  of 
the  surrounding  medium  and  many  other  accessory  conditions  must  be  essential  in 
determining  the  action  of  acetylcholine  when  applied  externally.  These  variations  do 
not  permit  the  assumption  that  the  fundamental  physico-chemical  mechanism  of  the 
propagation  of  the  nerve  impulse  may  not  be  the  same.  In  view  of  the  physico-chemical 
properties  of  acetylcholine  and  similar  N-methylated  compounds,  the  difficulties  will 
become  nearly  insurmountable  in  the  study  of  brain  and  spinal  chord  which  contain 
large  amounts  of  lipid.  It  is  not  surprising  that  the  painstaking  efforts  to  demonstrate 
or  to  disprove  the  "cholinergic"  nature  of  synapses  .in  brain  and  spinal  chord  have 
resulted  in  a  most  unsatisfactory  and  confusing  picture. 

In  contrast  the  conflicting  results  obtained  when  the  "cholinergic"  nature  of 
synapses,  especially  in  brain,  is  tested  by  the  usual  criteria  of  chemical  mediation,  the 
approach  based  on  the  study  of  the  enzymes  connected  with  acetylcholine  metabolism 
and  their  correlation  with  function  did  not  encounter  comparable  difficulties.  All 
results  obtained  in  this  way  indicate  the  generality  of  the  role  of  acetylcholine  in  all 
conducting  tissues,  including  that  of  brain  and  spinal  chord^^. 

c)  Basic  similarity  between  conduction  and  transmission.  At  the  Symposium  on  the 
synapse,  in  1939,  Erlanger^*  scrutinized  the  problem  whether  the  electrical  charac- 
teristics of  synaptic  transmission  are  basically  different  from  those  which  may  be 
observed  on  the  axon.  His  data  indicate  that  the  electrical  phenomena  considered  to  be 
pecularities  of  the  synapse  may  be  demonstrated  on  fibres,  z;z^.,  latency,  one-way  trans- 
mission, repetition,  temporal  summation  and  facilitation,  and  transmission  of  the  action 
potential  across  a  non-conducting  gap.  The  facts  based  on  the  electrical  signs  of  nerve 
activity  make  it  unnecessary  to  assume  that  any  condition  exists  at  the  synapse  which 
differs  in  principle  from  that  found  in  the  peripheral  axon,  except  in  quantitative  respect. 

Ten  years  have  passed.  During  that  time  extensive  investigations  have  been  made 
on  the  electrical  characteristics  of  tranrmission  across  the  natural  and  artificial  synapse 
(ephapse).  From  the  work  of  many  investigators,  mainly  Arvanitaki^^'  ^^,  Bullock^^ 
EccLES^^,  Granit  and  Skoglund^  and  others  considerable  evidence  has  accumulated 
References  p.  9 3(9 5. 


90  D.  NACHMANSOHN  VOL.  4  (1950) 

in  support  of  Erlanger's  views  that  the  basic  mechanism  of  transmission  and  con- 
duction is  the  same,  the  propagating  agent  being  in  both  cases  the  flow  of  current. 
According  to  Eccles^^,  impulses  travelling  down  the  pre-synaptic  fibre,  generate  a 
current  which  produces  in  the  synaptic  membrane  of  the  post-synaptic  cell  an  anodal 
focus  with  cathodal  surround;  this  is  followed  in  a  second  phase  by  a  more  intense 
cathodal  focus  with  anodal  surround.  The  cathodal  focus  sets  up  a  local  response  from 
which  a  catelectrotonus  spreads  over  the  post-synaptic  cell  membrane.  The  catelectro- 
tonus,  the  end  plate  potential,  sets  up  a  propagated  impulse  in  the  post-synaptic  cell 
as  soon  as  a  certain  threshold  is  reached.  The  sequence  of  events  is  similar  to  that 
observed  on  artificial  synapses  and.  on  a  single  unit  preparation  of  the  synapse,  the 
stellate  ganglion  of  Squid  (Bullock^').  Since  the  electrical  signs  and  the  biochemical 
data  favor  the  assumption  that  the  mechanism  of  transsynaptic  transmission  is  basically 
the  same  as  that  of  conduction,  it  follows  that  the  role  of  acetylcholine  in  these  mecha- 
nisms is  most  likely  the  same.  In  both  cases  the  propagating  agent  is  the  flow  of  current, 
but  the  release  and  the  removal  of  acetylcholine  must  be  essential  events  in  the  alteration 
of  the  pre-  and  post-synaptic  membrane  during  the  flow  of  current  across  the  synaptic 
region  and  the  generation  of  the  end  plate  potential.  It  would  be  difficult  to  picture 
these  currents  as  being  different  in  nature  from  those  in  the  axons.  A  few  biochemical 
data  may  be  mentioned  in  this  connection  which  support  the  assumption  of  a  high  rate 
of  acetylcholine  metabolism  in  the  post-synaptic  membrane  of  the  motor  end  plate. 
CouTEAUX  AND  Nachmansohn^^'  ®^^  found  that,  following  the  section  of  the  sciatic 
nerve  of  guinea  pigs,  the  high  concentration  of  acetylcholine-esterase  of  the  motor  end 
plates  of  the  gastroncemius  decreases  only  slightly.  Within  three  to  four  weeks  after 
the  operation  one-fourth  or  possibly  less  of  the  enzyme  concentration  had  disappeared. 
Then  the  activity  remains  constant  for  many  months.  This  result  suggests  that  three 
quarters  of  the  enzyme  or  more  is  localized  in  the  post-synaptic  membrane,  the  "sole 
plate"  of  KiJHNE,  a  pure  muscular  element  which  persists  after  the  disappearance  of  all 
nerve  elements. 

The  electric  organs  have  physiologically  evolved  from  striated  muscle.  The  electric 
plates  are  homologous  with  the  motor  end  plate.  The  discharge  of  these  organs  is  homo- 
logous with  the  end  plate  potential.  Recent  studies  of  Couteaux^^^  have  revealed  that 
the  post-synaptic  membrane  of  the  motor  end  plate  is  morphologically  a  very  peculiar 
structure.  By  using  Janus  green  or  methyl  violet,  he  demonstrated  a  striking  similarity 
with  the  electrolemma  of  the  electric  plate  surrounding  the  nerve  endings.  The  direct 
proportionality  between  the  voltage  developed  during  the  discharge  and  the  concen- 
tration of  acetylcholine-esterase  observed  in  the  electric  tissue  suggests  a  high  rate  of 
acetylcholine  metabolism  associated  with  the  end  plate  potential. 

These  findings  alone  without  all  the  other  evidence  accumulated  would  not  neces- 
sarily imply  that  the  acetylcholine  is  released  in  the  post-synaptic  membrane  itself. 
The  following  observations  are,  however,  of  interest  in  this  connection.  The  discovery 
of  the  extraordinarily  high  concentration  of  acetylcholine-esterase  in  electric  tissue  made 
possible  the  assumption  that  acetylcholine  might  be  the  agent  that  produces  the  depolari- 
zation presumably  occurring  during  the  action  potential.  The  possibility  of  a  depolarizing 
action  of  acetylcholine  has  been  considered  by  Dubuisson  and  Monnier'^"  and  Cowan'^. 
In  1938,  when  the  prerequisite  for  such  a  theory,  namely  the  high  speed  of  destruction 
of  the  active  agent  appeared  established.  Auger  and  Fessard  tested  the  effect  of  eserine 
on  the  discharge  of  the  electric  tissue  of  Torpedo  marmorata'^.  As  may  be  seen  in  Fig.  3 
References  p.  93/95. 


VOL.  4  (1950) 


PERMEABILITY   AND    NERVE    FUNCTION,  I 


91 


the  height  of  the  potential  is  markedly  depressed  in  presence  of  eserine.  The  duration 
of  the  descending  phase  is  considerably  prolonged.  This  effect  of  eserine  on  the  end  plate 
potential  is  consistent  with  the  assumption  that 
the  appearance  and  the  removal  of  acetylcholine 
within  the  post-synaptic  membrane  may  be  essen- 
tial for  the  generation  of  the  potential. 

In  view  of  their  corresponding  biochemical  and 
bioelectrical  findings,  Fessard  and  Nachmansohn 
decided  then  to  test  whether  acetylcholine  injected 
into  the  electric  organ  may  produce  an  action 
potential.  Such  an  electrogenic  effect  might  be 
expected  if  acetylcholine  is  the  compound  which  is 
responsible  for  the  alterations  of  the  membrane, 
occurring  during  the  discharge.  In  experiments 
carried  out  at  Arcachon  in  1939  on  Torpedo  mar- 
mot ata,  in  which  they  were  joined  by  Feldberg, 
they  were  able  to  demonstrate  that  acetylcholine  has  an  electrogenic  effect'^'  '*.  The 
arterial  injection  of  acetylcholine  caused  potential  changes  similar  to  the  natural  dis- 
charge. However,  the  changes  were  small  and  slow  and  very  large  amounts  were  neces- 
sary for  the  effect.  Fig.  4  illustrates  the  effects  of  acetylcholine  injected  in  amounts 
varying  between  5  and  200  //g.  5  jjg  had  no  effect.  With  200  //g  the  potential  difference 
was  about  0.7  millivolts  and  the  descending  phase  had  not  yet  reached  the  base  line 
after  several  seconds.  If  the  acetylcholine  is  injected  in  presence  of  eserine,  preventing 
a  too  rapid  destruction  of  the  ester,  the  effects  are  greatly  enhanced.  Fig.  5  shows  that 
under  these  conditions  an  effect  may  be  obtained  even  with  2.5  //g  of  acetylcholine. 
With  10  /<g  the  potential  change  produced  is  greater  than  3  millivolts,  although  the 

duration  is  still  about  3  seconds. 


Fig.  3.  Effect  of  eserine  on  the  dis- 
charge of  electric  tissue  of  Torpedo 
marmorata.  The  fully  drawn  line  shows 
the  discharge  in  absence,  the  dotted 
line  in  presence  of  eserine^. 


I ' ^  J.     L 


m 


T 


Fig.  4.  Potential  changes  produced  by 
intraarterial  injection  of  acetylcholine  into 
the  electric  organ  of  Torpedo  marmorata. 
I,  II,  IV  and  V  correspond  to  the  injection 
of  200,  100,  20  and  5  /<g  of  the  ester; 
whereas  at  III  only  perfusion  fluid  was 
injected.  Between  II  and  III  the  sensi- 
tivity has  been  increased  fourfold.  0.5 
millivolt  indicated  at  I,  o.i  millivolt  at 
IV.  Time  in  seconds. 


Fig.  5.  Potential  changes  produced  in  the 
same  way  as  in  Fig.  4  but  in  presence  of 
eserine.  I,  II  and  IV  correspond  to  the  injec- 
tion of  10,  5  and  2.5  /ig  of  acetylcholine;  at 
III  only  perfusion  fluid  was  injected.  0.5 
millivolt  indicated  at  II.  Time  in  seconds. 


The  experiments  show  that  the  ester  may  produce  an  alteration  of  the  membrane 
preceding  the  flow  of  current.  They  support  the  view  that  the  ester  plays  an  essential 
role  in  the  generation  of  the  current  and  make  it  difficult  to  assume  that  the  release  of 
References  p.  93I95. 


92  D.  NACHMANSOHN  VOL.  4  (1950) 

acetylcholine  may  occur  in  the  recovery  period.  In  that  case  it  would  be  hard  to  under- 
stand how  the  compound  produces  current.  Although  the  potential  changes  resemble 
the  normal  discharge,  there  is,  however,  a  most  striking  contrast  in  two  respects:  the 
smallness  of  the  voltage  and  the  1 000  fold  increase  of  the  duration.  The  normal  discharge 
occurs  in  2  to  3  milliseconds;  the  voltage  of  a  single  unit  is  about  100  millivolts.  Although 
a  quantitative  evaluation  is  impossible  since  the  number  of  units  in  series  reached  by  the 
intraarterial  injection  is  uncertain,  the  discrepancy  as  to  duration  and  strength  is  enor- 
mous, even  in  presence  of  eserine.  The  method  used  is  crude  compared  to  the  effect  which 
might  be  expected  if  the  compound  were  released  from  the  nerve  ending.  In  that  case  it 
would  reach  the  opposite  surface  much  faster,  but  in  view  of  the  relatively  large  amounts 
injected,  of  which  apparently  at  least  a  fraction  reaches  the  active  membrane,  the 
response  is  small  beyond  all  proportion.  It  thus  becomes  difficult  to  conceive  that 
physiologically  the  substance  is  released  from  the  nerve  ending  and,  penetrating  the  inter- 
cellular space,  produces  the  end  plate  potential.  This  difficulty  does  not  arise  if  it  be 
assumed  that  the  release  and  the  removal  of  the  ester  are  intracellular  events  which 
do  not  involve  any  diffusion  but  occur  in  the  post-synaptic  membrane  and  generate 
there  the  flow  of  current. 

If  locally  supplied  energy  is  necessary  for  the  small  electric  currents  which  propagate 
the  impulse  along  the  axon  as  postulated  by  Keith,  Lucas,  and  Adrian,  it  appears 
almost  certain  that  such  energy  will  be  required  for  the  generation  of  a  potential  in  the 
second  unit.  The  flow  of  current  reaching  the  post-synaptic  membrane  may  result  in 
a  release  of  acetylcholine  which  may  act  as  a  trigger  in  the  chain  of  events  and  supplj' 
the  energy  for  building  up  the  end  plate  potential.  It  is  remarkable  that  exactly  this 
mode  of  action  has  been  proposed  by  Lapicque'^  in  1936 — "I'etat  d'excitation  suscite 
dans  la  sole  nucleee  pent  y  declencher  une  reaction  auxiliaire  venant  fournir  le  supple- 
ment de  puissance  requise.  Tel  serait  le  role  de  I'acetylcholine;  c'est  exactement  le  role 
que  joue  I'amorce  dans  la  technique  des  explosifs  ...  La  production  de  I'acetylcholine 
serait,  dans  cette  conception,  situee,  non  entre  le  nerf  et  le  muscle,  mais  dans  le  muscle 
lui-meme,  auquel  appartient  sans  conteste  la  sole  nucleee.  II  s'agirait  done  strictement 
parlant,  non  d'un  intermediaire  dans  la  transmission  de  I'excitation  entre  nerf  et  muscle, 
mais  d'un  premier  stade,  formant  relais  dans  I'excitation  musculaire  pour  assurer  sa 
generalisation  a  toute  la  masse  du  myone". 

The  electrogenic  effect  of  acetylcholine  injected  into  the  electric  tissue  is  another 
illustration  of  the  fact  that  the  post-synaptic  membrane  is  not  protected  against  the 
ester.  It  is  interesting  that  the  effect  of  curare  on  electric  tissue  was  a  controversial  issue 
for  a  long  time.  Recently,  however.  Auger  and  Fessard'^  have  shown  that  the  effect 
of  curare  is  regularly  reproducible  if  the  permeability  factor  is  taken  into  account  and 
the  drug  is  applied  in  adequate  form. 

Curare,  being  a  methylated  quaternary  ammonium  salt,  may  act  upon  the  protein 
of  the  active  membrane  as  a  competitor  of  acetylcholine.  The  effect  persists  since  the 
compound  cannot  be  hydrolyzed  but  must  be  removed  by  diffusion.  If  the  rapid  removal 
of  acetylcholine  is  inhibited  by  eserine,  the  result  is  strikingly  similar  to  that  obtained 
with  partial  curarization  of  the  end  plate,  as  the  experiments  of  Auger  and  Fessard 
have  shown.  The  depression  and  prolongation  of  the  potential  in  Fig.  3  must  obviously 
be  attributed  to  the  persistence  of  acetylcholine  and  with  still  higher  concentrations  of 
eserine  a  complete  "curarization"  will  be  obtained. 

As  pointed  out  by  Erlanger,  conduction  along  the  axon  and  transmission  across 
References  p.  93I95. 


VOL.  4  (1950)  PERMEABILITY   AND   NERVE    FUNCTION,  I  93 

synapses  may  vary  as  to  quantitative  aspects.  This  is  not  surprising  in  view  of  the 
discontinuity  and  other  structural  differences.  Although  the  time  relations  are  similar, 
there  is  a  synaptic  delay  of  the  order  of  a  millisecond.  This  may  be  the  result  of  several 
factors,  as  e.g.,  the  decreased  diameter  of  the  nerve  fibre  near  the  ending  which  may 
lead  to  a  decreased  rate  of  conduction.  Exact  measurements  of  these  various  factors  are 
difficult,  due  to  obvious  technical  reasons.  However,  the  quantitative  differences 
between  intracellular  and  transsynaptic  propagation  are  well  in  the  expected  range, 
and  none  of  them  requires  the  assumption  of  a  fundamentally  different  mechanism. 

In  conclusion,  no  convincing  evidence  exists  supporting  the  idea  that  acetylcholine 
assumes  a  function  at  the  synapse  entirely  different  from  that  in  the  axon,  i.e.  is  released 
from  the  nerve  ending,  penetrates  the  intercellular  space  and  acts  on  the  post-synaptic 
membrane,  thus  substituting  the  flow  of  current  as  a  "chemical  mediator".  A  funda- 
mental rule  of  scientific  thinking  requires  that  one  should  not  assume  two  different 
principles  without  necessity.  Work  and  Work'^  have  recently  quoted  the  excellent 
formulation  of  this  rule  by  David  Hume  in  his  Treatise  of  Human  Nature:  "To  invent 
without  scruple  a  new  principle  to  every  new  phenomenon,  instead  of  adapting  it  to 
the  old;  to  overload  our  hypothesis  with  a  variety  of  this  kind,  are  certain  proofs,  that 
none  of  these  principles  is  the  just  one,  and  that  we  only  desire,  by  a  number  of  false- 
hoods, to  cover  our  ignorance  of  the  truth".  Neither  the  so-called  "electrical"  nor  the 
"chemical"  concept  of  synaptic  transmission  is  satisfactory.  The  interpretation  pro- 
posed harmonizes  both  concepts  by  integrating  the  progress  achieved  concerning  the 
structure,  the  biochemical  data  and  the  electrical  signs  of  activity. 

The  earlier  observations  on  acetylcholine  deserve  credit  for  having  drawn  the 
attention  of  physiologists  to  this  compound  in  connection  with  nerve  activity.  However 
whereas,  the  ester  was  first  associated  with  one  type  of  nerve  endings,  then  with  a  few 
others,  the  study  of  its  role  by  the  combination  of  chemical  and  physical  methods  has 
shown  its  essentiality  in  the  conduction  of  nerve  and  muscle  impulses  throughout  the 
animal  kingdom.  The  type  of  approach  applied  by  Otto  Meyerhof  to  studying  muscular 
contraction  has  proved  valuable  in  obtaining  a  better  understanding  of  fundamental 
principles  underlying  the  mechanism  of  another  cellular  function  vital  for  life. 


REFERENCES 
^  G.  Hevesy,  Radioactive  Indicators,  Interscience,  New  York  1948. 

'^    R.   SCHOENHEIMER  AND   D.    RiTTENBERG,    Pliysiol.   RcVS,   20   (1940)   2l8. 

3  A.  Krogh,  Proc.  Roy.  Soc,  B  133  (1946)  140. 

■•  E.  Du  Bois  Reymond,  Gesammelte  Abhandlungen  zur  allgemeinen  Muskel-  und  Nervenphysik, 

Leipzig,  Veit  and  Co,  1877. 
^  W.  OsTWALD,  Z.  physik.  Chem.,  6  (1890)  71. 
^  J.  Bernstein,  Pjliigers  Arch.  Physiol.,  92  (1902)  521. 
'  H.  J.  Curtis  and  K.  S.  Cole,  /.  Cellular  Conip.  Physiol.,  19  (1942)  135. 

*  A.  L.  HoDGKiN  AND  A.  F.  HuxLEY,  /.  Phvsiol.,  104  (1945)  176. 

*  A.  L.  HoDGKiN  AND  A.  F.  HuxLEY,  /.  Physiol.,  106  (1947)  341;  Pyoc.  Intern.  Congr.  Physiol., 
Oxford  1947. 

10  R.  D.  Keynes,  /.  Physiol.,  107  (1948)  P. 

1^  M.  A.  RoTHENBERG  AND  E.  A.  Feld,  /.  Biol.  Chem.,  168  (1947)  223. 

^2  R.  Schoenheimer,  The  dynamic  state  of  body  constituents.  Harvard  University  Press,  1942. 
^^  H.  J.  Curtis  and  K.  S.  Cole,  /.  Gen.  Physiol.,  22  (1938)  37. 

^*  K.  Lucas,  The  conduction  of  the  nervous  impulse.  Revised  by  E.  D.  Adrian,  London,   Longmans. 
1917. 


94  D.  NACHMANSOHN  VOL.  4  (1950) 

15  A.  C.  Downing,  R.  W.  Gerard,  and  A.  V.  Hill,  Proc.  i?o>'.  5oc.,  B  100  (1926)  223. 

15a  A.  V.  Hill,  Chemical  wave  transmission  in  nerve,  Cambridge  University  Press,  1932. 

"  R.  W.  Gerard  and  O.  Meyerhof,  Biochem.  Z..  191  (1927)  125. 

1'  R.  Hunt  and  R.  de  M.  Taveau,  Brit.  Med.  J.,  2  (1906)  1788. 

1*  J.  F.  Fulton,  Physiology  of  the  Nervous  System,  Oxford  Univ.  Press  (1938,  I943)- 

19  J.  C.  EccLES,  Ann.  N.Y.  Acad.  Sci.,  47  (1946)  429. 

20  D.  Nachmansohn,  In:  R.  S.  Harris  and  K.  V.  Thimann,  Vitamins  and  hormones,  3  (x945)  337- 

21  D.  Nachmansohn,  In:  D.  E.  Green,  Currents  in  Biochem.  Research,  New  York,  335  (1946). 

22  D.  Nachmansohn,  Ann.  N.Y.  Acad.  Sci.,  47  (1946)  395- 

23  D.  Nachmansohn,  Bull.  John  Hopkins  Hosp.,  83  (1948)  463- 

2*  D.  Nachmansohn,  Acetylcholine  in:  G.  Pincus  and  K.  V.  Thimann,  The  Hormones,  Academic 

Press,  New  York,  in  press. 
25  M.  A.  Rothenberg  and  D.  Nachmansohn,  /.  Biol.  Chem.,  168  (1947)  223. 
28  D.  Nachmansohn  and  M.  A.  Rothenberg,  /.  Biol.  Chem.,  158  (1945)  653. 

27  T.  H.  Bullock,  H.  Grundfest,  D.  Nachmansohn,  and  M.  S.  Rothenberg,  /.  Neurophysiol., 

10  (1947)  "• 

28  D.  Nachmansohn  and  H.  B.  Steinbach,  /.  Neurophysiol.,  5  (1942)  109. 

29  K.  B.  Augustinsson,  Arch.  Biochem.,  in  press. 

30  K.  B.  Augustinsson  and  D.  Nachmansohn,  Science,  no  (i949)  98- 

31  D.  Nachmansohn,  C.  W.  Coates,  and  M.  A.  Rothenberg,  /.  Biol.  Chem.,  163  (1946)  39- 

32  D.  Nachmansohn  and  A.  L.  Machado,  /.  Neurophysiol.,  6  (1943)  397- 

33  D.  Nachmansohn  and  M.  S.  Weiss,  /.  Biol.  Chem.,  172  (1948)  677. 

3*  T.  H.  Bullock,  D.  Nachmansohn,  and  M.  A.  Rothenberg,  /.  Neurophysiol.,  9  (1946)  9- 
35  D.  Nachmansohn,  M.  A.  Rothenberg,  and  E.  A.  Feld,  Arch.  Biochem.,  14  (1947)  i97- 
38  T.  H.  Bullock,  H.  Grundfest,  D.  Nachmansohn,  and  M.  A.  Rothenberg,  /.  Neurophysiol., 
10  (1947)  63. 

37  E.  a.  Feld,  H.  Grundfest,  D.  Nachmansohn,  and  M.  A.  Rothenberg,  /.  Neurophysiol.,  11 
(1948)  125. 

38  D.  Nachmansohn  and  E.  A.  Feld,  /.  Biol.  Chem.,  171  (1947)  7i5- 

39  R.  W.  Brauer  and  M.  a.  Boot,  Federation  Proc,  4  (i945)  ii3- 
*°  M.  E.  Greig  and  W.  C.  Holland,  Federation  Proc,  8  (1949)  297. 

*i  M.  A.  Rothenberg,  D.  B.  Sprinson,  and  D.  Nachmansohn,  /.  Neurophysiol.,  11  (1948)  m- 

"  H.  King,  /.  Chem.  Soc,  2  (1935)  1381. 

*3  O.  Wintersteiner  and  J.  D.  DuTCHER,  Science,  97  (1943)  467. 

"  C.  W.  Coates  and  R.  T.  Cox,  Zoologica,  27  (1942)  25. 

"  D.  Nachmansohn,  K.  B.  Augustinsson,  M.  A.  Rothenberg,  and  LA.  Freiberger,  in  preparation. 

■•8  K.  B.  Augustinsson  and  D.  Nachmansohn,  /.  Biol.  Chem.,  179  (i949)  543- 

^7  R.  W.  Gerard,  B.  Libet,  and  D.  Cavanaugh,  Federation  Proc,  8  (1949)  55- 

^8  S.  Hestrin,  J.  Biol.  Chem.,  in  press. 

*9  K.  D.  RoEDER,  N.  K.  Kennedy,  and  E.  A.  Samson,  /.  Neurophysiol.,  10  (1947)  ^■ 

50  D.  Nachmansohn  and  H.  Schneemann,  /.  Biol.  Chem.,  159  (i945)  239- 

51  K.  V.  Thimann,  Arch.  Biochem.,  2  (1943)  87. 

52  A.  L.  Bennet  and  K.  G.  Chinberg,  /.  Pharmacol.,  88  (1946)  72. 

53  L.  AsHER,  PflUgers  ges.  Physiol.,  210  (1925)  689. 
5*  K.  Nakayama,  Z.  Biol.,  82  (1925)  581. 

55  A.  W.  KiBjAKOW,  PflUgers  ges.  Phys.,  232  (i933)  432- 
58  Q.  Calabro,  Riv.  Biol.,  15  (1933)  299. 

57  L.  BiNET  AND  B.  MiNZ,  Compt.  rend.  soc.  bioL,  117  (i934)  1029. 

58  G.  Bergami,  Arch.  ist.  biochim.  ital.,  8  (1936)  3. 

59  E.  B.  Babsky,  Bull.  biol.  med.  exptl  URSS,  5  (1938)  5i- 

80  E.  B.  Babsky  and  B.  M.  Kisljuk,  Fiziol.  Z.,  24  (1938)  746- 

81  A.  V.  MuRALT,  Proc.  Roy.  Soc,  B  123  (i937)  399- 

82  A.  V.  MuRALT,  Pfltigcrs  gcs.  Physiol.,  245  (1942)  604. 

83  K.  Brecht  and  M.  Corsten,  PflUgers  ges.  Physiol.,  245  (1942)  160. 
8*  J.  Erlanger,  /.  Neurophysiol.,  2  (1939)  370. 

85  A.  Arvanitaki,  J.  Neurophysiol.,  5  (1942)  89. 

88  A.  Arvanitaki,  /.  physiol.  path,  gen.,  38  ((1943)  147. 

87  T.  H.  Bullock,  7.  Neurophysiol.,  11  (1948)  343. 

88  R.  Granit  and  C.  R.  Skoglund,  /.  Physiol.,  103  (i945)  435- 

88a  R.  Granit,  R.  Leksell,  and  C.  R.  Skoglund,  Brain,  67  (1944)  125. 

89  R.  Couteaux,  H.  Grundfest,  D.  Nachmansohn,  and  M.  A.  Rothenberg,  Science,  104  (1946)  317- 
89a  R.  Couteaux,  Bull,  biol.,  76  {1942)  14. 

89b  R.  Couteaux,  Rev.  canad.  biol.,  6  (1947)  563. 

70  DuBuissoN  AND  MoNNiER,  Arch.  intcm.  Physiol.,  38  (1934)  180. 

71  S.  Covi'AN,  /.  Physiol.,  88  (1936)  4  P. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  I  95 

'2   D.  Auger  and  A.  Fessard,  Livro  Homnagem  aos  Professores  Alvaro  e  Miguel  Ozorio  de  Almeida, 

Rio  de  Janeiro  1939. 
"^  \V.  Feldberg,  a.  Fessard,  and  D.  Nachmansohn,  /.  Physiol.,  97  (1940)  3  P. 
'*  A.  Fessard,  Ann.  N.Y.  Acad.  Sci.  47  (1946)  501. 
^5  L.  Lapicque,  Compt.  rend.  soc.  biol.,  Paris  122  (1936)  990. 
'*  T.  S.  Work  and  E.  Work,  The  Basis  of  Chemotherapy,  Interscience,  New  York  1948. 

Received  May  31,  1949 


^6  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


STUDIES  ON  PERMEABILITY   IN   RELATION  TO  NERVE  FUNCTION 

II.  IONIC  MOVEMENTS  ACROSS  AXONAL  MEMBRANES* 

by 

M.  A.  ROTHENBERG** 
Department  of  Neurology  and  Biochemistry,  College  of  Physicians  and  Surgeons,  Columbia  University, 

New  York,  N.Y.  (U.S.A.) 


INTRODUCTION 

The  ionic  concentration  gradients  which  exist  between  the  inside  and  the  outside 
of  nerve  fibres  and  their  possible  role  in  nerve  function  have  been  discussed  in  the  preced- 
ing paper.  In  spite  of  the  importance  of  this  question  very  little  information  is  available 
as  to  the  ionic  movements  across  axonal  surface  membranes  in  rest  and  during  activity. 
The  investigations  on  the  giant  axon  of  Squid  have  demonstrated  that  this  material  is 
most  suitable  for  permeability  studies.  With  the  increased  availability  of  radioactive 
ions  from  the  Oak  Ridge  pile  a  more  direct  approach  to  the  problem  became  feasible. 
It  was  thought  that  precise  and  more  quantitative  data  might  be  obtained  by  subjecting 
the  giant  axon  of  Squid,  Loligo  peallii,  to  artificial  environments  in  which  all  or  part  of 
a  given  ionic  constituent  was  replaced  in  isomolar  concentration  with  its  radioactive 
isotope. 


METHODS 

Chemical.  Na^*  and  K'*^,  available  from  the  Oak  Ridge  pile  in  the  form  of  the  carbonates,  were 
dissolved  in  the  smallest  possible  volume  of  distilled  water  and  then  converted  to  the  chlorides  by 
the  addition  of  equivalent  quantities  of  dilute  HCl.  Aliquots  of  the  neutral  solution  were  then  trans- 
ferred to  tared  vials  and  evaporated  to  dryness  under  infra-red  heating  lamps.  The  quantity  of  salt 
per  vial  was  determined  by  weighing  and  artificial  sea  water  was  prepared  from  these  as  described 
below.  All  necessary  precautions  were  maintained  {i.e.,  remote  control  pipetting  behind  thick  lead 
shields,  etc.)  in  carrying  out  the  conversions  of  carbonates  to  chlorides***. 

The  Ca*^  employed  in  our  earliest  experiments  was  that  obtained  from  the  Oak  Ridge  pile  in 
the  form  of  CaCOg  (AEC  Catalog  Item  41=  13  A).  Since  this  material  contained  A''  in  addition  to 
Ca*^,  it  was  deemed  necessary  to  pump  out  the  A^'  under  high  vacuum  before  carrying  out  the  con- 
vei'sion  of  the  carbonate  to  chloride.  In  general,  the  latter  conversion  was  carried  out  in  a  manner 
similar  to  that  for  Na^"*  and  K*^  above.  In  later  experiments,  high  specific  acitivity  Ca*^  was  employed 


*  These  investigations  were  supported  by  a  research  grant  from  the  Atomic  Energy  Commission. 
From  a  dissertation  submitted  in  partial  fulfilment  of  the  requirements  for  the  degree  of 
Doctor  of  Philisophy  in  the  Faculty  of  Pure  Science  of  Columbia  University. 

***  We  are  indebted  to  Tracerlab,  Inc.,  Boston,  Mass.,  for  carrying  out  the  carbonate  to 
chloride  conversions. 

References  p.  114. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  II  97 

( AEC  Catalog  Item  #  S-5)*.  Aliquots  of  the  Ca'*^  solution  were  pipetted  into  the  appropriate  volumes 
of  Ca-free  artificial  sea  water  to  give  the  correct  Ca  concentration  (0.012  M). 

Preparation  of  biological  material.  The  last  stellar  nerves  (containing  a  giant  axon)  were  excised 
from  specimens  of  Loligo  peallii,  after  first  tying  both  ends  of  the  portion  desired.  Nerve  sections  were 
then  kept  in  fresh  natural  sea  water  for  i^  to  2  hours  before  use.  The  results  of  Steinbach  and 
Spiegelman^  had  indicated  that  during  the  first  2  hours  after  excision  of  stellar  nerves,  the  chemically- 
determined  values  for  Na  vary  considerably  and  it  is  only  after  this  time  has  elapsed  that  the  axoplasm 
comes  into  equiUbrium  with  its  outer  environment.  The  value  for  Na  reaches  its  maximum  value  of 
10  meq.  per  cent  within  this  period. 

The  nerves  were  then  exposed  to  artificial  sea  water  prepared  according  to  Pantin^  in  which  all 
or  part  of  a  given  ion  species  had  been  replaced  in  isomolar  concentration  with  radioactive  material. 
The  sea  water  contained  0.52  M  NaCI,  0.013  ^  KCl,  0.012  M  CaClj,  and  0.024  M  MgClj.  The  pn  was 
adjusted  to  7.7-8.0  by  the  addition  of  a  small  volume  of  bicarbonate  or  NaOH,  the  latter  in  those 
cases  where  the  adjustment  required  considerable  amounts  of  alkali.  After  the  desired  period  of 
exposure,  the  nerves  were  removed  and  rinsed  several  times  in  a  few  changes  of  fresh  natural  sea 
water.  After  blotting  of  filter  paper,  the  proximal  end  was  cut  off.  The  axoplasm  (nerve  cytoplasm) 
was  extruded  by  the  application  of  gentle  but  gradually  increasing  pressure  with  a  pair  of  forceps 
in  the  direction  of  the  cut  end.  The  extruded  axoplasm  was  collected  on  a  tared  aluminum  planchet 
(130-150  mg  each  and  about  one  inch  in  diameter)  and  weighed  quickly  with  a  torsion  balance.  One 
ml  of  distilled  water  was  then  added  to  each  planchet  to  insure  even  distribution  of  the  radioactive 
substance  over  the  entire  area  of  the  planchet. 

Determination  of  radioactivity.  Samples  were  then  evaporated  to  dryness  under  infra-red  lamps 
and  the  radioactivity  measured  with  a  Tracerlab  64  Scaler**.  Measured  radioactivities  were  recal- 
culated to  zero  time  from  the  decay  curve  of  the  individual  ion  under  investigation  in  order  to  correct 
for  the  decomposition  which  occurred  during  the  measurement  of  sample  activities.  This  correction 
becomes  appreciably  large,  when  using  Na^*  and  K^^  which  have  half-lives  of  14.8  and  12.4  hours 
respectivelv.  Comparison  of  the  activities  of  the  samples  with  standards  prepared  from  aliquots  of 
the  radioactive  artificial  sea  water  (and  analysed  at  the  same  level  in  the  counting  chamber)  enabled 
the  calculation  of  the  ion  content  of  the  axoplasm  samples. 

The  method  of  preparation  of  the  standards  for  Tables  I,  II,  and  III  are  given  at  the  top  of 
each  of  these  tables.  The  Na  standards  for  the  data  given  in  Tables  IV,  VI,  IX,  and  X  were  prepared 
by  diluting  the  sea  water  (containing  0.39  M  Na^^Cl  -f  0.13  M  Na^^Cl)  250  times  with  distilled  water. 
0.5  ml  aliquots  were  then  evaporated  to  dryness  in  duplicate  on  aluminum  planchets  (1.04  micromoles 
Na/0.5  ml).  For  Tables  VII  and  VIII,  Na  standards  were  prepared  by  this  same  method.  However, 
since  a  reduction  in  the  total  NaCl  concentration  had  been  made  in  order  to  maintain  the  isotonicity 
in  the  presence  of  added  inhibitors  of  cholinesterase,  the  0.5  ml  aliquots  contained  only  i.oo  micro- 
mole  Na/0.5  ml.  The  K  standards  for  the  data  given  in  Tables  V  and  VII  were  prepared  by  diluting 
the  sea  water  (containing  0.013  M  K^^Cl)  100  times  and  then  evaporating  i.o  ml  aliquots  in  duphcate 
as  above  (0.13  micromole  K/i.o  ml).  Radioactivities  recorded  in  Tables  IV  through  X  have  all  been 
corrected  to  zero  time. 

Electrical.  Nerves  were  tested  for  normality  of  conduction  both  before  and  after  exposure  to 
radioisotope  containing  sea  water.  The  nerves  were  stimulated  through  a  pair  of  silver  wire  electrodes 
by  condenser  discharge  shocks  of  a  time  constant  less  than  0.2  milliseconds.  Action  potentials  were 
led  off  by  means  of  a  second  pair  of  silver  wire  electrodes  to  a  condenser  coupled  amplifier  of  a  modified 
Toeney  differential  type  circuit  and  then  recorded  on  a  DuMont  No.  279  Dual  Beam  Oscilloscope. 
Only  those  nerves  were  used  which  still  exhibited  normal  conduction  at  the  end  of  the  experiment. 
Studies  of  the  rates  of  ion  exchange  during  electrical  activity  of  the  nerves  were  carried  out  in 
the  following  manner:  Nerve  chambers  were  used  of  narrow  bore  polystyrene  tubing  (2  mm  i.d.)  into 
which  were  sealed,  at  right  angles  to  the  length  and  at  5  mm  intervals,  0.0156"  diameter  Pt  wire  as 
described  previously  (II).  Nerves  were  mounted  in  the  chamber  by  threading  a  long  thin  wire  through 
the  polystyrene  tube  (one  end  of  the  wire  having  previously  been  tied  to  the  thread  attached  to  the 
nerve).  The  nerve  was  then  carefully  drawn  into  the  tube.  By  slipping  a  piece  of  narrow  bore  rubber 
tubing  over  that  end  of  the  polystyrene  tube  from  which  the  thread  issued,  the  thread — and  thereby 
the  nerve — was  fixed  in  position.  The  rubber  tubing  was  then  connected  to  a  perfusion  bottle  filled 
with  sea  water  containing  the  radioactive  ions.  Perfusion  of  the  nerve  preparation  was  carried  out  by 
means  of  gravity.  The  diameter  of  the  plastic  tubing  chosen  was  such  that  only  a  very  thin  layer  of 
sea  water  remained  between  the  nerve  and  the  wall  of  the  polystyrene  tube.  Thus,  the  difficulty  of 
excessive  shunting  by  the  sea  water  was  largely  eliminated  and  stimulation  of,  and  recording  from, 
the  nerve  was  possible  throughout  the  period  of  exposure  to  the  isotope  containing  sea  water. 


*  We  are  indebted  to  Dr  G.  Failla  and  Dr  P.  Aebersold  for  making  the  high  specific  activity 
Ca^^  (carrier  free)  available  to  us. 

**  We  are  indebted  to  Dr  G.  Failla  and  the  Marine  Biological  Laboratory,  Woods  Hole, 
Mass.,  for  making  the  Scaler  available  to  us. 

References  p.  114. 
7 


3.0 


S2.5 


2.0 


g8  M.  A.  ROTHENBERG  VOL.  4  (1950) 

RESULTS 
A.  ION    EXCHANGES    AT    REST 

I.  Potassium.  In  one  series  of  experiments  the  stellar  nerves  were  exposed  to  arti- 
fical  sea  water  in  which  the  K^^  had  been  replaced  by  K*^  in  the  usual  sea  water  concen- 
tration (0.013  M)-  Analysis  of  axoplasm  samples  indicated  that  there  was  a  rapid  ex- 
change of  potassium  under  these  conditions.  Table  I  gives  a  few  examples  illustrating 
the  size  of  the  axoplasm  samples,  the  magnitude  of  the  radiation  measured  and  the 
manner  in  which  the  standards  were  prepared.  All  of  the  data  obtained  in  this  way  are 
presented  in  Fig.  i.  Each  point  on  the  graph  represents  a  single  experiment.  The  number 
of  millimoles  (mM)  of  K'*^  which  penetrated  per  100  gm  axoplasm  (wet  weight)  is  plotted 
against  time  of  exposure  of  the  nerve  fibre  to  the  radioisotopic  sea  water.  It  will  be 
noted  from  Fig.  i  that  the  rate  of  penetration  of  K*^  through  the  nerve  membrane  is 
initially  quite  high  but  it  then  slows  markedly  and  within  60  min,  analyses  indicate  an 

approach  to  a  maximal  value  or  2.5  mil- 
limoles/ioo  g  asymptotically.  If  one  ac- 
cepts the  values  for  the  potassium  content 
of  the  axoplasm  found  in  the  literature 
(Steinbach  and  Spiegelman,  32.1  meq. 
per  cent^ ;  Baer  and  Schmitt,  27  meq.  per 
cent^;  Webb  and  Young  25.3  meq.  per 
cent*)  it  can  be  seen  that  the  maximum 
exchange  obtainable  under  these  con- 
ditions is  approximately  one  tenth  of  the 
total  K  concentration  of  the  axoplasm. 
In  all  probability,  the  curve  in  Fig.  i 
is  a  composite  of  at  least  two,  or  possibly 
more,  distinct  reactions.  The  first  part 
of  the  curve,  with  the  steepest  slope,  is, 
in  all  probability,  a  true  measure  of  the 
rate  of  exchange  of  K  across  the  nerve  membrane.  The  second  phase  in  which  the 
rate  of  exchange  has  slowed  down  may  possibly  be  ascribed  to  a  movement  of  the 
radioactive  ions  from  the  inside  to  the  outside  after  having  reached  a  certain  level. 
Finally,  when  the  inside  concentration  is  about  twice  that  of  the  outside,  there  ap- 
pears to  be  an  equilibrium  of  the  movements  in  the  two  directions. 

The  expeiiments  show  that  even  at  rest,  there  is  a  dynamic  equilibrium  between  the 
K  inside  the  fibre  and  that  in  its  outer  environment^.  Within  50  min  an  equilibrium  is 
established.  Under  such  conditions  only  about  one  tenth  of  the  total  K  inside  the  fibre 
has  exchanged  for  K*^  in  the  bathing  medium.  The  K*^  concentration  inside  the  fibre 
is  2.5  millimoles/ioo  g  axoplasm  against  1.3  millimoles/ioo  ml  for  the  sea  water.  When 
a  steady  state  of  exchange  has  been  attained,  it  is  possible  to  calculate  the  permeability 
constant  for  this  exchange  of  K  at  rest  by  means  of  Collander's  equation  as  modified 
by  Krogh''.  According  to  Krogh  where  d  is  the  diameter  of  the  cell  (cm),  t  is 


1.5 


1.0 


0.5 


"^  • 

.X 

^.' 

• 

/ 

A 

• 

15 


30 


60 


90 
Min.  of  exposure 

Fig.    I.    K  penetration  across   the    membrane    of 

the  giant  axon  of  Squid  when  exposed  to  0.013 

K^^Cl    in    artificial    sea    water.    The    horizontal 

broken   line   on   the   ordinate    indicates    the    K^^ 

concentration    outside.    The    penetration    of    K*^ 

in  millimoles  (mM)/ioo  g  axoplasm  (wet  weight) 

is  plotted  against  time  in  minutes. 


P  -  0.576  —  logio 


C. 


U<-  V^n 


References  p.  114. 


VOL.  4  (1950) 


PERMEABILITY   AND    NERVE    FUNCTION,  II 


99 


time  (hours),  Cg  and  Q  concentrations  of  the  ion  inside  and  outside  respectively,  and  a, 
and  a-o  are  the  corresponding  activities,  d  may  be  assumed  to  be  =  0.05  cm,  Cj  =  0.32  M 
(Steinbach  and  Spiegelman)  and  Q  =  0.013  M.  Substituting  40400  cts/min/ml  for 
Rq  (from  Table  I)  and  77700  cts/min/g  for  a^  (from  Fig.  i)  when  t  =  0.83  h,  one  obtains 
a  value  of  1.25-10"^  cm/h  for  P,  the  permeability  constant,  from  the  equation  above. 

TABLE  I 

K'*^    PENETRATION 

Nerves  exposed  to  sea  water  containing  0.013  M  K^^Cl  for  varying  periods  of  time.  Standards  (S^ 
and  Sg) :  sea  water  diluted  1:10  and  then  0.5  ml  evaporated  to  dryness  in  duplicate  (0.65  micromole 
K*^/o.5  ml).  Counts  per  min  indicate  the  actual  count,  uncorrected  for  time  decay  of  radioactivity. 


Time  of  exposure 

Axoplasm 

Counts 

Millimoles 

Micromoles 

(min) 

(mg) 

per  min 

per  100  g 

per  100  gper  min 

4 

9.2 

90 

0.30 

75 

9 

7.6 

135 

0.52 

58 

14 

II. 7 

466 

1. 19 

85 

19 

11.6 

570 

I-5I 

80 

24 

8.3 

430 

1.60 

67 

30 

9.2 

570 

1.91 

64 

45 

12.9 

930 

2.22 

49 

55 

16.0 

1247 

2.49 

45 

65 

13-6 

1007 

2.38 

37 

80 

18.8 

1490 

2-54 

25 

Si 

2017  1  average 

t 

S2 

2022  1     2020 

Fig.  2  shows  the  rates  of  exchange  of  K  against  time.  It  will  be  noted  that  the  rate 
is  initially  high  but  then  drops  to  a  value  which  is  only  about  one  fourth  of  that  of  the 
initial  rate.  The  rate  of  penetration  ap-  .s 
proaches  a  limiting  value  of  20  millimoles/  "^^^^ 
100  g/min  (or  2.5  •  io~^  mole/cm^/min  ^^q 
assuming  an  average  diameter  of  500  ju).  -^ 

In  a  second  series  of  experiments,  ^^'^ 
the  nerves  were  exposed  to  0.026  M  K*^  ^  20 
CI  in  the  bathing  sea  water  (twice  the 
normal  K  concentration).  In  carrying 
out  these  experiments,  a  decrease  in  NaCl 
concentration  was  made  equivalent  to  the 
increase  in  KCl  in  order  to  maintain  the 
isotonicity  of  the  sea  water.  The  data  ob- 
tained are  plotted  in  Fig.  3. 

It  is  evident  from  a  comparison  of  Figs  i  and  3  that  the  shapes  of  the  curves  ob- 
tained for  0.013  M  and  0.026  M  KCl  are  very  much  alike.  However,  since  the  ordinate 
in  Fig.  3  is  greater  by  a  factor  of  two,  it  can  be  seen  that  in  the  latter  case  the  penetra- 
tion of  K*2  into  the  fibre  reaches  a  maximal  value  of  5.3  millimoles/ioo  g  axpolasm. 
As  in  the  case  of  the  experiments  with  0.013  ^  KCl,  exchange  of  K^^  inside  for  K^^ 
outside  reaches  an  equilibrium  when  the  inside  concentration  of  K^^  is  twice  that  of 
the  outside. 

As  in  the  case  of  Fig.  i.  Fig.  3  should  probably  have  been  resolved  into  three 
distinct  phases.  The  considerations  applied  to  the  segments  of  Fig.  i  are  also  applicable 
References  p.  114. 


90 

Min.  of  exposure 
Fig.  2.  Rate  of  K  penetration  across  the  mem- 
brane of  the  giant  axon  of  Squid  when  exposed 
to  0.013  M  K*^C1  in  artificial  sea  water.  The  rate 
of  penetration  of  K*^  in  micromoles  (/<M)/ioo 
g/min  is  plotted  against  time  of  exposure  in  min. 


100 


M.  A.  ROTHENBERG 


VOL.  4  (1950) 


6.0 


^5.0 
6«.0 


3.0 


2.0 


1.0 


•^^ 

• 

y 

.... 

/ 

•/ 

•• 

tt 


JO 


60 


90 
Min.  of  exposure 

Fig.  3.  K  penetration  across  the  membrane  of  the 
giant  axon  of  Squid  when  exposed  to  0.026  M  K^^Cl 
in  artificial  sea  water  (twice  the  normal  K  concen- 
tration). The  horizontal  broken  line  on  the  ordinate 
indicates  the  K*-  concentration  outside.  The  pene- 
tration of  K*^  in  millimoles  (mM/ioo  g  axoplasm 
(wet   weight)    is   plotted   against  time   in   minutes. 

|2A0 
%,200 


<-  160 

^120 

80 


\ 

\ 

. 

\ 

•] 

' 

-: — -  . 

• 

40 


15  30  60  90 

Min.  of  exposure 

Fig.  4.  Rate  of  K  penetration  across  the  membrane 
of  the  giant  axon  of  Squid  when  exposed  to  0.026  M 
K-'^Cl  in  the  artificial  sea  water  (twice  the  normal 
K  concentration).  The  rate  of  penetration  of  K'*^ 
in  micromoles  (^M)/ioo  g/min  is  plotted  against 
time  of  exposure  in  minutes. 


o20 


10 


15  30  60  DO 

Min.  of  exposure 

Fig.  5.  Na  penetration  across  the  membrane  of  the 
giant  axon  of  Squid  when  exposed  to  artificial  sea 
water  containing  either  0.13  M  or  0.065  M  Na^^Cl. 
Total  NaCl  concentration  is  0.52  M.  The  penetra- 
tion of  Na  in  millimoles  (mM)/ioo  g  axoplasm 
(wet  weight)  is  plotted  against  time  of  exposure  in 
minutes. 


• 

•       r                - 

..••^ 

•        • 

'.    ••     • 

.IT 

to  those  of  Fig.  3.  The  rates  of  K*^ 
penetration  against  time  with  0.026 
M  KCl  outside  are  given  Fig.  4.  From 
a  comparison  of  Figs  2  and  4,  it  is 
evident  that  the  initial  rate  of  K*^ 
penetration,  using  0.026  M  KCl  out- 
side, is  greater  than  that  of  the  initial 
penetration  rate  obtained  with  0.013 
M  KCl  outside.  Also,  in  the  case  of 
0.026  M  KCl  outside,  the  rate  of 
penetration  falls  more  rapidly  than 
in  Fig.  I.  However,  the  limiting  rate 
of  penetration  finally  attained  is 
twice  that  of  Fig.  2. 

2.  Sodium.  The  problem  of  Na 
penetration  into  the  giant  axons  of 
Squid  was  investigated  in  a  manner 
similar  to  that  employed  for  K*^.  In  this 
case,  however,  either  one  fourth  or  one 
eighth  of  the  Na^^  in  the  sea  water 
(normally  0.52  M)  was  replaced  by 
Na-*.  The  remainder  of  the  Na,  neces- 
sary for  maintainence  of  isotonicity 
of  the  sea  water,  was  made  up  with 
ordinary  Na^^.  All  other  ions  were 
maintained  in  their  normal  concentra- 
tions. Calculation  of  the  Na  pene- 
trating the  fiber  was  made  on  the 
assumption  that  there  was  no  inherent 
difference  in  the  case  of  Na^^  and  Na^* 
penetrations.  Some  typical  data  ob- 
tained are  illustrated  in  Table  II. 

Fig.  5  represents  all  of  the  Na 
penetration  data  accumulated.  It  will 
be  noted  that  Na  enters  the  fibres  at  a 
rather  high  initial  rate  which  falls 
markedly  quite  quickly.  The  Na  pene- 
tration reaches  a  maximum  of  ap- 
proximately 17.0  millimoles/ioo  g. 
This  value  is  in  good  agreement  with 
the  value  of  16.2  meq.  per  cent  (16.2 
millimoles/ioog)  calculated  by  Stein- 
bach  AND  Spiegelman^^  from  the 
data  of  Webb  and  Young.  Our  value 
for  the  Na  penetrating  would,  there- 
fore, seem  to  indicate  that  exchange 
of  Na  across  the  nerve  membrane  is 


References  p.  114. 


VOL.  4  (1950) 


PERMEABILITY   AND   NERVE    FUNCTION,  II 


lOI 


complete  within  about  30  min.  Attainment  of  the  steady  state  is  accompHshed  when 
all  of  the  Na  inside  the  nerve  has  been  exchanged  for  Na^*.  Under  such  conditions, 
substituting  in  the  permeability  equation,  the  values  of  0.162  M  for  Cg  (Webb  and 
Young),  0.52  M  for  Q,  934.3  cts/min//:d  for  a^  (Table  II)  and  293.6  cts/min/^/.g  for 
a^  (Fig.  3)  with  t  =  0.5  h  and  d  =  0.05  cm,  gives  a  value  for  the  permeability  constant 
of  5.76-10-2  cm/h. 

TABLE  II 

Na^*    PENETRATION 

Nerves  exposed  to  sea  water  containing  0.39  M  Na^^Cl  +0.13  Na^^Cl  for  var^'ing  periods  of  time. 
Standards  (S^  and  Sj) :  sea  water  diluted  1:100  and  then  0.4  ml  evaporated  in  duplicate  (2.1  micro- 
moles/0.4  ml).  Counts  per  min  indicate  the  actual  count,  uncorrected  for  time  decay  of  radioactivity. 


Time  of  exposure 

Axoplasm 

Counts 

Alillimoles 

Micromoles 

(min) 

(mg) 

per  mm 

per  100  g 

per  100  g  per  mm 

3 

11.4 

1014 

4-7 

1-57 

9 

12.0 

2090 

9-7 

1.08 

II 

15-2 

3550 

12.4 

I-I3 

20 

13-8 

3234 

12.8 

0.64 

35 

152 

3924 

14-3 

0.41 

42 

14.6 

3420 

13.0 

0.31 

50 

12. 1 

2770 

12.7 

0.25 

55 

10.2 

4834 

26. S 

0.49 

60 

10.4 

2160 

II. 7 

0.20 

80 

II. I 

3464 

17.9 

0.23 

Sx 

3720  1  average 

S2 

3754/     3737 

c:   6.0 
6  5.0 


The  degree  of  scattering  appears  to  be  slightly  larger  in  the  case  of  Na  than  of  K. 
This  could,  to  some  extent,  be  due  to  a  slight  contamination  of  the  samples  with  radio- 
active sea  water  since  the  sea  water  contained  such  a  high  concentration  of  radioactive 
Na.  Another  factor  may  be  the  individual  variations  in  Na  content  of  these  nerves.  The 
data  of  Steinbach  and  Spiegelman  indicate  that  the  values  vary  considerably  from 
one  nerve  to  the  next :  3  to  4  hour  exposure  of  axons  to  sea  water  gave  Na  values  varying 
from  7.8  to  17.4  meq.  per  cent.  No  apparent  effort  was  made  in  their  work  to  determine 
whether  or  not  all  of  these  nerves  main- 
tained conduction.  It  is,  therefore,  not 
certain  that  such  large  deviations  are 
actually  within  the  normal  range  of  s  4.0 
variation.  Nevertheless,  it  is  quite  con- 
ceivable that  marked  individual  devia- 
tions occur. 

The  rates  of  penetration  of  Na  into 
Squid  nerves  are  plotted  against  time 
in  Fig.  6.  It  will  be  noted  that  the  initial 
rate  of  penetration  of  Na  into  fibres  is 
extremely  high  but  falls  to  a  very  low 
level  within  15  to  20  min.  The  rate  of 
penetration  after  40  min  of  exposure 
has  fallen  to  a  value  about  one  twen- 


3.0 


2.0 


1.0 


\ 

\ 

[ 

'x 

^ 

^^^ 

^^—U-".,.' 

15 


30 


60 


90 
Min.  of  exposure 

Fig.  6.  Rate  of  Na  penetration  across  the  mem- 
brane of  the  giant  of  Squid  when  exposed  to  arti- 
ficial sea  water  containing  either  0.13  M  or  0.065  ^I 
Na^^Cl.  Total  NaCl  concentration  is  0.52  M.  The 
rate  of  penetration  of  Na^*  in  millimoles  (mM)/ioo 
g/min  is  plotted  against  time  of  exposure  in  min. 


References  p.  114. 


102 


M.  A.  ROTHENBERG 


VOL.  4  (1950) 


6 


CiOA 


tieth  of  that  of  the  initial  rate.  This  rapid  fall  in  the  rate  of  penetration  is  further 
support  for  the  assumption  that  complete  exchange  of  Na  across  the  membrane  occurs 
within  a  short  period  of  time. 

Extrapolation  of  the  curve  in  Fig.  6  to  zero  time  gives  a  value  of  5.8  millimoles/ioo 
g/min  for  the  initial  rate  of  Na  exchange  in  these  nerves.  If  one  carries  out  a  similar 
operation  for  the  curve  of  Fig.  2,  a  value  of  0.082  millimole/ioo  g/min  for  K  is  obtained. 
These  results  seem  to  indicate  that  the  initial  rate  of  exchange  of  Na  is  many  times 
greater  than  of  K.  These  findings  do  not  support  the  concepts  of  Conway^  that  nerve 
membranes  are  impervious  to  Na  although  it  has  to  be  kept  in  mind  that  the  obser- 
vations are  limited  to  the  giant  axons  of  Squid.  The  observations  presented  are  consistent 
with  those  of  Steinbach  and  Spiegelman  who  have  been  able  to  demonstrate  that  Na 

enters  these  nerves. 

3.  Calcium.  Table  III  gives  some  of 
the  date  obtained  when  nerves  were 
exposed  to  high  specific  activity  of  Ca*^ 
(0.012  M)  in  artificial  sea  water  for  varying 
periods  of  time.  All  of  the  date  obtained 
are  plotted  in  the  curve  of  Fig.  7.  As  in 
the  cases  of  Na  and  K,  each  point  on 
the  curve  represents  a  single  nerve.  The 
curve  has  been  drawn  through  the  mean 
of  the  several  values  at  a  given  time  of 
exposure.  The  data  obtained  were  the 
same  when  low  specific  activity  Ca*^  was 
used. 


1.0 


.0.8 


0.2 


1 1 
"  II 

/I  I    I       1 


10      20 


50 


100 
Mil),  of  exposure 


Fig.  7.  Ca  penetration  across  the  membrane  of 
the  giant  axon  of  Squid  when  exposed  to  artifical 
sea  water  containing  0.012  M  Ca'^^Clj.  The  pene- 
tration of  Ca**  inmilHmoles  (m!\I)/ioo  g  axoplasm 
(wet  weight)  is  plotted  against  time  in  minutes. 


TABLE  III 

Ca''*    PENETRATION 

Nerves  exposed  to  sea  water  containing  0.012  M  Ca^^CIj  (high  specific  activity)  for  varying  periods 
of  time.  Standards  (S^  and  Sj) :  sea  water  diluted  1:200  and  then  0.5  ml  evaporated  in  duplicate 
(0.03  micromole  Ca**/o.5  ml). 


Time  of  exposure 

Axoplasm 

Counts 

Millimoles 

Micromoles 

(min) 

(mg) 

per  min 

per  100  g 

per  100  g  per  mm 

50 

19.2 

10167 

0.79 

15-6 

50 

8.2 

4762 

0.87 

17.2 

50 

6.0 

2829 

0.71 

14.2 

50 

6.6 

3271 

0.74 

14.6 

100 

9.4 

1607 

0.26 

2.7 

100 

4.6 

1139 

0.37 

3-8 

Si 

1997  1  average 

Si 

2010 1     2004 

It  will  be  noted  from  Fig.  7  that  the  Ca*^  inside  the  nerve  seems  to  reach  a  maximum 
value  of  0.82  millimole/ioo  g  within  45  min  and  then  decreases  to  a  value  of  0.45  milli- 
mole/ioo  g  at  100  min  of  exposure.  It  is  evident,  therefore,  that  the  Ca  penetrates  into 
these  nerve  fibres.  The  values  obtained  seem  to  indicate  that  the  concentration  of  Ca^^ 
at  100  min  is  lower  than  at  50  min.  Further  investigations  are  desirable  for  an  inter- 
pretation of  this  observation. 
References  p.  114. 


VOL.  4  (1950) 


PERMEABILITY   AND    NERVE    FUNCTION,  II 


103 


Fig.  8  is  a  curve  obtained  by  plotting  the  rates  of  penetration  of  Ca*^  into  the  nerves 
against  time  of  exposure.  It  will  be  noted  that  the  initial  rate  of  exchange,  extrapolated 
to  zero  time,  is  quite  high  and  com- 
parable to  the  initial  extrapolated  «s  50 
value  for  K  (50  micromoles/ioo  gm/ 
min  and  82  micromoles/ioo  gm/min 
respectively).  _^  jo 


6^0 


Fig.  8.  Rate  of  Ca  penetration  across  the 
membrane  of  the  giant  axon  of  Squid  when 
exposed  to  artificial  sea  water  containing 
0.012  M  Ca^^Cl.  The  rate  of  penetration  of 
Ca**  in  micromoles  (^M)/ioo  g/min  is 
plotted  against  time  of  exposure  in 
minutes. 


5: 


20 


10 


xr: — 

<i  >. 


10  20 


50 


100 
Min.  of  exposure 


B.  FACTORS   INFLUENCING   EXCHANGE    OF   Na   AND    K 

In  view  of  the  considerable  individual  variations  of  the  ion  content  of  these  nerves, 
it  appeared  advisable  to  modify  the  method  of  accumulation  of  data  in  studying  the 
effects  of  a  number  of  factors  on  the  ion  exchanges  across  the  nerve  membrane.  Instead 
of  collecting  single  values  at  varying  periods  of  exposure,  a  large  number  of  nerves 
were  exposed  simultaneously  under  identical  conditions  and  for  the  same  period  of 
time.  At  least  five  values  were  obtained  for  a  given  condition  and  only  the  average  values 
utilized  in  carrying  out  comparisons.  All  exposures  were  limited  to  30  min.  They  were 
carried  out  at  room  temperature  (22°  C),  except  for  the  cases  in  which  the  Qjq  of  Na 
and  K  exchange  were  studied. 

I-  Qio^f  -^^  ^^^  ^  exchange.  Table  IV  contains  the  data  obtained  when  nerves  were 
exposed  to  0.39  M  Na^^  CI  +  0.13  M  Na^*  CI  in  artificial  sea  water  for  30  min  at  22°  and 
13°  C  respectively.  At  22°  C,  the  average  of  eight  nerves  gave  a  value  of  9.5  millimoles/ 
100  g  while  at  13°  C  the  average  of  eight  nerves  was  8.6  millimoles/ioo  g.  This  would 
correspond  to  a  Q^  of  1.22. 

TABLE  IV 

EFFECT  OF  TEMPERATURE  ON  THE  RATE  OF  PENETRATION  OF  Na 

Nerves  exposed  for  30  min  to  sea  water  at  22°  and  13°  C  containing  0.39  M  Na^^Cl  +  0.13  M  Na^^Cl. 
Sj  and  Sj  =  standards. 


22°  c 

Axoplasm 

(mg) 

Counts 
per  min 

Millimoles 
per  100  g 

13°  c 

Axoplasm 
(mg) 

Counts 
per  min 

Millimoles 
per  100  g 

Si 
S2 

1324  1  average 
1285        1305 

10.2 

1273 

9-9 

11.4 

1405 

9.8 

9-4 

1321 

II. 2 

16.6 

1964 

9.4 

5-2 

590 

9-1 

14.6 

1624 

8.9 

8.8 

996 

9.0 

20.4 

2100 

8.2 

5-8 

599 

8.2 

12.6 

1 199 

7.6 

12.0 

1341 

8.9 

8.8 

lOOI 

9.0 

14.2 

1681 

9.4 

16.6 

1694 

8.1 

12.2 

1568 

10.4 

18.8 

1864 

7-9 

Average 

9-5 

Average 

8.6 

References  p.  114. 


104 


M.  A.  ROTHENBERG 


VOL.  4  (1950) 


The  exchange  of  K  was  studied  under  identical  conditions  (30  min  exposure  at 
22°  and  13°  C)  using  0.013  M  K^2(;;i  instead  of  K39(3i  ^j^  ^j^g  ggg^  water.  At  22°  C  the 
average  of  seven  nerves  was  1.31  milhmoles/ioo  g  and  at  13°  C  the  average  of  the  same 
number  of  nerves  was  1.09  milhmoles/ioo  g  (Table  V).  This  would  correspond  to  a 
Qio  of  1.33- 


TABLE  V 

EFFECT  OF  TEMPERATURE  ON  THE  RATE  OF  PENETRATION  OF  K 

Nerves  exposed  for  30  min  to  sea  water  at  22°  C  and  13°  C  containing  0.013  M  K^^ci.  Sj  and  Sg 
=  standards. 


22°  c 

Axoplasm 

(mg) 

Counts 
per  min 

Millimoles 
per  100  g 

13°  c 

Axoplasm 
(mg) 

Counts 
per  min 

Millimoles 
per  100  g 

Si 

S2 
10.8 
10.4 

7.2 

1014^  average 
1051  1     1033 
1230 
1152 
758 

1-43 
1-39 
1-33 

6.4 

8.4 

4.2 

10.4 

525 

732 

395 

1030 

1.03 

I. ID 
1. 18 
1-25 

Si 
S2 

7.2 

15-8 
12.4 
22.0 

482  \  average 
488 1      485 
372 
659 
586 
1000 

1-39 
1. 12 
1.27 
1.22 

6.4 

13.2 

9.0 

Average 

272 
456 
328 

1. 14 
0.92 
0.98 

Average 

I-3I 

1.09 

The  values  for  the  Q^q  obtained  above  for  both  Na  and  K  are  in  good  agreement 
with  the  theoretical  value  of  1.25  calculated  from  ionic  conductivity  measurements. 
The  ionic  velocities  increase  by  about  2  to  2.5%  for  every  degree  rise  of  temperature''. 
It  is,  therefore,  possible  that  no  important  energy  yielding  chemical  reactions  are 
involved  in  the  exchange  of  ions  across  the  nerve  membrane  under  these  experimental 
conditions. 

2.  Electrical  activity  and  Na  exchange.  Stimulation  of  nerves  by  supramaximal 
shocks  while  being  perfused  with  sea  water  containing  0.39  M  Na^^Cl  +  0.13  M  Na^^Cl 
produced  a  marked  alteration  in  the  rate  of  exchange  of  Na  when  compared  to  resting 
nerves.  As  described  under  Methods,  nerves  were  mounted  in  plastic  chambers  in  which 
stimulating  and  recording  electrodes  were  imbedded.  The  nerves  were  stimulated  at  a 
rate  of  100  times  per  second  for  30  min.  Only  those  nerves  which  exhibited  normal 
responses  throughout  this  period  of  stimulation  were  analysed.  Analysis  of  the  axoplasm 
of  six  of  these  nerves  indicated  that  15.9  millimoles  Na/ioo  g  (mean  value)  had  exchanged 
within  30  min  as  compared  with  9.5  millimoles/ioo  g  at  rest.  This  would  correspond  to 
an  increase  in  the  rate  of  exchange  of  approximately  67%  above  that  at  rest.  The  results 
of  the  individual  analyses  are  recorded  in  Table  VL 

If  the  cation  molarity  (Na  plus  K)  of  the  Squid  axoplasm  is  a  constant,  as  is  sug- 
gested by  the  work  of  Steinbach  and  Spiegelman,  then  it  is  evident  that  during  nerve 
References  p.  114. 


VOL.  4  (1950) 


PERMEABILITY   AND    NERVE    FUNCTION,  II 


105 


TABLE  VI 

EFFECT    OF   ELECTRICAL    ACTIVITY    OF    THE    NERVE    ON    THE    RATE    OF    PENETRATION    OF    Na 

Nerves  were  stimulated  at  a  rate  of  100  times  per  second  for  a  period  of  30  min  in  sea  water  containing 
0.39  M  Na23Cl  +  0.13  M  Na^^Cl  at  22°  C.  S^  and  Sj  =  Standards. 


Axoplasm 

Counts 

Millimoles 

(mg) 

per 

min 

per  100  g 

Si 

2045  1  a 
2042 

verage 

S2 

2044 

II. 2 

3420 

15-5 

20.4 

6490 

16.2 

16.0 

5340 

17.0 

12.2 

4380 

18.3 

6.2 

1803 

14.8 

11.4 

3002 

13.6 

Average 

15-9 

Control  (see 

Table  IV) 

9-5 

activity,  a  quantity  of  K  has  been  lost  by  the  nerve  to  the  sea  water  equivalent  to  the 
Na  which  penetrated  during  the  same  period.  In  the  case  under  consideration,  this  would 
be  equivalent  to  a  loss  of  6.4  millimoles  K/ioo  g  of  axoplasm.  This  loss  appears  to  be 
very  high  since,  as  discussed  earlier,  at  rest  a  maximum  of  2.5  milHmoles  K/ioo  g  are 
easily  exchangeable. 

A  few  calculations  concerning  the  exchange  of  ions  during  activity  of  the  nerve 
may  be  of  interest.  The  average  diameter  of  the  stellar  nerve  may  be  assumed  to  be  of 
the  order  of  500  fx.  An  axoplasm  cylinder  of  r  =  0.025  cm  and  weighing  i  g  would 
have  a  surface  area  of  80  cm^.  Since  an  increased  exchange  of  6.4  millimoles  Na/ioo  g 
(or  6.4-10"^  mole/g)  has  been  demonstrated  for  a  nerve  which  had  been  stimulated 
1.8 -lo^  times  (100  per  second  for  30  min),  it  follows  that  6.4-10"^  mole/g  divided 
by  1.8-10^  or  3.6-  io~i°  mole/g/impulse  of  Na  penetrated  into  the  axoplasm  of  the  nerve 
from  the  sea  water.  This  value  corresponds  to  4.5  •  iq-^^  x^q\q  of  Na  penetrating/cm^/im- 
pulse.  It  has  been  reported  by  Pumphrey  and  Young^  that  the  diameters  of  these 
giant  nerve  fibres  of  Squid  usually  vary  from  280  to  720  //  in  diameter  and  may  in  some 
cases  by  as  large  as  1000  /t  (i  mm).  If  one  calculates  the  values  of  Na  which  would 
penetrate  per  cm^  per  impulse  for  the  usual  extremes  in  the  size  of  the  fibres  under  the 
above  conditions,  one  obtains  the  values  2.6-10-^-  and  6.5  •lO"!^  mole/cm-/impulse  for 
the  smaller  and  larger  diameters  respectively.  If  one  assumes  that  the  increased  Na 
penetration  during  activity  is  equivalent  to  the  K  loss  during  the  same  period,  as  the 
work  of  several  investigators  indicates,  then  it  follows  that  the  transfer  of  4.5 -10"^^ 
mole/cm^/impulse  of  K  has  occurred  during  the  period  of  nerve  activity.  This  value 
is  in  excellent  agreement  with  that  indirectly  calculated  by  Hodgkin  and  Huxley^ 
on  the  basis  of  the  changes  in  membrane  conductivity  which  occur  in  single  fibre 
preparations  of  Carcinus  maenus  nerves  during  normal  conduction.  They  obtained  a 
value  of  i.7-io~i2  mole/cm^/impulse.  The  value  is  also  in  good  agreement  with  that 
obtained  by  Keynes^".  This  investigator  soaked  multifibre  preparations  of  Carcinus 
nerves  in  K^2_  Upon  stimulation  he  found  the  leakage  of  2.1-10-12  mole/cm^/impulse. 
The  data  with  Na^*,  Hke  those  of  Keynes,  are  direct.  The  method  of  Hodgkin  and 
Huxley,  although  most  ingenious,  necessitates  numerous  assumptions  and  is  therefore 
References  p.  114. 


io6 


M.  A.  ROTHENBERG 


VOL.  4  {1950) 


inherently  indirect.  In  spite  of  the  fact  that  the  methods  and  materials  employed  are 
different,  the  agreement  is  surprisingly  close  in  the  three  cases. 

3.  Effect  of  inhibitors  of  acetylcholine-esterase  on  the  ion  exchange.  The  effects  of  two 
inhibitors  of  acetylcholine-esterase  were  studied  on  the  rate  of  exchange  of  Na  and  K 
in  these  fibres.  In  Table  VII  are  given  the  results  obtained  when  giant  axons  were 
exposed  for  30  min  to  0.022  M  diisopropyl  fiuorophosphate  (DFP)  in  sea  water  containing 
0.013  M  K*2C1.  DFP  at  this  concentration  is  capable  of  abolishing  nerve  conduction 
within  approximately  2  min^^  and  the  action  of  this  compound  can  probably  be  attri- 
buted exclusively  to  the  inactivation  of  the  enzyme^^.  The  average  of  five  nerves  exposed 
to  sea  water  containing  DFP  and  K^^  gave  a  value  of  1.08  milHmoles  K/ioo  g  while 
exposure  to  sea  water  for  the  same  period  of  time  in  the  absence  of  DFP  gave  a  value 
of  1. 31  millimoles/ioo  g.  Assuming,  as  above,  that  the  average  diameter  of  these  fibres 
is  500  [X  (area  of  i  g  cylinder  of  axoplasm  being  equal  to  80  cm^),  then  one  obtains  a  value 
of  5.5-10"^  mole/cm^/min  as  the  rate  of  exchange  of  K  in  sea  water  at  rest.  In  the  pre- 
sence of  DFP  this  rate  falls  to  4.5 -lO"-^  mole/cm^/min.  This  would  correspond  to  a 
decrease  of  i.c-io"^  mole/cm^/min  in  the  presence  of  DFP.  Although  the  concentration 
of  K*2  in  the  axoplasm  is  smaller  in  the  presence  of  DFP  than  in  its  absence,  this  result 
does  not  indicate  a  deci  eased  permeability.  In  view  of  the  concentration  gradient  be- 
tween the  inside  of  the  axon  and  its  outer  environment  an  increase  in  permeabihty 
may  lead  to  an  increase  of  the  K  outflow  from  the  interior.  The  K*^  penetrating  from 
the  outside  may  share  the  same  fate  and  the  final  inside  concentration  will  eventually 
be  smaller  than  that  under  normal  conditions. 


TABLE  VII 

EFFECT  OF  DFP  ON  THE  RATE  OF  PENETRATION  OF  K  AND  Na 

Nerves  exposed  to  0.022  M  DFP  in  sea  water  containing  either  0.013  M  K'l^Cl  or  0.37  M  Na'^'Cl  -f- 
0.13  M  Na^^Cl.  Si  and  Sj  =  standards. 


K 

Counts 

Millimoles 

Na 

Counts 

Millimoles 

Axoplasm 
(mg) 

per  min 

per  100  g 

Axoplasm 
(mg) 

per  min 

per  100  g 

Si 

1014  1  average 
1051  1     1033 

Si 

1324  )  average 

S2 

S2 

1285  1     1305 

5-8 

472 

1.02 

10.6 

2319 

16.8 

8.0 

776 

1. 21 

8.6 

1589 

14.2 

4.6 

398 

1 .09 

12  0 

2535 

16.2 

6.0 

447 

0.93 

10.2 

2480 

18.6 

5-4 

493 

115 

16.0 

2990 

143 

7.0 

1649 

18.0 

Average 

1.08 

13.8 

2970 

16.5 

9.6 

2055 

16.6 

14.8 

2975 

15-4 

Average 

16.4 

Control  (see 

Table  V) 

I-3I 

Control  (see 

Table  IV) 

9-5 

This  view  is  confirmed  by  the  effect  of  the  DFP  on  the  Na  movement.  Table  VII 
gives  the  results  obtained  when  nerves  were  exposed  to  DFP  in  the  same  concentration 
as  above  (0.022  M)  in  the  presence  of  0.13  M  Na^^Cl  +  0.37  M  Na^^Cl  in  the  sea  water. 
The  mean  of  nine  nerves  exposed  to  DFP  in  sea  water  gave  a  values  of  16.4  millimoles 
References  p.  114. 


VOL.  4  (1950) 


PERMEABILITY   AND    NERVE    FUNCTION,  II 


10'; 


Na/ioo  g  as  compared  to  9.5  millimoles/ioo  g  when  exposed  to  sea  water  in  the  absence 
of  DFP.  This  would  correspond  to  a  rate  of  penetration  of  Na  of  4.o-io~^/cm2/min  in 
the  absence  of  DFP  and  a  penetration  of  6.9-10"^  mole/cm^/min  in  the  presence  of 
DFP,  assuming  the  average  fibre  diameter  to  be  500  /<.  The  rate  of  Na  penetration  has 
increased  markedly.  This  could  be  expected  on  the  basis  of  the  concentration  gradient 
in  the  event  of  increased  permeability.  It  may  be  noted  that  the  Na  penetration  has 
increased  to  a  greater  extent  than  the  K  penetration  has  decreased.  Considering  the 
difference  in  the  rates  of  entrance  of  Na  and  K,  it  has  to  be  kept  in  mind  that  in  the 
experiments  described,  only  the  penetration  of  ions  into  the  interior  has  been  determined. 
No  measurements  have  been  carried  out  in  respect  to  the  leakage  of  K.  If  the  amount 
of  K  actually  passing  from  the  inside  to  the  outside  were  considerably  increased,  this 
would  not  be  indicated  by  the  method  used. 

The  effect  of  eserine,  another  inhibitor  of  acetylcholine-esterase,  on  the  rate  of  Na 
penetration  into  the  nerve  was  also  studied.  The  results  are  given  in  Table  VIII.  It  will 
be  noted  that  13.2  millimoles  Na/ioo  g  enter  these  nerves  in  the  presence  of  0.019  ^ 
eserine  in  the  sea  water  containing  0.13  M  Na^'^Cl  +  0.37  M  Na^^Cl.  This  would  corre- 
spond to  a  rate  of  exchange  of  Na  of  5.5-10"^  mole/cm^/min  in  the  presence  of  eserine 
as  compared  to  4.0-10"^  mole/cm^/min  in  its  absence,  again  assuming  the  average 
fibre  diameter  to  be  500  /n.  The  above  value  is  the  average  of  ten  nerves  and,  as  in  the 
other  experiments,  nerves  were  exposed  for  30  min  to  the  eserine-containing  sea  water. 
Eserine,  in  the  concentration  used,  abolishes  nerve  conduction  reversibly  within  5-15 
min.  The  time  required  to  abolish  the  action  potential  of  these  nerves  shows  considerable 
variation  in  the  case  of  eserine  and  is  closely  dependent  upon  the  p^  and  other  factors^^. 
Air  o-xidation  of  the  eserine  proceeds  rapidly  at  the  p^  employed  (7.7-8.0)  and  therefore 


TABLE  VIII 

EFFECT    OF    ESERINE    ON    THE    RATE    OF    PENETR.\TION    ON    Na 

Xerves  exposed  to  0.019  M  eserine  in  sea  water  (pn  7.7-8.0)  containing  0.37  M  Na^'^Cl  -\-  0.13  M 


Na^*Cl.  S,  and  S, 


Standards. 


Axoplasm 

Counts 

Millimoles 

(mg) 

per  min 

per  100  g 

Si 

2002  \  average 
1910  1     1956 

S2 

22.0 

5307 

12.3 

15.0 

3705 

12.6 

19.4 

5550 

14-7 

3.8 

1019 

137 

Si 

1820  \  average 

s. 

1861        1841 

9.2 

2458 

I5-I 

22.4 

4950 

12.0 

8.4 

1946 

12.6 

14.8 

3660 

134 

20.8 

4720 

12-3 

26.0 

6490 

13-5 

Average 

132 

Control  (see 

Table  IV) 

9.5 

References  p.  114. 


io8 


M.  A.  ROTHENBERG 


VOL.  4  (1950) 


a  given  solution  cannot  be  used  for  a  prolonged  period  of  time.  The  results  presented 
were  obtained  with  fresh  eserine  solutions.  Although  there  is  a  marked  increase  in  Na 
exchange,  the  effect  of  eserine  is  not  as  large  as  that  obtained  with  DFP. 

4.  Cocaine  and  Na  exchange.  The  effects  of  cocaine  in  0.005  M  in  sea  water  have  been 
studied  using  0.13  M  Na^^Cl  +  0.39  M  Na^^Cl  in  the  bathing  fluid.  Nerves  were  exposed 
to  this  solution  for  30  min.  The  results  are  reported  in  Table  IX.  No  decrease  in  mem- 
brane permeability  is  evident  from  the  data.  The  Na  exchange  amounted  to  11. 2  milli- 
moles/ioo  g  (average  of  six  nerves).  Again  assuming  a  fibre  diameter  of  500  /i,  this  would 
correspond  to  a  rate  of  Na  exchange  of  4.6-10"^  mole/cm^/min,  a  slight  increase  com- 
pared with  the  control. 


TABLE  IX 

EFFECT  OF  COCAINE  ON  THE  RATE  OF  PENETRATION  OF  Na 

Nerves  exposed  to  0.005  M  cocaine  in  sea  water  containing  0.39  M  Na^^Cl  +  0.13  M  Na^^Cl.  S^  and 
S,  =  standards. 


Axoplasm 

Counts 

Millimoles 

(mg) 

per  min 

per  100  g 

s.        * 

2045  )  average 

S2 

2042  1     2044 

6.4 

1301 

10.5 

5-8 

1102 

9-9 

4.8 

995 

10.8 

12.8 

2983 

12. 1 

4.8 

1142 

12.3 

7.2 

1616 

II. 6 

Average 

II. 2 

Control  (see 

Table  IV) 

9-5 

5.  Effect  of  X-ray  irradiation.  The  effects  of  high  intensity  X-ray  irradiation  on  the 
membrane  permeability  to  Na  was  studied.  Nerves  were  irradiated  with  50000  R  and 
125000  R  while  immersed  in  a  shallow  dish  containing  natural  sea  water  (water  layer 
about  5  mm  thick).  Immediately  after  irradiation,  the  nerves  were  transferred  to 
artificial  sea  water  containing  0.39  M  Na^^Cl  +  0.13  M  Na^^Cl.  After  30  min  exposure  to 
sea  water  the  nerves  were  analysed.  Only  those  nerves  which  still  exhibited  normal 
conduction  upon  stimulation  were  used.  The  results  are  given  in  Table  X. 

In  the  axoplasm  of  nerves  irradiated  with  125000  R,  an  average  value  of  14.1 
miUimoles/ioo  g  was  found  (average  of  seven  values).  This  corresponds  to  a  penetration 
of  5.9-10"^  mole/cm^/min.  Consequently,  the  rate  of  penetration  had  markedly  increased. 
The  findings  suggest  that  irradiation  had  strongly  increased  the  permeability. 

Irradiation  with  50000  R  gave  an  average  value  of  10.9  millimoles  Na/ioo  g  (average 
of  eight  nerves).  This  corresponds  to  a  rate  of  penetration  of  Na  of  4.7-10""^  mole/cm^/ 
min.  The  increase  in  the  rate  of  penetration  is  relatively  small  but  appears  significant, 
especially  in  connection  with  the  high  increase  observed  with  the  larger  dose  of  irra- 
diation. It  may  be  noted  that  the  effect  was  obtained  immediately  after  irradiation. 


References  p.  114. 


VOL.  4  (1950) 


PERMEABILITY   AND    NERVE    FUNCTION,  II 


109 


TABLE   X 

EFFECT    OF    X-RAY    IRRADIATION    ON    THE    RATE    OF    PENETRATION    OF    Na 

Nerves  irradiated  with  50000  R  and  125000  R  respectively  in  natural  sea  water  and  then  exposed 
for  30  min  to  artificial  sea  water  containing.  0.39  M  Na^^Cl  +0.13  M  Na^^Cl.  Sj  and  S2  =  standards. 


50000  R 

Axoplasm 

(mg) 

Counts 
per  min 

Millimoles 
per  100  g 

125000  R 

Axoplasm 
(rag) 

Counts 
per  min 

Millimoles 
per  100  g 

Si 

S2 
10.4 

13-4 
13-8 

2002  1  average 
1910  1     1956 

2425 
2620 

2375 

12.4 

10.4 

91 

7.0 
5-0 
3-8 
6.2 

1610 
1248 

1033 
1700 

12.2 
133 
14-5 
14.6 

Si 
S2 
6.6 
7.6 
8.4 
9.8 
II. 8 

1820    average 

1861  1     1841 

1 198 

1565 

1576 

1696 

2725 

Table  IV) 

10.3 
II. 6 
10.6 

9-7 
13.0 

Si 
S2 
5-6 
4.0 

5-4 
Average 

2045  \  average 

2042  J     2044 

1380 

1225 

1711 

12.5 
15.6 
16. 1 

Average 
Control  (see 

10.9 

14. 1 
9-5 

DISCUSSION 

From  the  results  obtained  upon  exposure  of  nerves  to  sea  water,  at  rest,  containing 
radioactive  K*^,  it  can  be  seen  that  part  of  the  K  of  the  nerve  interior  is  in  dynamic 
equihbrium  with  that  in  the  outer  bathing  medium.  The  lack  of  exchange  of  approxi- 
mately 90%  of  the  K^^  under  these  conditions  is  unexplained.  It  appears  that  most  of 
the  K  inside  the  nerve  is  not  easily  lost  by  the  cell.  Once  the  free,  easily  diffusible  K 
has  been  exchanged  for  K''^,  the  rate  of  K  exchange  falls  to  a  very  low  level.  This  is  in 
good  agreement  with  the  observations  of  Hevesy  and  Hahn  on  rabbit  muscle  and  red 
blood  cells^*,  of  Steinbach  on  Thyone  briareus  muscle^^,  and  of  Heppel  on  rat  muscle^^. 
In  all  of  these  investigations  no  more  than  10-30%  of  the  total  K  content  of  the  tissues 
under  investigation  was  exchangeable  at  rest. 

In  an  effort  to  explain  the  difficulty  of  incomplete  K  exchange  essentially  two 
theories  have  been  discussed.  The  one  considers  the  possibility  that  the  K  is  present 
in  bound  form.  The  idea  has  been  proposed  that  a  K  salt  of  an  unknown  organic  acid 
with  a  very  low  dissociation  constant  exists.  As  emphasized  by  Krogh^,  there  is  no 
evidence  foi  the  existence  of  bound  K  and  from  a  theoretical  basis,  it  appears  doubtful 
that  it  can  exist.  Hill  and  Kupalov^'  have  shown  that  all  the  K  inside  the  muscle  cell 
is  required  to  be  in  ionic  form  in  order  to  account  for  the  osmotic  pressure.  Moreover, 
its  presence  in  ionic  form  is  necessary  to  insure  the  neutral  reaction.  Another  possibility 
discussed  is  the  presence  of  K  impermeable  barriers  inside  the  cell.  No  such  structures 
are  known.  The  reasons  for  exchange  of  only  a  small  fraction  of  the  total  K  cannot 
be  resolved  at  present. 

References  p.  114. 


no  M.  A.  ROTHENBERG  VOL.  4  (1950) 

The  values  for  the  Qiq  for  K  and  Na  exchange  obtained,  1.22  and  1.33  respectively, 
are  in  good  agreement  with  the  value  of  1.25  calculated  theoretically  from  ionic  conduc- 
tively  measurements.  These  figures  do  not  support  the  assumption  that  important  energy 
yielding  reactions  are  involved  in  the  transport  of  ions  across  these  nerve  membranes 
in  resting  condition.  Krogh  discusses  the  possibility  that  the  extrusion  of  Na  from  the 
cell  interior  is  an  active  process  requiring  energy.  In  support  of  this  hypothesis,  he  cites 
experiments  of  Harris^^  and  Danowski^^  with  rabbit  and  human  erythrocytes  in  which 
it  had  been  shown  that,  at  low  temperature  and  at  body  temperature  in  the  absence  of 
glucose,  K  is  lost  to  the  bathing  medium  and  replaced  by  Na.  When  glycolysis  is  restored, 
the  normal  K  balance  is  restablished,  even  in  vitro,  with  a  resumption  of  rapid  Na 
extrusion.  If  the  extrusion  of  Na  is  an  active  process  in  the  nerve  preparation  tested, 
under  resting  condition,  one  would  have  expected  to  obtain  a  larger  value  for  the  Q^q. 
Lowering  the  temperature  of  these  nerves  by  ten  degrees  should  have  produced  a  marked 
effect  on  the  glycolytic  processes  and  should  have  been  expected  to  yield  larger  Na  values 
than  those  obtained. 

The  fact  that  in  resting  condition  no  expenditure  of  energy  seems  to  be  required 
for  the  ionic  movements  does  by  no  means  preclude  the  possibility  that  under  other 
conditions  these  movements  may  require  energy.  It  appears  likely  that  during  the 
early  growth  stage  of  these  nerves  chemical  reactions  are  in  operation  which  are  respon- 
sible for  the  establishment  of  the  large  concentration  gradient  between  the  potassium 
inside  the  fibre  and  that  in  the  outer  bathing  fluid.  The  same  is  true  for  the  disequilibrium 
observed  after  activity.  The  extra  oxygen  uptake  observed  after  activity  indicates  that 
energy  yielding  reactions  are  involved  in  the  restoration  of  the  resting  condition. 

The  present  studies  of  the  ion  exchange  occurring  in  nerve  during  activity  have 
indicated  that  the  Na  content  increases  markedly.  Similar  results  have  been  obtained 
with  muscle  tissue  by  Fenn  et  al.  on  frog,  and  rat^"'  2^'  ^^,  Wood,  Collins  and  Moe  on 
dog  gastrocnemius^^,  Tipton  on  cat  muscle^*,  Heppel  on  K-deprived  rats^^  and  Hahn 
AND  Hevesy  on  rats^*.  All  of  these  investigations  show  that  in  contracting  muscles 
the  permeability  to  ions  is  increased.  K  is  lost  from  the  fibres  and  is  replaced  by  Na. 
Steinbach  and  Spiegelman^  have  demonstrated  that  the  cation  molarity  of  the  Squid 
axoplasm  is,  under  a  variety  of  conditions,  constant  at  rest.  It  appears,  therefore, 
justifiable  to  assume  that  during  nerve  activity  K  loss  is  compensated  for  by  the  pene- 
tration of  an  equivalent  quantity  of  Na  into  these  fibres. 

This  idea  is  supported  by  the  demonstration  of  the  penetration  of  4.5  •lO"^^  mole 
Na/cm^/impulse,  a  value  which  is  in  close  agreement  with  the  value  of  1.7  •lO"^^  mole 
K/cm^/impulse  found  by  Hodgkin  and  Huxley^  and  2.1 -10"^^  mole  K/cm^/impulse 
reported  by  Keynes^".  The  value  reported  here  indicates  that  during  activity  a  con- 
siderable increase  of  Na  inside  takes  place.  6.4  millimoles  per  100  g  were  found  after 
30  min  stimulation  at  100  per  second  as  compared  with  1.3  millimoles  per  100  g  at  rest. 
If  an  equivalent  amount  of  K  has  leaked  out,  21%  of  the  total  K  content  has  been 
exchanged  during  this  stimulation  period.  It  should  be  noted  here  that  the  period  of 
stimulation  employed  is  by  no  means  the  maximum  possible  with  these  nerves.  Much 
more  prolonged  periods  of  stimulation  at  100  per  second  are  possible  and  one  would 
expect  an  even  greater  ion  exchange.  It  should  be  borne  in  mind  that  the  above  changes 
are  completely  reversible  and  cessation  of  stimulation  should  result  in  restoration  of 
the  normal  balance.  From  the  above  considerations,  it  may  be  concluded  that,  even 
though  90%  of  the  K  content  of  the  nerve  is  not  exchangeable  at  rest,  during  activity 
References  p.  114. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  II  III 

some  reactions  have  occurred  which  facihtate  the  more  rapid  loss  of  K  by  these  libres. 

A  short  discussion  of  the  methods  employed  in  the  papers  of  Hodgkin  and  Huxley 
AND  Keynes  as  compared  with  the  present  investigations  might  be  of  interest.  The 
method  used  by  Hodgkin  and  Huxley  involves  measurement  of  the  small  changes 
in  the  ionic  conductivities  over  small  areas  of  the  nerve  membranes  before  and  after 
activity.  Both  the  electrical  recording  equipment  and  the  electrode  assemblies  are 
complex  and  the  method  employed  necessitates  numerous  assumptions.  The  method 
employed  by  Keynes  is  more  direct.  However,  he  has  used  multifibre  preparations. 
Under  such  circumstances,  one  could  expect  a  retarded  diffusion  of  K*^  away  from  the 
nerve  preparation  because  of  the  possible  trapping  of  K  in  the  intracellular  fluids.  Since 
only  the  radioactivity  of  the  K*-  remaining  in  the  nerve  preparation  was  measured 
in  these  investigations,  one  would  expect  that  values  obtained  in  this  manner  would 
be  higher  than  the  actual  intracellular  K*^  content  of  the  fibres.  The  calculated  value 
for  the  K  leakage  per  cm^  per  impulse  would  therefore  be  expected  to  be  smaller  than 
the  true  value. 

The  method  employed  in  the  present  investigation  is  direct.  Since  it  is  possible 
to  analyse  directly  the  axoplasm  of  the  single  nerve  fibre,  the  values  obtained  must  be 
considered  to  be  more  precise  than  those  obtained  by  either  of  the  above  methods. 
The  only  assumption  involved  is  the  exact  size  of  the  individual  fibres  employed.  How- 
ever, since  all  of  the  Squid  used  were  of  approximately  the  same  size,  it  is  safe  to  assume 
that  the  fibres  were  all  of  approximately  the  same  diameters.  For  medium  size  Squid 
this  is  approximately  500  fi  (0.05  cm).  It  is  justifiable  to  assume  that  the  average  value 
is  close  to  this  figure. 

The  investigation  of  the  effect  of  inhibitors  of  acetylcholine-esterase  on  the  rates 
of  the  ion  exchange  across  the  nerve  membrane  requires  some  comment.  It  has  been 
shown  that  exposure  of  nerves  to  sea  water  for  30  minutes  containing  K^^  pj^s  DFP 
causes  a  decrease  in  the  rate  of  K  exchange  from  1.31  to  1.08  millimoles  per  100  g.  The 
exposure  of  nerves  to  DFP  has  apparently  altered  the  permeability  of  the  nerve  mem- 
brane. The  DFP  could  conceivably  have  affected  the  membrane  by  decreasing  its 
permeability.  However,  the  effect  of  DFP  on  the  rate  of  Na  penetration  excludes  this 
interpretation.  The  value  for  the  Na  penetration  markedly  increased  from  9.6  millimoles 
Na  per  100  g  to  16.4  milUmoles  upon  the  addition  of  0.022  M  DFP.  If  the  DFP  had  had 
the  effect  of  decreasing  the  membrane  permeability  one  would  have  expected  a  de- 
creased Na  exchange.  It  might  have  been  expected  that  with  increased  ion  permeability 
the  K  could  penetrate  into  the  fibre  more  readily.  However,  since  the  concentration 
of  K  inside  of  these  nerves  is  approximately  20  times  that  of  sea  water,  it  is  likely  that 
the  easily  exchangeable  K  will  rapidly  diffuse  out  into  the  sea  water  in  an  attempt  to 
equalize  the  adverse  concentration  gradient  across  the  nerve  membrane.  The  K,  in 
this  case,  will  be  replaced  by  the  entrance  of  Na  in  order  to  maintain  the  electrical 
neutrality  of  the  axoplasm.  In  such  an  event,  the  exchange  of  K*^  would  proceed  at  a 
decreased  rate  and  this  obviously  accounts  for  the  decreased  K  exchange  in  the  presence 
of  DFP.  Thus,  the  Na  and  K  exchange  measurements  are  consistent  with  the  concept 
that  the  membrane  permeability  had  been  increased  by  the  DFP. 

The  probability  of  the  exchange  of  K^^  for  radioactive  Na^*  was  discussed  befoie. 
Another  factor  to  be  considered  is  the  constancy  of  the  total  cation  content  of  these 
nerves.  It  has  been  demonstrated  by  Steinbach  and  Spiegelman^  that  under  normal 
resting  conditions  the  cation  content  (Na  -f  K)  of  these  nerves  is  a  constant.  However, 
References  p.  114. 


112  M.  A.  ROTHENBERG  VOL.  4  (1950) 

it  is  not  known  whether  nerves  in  which  the  permeability  has  been  increased  still  main- 
tain their  normal  total  cation  concentration.  It  is  possible  that  under  these  conditions 
Na  as  well  as  CI  may  diffuse  into  the  cell.  This  would  result  in  increased  total  base 
content.  Since  the  total  base  content  of  the  axoplasm  samples  has  not  been  measured, 
the  contribution  by  the  NaCl  diffusion  into  the  nerve  cannot  be  evaluated.  This  problem 
has  to  be  investigated  further. 

The  effect  of  eserine,  another  inhibitor  of  acetylcholine-esterase  had  a  similar  but 
less  marked  effect  than  DFP  in  increasing  the  membrane  permeability  to  Na.  It  may 
be  noted,  that  in  the  case  of  DFP  conduction  was,  on  the  basis  of  previous  experience, 
abolished  irreversibly.  In  the  case  of  eserine  the  effect  was  almost  certainly  still  rever- 
sible. 

The  result  obtained  with  acetylchoHne-esterase  inhibitors,  suggest  that  these  sub- 
stances may  be  capable  of  altering  the  membrane  permeability.  Since  the  only  known 
action  of  these  compounds  is  the  inhibition  of  the  enzyme  acetylcholine-esterase^^ 
which  is  known  to  be  closely  connected  with  nerve  conduction,  it  is  possible  that  the 
effect  observed  is  a  manifestation  of  the  inactivation  of  the  enzyme.  These  experiments 
do  not  permit  any  definite  conclusion,  especially  in  view  of  the  irreversible  action  of 
DFP  during  the  long  exposure  period  used.  However,  they  may  open  a  new  approach  to 
the  importance  of  the  acetylcholine-esterase  system  in  the  permeability  of  the  surface 
membrane  to  ions. 

The  study  of  effects  of  cocaine  on  the  membrane  permeability  to  Na  has  indicated 
a  small  increase  in  the  rate  of  exchange.  The  data  are  inadequate  to  judge  whether  or 
not  this  increase  is  significant.  Employing  the  same  concentration  of  cocaine  (5  •  io~^  M), 
Shanes",  from  membrane  potential  measurements,  came  to  the  conclusion  that  a 
decrease  in  permeability  had  been  accomplished.  The  results  obtained  here  fail  to  con- 
firm his  reports. 

The  study  of  effects  of  irradiation  of  nerves  with  large  doses  of  X-rays  (50000  R 
and  125000  R)  indicates  that  immediately  following  exposure,  marked  alterations  in 
membrane  permeability  are  evident.  Exposure  to  125000  R  caused  a  large  increase 
in  membrane  permeability  while  50000  R  caused  only  a  small  but  significant  increase. 
It  should  be  noted  that  these  studies  were  carried  out  immediately  after  irradiation. 
It  is  possible  that  a  more  marked  effect  would  be  evident  with  smaller  doses  of  irradiation 
if  longer  periods  of  time  were  permitted  to  elapse  between  irradiation  and  exposure  to 
radioactive  ions.  From  our  present  knowledge,  it  is  clear  that  the  most  notable  effects 
of  exposure  to  radiation  occur  after  prolonged  periods  of  time  so  that  a  longer  time 
lapse  than  that  used  in  these  experiments  might  be  preferable.  It  appears  significant  that 
it  has  been  possible  to  demonstrate  increased  membrane  permeability  as  result  of  X-ray 
irradiation. 

I  wish  to  express  my  gratitude  to  Dr  David  Nachmansohn  for  suggesting  these 
investigations  and  for  the  guidance  and  encouragement  he  has  given  throughout  the 
course  of  this  research.  I  am  indebted  to  Mrs  Emily  Feld-Hedal  and  Mrs  Heidi 
Richards  for  their  assistance  in  the  experiments. 

SUMMARY 

I.  Studies  on  the  permeability  of  the  surface  membranes  of  the  giant  axon  of  Squid  to  K  indicate 
that  a  dynamic  rather  than  a  static  equilibrium  exists  at  rest.  Approximately  10%  of  the  total  K 

References  p.  114. 


VOL.  4  (1950)  PERMEABILITY   AND    NERVE    FUNCTION,  II  II3 

in  the  fibre  is  replaced  by  K*^  from  the  bathing  medium  within  one  hour.  When  the  nerve  is  bathed 
in  twice  the  normal  K  concentration  (0.026  M)  the  K  content  of  the  axoplasm  reaches  a  maximum 
twice  that  obtained  with  the  normal  K  concentration  outside. 

2.  Exposure  of  nerves  to  sea  water  containing  Na^*  results  in  a  total  exchange  of  all  of  the  Na 
in  the  axoplasm  for  its  radioactive  isotope  within  20  to  30  minutes. 

3.  Studies  with  Ca''^  in  the  outer  bathing  fluid  indicate  an  uptake  of  Ca**  to  the  extent  of  0.85 
millimoles  per  100  g  within  45  minutes  and  then  a  decrease  to  0.45  millimoles  per  100  g  at  100  minutes 
of  exposure. 

4.  The  temperature  coefficient  (Qio)  obtained  from  the  rates  of  exchange  of  Na  and  K  does  not 
indicate  that  there  are  important  energy  yielding  chemical  reactions  involved  in  the  exchange  of 
ions  across  the  membrane  at  rest.  The  values  obtained  (1.22  for  K  and  1.33  for  Na)  are  in  good 
agreement  with  the  theoretical  value  (1.25)  calculated  from  ionic  conductivity  measurements. 

5.  Electrical  activity  causes  an  increased  rate  of  Na  penetration  into  the  fibre.  4.5-10—12  mole 
of  Na  enter  per  cm^  per  impulse. 

6.  Inhibitors  of  cholinesterase,  e.g.,  eserine  and  DFP,  seem  to  produce  an  increase  in  membrane 
permeability.  The  rate  of  K*^  penetration  is  decreased,  that  of  Na^^  increased. 

7.  Exposure  to  cocaine  (0.005  ^^)  does  not  affect  markedly  the  rate  of  Na^*  penetration. 

8.  X-ray  irradiation  with  125000  R  produces  a  large  and  immediate  increase  in  membrane 
permeability  to  Xa^*  whereas  50000  R  produces  a  smaller  effect  but  in  the  same  direction. 

RfiSUMfi 

1.  L'^tude  de  la  permeabilite  au  potassium  de  la  membrane  du  cordon  nerveux  principal  de 
Seiche  indique  I'existence  au  repos  d'un  equilibre  dynamique  plutot  que  statique.  Environ  le  10% 
du  K  total  de  la  fibre  est  remplace  par  K*^  du  milieu  environnant  en  une  heure.  Si  le  nerf  est  immerge 
dans  une  solution  de  concentration  de  K  deux  fois  plus  grande  que  la  concentration  normale  (0.026  M) 
la  teneur  en  K  de  I'axoplasme  atteint  un  maximum  qui  est  egal  au  double  de  la  valeur  obtenue  avec 
une  concentration  externe  normale  de  K. 

2.  Si  Ton  expose  un  nerf  a  I'eau  de  mer  contenant  Na^"*  un  echange  total  a  lieu  entre  le  Na  de 
I'axoplasme  et  son  isotope  radioactif  en  20  a  30  minutes. 

3.  Si  le  bain  exterieur  contient  Ca*^,  celui-ci  est  absorbe  jusqu'a  0.85  millimoles  par  100  g  en 
45  minutes,  puis  la  concentration  de  Ca*^  decroit  jusqu'a  une  valeur  de  0.45  millimoles  par  100  g 
au  bout  de  100  minutes. 

4.  Le  coefficient  de  temperature  (Qxq)  obtenu  a  partir  des  vitesses  d'echange  de  Na  et  K  ne 
semble  pas  indiquer  que  des  reactions  chimiques  degageant  d'importantes  quantites  d'energie  soient 
liees  a  I'echange  des  ions  a  travers  la  membrane.  Ses  valeurs  obtenues  (1.22  pour  le  K  et  1.33  pour 
le  Na)  sont  en  accord  avec  la  valeur  theorique  (1.25)  calculee  a  partir  de  mesures  de  conductivite 
ionique. 

5.  L'activite  electrique  augmente  la  vitesse  de  penetration  du  Na  dans  la  fibre.  4.5- 10—^2  mols 
de  Na  penetrent  par  cm^  et  par  influx. 

6.  Les  inhibiteurs  de  I'acetylcholine  esterase,  p.  ex.  I'eserine  et  le  DFP  semblent,  augmenter 
la  permeabilite  de  la  membrane.  La  vitesse  de  penetration  de  K*^  diminue  tandis  que  celle  de  Na^* 
augmente. 

7.  Une  exposition  a  la  cocaine  (0.005  M)  n'affecte  pas  considerablement  la  vitesse  de  penetration 
de  Na^^. 

8.  L 'irradiation  aux  rayons-X  de  125000  R  produit  une  augmentation  importante  et  immediate 
de  la  permeabilite  de  la  membrane  au  Na^*.  50000  R  produisent  un  effet  moindre  dans  le  meme  sens. 


ZUSAMMENFASSUNG 

1.  Die  Permeabilitat  der  Membranen  des  Hauptnervenstranges  vom  Tintenfisch  (Loligo  peallii) 
fiir  K  wurde  untersucht  und  gefunden,  dass  in  der  Ruhe  eher  ein  dynamisches  als  ein  statisches 
Gleichgewicht  zu  bestehen  scheint.  Ungefahr  10%  des  gesamten  K-Gehaltes  der  Faser  werden  inner- 
halb  einer  Stunde  durch  K*^  aus  der  umgebenden  Losung  ersetzt.  1st  der  K-Gehalt  des  Bades  zweimal 
so  gross  wie  die  normale  Konzentration  (0.026  M),  dann  ist  auch  der  maximale  K-Gehalt  des  Ncrven- 
stranggewebes  zweimal  so  gross  wie  bei  normaler  ausserer  Konzentration. 

2.  In  Xa^^-haltigem  Meerwasser  findet  ein  voUkommener  Austausch  des  im  Gewebe  enthaltenen 
Na  gegen  sein  radioaktives  Isotop  innerhalb  20  bis  30  Minutes  statt. 

3.  Enthalt  das  aussere  Bad  Ca**,  so  wird  dieses  bis  zu  0.84  Millimol  per  100  g  in  45  Minuten 
aufgenommen;  dann  nimmt  der  Ca^^-Gehalt  wieder  ab  und  betragt  noch  100  Minuten  0.45  Millimol  per 
100  g. 

4.  Der  aus  den  Austauschgeschwindigkeiten  fiir  Na  und  K  errechnete  Temperaturkoeffizient 


114  M-  -'^-  ROTHENBERG  VOL.  4  (1950) 

(Qio)  weist  nicht  darauf  hin,  dass  in  der  Ruhe  stark  exothermische  chemische  Reaktionen  an  dem 
lonenaustausch  durch  die  Membrane  beteiligt  sind.  Die  erhaltenen  Werte  (1.22  fiir  K  und  1.33  fiir 
Na)  stimmen  gut  mit  dem  aus  Messungen  der  lonenleitfahigkeit  errechneten  theoretischen  Werte 
(1.25)  iiberein. 

5.  Durch  elektrische  Arbeit  wirddas  Eindringen  von  Na  beschleunigt.  4.5-  lo-^^  Mol  Na  per  cm^ 
dringen  bei  jeder  Anregung  ein. 

6.  Hemmstoffe  der  Acetylcholinesterase,  wie  Eserin  und  DFP  scheinen  die  Permeabilitat  der 
Membrane  zu  erhohen.  K-*^  wird  langsamer,  Na^^  rascher  aufgenommen. 

7.  Cocain  (0.005  M)  beeinflusst  die  Aufnahmegeschwindigkeit  von  Na^"*  nicht  merklich. 

8.  Bestrahlung  mit   Rontgen-Strahlen   (125000)   erhoht  R  die  PermeabUitat  fiir  Na^*  augen- 
blicklich  stark,  mit  50000  R  ist  dieser  Effekt  gleichgerichtet  aber  geringer. 

REFERENCES 

1  H.  B.  Steinbach  and  S.  Spiegelman,  /.  Cellular  Comp.  Physiol.,  22  (1943)  187. 

2  C.  F.  A.  Pantin,  /.  Exptl  Biol.,  11  (1934)  11. 

3  R.  S.  B.AER  AND  F.  O.  ScHMiTT,  /.  Cellular  Comp.  Physiol.,  14  (1939)  205. 

*  D.  A.  Webb  and  J.  Z.  Young,  /.  Physiol.,  98  (1940)  299. 

■iaM.  A.  Rothenberg  and  E.  A.  Feld,  /.  Biol.  Chem.,  i-jz  (1948)  345. 
5  A.  Krogh,  Proc.  Roy.  Soc,  B  133  (1946)  140. 

*  E.  J.  Conway,  Irish  J.  Med.  Science,  Oct.-Nov.  (1947)  593. 

^  S.  Glasstone,  Textbook  of  Physical  Chemistry,  D.  van  Nostrand  Co  (1941)  895. 
s  R.  J.  Pumphrey  and  J.  Z.  Young,  /.  Exptl  Biol.,  15  (1938)  453. 
8  A.  L.  HoDGKiN  and  a.  F.  Huxley,  /.  Physiol.,  106  (1947)  34i- 
1°  R.  D.  Keynes,  /.  Physiol,  107  (1948)  35  P. 

11  T.  H.  Bullock,  H.  Grundfest,  D.  Nachmansohn,  and  M.  A.  Rothenberg,  /.  Neurophysiol., 

ID  (1947)  63. 

12  H.  Grundfest,  D.  Nachmansohn,  and  M.  A.  Rothenberg,  /.  Neurophysiol.,  10  (1947)  155. 

13  T.  H.  Bullock,  D.  Nachmansohn,  M.  A.  Rothenberg,  and  K.  Sterling,  /.  Neurophysiol.,  9 
(1946)  253. 

1*  G.  Hevesy  and  L.  Hahn,  Kgl.  Danske.  Videnskab.  Selskabs  Biol.  Medd.,  16  (1941)  i- 

1^  H.  B.  Steinbach,  /.  Cellular  Comp.  Physiol.,  9  (1937)  429. 

18  L.  A.  Heppel,  Am.  J.  Physiol.,  127  (1939)  385. 

"  A.  V.  Hill  and  P.  S.  Kupalov,  Proc.  Roy.  Soc,  B  106  (1930)  445. 

18  J.  Harris,  Biol.  Bull.,  79  (1940)  373. 

1^  T.  S.  Danowski,  J.  Biol.  Chem.,  139  (1941)  693. 

20  W.  O.  Fenn,  Physiol.  Revs,  16  (1936)  450. 

21  W.  O.  Fenn  and  D.  M.  Cobb,  Am.  J.  Physiol.,  115  (1936)  345- 

22  W.  O.  Fenn,  D.  M.  Cobb,  J.  F.  Manery,  and  W.  R.  Bloor,  Am.  J.  Physiol.,  121  (1937)  595- 

23  E.  H.  Wood,  D.  A.  Collins  ,and  G.  K.  Moe,  Am.  J.  Physiol.,  128  (1940)  635. 
2*  S.  R.  Tipton,  Atn.  J.  Physiol.,  124  (1938)  322. 

25  L.  A.  Heppel,  Am.  J.  Physiol.,  128  (1939)  440. 

26  M.  Dixon  and  D.  M.  Needham,  Nature,  158  (1946)  432. 
2'  A.  M.  Shanes,  Science,  107  (1948)  679. 

Received  May  17th,  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  II; 


NERVE  CONDUCTION 

WITHOUT  INCREASED  OXYGEN  CONSUMPTION;  THE  ACTION  OF 

AZIDE  AND  FLUOROACETATE* 

by 

R.  W.  GERARD  and  R.  W.  DOTY 

Department  of  Physiology,  University  of  Chicago  (U.S.A.) 


The  precise  correlation  of  an  extra  oxygen  consumption  of  active  nerve  with  an 
extra  heat  production  was  estabhshed  nearly  a  quarter  of  a  century  ago  by  one  of  us  in 
Prof.  Meyerhof's  laboratory.  It  is  an  especial  pleasure  to  report  the  present  extension 
of  such  studies,  in  his  honour.  Nor  can  we  refrain  from  an  expression  of  admiration  for 
his  continued  vigour  of  thought  and  research  despite  a  weight  of  personal  disaster  that 
would  have  crushed  most  men. 

That  the  extra  energy  release  of  nerve  activity  is  essential  to  conduction  and 
recovery  was  taken  for  granted  since  its  discovery.  With  energy  sources  blocked  by 
oxygen  lack  or  lAA  poisoning,  conduction  failed.  With  tetanization  at  a  rate  to  limit 
full  development  of  the  delayed  heat  and  oxygen  consumption,  conduction  was  de- 
pressed. Restoration  of  full  metabolism  restored  full  conduction  in  all  cases.  The  actual 
fuel  burned  proved  not  identical  for  rest  and  activity.  True,  both  resting  and  active 
metabolism  seemed  to  focus  on  the  production  of  energy-rich  phosphate  bonds,  especi- 
ally as  creatine  phosphate.  And  true,  also,  that  the  procedures  that  blocked  conduction 
affected  resting  as  well  as  active  respiration.  Nonetheless,  there  seemed  no  reason  to 
question  the  essential  contribution  of  the  active  respiration  to  actual  conduction.  A 
tentative  report  by  Schmitt,  of  a  fall  in  oxj^gen  consumption  on  stimulation  of  yohim- 
binized  nerve,  was  given  little  weight ;  and  Lorente  de  No's  finding,  that  excitation 
could  be  restored  in  a  nerve  blocked  by  anoxia,  with  the  aid  of  a  repolarizing  current, 
did  not  really  question  the  necessity  of  the  metabolism  as  a  normal  source  of  membrane 
polarization. 

Yet  it  was  early  shown  by  Feng  and  in  this  laboratory  that  lactate,  indifferent  to 
nerve  conduction  and  metabolism  under  normal  conditions,  could  restore  resting  oxygen 
consumption  and  active  conduction  after  lAA  poisoning  —  suggesting  some  interchange- 
ability  of  resting  and  active  metabolic  energy.  Further,  90  to  97%  of  the  energy  of 
activity  is  liberated  after  an  impulse  has  traveled  and  the  nerve  again  reset  for  action. 
Moreover,  a  factor  of  safety  of  live  for  the  resting  metabolism  could  be  estimated. 
Activity  might,  then,  be  supported  under  emergency  conditions  by  a  portion  of  the 
resting  metabolism.  Sodium  azide,  found  by  Stannard  to  eHminate  the  contraction 
respiration  of  muscle,  was  tested  on  nerve  in  Bronx's  laboratory  and  here  and  found 
indeed  able  to  abolish  the  extra  oxygen  consumption  of  active  nerve  while  leaving 
conduction  intact  and  resting  respiration  largely  so.  We  found,  further,  that  methyl 


This  work  was  performed  under  contract  with  the  Office  of  Naval  Research. 


Il6  R.  W.  GERARD,  R.  W.  DOTY  VOL.  4  (1950) 

fluoroacetate  can  reduce  the  resting  oxygen  consumption  below  half  normal  while 
leaving  conduction  and  the  attendant  respiration  increase  intact.  Resting  and  active 
respiration  are  thus  sharply  separable,  yet  they  are  effectively  interchangeable  in  support 
of  function. 

For  these  studies,  a  modified  Gerard-Hartline  capillary  respirometer  was  developed.  Ten 
slots  in  a  plexiglass  block  served  as  nerve  chambers,  each  fitted  with  stimulating  and  lead-off  elec- 
trodes. Capillaries  led  from  each  into  a  large  chamber  machined  in  the  same  block,  the  whole  being 
covered  with  a  plexiglass  sheet  and  mounted  in  a  glass-walled  water  bath.  The  movement  of  dodecane 
indicator  drops  in  the  capillaries  was  followed  with  a  horizontal  microscope  mounted  on  the  compound 
rest  of  an  II  inch  lathe.  Stimuli  at  120/sec  gave  an  action  spike  of  about  25  mm  measured  on  the 
cathode  ray  tube  face. 

The  resting  Qq^  of  twenty  four  pairs  of  frog  sciatics  at  24°  C  (22  to  26)  centered 
around  65  and  the  two  nerves  of  a  pair  agreed  within  12%  (aver.  4%)  in  all  but  three 
cases.  The  increased  Q02  on  maximal  stimulation  averaged  21,  but  with  an  average 
difference  between  members  of  a  pair  of  nearly  30%.  The  coefficient  of  correlation 
between  spike  height  and  activity  Qq^  was  only  0.4  for  67  normal  nerves,  and  that 
between  resting  Qq^  and  the  active  increase,  — o.i.  Even  allowing  for  methodological 
errors,  these  data  suggest  some  real  independence  of  the  three  variables. 

In  ten  experiments  with  Na  azide  (o.i  or  0.3  mM,  Ph7-5,  i  hour  soak),  spike  height 
of  the  exposed  nerves  averaged  88  %  of  their  undrugged  partners,  while  the  Q02  increase 
on  tetanization  was  only  12%  of  the  normals.  In  four  experiments  with  spike  height  in 
both  nerves  of  a  pair  alike,  the  Qq^  increase  in  the  azide  member  was  o  or  i.  Even  these 
azide  concentrations  do  not  fully  spare  the  resting  metabolism,  which  was  depressed  by 
o  in  4  experiments  to  some  50%  in  2.  When  resting  oxygen  was  cut  in  two  and  the  active 
increase  abolished,  spike  height  was  greatly  reduced.  Stronger  azide  (5  or  10  mM)  cut 
resting  Q02  to  20-35%  of  normal  and  stopped  conduction.  Full  conduction  without 
increased  Q02  is  possible  for  at  least  4  hours. 

In  II  experiments  with  MFA  (i  to  2.5  mM),  the  spike  height  and  the  extra  Q02  of 
activity  remained  entirely  normal  in  the  exposed  nerves,  while  the  resting  Q02  was 
depressed  25%  on  the  average,  one  third  maximum.  This  depression  cannot  be  solely 
of  non-axonal  tissue  {e.g.,  Schwann  cells),  for  fiber  thresholds  rise  acutely.  With  stronger 
MFA  (13  experiments  at  5  or  7.5  mM),  resting  and  active  Q02  were  both  cut  to  about 
half  and  spike  height  to  under  two-thirds  normal.  In  individual  cases,  the  active  spike 
and  Q02  were  essentially  normal  with  resting  Q02  depressed  to  one-third;  in  one  case 
activity  responses  remained  normal  for  7  hours  with  resting  Q02  at  50%.  More  usually 
with  resting  Qq^  cut  in  half  the  active  increase  was  also  abolished  while  spike  height 
remained  close  to  normal. 

A  nerve  can  thus  continue  to  conduct  for  hours  with  no  increase  in  oxygen  con- 
sumption and  even  with  some  half  its  resting  respiration  lost.  Whether  other  energy 
sources  are  being  tapped  or  even  whether  the  small  initial  heat  persists  without  delayed 
heat  under  such  drug  action,  could  be  determined  by  heat  measurements;  but  it  seems 
most  likely  that  the  extra  energy  for  activity  is  somehow  derived  from  the  resting 
metabolism  by  virtue  of  the  considerable  safety  factor  normally  present. 

SUMMARY 

Using  a  modified  Gerard-Hartline  capillary  respirometer  the  resting  respiration  of  frog  nerve 
at  24°  C  was  measured,  Qog  65,  as  well  as  the  increase  on  tetanization  at  120/sec,  Qoj  21,  and  the 


VOL.  4  (1950)  NERVE    CONDUCTION   WITHOUT   INCREASED    QOj  II7 

action  spike.  Azide  (0.1-0.3  mM)  can  abolish  the  activity  increase  of  oxygen  consumption  while 
leavang  intact  (sometimes)  the  resting  level  and  conduction.  Methylfluoroacetate  (2  mM),  conversely, 
can  reduce  the  resting  oxygen  consumption  below  half  while  leaving  intact  the  activity  increase  and 
conduction.  Resting  and  active  metabolism  are  thus  separable  and  conduction  can  continue  at  least 
seven  hours  with  no  extra  respiration  and  even  with  half  depression  of  the  resting  level. 

RfiSUMfi 

Au  moyen  d'un  respirometre  capillaire  Ger.\rd-H.\rtline  modifie,  on  a  mesure  a  24°  la  respi- 
ration de  nerfs  de  grenouille  au  repos  (Qoj  65),  son  augmentation  par  tetanisation  a  120/sec  (Qog  21), 
et  la  "pointe"  d'action.  L'ion  N3  (0.1-0.3  mM)  pent  abolir  I'accroissement  de  consommation  d'oxygene 
du  a  I'activite,  tout  en  laissant  intacts  (parfois)  le  niveau  du  repos  et  la  conduction.  Le  fluorac^tate 
de  methyle  (2  mM)  par  contre  peut  reduire  la  consommation  d'oxygene  au  repos  de  plus  de  la  moitie 
tout  en  laissant  intacts  I'accroissement  du  a  I'activite  et  la  conduction.  Le  metabolisme  au  repos  et 
pendant  I'activite  sont  ainsi  separables,  et  la  conduction  peut  continuer  pendant  au  moins  7  heures 
sans  respiration  supplementaire  et  meme  avec  un  abaissement  de  moitie  du  niveau  du  repos. 

ZUSAMMENFASSUNG 

Mittels  eines  abgeanderten  Ger.'V.rd-H.\rtline  Kapillar-Respirometers  wurde  die  Atmung  des 
ruhenden  Frischnervs  bei  24°  gemessen  (Qog  65),  desgleichen  die  Steigerung  durch  Tetanisierung 
bei  120/sek.  (Qoj  21)  und  die  "Wirkungspitze".  Azid  (0.1-0.3  mM)  kann  die  Steigerung  des  Sauer- 
stoffverbrauchs  bei  der  Arbeit  unterdriicken,  wahrend  der  Verbrauchsspiegel  bei  Ruhe  (manchmal) 
und  die  Ubertragung  unverandert  bleiben.  Methyl-fluoracetat  (2  mM)  dagegen  kann  den  Sauerstofi- 
verbrauch  bei  Ruhe  unter  die  Halfte  herabdriicken,  wahrend  die  Steigerung  bei  Arbeit  und  die 
Ubertragung  unberiihrt  bleiben.  Ruheumsatz  und  Arbeitsumsatz  sind  also  trennbar,  und  die  t)ber- 
tragung  kann  mindestens  7  Stunden  lang  fortbestehen  ohne  zusatzliche  Atmung,  und  sogar  mit  einem 
auf  die  Halfte  herabgeminderten  Ruhespiegel. 

Received  May  4th,  1949 


Il8  BIOCHIMICA   ET   BIOPHYSICA   ACTA  VOL.  4  (1950) 


SOME  EVIDENCE  ON  THE 
FUNCTIONAL  ORGANIZATION  OF  THE  BRAIN 

by 

HAROLD  E.  HIMWICH 

Medical  Division,  Army  Chemical  Center,  Maryland  {U.S.A.) 


"With  health,  the  assertion  is  that  each  person's  normal  thought  and  conduct  are, 
or  signify,  survivals  of  the  fittest  states  of  what  we  may  call  the  topmost  "layers".  Now 
suppose  that  from  disease  the  normal  highest  level  of  evolution  (the  topmost  layer)  is 
rendered  functionless.  This  is  the  dissolution  ...  I  contend  that  his  mental  symptoms 
are  survivals  on  the  lower,  but  then  highest,  level  of  evolution"  (remaining  in  function). 

So  wrote  Hughlings  Jackson  in  1884^.  One  type  of  evidence  for  such  an  evolution- 
ary concept  involving  a  hierarchy  of  levels  is  observed  by  studying  behaviour  following 
a  series  of  surgical  sections  of  the  brain.  A  transection  below  the  medulla  gives  rise  to 
the  spinal  animaP,  a  decapitated  preparation  kept  alive  by  artificial  respiration  but 
still  responding  to  stimulation  with  primitive  though  appropriate  muscular  actions. 
A  painful  stimulus  applied  to  the  foot  pad,  for  example,  evokes  flexion  of  that  leg,  a 
movement  that  makes  for  survival  as  the  leg  is  withdrawn  from  harm. 

The  decerebrate  animal  produced  by  cutting  through  a  higher  level^,  namely  the 
lower  portion  of  the  midbrain  and  therefore  retaining  the  medulla  reveals  a  release  of 
the  antigravity  muscles  permitting  an  abnormal  sort  of  erect  standing  called  decerebrate 
rigidity.  The  decorticate  animal  with  extirpation  of  the  highest  portion  of  his  brain 
only,  expresses  sham  rage,  a  release  of  emotional  patterns  from  cortical  control^.  Both 
decerebrate  rigidity  and  sham  rage  may  appear  spontaneously  or  may  be  evoked.  These 
three  sections  of  the  neuraxis  reveal  patterns  of  behaviour  which  are  functional  in  the 
intact  organism  but  are  modified  by  anatomically  higher  areas,  of  later  development 
which  facilitate  more  delicate  sensory  perception  and  finer  execution  of  movement. 
For  the  organism  to  take  advantage  of  these  improved  capacities  the  behaviour  of  the 
lower  portions  of  the  brain  must  be  subjected  to  the  inhibition  as  well  as  the  reinforce- 
ment of  the  higher  planes  and  when  their  influence  is  removed  we  see  a  release  of  function 
in  the  lower  areas,  a  result  of  loss  of  restraint.  Strong  support  for  the  observation  that 
inhibition  is  a  function  of  the  brain  has  been  afforded  by  the  physiological  experiments 
of  DussER  DE  Barenne  AND  McCuLLOCH^  who  demonstrated  thst  stimulation  of  one 
cerebral  area  suppresses  activity  in  another. 

For  another  type  of  evidence  we  must  turn  to  an  examination  of  man  for  an  oppor- 
tunity is  afforded  to  study  the  human  brain  when  sections  are  made  in  a  functional 
manner.  An  example  is  observed  during  hypoglycemia  when  a  temporary  "dissolution" 
of  the  brain  is  a  result  of  excessive  insulin^.  The  behavioural  phenomena  observed  may 
be  allocated  to  certain  cerebral  areas.  In  fact,  the  signs  exhibited  are  those  that  might 
References  p.  125. 


VOL.  4  (1950)  FUNCTIONAL   ORGANIZATION    OF   THE   BRAIN  II9 

be  expected  if  successive  surgical  sections  were  made  at  different  levels  of  the  brain. 

In  order  to  explain  the  changes  observed  in  hypoglycemia  it  must  be  recalled  that 
glucose  is  no  longer  available  to  the  brain.  Since  glucose  is  the  chief  foodstuff  of  the 
brain^' '» ^  the  metabolic  fires  falter  because  of  the  decrease  in  the  coal  to  be  burned*. 
A  decrease  to  52  %^°  and  40  %^^  respectively  of  the  normal  rate  have  been  reported 
in  hypoglycemia.  With  the  most  profound  metaboHc  depression  {i.e.,  in  the  5th  phase, 
see  below)  cerebral  metabolic  rate  may  be  reduced  to  25  %  of  the  normaP^.  But  not  all 
parts  of  the  brain  are  effected  to  an  equal  degree.  Though  the  brain  possesses  a  high 
rate  of  metabolism,  the  rate  is  not  the  same  in  all  regions  but  in  general  exhibits  a 
quantitative  gradient  along  the  neuraxis,  most  intense  anteriorally  and  superiorally  in 
the  cerebral  hemispheres  and  less  so  posteriorally  and  inferiorally  until  it  reaches  its 
lowest  level  in  the  medulla  oblongata.  This  conception  is  borne  out  by  the  observation 
of  excised  cerebral  tissues  which  show  a  decreasing  rate  of  oxygen  intake  as  the  neuraxis 
is  descended^^'  ^^.  The  oxygen  consumption  of  various  parts  in  the  human  brain  in 
vivo  will  not  be  considered  at  this  time  because  of  conflicting  results^*'  ^^.  Pending 
the  solution  of  this  discrepancy  we  may  point  to  another 
bit  of  evidence  of  a  hierarchy  in  metabolic  rate.  In  order 
to  combat  hypoglycemic  coma  carbohydrate  must  be  admi- 
nistered and  it  has  been  observed  that  a  larger  amount  of 
glucose  is  required  to  restore  the  functions  of  the  cerebral 
hemispheres  than  for  the  subcortical  areas^^.  Presumably 
a  greater  amount  of  foodstuff  is  necessary  to  support  a 
higher  rate  of  metabolism. 

If  we  accept  the  concept  of  dissimilar  metabolic  rates 

it  must  follow  that  all  parts  of  the  brain  will  not  be  equally      ^'S-  i-  Representation  (trans- 

rr     ,     1    ,        T  1  -li,!,,!  •  -,1         versc    sectlon)    of    the    brain 

affected   by  hypoglycemia   but   that   those  regions  with      disclosing  the  five  phyletic 

fastest    rates    would     succumb    first    and    those    with    the       areas:    i.  cerebral  cortex;   2. 

slowest,  for  example  the  medulla,   last.  Then  in  accord      subcorticodiencephalon ;      3. 

.  ^  midbrain;  4.  pons  and  upper 

With    HUGHLINGS    Jackson's    idea^    that    the    brain    is    so       medulla;  5.  medullary  centers 

constructed  that  the  higher  anatomic  and  newer  phyletic 

portions  contain  areas  which  regulate  and  control  the  lower  anatomic  and  older  phyletic 

regions  we  might  expect  a  series  of  release  phenomena  as  each  area  in  turn  succumbs 

to  an  increasingly  severe  degree  of  carbohydrate  deprivation^^.  Such  a  series  is  seen 

in  the  insulin  h5Apoglycemia  repeatedly  produced  in  the  pharmacologic  treatment  of 

schizophrenia^^. 

Following  the  injection  of  insulin  the  first  phase  involves  the  depression  of  the 
cerebral  cortex  (area  i,  Fig.  i).  Sensations  become  dull  and  abnormal,  understanding  is 
impaired  and  motor  activity  poor  in  execution.  Contact  with  the  environment  is  gra- 
dually lost  as  the  patient  becomes  unconscious,  the  beginning  of  the  second  stage.  The 
second  group  of  signs  proves  to  be  due  to  a  release  of  the  functions  in  area  2,  the  sub- 
corticodiencephalon.  Three  types  of  phenomena  are  observed  in  this  stage.  First  are 
changes  in  motility  reminiscent  of  those  seen  in  a  newborn  baby  with  motor  restlessness 
and  primitive  movements  of  many  types  such  as  involuntary  sucking  and  involuntary 
grasping.  Second  there  is  increased  sensitivity  so  that  responses  to  stimuli  become  in- 
tense, excessive  and  at  the  same  time  lose  direction.  Finally,  alterations  in  the  autonomic 
system  are  seen  with  sympathetic  predominance  indicated  by  dilatation  of  the  pupils, 
bulging  of  the  eyeballs  from  their  sockets,  acceleration  of  the  heart  rate  and  rise  of  blood 
References  p.  125. 


120  H.  E.  HIMWICH  VOL.  4  (1950) 

pressure.  This  stage  is  not  unlike  that  of  sham  rage  exhibited  by  the  decorticate  animal. 
The  third  constellation  (area  3)  represents  functions  allocated  to  the  midbrain.  For 
example  the  body  is  seized  by  violent  (tonic)  spasms  during  which  the  legs  become  ligidly 
extended,  the  trunk  is  arched  while  the  arms  are  thrust  forward,  bent  at  the  elbows. 
The  fourth  group  of  manifestations,  referable  to  the  pons  and  upper  portion  of  the 
medulla  (area  4),  begins  when  the  arms  are  no  longer  held  in  front  of  the  body  but  are 
slowly  forced  back  over  the  head  (extensor  spasm).  The  back  however  is  arched  the  legs 
are  extended  as  in  the  third  stage  and  the  entire  picture  is  similar  to  that  of  a  decerebrate 
animal.  Finally  in  the  fifth  stage  (area  5)  the  cold,  gray,  clammy  skin,  the  slow  and 
feeble  heart,  the  greatly  depressed  respiration,  the  muscular  flaccidity,  and  the  con- 
tracted pupils  all  give  evidence  that  the  metabolic  depression  is  now  affecting  the  vital 
medullary  centers. 

Soon  after  the  fifth  group  of  signs  appear  it  is  necessary  to  give  the  patient  sugar. 
The  blood  glucose  values  rapidly  rise  and  the  brain  once  more  obtains  adequate  supplies. 
The  alterations  in  behaviour  during  recovery  conform  to  the  same  plan  as  those  seen 
during  their  development  but  this  time  their  order  is  reversed. 

It  is  well  to  make  comparisons  with  the  results  of  metabolic  depression  other  than 
those  produced  by  hypoglycemia.  If  the  signs  are  due  to  a  metabolic  deficit  then  the 
same  or  at  least  a  similar  series  of  signs  should  be  produced  irrespective  of  the  manner 
by  which  the  metabolic  deficit  is  created.  As  an  example  let  us  consider  anoxia,  a  con- 
dition in  which  oxygen  is  no  longer  available  to  the  brain  in  common  with  the  other 
organs. 

It  is  true  that  energy  may  be  provided  in  the  absence  of  oxygen,  an  anaerobic 
mechanism  of  great  biological  importance,  for  example,  in  sudden  muscular  activity. 
In  the  brain  however,  though  not  without  significance^^'  ^^'  2^,  the  anaerobic  release  of 
energy  is  strictly  limited  for  most  of  the  energy  usually  available  in  the  carbohydrate 
foodstuff  of  the  brain,  glucose,  cannot  be  realized.  For  that  reason  the  brain  is  highly 
sensitive  to  oxygen  lack  and  when  thus  bereft  of  energy,  can  no  longer  support  its  own 
functions. 

Whereas  the  signs  of  hypoglycemia  may  be  observed  over  a  period  of  5  hours 
those  of  acute  anoxia  are  more  fleeting  and  must  be  limited  to  a  period  of  as  many 
minutes.  Nevertheless  the  changes  in  behaviour  follow  the  same  general  path  of  those 
of  hjrpoglycemia  and  indicate  a  downward  progression  during  anoxia  and  the  reversed 
direction  on  recovery.  These  signs  were  demonstrated  in  a  series  of  psychotic  patients 
who  respired  undiluted  nitrogen  administered  by  means  of  a  mask^^.  Early  is  seen  a 
brief  period  during  which  consciousness  becomes  impaired  as  the  cerebral  hemispheres 
are  the  first  to  suffer  from  the  decrease  in  available  energy  (area  1,  Fig.  i).  The  first 
phase  ends  as  environmental  contact  is  lost.  With  the  loss  of  consciousness  a  series  of 
dramatic  neuromuscular  reactions  occurs  beginning  with  a  period  of  aimless  motor 
restlessness  which  ensues  after  the  subcorticodiencephalon  acquires  freedom  from  cor- 
tical restraint  (area  2).  Next  come  strong  muscular  contractions  like  those  described  in 
the  third  phase  of  hypoglycemic  coma  (tonic  spasms)  as  the  midbrain  is  freed  from  higher 
control  (area  3).  Finally  emprosthotonos,  flexion  of  the  body,  or  opisthotonos,  extreme 
extension,  are  seen  in  the  fourth  stage  (area  4).  These  signs  are  release  phenomena  and 
indicate  a  decerebration  of  functional  origin.  At  this  point  the  inhalation  of  nitrogen 
is  stopped  to  prevent  involvement  of  the  medullary  centers.  With  the  subsequent  ad- 
ministration of  air  or  oxygen  the  normal  cerebral  integrations  are  rapidly  restored. 
References  p.  125. 


VOL.  4  {1950) 


FUNCTIONAL   ORGANIZATION    OF   THE   BRAIN 


121 


Supporting  data  for  such  a  sequence  of  changes  during  hypoglycemia^^  or  acute 
anoxia^'*  is  afforded  by  electroencephalographic  tracings  which  reveal  that  the  cortical 
rhythm  vanishes  before  the  subcortical.  Conversely  the  administration  of  glucose  or 
oxygen  restores  the  subcortical  waves  before  those  of  the  cortex,  additional  evidence 
that  the  cerebral  cortex  workes  at  a  higher  rate  of  activity  and  has  greater  demands  for 
energy  than  the  subcortex. 

Turning  to  the  problem  of  pentothal  anesthesia,  we  find  that  pentothal,  like  the 


stage 


m 


Planel 


PlaneH 


PlaneM 


IF 


Clouding 


Hyper 
sensitivity 


Light  surgical 


Moderate 
surgical 


Deep  surgical 


Impending 
failure 


Characterislics 


Euphoria  loss 

of 
discrimination 


impairment  of 

environmental 

contact 


Loss  of 
consciousness 


Hypoactivity  to 
painful  stimulus 


Loss  of  somatic 
response  to  pain 


Loss  of  visceral 
response  to  pain 


Fall  in 
pulse  pressure 


Site  of 
depression 


Slight 
depression 
of  cortex 


moderate 
depression 
of  cortex 


Predominant 
control  by 
subcortex 


Moderate 

depression 

of  subcortex 


Predominant 
control  by 
midbrain 


Moderate 

depression 

of  midbrain 


Moderate 
depression 
of  pons 


Brain 


Fig.  2.  A  correlation  between  the  stages  of  pentothal  anesthesia  and  the  outstanding  clinical  signs 

and  their  neuro-anatomic  allocations 

References  p.  125. 


122  H.  E.  HIMWICH  VOL.  4  (1950) 

other  barbiturates,  exerts  a  metabolic  inhibition  which  is  most  marked  in  the  brain 
and  relatively  unimportant  in  other  organs^^.  Measurements  of  brain  metabolism  made 
on  human  beings  in  the  second  and  third  stages  of  pentothal  anesthesia  disclose  a 
decrease  of  approximately  one-third^^. 

The  barbiturates  not  only  employ  metabolic  deprivation  but  also  act  on  nerve 
f  unction^^.  The  latter  action  may  be  described  as  an  elevation  of  the  synaptic  threshold^^ 
due  perhaps  to  impeded  recovery  after  impulse  propagation^^.  Despite  these  diverse 
influences  it  is  feasible  to  follow  the  events  caused  by  metabolic  depression. 

In  this  brief  exposition  it  is  impossible  to  review  the  signs  of  pentothal  anesthesia. 
Instead  an  explanatory  diagram  is  inserted  (Fig.  2).  The  figure  is  taken  from  a  paper^^ 
in  which  it  is  suggested  that  the  metabolic  inhibition  is  the  cause  for  certain  similarities 
between  barbiturate  anesthesia  and  hypoglycemia  or  anoxia  and  especially  so  for  the 
march  of  signs  down  the  neuraxis  with  deepening  anethesia.  On  the  other  hand  the 
distinguishing  characteristics  of  the  anesthesia  are  attributed  to  the  special  effects  which 
the  barbiturate  exert  upon  nerve  functions. 

Since  the  progression  of  the  changes  in  behaviour  observed  following  surgical  or 
pharmacologic  intervention  seem  to  depend  upon  the  hierarchy  of  metabolic  rates  in 
the  various  parts  of  the  brain  it  is  worth  while  to  examine  that  phenomenon  further. 
A  clue  as  to  its  origin  may  be  offered  by  a  study  of  the  changes  in  oxygen  intake  of  the 
various  parts  of  the  brain  during  early  growth.  Animals  which  are  born  in  an  immature 
state,  resembling  man  in  that  way,  are  appropriate  material  for  a  study  of  postnatal 
metabolic  changes.  The  newborn  rat,  blind,  poikilothermic  and  without  righting  re- 
flexes, essentially  a  bulbospinal  animal,  can  be  followed  through  early  growth  while  the 
later  developed  portions  of  the  brain  take  on  their  due  functions.  The  birth  process  marks 
the  passage  from  intrauterine  life  to  individual  independence  but  does  not  necessarily 
represent  a  definite  change  in  the  fundamental  patterns  of  growth  and  energy  production. 

Numerous  in  vitro  studies  of  oxygen  intake  reveal  a  higher  rate  of  metabolism  in 
the  adult  than  in  the  infant.  This  was  first  observed  in  infant  rat  brain^",  and  later 
confirmed  on  the  dog^^.  These  results  indicate  a  rapid  rise  of  cerebral  metabolism  in 
early  life.  The  metabolic  changes  are  the  resultants  of  the  distinctive  rates  in  the  discrete 
parts  of  the  brain.  It  has  been  experimentally  established  that  the  metabolic  rates  are 
not  equally  affected  by  growth,  but  that  each  area  possesses  its  own  pattern  of  devel- 
opment. In  experiments  on  the  rat^^  and  the  dog^^  (Fig.  3)  it  was  found  that  the  lower 
parts  of  the  brain  are  relatively  more  active  than  the  higher  ones  at  birth,  and  as 
development  continues,  the  wave  of  metabolism  presses  forward  so  that  the  lower 
portions  of  the  central  nervous  system  are  surpassed  by  the  anatomically  higher  and 
phyletically  more  recently  developed  regions.  The  increasing  rate  of  metabolism  of  the 
brain  as  a  whole  must  therefore  be  attributed  chiefly  to  the  increasing  rate  in  the  newer 
parts  of  the  brain  during  early  life. 

Additional  evidence  for  this  phyletic  sequence  can  be  observed  by  a  study  of  the 
anaerobic  metabolism.  The  short  period  of  survival  in  anoxia  observed  in  the  mammal 
is  made  possible  by  the  anaerobic  production  of  energy  which  includes  the  splitting  of 
carbohydrate  to  form  lactic  acid.  The  cerebral  glycolytic  rates  are  slowest  in  the  new- 
born and  increase  to  a  maximum  in  early  life^^,  ^^.  In  order  to  determine  the  contribution 
of  each  area  in  the  brain  making  for  this  changing  rate  of  glycolysis  both  dogs  and  cats 
were  employed^*  and  in  several  age  groups:  newborns  to  one  week,  three  to  seven  weeks, 
three  months,  and  adult.  In  general,  the  results  of  the  experiments  on  dogs  and  cats 
References  p.  125. 


VOL.  4  (1950) 


FUNCTIONAL   ORGANIZATION    OF   THE   BRAIN 


123 


were  similar.  At  birth  the  medulla  oblongata  revealed  the  highest  glycolysis.  In  the 
adult,  however,  it  is  the  cortex  that  shares  the  most  rapid  metabolic  rate  with  the  caudate 
nucleus. 

The  developmental  progression  observed  in  oxidation  and  glycolysis  has  also  been 
found  in  the  distribution  of  cerebral  glycogen.  Chemical  determinations  demonstrate 
that  glycogen  concentrations  of  the  cerebral  cortex  and  caudate  nucleus  increase  with 
age.  The  percentage  of  glycogen  in  the  lower  parts,  however,  the  cerebellum,  medulla 
and  spinal  cord  diminish  progressively  and  are  least  in  the  adult^^. 

The  quantitative  analyses  presented  above  show  that  both  aerobic  and  anaerobic 
mechanisms  are  accelerated  after  birth.  It  seems  probable  that  the  more  rapid  rates 
are  an  expression  of  an  increased  concentration  of  enzymes.  Such  an  increase  can  be 
accounted  for  by  the  growing  capac- 
ities of  phosphorylase,  phospho- 
glucomutase^^,  adenosine  triphos- 
phatase^ and  the  cytochrome-cyto- 
chrome oxidase  system^"'  ^  occur- 
ring in  the  brain  during  the  early 
postnatal  growth  of  the  rat.  Carbonic 
anhydrase  though  not  found  in  the 
fetal  rat  is  present  in  the  adult  where 
it  is  more  plentiful  in  the  function- 
ally dominant  cerebral  areas  than 
in  the  cord^^.  A  study  of  fetal  sheep 
proved  that  the  enzyme  cholin- 
esterase  is  present  in  greater  concen- 
tration in  the  spinal  cord  than  the 
brain  during  early  gestation.  This 
relationship  however  is  reversed  in 
the  last  weeks  before  birth  as  the 
cholinesterase  activities  of  the  cord 
diminish  while  those  of  the  brain  far 
outstrip  it***.  This  enzymatic  evolu- 
tion which  appears  earlier  in  the 
sheep  than  in  the  rat  is  not  to  be 
attributed  solely  to  a  difference  in 
the   enzyme   studied    in   these   two 

species  but  it  must  also  be  remembered  that  the  sheep  is  further  advanced  in  the 
development  of  behavioural  patterns  at  the  time  of  birth. 

To  summarize,  the  increase  in  metabolic  intensity  does  not  occur  in  all  parts  of 
the  brain  simultaneously,  but  appears  in  the  various  portions  at  different  times.  The 
order  of  appearance  is  not  a  haphazard  one  but  develops  first  in  the  posterior  portions 
of  the  neuraxis  and  then  progresses  in  an  anterior  direction.  Such  a  stepwise  passage 
advancing  from  the  older  to  the  newer  parts  of  the  brain  recapitulates  its  phyletic 
development.  Since  many  of  the  metabolic  studies  reviewed  were  made  on  newborns. 
It  would  seem  that  Haeckel's  dictum  that  ontogeny  recapitulates  phylogeny^^  should 
be  broadened,  in  the  case  of  the  brain,  and  the  time  extended  to  include  early  postnatal 
growth  with  prenatal  development. 
References  p.  125. 


5        6        7 
Age  in  wfeks 


Adu'J 


Fig.  3.  Oxygen  consumption  vs.  Age  Dog  Brain  Parts. 
In  the  first  week  of  life  the  highest  rate  of  metabolism 
in  the  puppy's  brain  is  found  in  the  meduUa;  during 
the  third  week  the  midbrain  assumes  the  highest 
oxj'gen  consumption.  From  the  fifth  to  the  seventh 
week,  the  respiratory  metabolism  of  all  parts,  with 
the  exception  of  the  medulla,  is  higher  than  the  cor- 
responding values  for  the  first  week  of  life  and  the 
caudate  nucleus  has  advanced  to  the  greatest  oxygen 
intake  up  to  this  time.  In  the  adult  dog  the  latter  still 
retains  its  prime  position,  while  the  cerebral  cortex 
ascends  to  second  place.  The  cerebellum,  thalamus, 
midbrain    and    medulla    follow    in    descending   order. 


124  ^-  ^-  HIMWICH  VOL.  4  (1950) 

To  climb  the  phyletic  ladder  from  our  remotest  ancestors  through  the  fish,  am- 
phibia, reptiles  and  mammals,  would  entail  a  tremendous  volume  of  description,  which 
is  not  the  point  of  this  contribution.  The  general  trend  of  this  process  of  cephalization, 
or  concentration  of  neural  functions  in  the  oral  end  of  the  animal,  may  be  described 
briefly:  as  far  back  as  the  fish,  brain  is  divided  into  five  portions  as  it  is  in  man,  but  in 
the  fish  and  amphibia  the  chief  site  of  integration  for  sensory  and  motor  impulses  lies 
in  the  midbrain.  In  these  species  the  highest  portion  of  the  brain  consists  chiefly  of  the 
olfactory  bulb,  and  the  cerebral  cortex  which  becomes  all-important  in  man,  is  repre- 
sented only  by  a  thin  layer  of  cells.  On  further  ascending  the  phyletic  scale  to  reptiles 
and  birds  as  well  as  mammals,  the  subcortical  structures  immediately  anterior  to  the 
midbrain  become  more  prominent,  as  the  organism  achieves  greater  coordinating 
control.  Lastly,  the  cerebral  cortex,  though  getting  off  to  a  late  start,  gradually  attains 
more  complexity  of  structure  and  diversity  of  function  until  in  the  lower  mammals  it 
surpasses  all  other  regions,  and  in  the  primates,  especially  in  man,  forms  the  largest  and 
most  comple:^  part  of  the  cerebral  tissue.  As  this  process  of  phylogeny  is  carried  on 
from  one  species  to  another,  no  part  of  the  neuraxis  is  scrapped,  but  each  older  part, 
in  turn,  comes  under  the  influence  of  a  later  developed  portion,  which  not  only  possesses 
finer  discrimination  and  analj'sers  but  also  plays  a  role  in  determining  the  motor 
expression  of  the  older  areas. 

Though  the  brain  of  man  as  we  see  it  today  looks  like  a  static  structure,  when  it  is 
examined  more  closely  in  the  light  of  the  phyletic  conception,  we  see  that  it  has  come 
to  its  present  construction  as  a  result  of  a  long  series  of  accretions,  beginning  with  the 
spinal  cord  and  medulla  oblongata  and  spreading  in  a  cephalad  direction,  layer  upon 
layer,  until  the  cerebral  hemispheres  form  the  greatest  mass  of  the  brain.  It  is  not  to 
be  supposed  that  each  level  is  independent  of  its  predecessors,  but  rather  that  it  exists 
with  a  specific  relation,  both  anatomically  and  physiologically,  to  the  phyletically 
older  portions'*^.  Owing  to  this  relation,  the  central  nervous  system  may  function  as  a 
unit,  but  a  unity  which  is  brought  to  a  higher  plane  of  integration  with  each  successive 
step.  The  human  brain  is  undoubtedly  the  latest  arrangement  of  the  central  nervous 
system,  but  not  necessarily  the  final  one. 

Sir  Charles  Sherrington^^  has  expressed  vividly  Hughlings  Jackson's  con- 
ception. "That  leading  end,  the  head,  has  receiving  stations  signalling  from  things  at 
a  distance,  things  which  the  animal  in  its  forward  movement  will  next  meet.  A  shell 
of  its  immediate  future  surrounds  the  animal's  head.  The  nerve-nets  in  the  head  arc 
therefore  busy  with  signals  from  a  shell  of  the  outside  world  which  the  animal  is  about 
to  enter  and  experience.  The  brain  has  thus  arisen  where  signalling  is  busiest  and  is 
fraught  most  with  the  germ  of  futurity.  Small  wonder  then  that  the  brain  plays  a  great 
role  in  the  motor  management  of  the  muscle.  Nerve  management  of  muscle  resolves 
itself  largely  into  management  of  nerve  by  nerve,  especially  by  brain,  more  and  more 
so  as  evolution  proceeds.  With  no  greater  equipment  of  muscle  the  superimposed 
amount  of  nerve  becomes  greater  and  greater;  each  new  nerve-growth  seems  to  entail 
further  nerve-growth.  Fresh  organization  roofs  over  prior  organization.  Brain  is  an 
example.  'So  on  our  heels  a  fresh  perfection  treads'.  But  were  it  a  government  ofiice  we 
might  be  suspicious.  This  brain  of  ours  is  a  perfect  excrescence  although  our  endowment 
of  muscle  remains  but  moderate". 


References  p.  125. 


VOL.  4  (1950)  FUNCTIONAL   ORGANIZATION    OF   THE   BRAIN  125 

REFERENCES 

1  J.  H.  Jackson,  Brit.  Med.  J.,  i  (1884)  591-593,  660-663,  7°3~7^7- 

'  C.  S.  Sherrington,  The  integrative  action  of  the  nervous  system,  New  Haven,  Yale  Univ.  Press,  191 1. 

3  P.  B.\RD,  A.  Research  Nervous  Mental  Diseases,  Proc,  19  (1939)  190—218. 

*  J.  G.  DussER  DE  Barenne,  W.  S.  McCulloch,  /.  Neurophysiol.,  4  (1941)  311-323. 

*  H.  E.  HiMwiCH,  Am.  J.  Digestive  Diseases,  11  (1944)  1-8. 

*  H.  E.  HiMwicH  AND  L.  H.  Nahum,  Proc.  Soc.  Exptl  Biol.  Med.,  26  (1929)  496-497;   Am.  J. 
Physiol.,  loi  (1932)  446-453. 

'  W.  G.  Lennox,  Arch.  Neurol.  Psychiat.,  26  (193 1)  719-724. 

*  Baker,  Zelma,  J.  F.  Fazekas,  and  H.  E.  Himwich,  /.  Biol.  Chem.,  125  (1938)  545-556. 

*  H.  E.  Himwich,  K.  M.  Bowman,  J.  Wortis,  and  J.  F.  Fazekas,  /.  Nervous  Mental  Disease,  89 
(1939)  273-293. 

1°  S.  S.  Kety,  R.  B.  Woodford,  M.  H.  Harmel,  F.  A.  Freyhan,  K.  E.  Appel,  and  C.  F.  Schmidt, 

A)n.  J.  Psychiat.,  104  (194S)  765-770. 
11  H.  E.  Himwich,  K.  M.  Bowman,  C.  D.\ly,  J.  F.  Fazekas,  J.  Wortis,  .and  W.  Gold  fare.  Am.  J. 

Physiol.,  132  (1941)  640-647. 
1*  H.  E.  Himwich,  P.  Sykowski,  and  J.  F.  Fazekas,  Am.  J.  Physiol.,  132  (1941)  293-296. 
"  H.  E.  Himwich  and  J.  F.  Fazekas,  Am.  J.  Physiol.,  132  (1941)  454-459. 
^*  S.  S.  Kety  and  C.  F.  Schmidt,  /.  Clin.  Invest.,  27  (1948)  476-483. 
^'  W.  A.  Himwich,  A.  Willi amina,  E.  Homburger,  R.  Maresca,  and  H.  E.  Himwich,  Am.  J. 

Psychiat.,  103  (1947)  689-696. 
1*  H.  E.  Himwich,  J.  P.  Frostig,  J.  F.  Fazekas,  and  Z.  Hadidian,  Am.  J.  Psychiat.,  96  (1939) 

731-785- 
1^  H.  E.  Himwich,  Psychiat.  Quart.,  18  (1944)  357-373. 
^*  M.  Sakel,  Nervous  and  Mental  Disease,  Monograph  series,  no.  62,  New  York  and  Washington 

(1938).  Authorized  translation  by  Joseph  Wortis,  M.D. 
1'  W.  A.  Himwich  and  H.  E.  Himwich,  /.  Neurophysiol.,  9  (1946)  133-136. 
-"  H.  E.  Himwich,  A.  O.  Bernstein,  H.  Herrlich,  A.  Chesler,  and  J.  F.  Fazekas,  Am.  J.  Physiol., 

135  (1942)  387-391- 
-^  J.  F.  Fazekas  and  H.  E.  Himwich,  Am.  J.  Physiol.,  139  (1943)  366-370. 
^2  F.  A.  D.  Alexander  and  H.  E.  Himwich,  Am.  J.  Psychiat.,  96  (1939)  643-655. 
-'  H.  Hoagland,  H.  E.  Himwich,  E.  Campbell,  J.  F.  Fazelas,  and  Z.  Hadidian,  /.  Neurophysiol., 

2  (1939)  276-288. 
-*  O.  Sugar  and  R.  W.  Gerard,  J.  Neurophysiol.,  i  (193S)  558-572. 
"  J.  H.  QuASTEL,  Physiol.  Revs,  19  (1939)  135-183. 

26  H.  E.  Himwich  and  B.  Etsten,  /.  Nervotis  Mental  Diseases,  104  (1946)  407-413. 
-"  P.  Heinbecker  and  S.  H.  Bartley,  J .  Neurophysiol.,  3  (1940)  219-236. 
2®  W.  H.  Marshall,  C.  N.  Woolsey,  and  P.  Bard,  /.  Neurophysiol.,  4  (1941)  1-24. 
2®  B.  Etsten  and  H.  E.  Himwich,  Anesthesiology,  7  (1946)  536-548. 
^°  H.  E.  Himwich,  Z.  Baker,  and  J.  F.  Fazekas,  Am.  J.  Physiol.,  125  (1939)  601-606. 
^^  D.  B.  Tyler  and  A.  Van  Harreveld,  Am.  J.  Physiol.,  136  (1942)  600-603. 
•■'^  H.  E.  Himwich,  A.  O.  Bernstein,  H.  Herrlich,  A.  Chesler,  and  J.  F.  Fazekas,  Am.  J.  Physiol., 

135  (1942)  387-391- 
2'  A.  Chesler  .and  H.  E.  Himwich,  Am.  J.  Physiol.,  141  (1944)  513-517. 
^*  A.  Chesler  and  H.  E.  Himwich,  Am.  J.  Physiol.,  142  (1944)  544-549. 
^  A.  Chesler  and  H.  E.  Himwich,  Arch.  Biochem.,  2  (1943)  175-181. 
^6  B.  Shapiro  and  E.  Wertheimer,  Biochem.  J .,  37  (1943)  397-403. 
•''"  H.  E.  Himwich,  A.  O.  Bernstein,  J.  F.  F.\zekas,  H.  C.  Herrlich,  and  E.  Rich,  Am.  J.  Physiol., 

137  (1942)  327-330. 
•'^  V.  R.  Potter,  B.  S.  Schneider,  and  G.  J.  Liebl,  Cancer  Research,  5  (1945)  21-24. 
•'"  W.  Ashby,  /.  Biol.  Chem.,  152  (1944)  235-240. 
''•'  D.  Nachmansohn,  /.  Neurophysiol.,  3  (1940)  396-402. 
^^  E.  Haeckel,  Generelle  Morphologic  der  Organismen,  Berlin  1866. 
**  F.  Tilney  and  H.  a.  Riley,  The  form  and  functions  of  the  central  nervous  system,  Paul  B.  Hoeber, 

Inc.,  New  York  1928. 
■*'  C.  S.  Sherrington,  The  brain  and  its  mechanism ,  The  Rede  lecture  delivered  Dec.  5,  1933,  Cam- 
bridge, England.  The  University  Press  (1933). 

Received  April  Sth,  1949 


126  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


THE  DEVELOPMENT  OF  MUSCLE-CHEMISTRY,  A  LESSON 
IN  NEUROPHYSIOLOGY 

by 

ALEXANDER  VON  MURALT 

Hallerianum,  Bern  [Switzerland] 


In  the  development  of  muscle-chemistry  four  different  periods  can  be  distinguished : 
the  pre-lactic  acid  era,  the  lactic  acid  era,  the  period  of  phosphorylations  and  the  myosin 
period.  The  name  of  Otto  Meyerhof  is  intimately  connected  with  three  of  them.  In  no 
field  of  physiology  has  knowledge  advanced  so  far  towards  the  fundamental  and  ele- 
mentary processes  of  function  as  in  muscle  chemistry.  This  advancement  is  mainly 
due  to  Otto  Meyerhof's  brilliant  conception  of  chemical  and  physical  aspects  and  to 
the  unparalleled  cooporation  of  two  masterminds  in  different  fields.  Otto  Meyerhof 
AND  A.  V.  Hill. 

In  the  prelactic  acid  era,  although  it  starts  paradoxically  with  Berzelius,  who 
discovered  in  1841  that  muscles  of  exhausted  deer  contained  more  lactic  acid  than 
muscles  of  animals  with  partially  paralysed  extremities^,  the  role  of  lactic  acid  was  quite 
unrecognized.  There  was  even  a  very  temperamental  discussion  as  to  what  might  be 
the  fuel  for  muscular  work.  Fick  and  Wislicenus^,  who  climbed  the  Faulhorn  (1956  m), 
between  the  lake  of  Brienz  and  the  valley  of  Grindelwald,  collected  their  urine  and 
showed  conclusively  in  a  famous  paper  in  1865  that  the  excreted  nitrogen  corresponded 
only  to  37  g  of  protein,  which  by  no  means  accounted  for  the  work  done.  This  statement 
caused  the  long-held  belief  of  Liebig,  that  protein  is  the  source  of  muscular  activity, 
to  be  discarded  and  attention  to  be  drawn  to  carbohydrates.  Six  years  later  Weiss^ 
showed  that  the  glycogen  content  of  muscle  decreases  with  the  work  done,  and  it  seems 
that  LucHSiNGER^  in  Ziirich  was  the  first  to  recognize  the  importance  of  nutrition  for 
the  maintenance  of  a  sufficient  glycogen  content  of  the  muscles,  and  to  point  out  that 
glycogen  is  the  intermediate  energy  carrier  between  ingested  foodstuffs  and  activity. 
The  next  step  was  only  reached  in  1893  when  Panormoff^  showed  that  glycogen  in 
muscle  is  hydrolysed  to  glycose.  Among  the  many  original  observations  which  Du  Bois- 
Reymond  made,  it  seems  that  he  was  the  first  to  recognize  that  a  muscle  becomes  acid 
with  activity  and  to  relate  this  finding  to  Berzelius's  observation  of  the  formation  of 
lactic  acid®.  It  is  quite  amazing  to  see  how,  as  early  as  1859,  a  very  clear  conception 
existed  and  how  it's  development  was  delayed  by  the  following  accumulation  of  a  great 
mass  of  very  unimportant  evidence  up  to  the  end  of  the  century.  This  is  even  more 
surprising  when  we  see  that  Heidenhain'  had  found  that  the  amount  of  lactic  acid 
increased  with  the  amount  of  work  done.  Nasse®  who  seems  to  have  had  great  influence 
at  this  time  however  believed  that  lactic  acid  was  only  formed  in  rigour  and  death,  and 
did  not  recognize  the  importance  of  Helmholtz's^  fundamental  finding  that  the  alco- 
holic extract  of  muscle  decreased  with  activity,  whereas  the  aqueous  extract  increased. 
References  p.  I2g. 


VOL.  4  (1950)  MUSCLE    CHEMISTRY   AND    NEUROPHYSIOLOGY  I27 

thus  giving  the  first  well  founded  evidence  for  chemical  events,  and  suggesting  that 
glucose  and  lactic  acid  increase  at  the  expense  of  glycogen.  It  seems  almost  unbehevable 
that  M.  v.  Frey  wrote  even  in  1909  about  chemical  changes  in  muscular  activity  .  .  . 
"which  acid  is  responsible  cannot  be  stated  to-day,  since  lactic  acid  seems  not  to  account 
for  it"  (referring  to  the  acidification  of  active  muscle!) 

The  importance  of  phosphates  seems  to  have  been  recognized  for  the  first  time 
by  Salkowski^",  who  described  the  liberation  of  inorganic  phosphate  from  an  organic 
compound  during  activiry,  a  finding  which  was  rejected  by  v.  Furth,  another  of  those 
most  unfortunate  cases  (which  occur  so  often!)  where  the  authority  of  one  man  has 
delayed  development. 

It  was  MacLeod^i  who  took  up  the  point  and  found  that  inorganic  phosphate  in- 
creased and  organic  phosphate  decreased,  and  Monari^^  f^^st  seems  to  have  observed 
that  the  creatine-content  of  muscle  increases  with  activity  (phosphagen  not  being  deter- 
mined  in  his  experiments).  These — in  our  present  point  of  view — most  important 
findings  could  not  be  corroborated  at  that  time  to  give  a  clear  conception  and  were 
almost  hurried  by  a  great  deal  of  other  chemical  evidence  which  we  consider  to-day  as 
entirely  uninteresting  and  which  filled  the  periodicals  of  the  time. 

The  lactic  acid  era  started  in  1907  with  the  classical  paper  of  Fletcher  and 
HoPKiNS^^,  in  which  they  definitely  established  that  fact  that  lactic  acid  is  formed 
during  activity  and  that  it  is  absent  (or  practically  absent)  in  resting  muscles.  This 
opened  up  a  vast  field  and  led  to  Meyerhof's  great  work,  which  is  summarized  in  a 
hypothesis,  which  was  called  the  lactic  acid  theory  of  Hill  and  Meyerhof.  The  mile- 
stones of  this  development  were  the  discoveries  of  the  Pasteur-Meyerhof  reaction, 
of  the  independence  of  initial  heat  of  oxygen,  the  very  accurate  measurements  of  muscle 
heat  by  A.  V.  Hill  and  his  colleagues,  and  their  relation  to  chemical  and  calorimetric 
values  obtained  by  Meyerhof,  the  extensive  study  of  lactic  acid  metaboHsm  in  muscle 
in  all  conditions  of  work,  rigour  and  death,  and  finally  the  brilliant  adaptation  of  this 
theory  to  muscular  work  in  man  by  A.  V.  Hill^^  and  his  conception  of  oxygen-debt. 
It  was  a  one-sided  picture — as  we  all  know  to-day — and  yet  it  is  one  of  the  golden  pages 
of  scientific  discovery,  because  every  new  finding  fitted  into  the  theory  and  led  to  a  very 
clear  conception  of  what  is  taking  place  in  a  working  muscle.  It  was  very  fortunate, 
that  Meyerhof  published  in  1930  his  famous  book  on  chemical  events  during  muscle 
contraction,  in  which  he  gave  an  admirable  account  of  the  lactic-acid  hypothesis^^. 

The  year  1930  brought,  what  A.  V.  Hill  called  the  revolution  in  muscle  physiology. 
Lundsgaard's^^  paper  on  mono-iodoacetic  acid  poisoned  muscles  and  the  absence  of 
lactic  acid  formation  in  these  muscles  was — as  it  seemed  at  first — a  heavy  blow  to  the 
lactic  acid  hypothesis.  It  is  very  interesting  to  read  to-day  the  conclusions  Bethe^' 
drew  at  that  time  and  it  is  equally  astonishing  to  see,  how  quickly  Meyerhof  reacted 
and  how  he  and  Lundsgaard  kept  the  lead.  The  conception  of  energetic  coupling 
between  different  reactions  was  worked  out  and  proved  to  be  a  new  and  extremely 
useful  aspect  in  the  classification  and  understanding  of  the  chemical  events  including 
adenylpyrophosphate,  creatinphosphate  and  fructosediphosphate  breakdown.  Ritchie^* 
introduced  the  idea  that  all  chemical  events  might  be  recovery  processes  and  therefore 
furnish  the  energy  for  the  next  contraction.  This  led  to  the  conception  that  energetically 
coupled  reactions  furnish  in  steps  the  necessary  free  energy  to  restore  the  energyloss 
which  occurs  in  an  explosive  way  during  contraction.  This  conception  has  been  recently 
summarized  by  Meyerhof^^  in  an  article  which  contains  all  the  classical  points  of  view 
References  p.  izg. 


128  A.  VON    MURALT  VOL.  4  (1950) 

of  the  era.  This  era  might  be  called  the  period  of  phosphorylations  and  it  is  character- 
ized by  the  discovery  of  the  PARNAS-reaction,  the  LoHMANN-reaction  and  the  complete 
series  of  steps  in  glycolysis  in  muscle,  with  the  isolation  of  the  corresponding  enzymes. 

In  1939  the  myosin  period  started  with  the  paper  of  Engelhardt  and  Ljubimova*°, 
which  was  followed  by  Szent-Gyorgyi  and  Banga's^^,  Needham's^^^  Bailey's^^  and 
Kleinzeller's^*  papers.  Myosin,  the  "muscle  machine"  or  what  A.  V.  Hill  has  always 
called  the  fundamental  process,  became  the  center  of  attention.  Myosin  had  been  known, 
of  course,  for  quite  a  long  time.  In  1930  my  friend  John  Edsall  and  I  published  experi- 
ments, which  showed  that  myosin  must  be  the  contractile  element  of  muscle.  The 
important  point  about  Engelhardt  and  Ljubimova's  paper  is,  however,  that  they 
found  that  the  enzyme  associated  with  the  breakdown  of  ATP  was  associated  with 
myosin.  With  this  it  became  evident  at  once  that  there  is  a  close  relation  between  the 
"muscle  machine"  and  the  whole  set  of  coupled  chemical  reactions.  Szent-Gyorgyi 
and  his  coworkers^^  have  added  a  great  deal  of  very  interesting  new  information 
about  the  nature  of  the  muscle  machine  and  thus  we  are  just  now  in  the  midst  of  a 
"myosin  era".  Meyerhof  has  attached  his  name  to  this  period  by  the  almost 
simultaneous  isolation  of  ATP-ase  from  myosin,  first  described  by  Price  and  Cori^^. 

What  is  the  lesson  neurophysiology  can  learn  from  this  development  ? 

1.  A  rather  long  period  of  widespread  chemical  research  has  to  precede  the  definite 
identification  of  those  chemical  reactions  which  are  really  essential.  I  am  afraid  that  the 
smallness  of  nerve  and  the  impossibility  to  accumulate  break-down  products  connected 
with  the  absence  of  fatigue  in  peripheral  nerve  has  prevented  any  extensive  chemical 
work.  Such  work  preceded  the  lactic  acid  era  in  muscle  chemistry.  The  ground  for 
neurophysiology  therefore  is  not  as  well  prepared  as  it  was  for  muscle-physiology  in  1907. 

2.  Once  the  importance  of  lactic  acid  was  established,  an  intensive  attack  was  made 
from  all  sides,  yielding  an  astounding  amount  of  information.  Looking  back  it  can  well 
be  said,  that  the  prejudiced  concentration  on  lactic  acid  was  very  much  worthwhile!  Is 
acetylcholine  in  neurophysiology  a  problem  which  will  prove  to  be  as  fruitful  as  lactic 
acid  was  in  muscle  physiology?  I  doubt  it  and  I  realize  that  in  this  respect  I  disagree 
with  my  colleague  Nachmansohn^'^  who  has  published  an  admirable  amount  of  work 
on  the  subject. 

3.  In  muscle  the  energy  expenditure  is  the  main  function.  In  nerve,  nature  gives 
us  an  opposite  example  of  maximal  economy  in  energy  expenditure  connected  with 
function.  The  energy  changes  are  so  small  that  it  took  even  A.  V.  Hill  15  years  to 
measure  them.  This  renders  the  task  of  corroboration  between  physical  and  chemical 
events  in  nervous  excitation  extremely  difficult  and  tedious. 

4.  In  muscle  physiology  it  was  possible  to  study  the  interesting  reactions  in  vitro, 
to  measure  the  various  steps  of  glycol^'-sis  and  to  isolate  the  important  enzyme-systems. 
Sodium  fluoride  and  isoacetic  acid  have  been  powerful  tools  in  this  work.  In  nerve- 
physiology  the  material  is  complex  and  there  is,  as  far  as  I  can  see,  no  definite  clue  to 
any  chemical  reaction  of  primary  importance.  Gerard^^  has  contributed  most  valuable 
studies  on  nerve-chemistry  by  working  along  lines  similar  to  those  used  by  muscle 
physiologists,  but  I  think  he  will  agree  with  me  in  saying,  that  our  knowledge  of  what 
is  going  on  chemically  in  order  to  restore  the  energy  expenditure  of  the  ionic  changes 
(potassium  going  "out",  sodium  going  "in"  and  vice  versa,  cf.  Hodgkin^^)  is  very  far 
from  being  satisfactory.  I  think  it  is  well  to  emphasize  that  brain-brei  is  in  no  way  a 

References  p.  izg. 


VOL.  4  (1950)  MUSCLE   CHEMISTRY   AND    NEUROPHYSIOLOGY  I29 

model  for  peripheral  nerve  chemistry  and  that  the  application  of  results  obtained  with 
brain-brei  must  be  regarded  with  caution. 

5.  Physical  phenomena,  accompanying  the  chemical  changes  have  been  a  great 
help  in  establishing  the  sequence  of  events  in  muscle.  Volume  change,  change  of  pn, 
variation  of  birefringence,  of  light  scattering  and  change  of  electrical  resistance  have 
been  studied  with  great  success,  and  it  is  one  of  the  outstanding  characteristics  of 
Meyerhof's  work  that  he  always  was  able  to  make  a  fruitful  correlation  between  these 
phenomena  and  the  chemical  aspect.  In  nerve,  all  these  effects — if  they  exist  at  all — are 
probably  extremely  small.  David  Hill  (personal  communication)  has  been  able  to 
detect  changes  of  light  scattering  and  volume  changes  in  certain  nerves.  This  may  be 
the  beginning  of  a  new  development.  But  on  the  whole, — except  for  action  potentials — 
the  nerve  does  not  offer  many  good  points  for  attack  from  the  physical  side. 

The  problem  of  the  function  of  nerve  remains,  as  A.  V.  Hill^°  has  stated  17  years 
ago,  intellectually  quite  a  respectable  one.  For  all  those  who  are  attracted  by  it  the 
study  of  the  development  of  muscle  chemistry  is  a  lesson  of  how  to  proceed.  Otto 
Meyerhof's  lifework  with  its  unique  combination  of  physical  and  chemical  aspects 
furnishes  the  pattern  which  must  be  followed,  if  we  want  to  understand  what  "excita- 
tion" really  means. 

REFERENCES 

1  C.  G.  Lehmann,  Lehrbuch  d.  physiol.  Chem.  I,  103  ,  Leipzig  1850. 

-  A.  Pick  and  J.  Wislicenus,   Vierteljahresschr.  naturforsch.  Ges.  Zurich,  10  (1865)  317. 

3  S.  Weiss,  Sitzber.  Akad.  Wiss.,  Wien,  64  (1871)  i. 

*  L.  LucHSiNGER,   Vierteljahresschr.  naturforsch.  Ges.  Ziirich,  20  (1875)  47. 
5  C.  Panormoff,  Z.  physiol.  Chem.,  17  (1893)  596. 

^  E.  Du  Bois-Reymond,  Monatsber.  Berl.  Akad.,  288  (1859). 

'  R.  Heidenhain,  Mechan.  Leistung  bei  der  Muskeltatigkeit,  Leipzig  1864. 

8  O.  Nasse,    Hdb.  d.  Physiol,  1  (1879)  288. 

'  H.  Helmholtz,  Arch.  Anat.  u.  Physiol.,  72  (1845). 

°  T.  Salkowski,  Z.  klin.  Med.,  17  (1890)  SuppL  21. 

1  I.  1.  R.  Macleod,  Z.  physiol.  Chem.,  28  (1899)  535. 

2  A.  MoNARi,  Jahresber.  Tierchem.,  296  (1889). 

^  W.  M.  Pletcher  and  P.  G.  Hopkins,  /.  Physiol.  (London)  35  (1907)  247. 
A.  V.  Hill,  Muscular  activity,  Baltimore  1926. 

*  O.  Meyerhof,  Die  chemischen  Vorgdnge  im  Muskel,  Berlin  1930. 
^  E.  LuNDSGAARD,  Biochcm.  Z.,  217  (1930)  162. 

'  A.  Bethe,  N aturwissenschaften,  18  (1930)  678. 

8  A.  D.  Ritchie,  Nature  (1932)  165. 

^  O.  Meyerhof,  Ann.  N.  Y.  Acad.  Sci.,  47  (1947)  815. 

20  V.  A.  Engelhardt  AND  M.  N.  LjUBiMOVA,  Nature,  144  (1939)  668. 

21  A.  Szent-Gyorgyi  and  I.  Banga,  Science,  93  (1941)  158. 

22  D.  M.  Needham,  Biochem.  J.,  36  (1942)  113. 

23  K.  Bailey,  Biochem.  J.,  36  (1942)  121. 

2*  A.  Kleinzeller,  Biochem.  J.,  36  (1942)  729. 

2'  A.  Szent-Gyorgyi,  Studies  Inst.  Med.  Chem.  Univ.  Szeged.,  Basel  1941-43. 

26a w.  H.  Price  and  C.  P.  Cori,  /.  Biol.  Chem.,  162  (1946)  393- 

26bB.  D.  PoLis  and  O.  Meyerhof,  /.  Biol.  Chem.,  163  (1946)  339. 

"  D.  Nachmansohn,  Ann.  N.  Y.  Acad.  Sci.,  47  (1946)  395. 

28  R.  W.  Gerard,  Physiol.  Revs.,  12  (1932)  469. 

2*  A.  Hodgkin,  /.  Physiol.,  108  (1949)  37. 

3"  A.  V.  Hill,  Chemical  wave  transmission  in  nerve,  Cambridge  1932. 

An  account  of  some  aspects  of  our  present  knowledge  in  neurophysiology  has  been 
given  by  the  author  in  his  book  Die  Signaliibermittlung  im  Nerven,  Basel  1946. 

Received  April  i6th,  1949 


PART  III 
DRUG  ACTION 


SUBSTRATE  SPECIFICITY  OF  AMINO-ACID  DECARBOXYLASES 

by 

H.  BLASCHKO 
Department  of  Pharmacology,  University  of  Oxford  {England) 


During  the  last  two  years  a  number  of  observations  on  substrates  of  amino-acid 
decarboxylases  have  been  recorded  from  this  laboratory.  In  this  review  the  attempt  is 
made  to  correlate  the  results  obtained  and  to  arrive  at  conclusions  of  a  more  general 
character.  The  experimental  data  and  the  methods  used  have  been  described  elsewhere 
(Blaschko,  Holton,  and  Sloane  Stanley^' 2-  Blaschko^;  Sloane  Stanley^*  s). 

The  decarboxylation  of  L-3 : 4-  dihydroxyphenylalanine  (DOPA)  is  catalysed  by 
two  enzymes:  the  mammahan  L-DOPA-decarboxylase  (Holtz,  Heise,  and  Ludtke^) 
:ind  the  bacterial  L-tyrosine  decarboxylase  (Epps').  The  two  enzymes  differ  in  their 
affinity  for  L-tyrosine:  this  is  probably  the  "natural"  substrate  of  the  bacterial  enzyme, 
but  it  is  not  attacked  by  the  mammalian  enzyme.  The  difference  in  substrate  specificity 
of  the  two  enzymes  has  been  studied  more  systematically. 

The  experimental  procedure  adopted  is  easily  described.  As  a  source  of  the  bacterial 
enzyme  we  used  an  acetone-dried  preparation  of  Streptococcus  f^ecalis  R  (ATCC  4083) ; 
we  owe  this  strain  to  Professor  I.  C.  Gunsalus.  The  bacteria  were  usualty  grown  in  a 
medium  free  of  vitamin  Bgi  in  these  preparations  the  tyrosine  apodecarboxylase  was 
present,  but  had  to  be  completed  by  the  addition  in  vitro  of  pyridoxal  and  ATP.  In 
some  of  the  experiments  we  used  a  "complete"  preparation  obtained  from  cells  grown 
in  the  presence  of  pyridoxal.  As  a  source  of  the  mammalian  DOPA  decarboxylase  we 
used  fresh  tissue  extracts,  from  guinea-pigs  kidney  or  from  rats  liver. 

The  enzymic  decarboxylation  of  each  amino-acid  was  measured  by  following  the 
time  course  of  CO2  formation  manometrically.  If  an  amino-acid  was  found  to  be  decarbo- 
xylated,  the  contents  of  the  manometer  flasks  were  used  for  a  determination  of  the 
pharmacological  activity  of  the  amine  formed.  The  activity  was  tested  on  the  arterial 
blood  pressure  of  the  spinal  cat;  the  pressor  activity  of  the  amine  formed  by  enzjmae 
action  was  compared  with  that  of  the  synthetic  amine. 

I.  monohydroxyphenylalanines 

Our  results  are  summarized  on  Table  I.  It  was  found  that  m-hydroxyphenylalanine 
(the  "meta-tyrosine"  of  Blum^)  was  a  substrate  of  the  mammalian  enzyme;  the  rate  of 
decarboxylation  was  slightly  less  than  with  3:4-dihydroxyphenylalanine  as  substrate. 
The  bacterial  preparation  also  acted  on  w-hydroxyphenylalanine,  at  about  one-third 
of  the  rate  of  decarboxylation  of  tyrosine. 

In  the  mammalian  tissue  extracts,  o-hydroxyphenylalanine  (Blum's®  "oitho- 
tyrosine")  was  decarboxylated  at  approximately  the  same  rate  as  the  meta  hydroxy 
References  p.  136I137.  130 


VOL.  4  (1950) 


SPECIFICITY   OF   DECARBOXYLASES 


131 


derivative.  With  the  bacterial  preparations,  the  rate  of  CO,  formation  from  o-hydroxy- 
phenylalanine  was  practically  zero. 

TABLE  I 

DECARBOXYLATION    OF    TYROSINE    AND    ITS    ISOMERS 

+  signifies  decarboxylation 
—  signifies  no  decarboxylation 


Substrates 

OH 

r^P" 

r> 

V 

kJ 

yoH 

CH2 

CHg 

CH2 

1 
CHXH2 

1 
CHNHg 

i 
CHNH2 

COOH 

COOH 

COOH 

Bacterial  preparation 

-r 

+ 

— 

Mammalian  preparation 

+ 

-f 

Results  of  competition  experiments  suggest  that  the  two  enzymes  responsible  for 
these  decarboxylation  reactions  are  the  bacterial  tyrosine  decarboxylase  and  the  mam- 
malian DOPA  decarboxylase.  One  molecule  of  each  DL-amino-acid  gives  one-half  of 
a  molecule  of  CO2  formed;  we  therefore  assume  that  only  one  of  the  two  steroisomers, 
the  L-form,  is  decarboxylated. 

These  findings  demonstrate  the  importance  of  the  phenolic  hydroxyl  groups  and 
their  positions  on  the  benzene  ring  for  the  reaction  between  enzyme  and  substrate.  It 
seems  safe  to  assume  that  these  groups  react  with  the  protein  part  of  the  decarboxylase 
system. 

The  nature  of  the  forces  which  are  at  work  between  enzyme  protein  and  substrate 
is  not  known.  In  the  case  under  consideration,  it  seems  possible  that  the  reaction 
between  the  phenolic  hydroxyl  groups  and  the  enzyme  involves  the  formation  of  a 
hydrogen  bond,  with  the  hydroxyl  group  either  as  a  "donor"  or  an  "acceptor".  At  any 
rate,  the  results  obtained  can  be  understood  if  it  is  assumed  that  the  substrate  must  be 
held  by  a  group  in  the  enzyme  situated  so  that  it  can  react  with  a  hydroxyl  group  in 

OH    *  * 

I  H  H  H 


HC    CH       HC    C^  HC    O^         HC    CH 


HC    CH 


HC    CH 


HC    CH 


HC 


\C^ 


\OH 


R  R  R  R 

Bacterial  enzyme  Mammalian  enzj'^me 

Fig.  I.  The  asterisk  marks  the  position  of  the  active  group  in  the  enzyme  in  relation  to  the  substrate. 
References  p.  136I1J7. 


132  H.  BLASCHKO  VOL.  4  (1950) 

one  of  two  adjacent  positions  on  the  benzene  ring.  The  position  of  this  group  in  the 
enzyme  would  be  different  for  the  bacterial  and  the  mammalian  enzyme,  as  shown  in 
Fig.  I. 

II.  2:5-DIHYDROXYPHENYLALANINE 

This  amino-acid  has  recently  been  synthetized  by  Neuberger^.  We  have  examined 
it  and  have  found  that  it  is  a  substrate  of  the  mammalian  enzyme,  but  that  it  is  not  a 
substrate  of  the  bacterial  enzyme. 

HO  HO 


io/     VcHa-CHNHg-COOH  ^     VcHg-CHNHa-COOH 

OH 
3 : 4-dihydroxyphenylalanine  •     2 : 5-dihydroxyphenylalanine 


That  2 : 5-dihydroxyphenylalanine  is  a  substrate  of  the  mammalian  decarboxylase 
is  easily  explained  by  the  hypothesis  outlined  above ;  the  lack  of  affinity  for  the  bacterial 
enzyme,  however,  is  not  obvious;  possibly  the  presence  of  the  hydioxyl  group  in  ortho 
position  interferes  with  the  attachment  to  the  enzyme. 

We  have  examined  both  the  l  and  the  D  forms  of  this  amino-acid ;  in  agreement 
with  expectation,  only  the  l  form  is  a  substrate  of  DOPA  decarboxylase.  The  product 
of  the  decarboxylation  reaction,  /3-2 : 5-dihydroxyphenylethylamine,  seems  to  be  a 
substrate  of  amine  oxidase ;  this  suggests  that  in  the  living  animal  it  is  metabolized  as 
follows : 

HO_ 

^  -CH,-CHNH„-COOH 


OH 

L-2 : 5-dihydroxyphenylalanine 

HO 
/"^CHg-CHa-NHg 

OH 
^-2 : 5-dihydroxyphenylethylamine 

HO 


-CH2-CHO 
OH 
homogentisic  aldehyde 

HO 

/     VcHg-COOH 

OH 

homogentisic  acid 


It  has  been  shown  that  the  amino-acid  gives  rise  to  homogentisic  acid  in  the  alcap- 
tonuric  subject  (Neuberger,  Rimington,  and  Wilson^").  In  normal  animals  and  human 
subjects,  both  the  amino-acid  and  the  corresponding  amine  are  fully  metabolized 
(Neuberger^;  Leaf  and  Neuberger^^).  This  aspect  of  our  findings  has  been  more  fully 
discussed  elsewhere  (Blaschko  et  al}). 
References  p.  1361137. 


VOL.  4  (1950)  SPECIFICITY   OF   DECARBOXYLASES  I33 

III.    3:4-DIHYDROXYPHENYLSERINE    (NORADRENALINE    CARBOXYLIC    ACID) 

The  study  of  this  compound  has  revealed  another  difference  between  the  mamma- 
Han  and  the  bacterial  decarboxylase^.  On  decarboxylation,  it  yields  noradrenaline : 

KO  HO 


HO<^      VCHOH-CHNH2-COOH "    HO(^      V-CHOH-CH2NH2  +  CO2 

3 : 4-dihydrox3rphenylserine  noradrenaline 

It  was  found  that  the  amino-acid  was  not  decarboxylated  by  extracts  of  mammalian 
tissues;  it  was,  however,  decarboxylated  by  the  bacterial  preparation.  The  rate  of  COg 
formation  with  dihydroxyphenylserine  was  much  slower  than  with  tyrosine  as  substrate, 
but  the  decarboxylation  was  almost  quantitative ;  approximately  one-half  of  the  racemic 
substance  was  decarboxylated.  The  biological  assay  on  the  arterial  blood  pressure  of 
the  spinal  cat,  together  with  the  measurement  of  the  amount  of  COg  formed,  showed  that 
the  amine  formed  was  laevo-noradrenaline. 


IV.  N-METHYLATED    AMINO-ACIDS 

Ten  years  ago,  the  observation  was  made  that  the  introduction  of  a  N-methyl  group 
abolished  the  substrate  specifity  for  DOPA  decarboxylase  (Blaschko^^).  Preparations 
of  mammalian  liver  and  kidney  which  had  DOPA  decarboxylase  activity  were  found 
not  to  act  on  N-methyl-3 : 4-dihydroxyphenylalanine : 

HO 


HO  /     y  CHOH-CHNH  (CH3)-COOH 


This  observation  was  made  the  basis  of  a  scheme  of  biosynthesis  of  sympathin  and 
adrenaline.  It  had  often  been  assumed  previously  that  the  formation  of  adrenaline 
involved  a  decarboxylation  reaction,  but  it  was  now  shown  that  the  body  was  not  able 
to  produce  a  secondary  amine  by  direct  decarboxylation  of  the  N-methyl-amino-acid, 
whereas  it  was  able  to  produce  the  corresponding  primary  amine.  Primary  amines  with 
sjmipathicomimetic  activity  were  therefore  postulated  as  intermediary  products  in 
adrenaline  synthesis.  Earlier  already,  pharmacologists  had  discussed  the  possibility  of 
the  identity  of  Cannon's  "sympathin  E"  with  noradrenaline  (Bacq^^;  Stehle  and 
Ellsworth^^).  The  biochemical  findings  gave  a  simple  explanation  for  the  occurrence 
of  this  substance. 

Two  amino-acids  were  studied  in  1939:  N-methyl-dihydroxyphenylalanine  and 
N-methyl-tyrosine.  One  important  methylamino-acid,  however,  was  not  available  at 
that  time ;  this  was  N-methyl-3 : 4-dihydroxyphenylserine.  Already  in  1906,  Friedmann^^ 
had  considered  this  acid  as  a  possible  precursor  ot  adrenaline ;  he  suggested  that  adren- 
aline was  formed  in  the  reaction: 

HO  HO 

Ho/     yCHOH-CHNH(CH3)-COOH ^      Ho/     yCHOH-CH2NH(CH8)  -^  CO2 

N-methyl-3 : 4-dihydroxyphenylserine  adrenaline 

(adrenaline  carboxylic  acid) 

This  suggestion  could  not  be  tested  by  experiment  until  the  synthesis  of  adrenaline 
References  p.  136113^. 


134  H.  BLASCHKO  VOL.  4  (1950) 

carboxylic  acid  was  achieved  by  Dalgliesh  and  Mann^^.  We  have  recently  examined 
this  compound.  It  was  found  not  to  be  decarboxylated  by  a  number  of  mammahan 
tissue  extracts  and,  unhke  the  corresponding  amino-acid,  dihydroxyphenylserine,  it  was 
not  a  substrate  of  the  bacterial  enzyme  preparation. 

The  substrate  specificity  of  DOPA  decarboxylase  in  connexion  with  pathways  of 
adrenaline  synthesis  has  recently  been  reviewed  elsewhere  (Blaschko^'').  Two  possible 
ultimate  precursors  of  adrenaline  were  discussed:  noradrenaline  and  N-methyl-3 : 4- 
dihydroxyphenylethylamine  (also  known  as  epinine) : 

HO  HO 

lio(      V-CHOH-CHjNHg  Ho/^^VcHa-CHj-NHiCHg) 


noradrenaline  epinine 

The  role  of  epinine  in  the  biosynthesis  of  adrenaline  has  recently  been  discussed 
by  Danneel^^  and  by  Holtz  and  Kroneberg^^.  The  presence  of  this  substance  in 
mammalian  tissue  has  never  been  demonstrated.  Recently,  noradrenaline  has  been 
found  in  human  tumours  of  the  suprarenal  medulla  (Holton^")  as  well  as  in  the  supra- 
renal gland  (Schumann^^).  Evidence  is  also  accumulating  that  both  adrenaline  and 
noradrenaline  are  released  from  the  suprarenal  medulla  (Meier  and  Bein^^;  Bulbring. 
and  Burn-^;  Holtz  and  Schumann^*). 

v.  DOPA    decarboxylase    AND    PYRIDOXINE    DEFICIENCY 

Like  the  mammalian  enzyme,  the  bacterial  enzyme  does  not  act  on  N-methyl- 
tyrosine  (Epps'^)  and  N-methyl-dihydroxyphenylserine.  This  suggests  that  the  inability 
to  act  on  N-methyl-amino-acids  is  due  to  a  property  common  to  both  enzymes. 

It  is  known  that  the  bacterial  codecarboxylase  (Gale  and  Epps^^),  the  prosthetic 
group  of  the  bacterial  tyrosine  decarboxylase,  is  pyridoxal  phosphate  (Gunsalus, 
Bellamy  and  Umbreit^^).  Green,  Leloir,  and  Nocito"  achieved  a  partial  purification 
of  DOPA  decarboxylase  and  a  reactivation  of  the  apoenzyme  by  pyridoxal  phosphate. 
It  is,  however,  not  generally  accepted  that  DOPA  decarboxylase  contains  pyridoxal 
phosphate  (see  Martin  and  Beiler^^;  Work  and  Work-^). 

When  the  DOPA  decarboxylase  activity  was  determined  in  liver  extracts  of  rats 
reared  on  a  diet  deficient  in  pyridoxine  (vitamine  Bg),  enzymic  activity  was  found  to  be 
low,  and  in  a  few  of  the  extracts  the  activity  had  practically  disappeared  (Blaschko, 
Carter,  O'Brien,  and  Sloane  Stanley^";  and  unpublished  observations).  Addition 
of  pyridoxal  plus  ATP  in  vitro  brought  about  a  partial  restoration  of  the  enzymic 
activity.  More  recently,  through  the  kindness  of  Dr  K.  Folkers,  we  have  been  able  to 
test  the  effect  of  synthetic  codecarboxylase:  we  have  found  that  it  is  possible  to  restore 
the  activity  of  the  extracts  from  Bg-deficient  animals  to  normal  values  by  the  addition 
in  vitro  of  10  /<g  of  synthetic  codecarboxylase  to  the  equivalent  of  550  mg  of  fresh  weight 
of  liver.  These  experiments  allow  us  to  conclude  that  DOPA  decarboxylase,  like  the 
bacterial  tyrosine  decarboxylase,  contains  pyridoxal  phosphate. 

There  is  experimental  support  for  a  suggestion  by  Snell^^  that  in  transamination 
the  initial  reaction  between  amino-acid  and  pyridoxal  phosphate  involves  the  formation 
of  a  -N  =  C(  bond.  In  analogy,  it  seems  likely  that  the  decarboxylation  requires  a 
reaction  between  the  amino  group  of  the  amino-acid  and  the  aldehyde  group  of  pyridoxal 
phosphate : 
References  p.  136J13J. 


VOL.  4  (1950) 


SPECIFICITY   OF   DECARBOXYLASES 


135 


R 

H— C— NH2      + 

I 
COOH 


0  =  C— H 

I 
C 

—  c    c  — 

II    I 


H— C— N  =  C— H 

I  I 

COOH     C 

—  C      C  — 

II       I 


+  H»0 


It  is  clear  that  this  reaction  will  only  occur  when  the  amino  group  is  unsubstituted. 
We  conclude  that  N-methyl-amino-acids  are  unable  to  react  with  the  formation  of  a 
-N  =  C(  bond.  This  inability  would  account  for  the  fact  that  N-methyl-amino-acids 
are  not  substrates  of  the  amino-acid  decarboxylases. 


VI.  THE    BASIS   OF   SUBSTRATE    SPECIFICITY 

The  experiments  discussed  have  shown  two  different  types  of  substrate  specificity. 
DOPA  decarboxylase  may  serve  to  demonstrate  these: 

a.  tyrosine  is  not  a  substrate  of  DOPA  decarboxylase,  because  it  does  not  react 
with  the  enzyme  protein ; 

b.  N-methyl-3 : 4-dihydroxyphenylalanine  is  not  a  substrate  of  DOPA  decarbo- 
xylase, because  it  does  not  react  with  the  coenzyme. 

DOPA  decarboxylase,  like  all  the  amino-acid  decarboxylases,  presents  a  third  type 
of  substrate  specificity:  specificity  for  the  members  of  the  L  series.  Holtz,  Heise,  and 
LtJDTKE^  suggested  already  that  DOPA  decarboxylase  was  specific  for  L-dihydroxy- 
phenylalanine ;  we  have  confirmed  this,  using  the  d  isomer  which  was  not  decarbo- 
xylated  (Bl.\schko^2)_ 

The  lack  of  affinity  for  the  d  form  is  easily  understood  in  the  light  of  the  evidence 
discussed  in  this  review.  If  we  consider  the  alpha  carbon  atom  of  the  amino-acid, 

la 

H— C— NH2 
COOH 

we  see  that  three  of  the  groups  attached  to  this  atom  take  part  in  the  decarboxylation 
reaction: 

a.  the  carboxy  group,  which  loses  carbon  dioxide, 

b.  the  amino  group  which  reacts  with  the  aldehyde  group  of  pyridoxal,  and 

c.  the  group  R  which  reacts  with  the  enzyme  protein. 

If  the  decarboxylation  requires  a  fixed  relationship  of  these  three  groups  relative 
to  the  enzyme,  it  is  clear  that  the  L  and  D  forms  are  not  equivalent ;  only  one  of  the 
stereoisomers  can  be  expected  to  fulfil  the  conditions  required  for  decarboxylation.  The 
stereospecificity  of  other  enzymes  dealing  with  amino-acids  may  have  a  similar  basis 
(see  Rydon^^),  but  the  conditions  of  specificity  are  not  so  completely  known. 

It  has  been  pointed  out  that  the  presence  of  a  third  polar  group  in  R  is  a  common 
feature  of  all  bacterial  amino-acid  decarboxylases  (Gale^*).  The  same  is  true  for  the 
mammalian  decarboxylases,  not  only  for  DOPA  decarboxylase,  but    also  for  the  l- 
cysteic  decarboxylase  of  mammalian  liver  (Blaschko^^). 
References  p.  136I13';. 


136  H.  BLASCHKO  VOL.  4  (1950) 

A  cknoidedgement 

The  author  and  his  colleagues,  Dr  G.  H.  Sloane  Stanley  and  Dr  Pamela  Holton, 
have  benefited  from  the  assistance  of  Dr  Ruth  Duthie,  Mrs  Isabella  Wajda,  Miss 
Alison  M.  Pickard,  Miss  Pamela  F.  Kordik  and  Mr  F.  A.  Holton  during  various 
stages  of  this  work.  We  are  also  grateful  to  all  those  who  have  supplied  us  with  the 
substances  used  in  our  experiments. 


SUMMARY 

1.  The  decarboxylation  by  bacterial  and  mammalian  enzymes  of  a  number  of  amino-acids 
structurally  related  to  tyrosine  has  been  studied. 

2.  The  position  of  the  phenolic  hydroxyl  group  in  tyrosine  and  its  isomers  is  shown  to  determine 
substrate  specificity.  This  is  explained  by  a  reaction  between  the  OH  group  of  the  substrate  and  the 
enzyme  protein. 

3.  Methylamino-acids  are  not  decarboxylated ;  this  is  explained  by  their  inability  to  react  with 
the  aldehyde  group  in  pyridoxal  phosphate  (codecarboxylase). 

4.  The  stereospecificity  of  the  amino-acid  decarboxylases  is  discussed  on  the  basis  of  these 
observations. 

RESUMfi 

1.  La  decarboxylation  de  quelques  acides  amines,  apparentes  a  la  tyrosine,  a  et6  etudiee  au 
moyen  de  ferments  bacteriens  et  animaux. 

2.  La  position  des  groupes  OH  dans  la  tyrosine  et  ses  isomeres  est  d^terminante  pour  la  sp6cificite 
des  decarboxylases.  Nous  en  deduisons  que  la  reaction  entre  I'apoferment  et  les  acides  amin6s  en 
question  a  lieu  au  niveau  du  groupe  OH. 

3.  Les  acides  methyl-amines  ne  sont  pas  d^carboxyles  en  presence  de  ces  ferments.  Ce  ph^nomene 
s'explique  par  I'impossibUite  du  groupe  N-m^thyUque  de  reagir  avec  Tald^hyde  du  phosphate  de 
pyridoxal  (codecarboxylase). 

4.  Les  resultats  de  ce  travail  nous  permettent  de  discuter  le  phenomene  de  la  stereospecificite 
des  decarboxylases. 

ZUSAMMENFASSUNG 

1.  Die  Decarboxylierung  einiger  dem  Tyrosin  verwandter  Aminosauren  durch  tierische  und 
bakterielle  Fermente  wurde  untersucht. 

2.  Die  Position  der  phenolischen  Hydroxylgruppe  des  Tyrosins  und  seiner  Isomeren  ist  fiir  die 
Substratspezifitat  von  Bedeutung.  Diese  Beobachtung  wird  erklart  durch  die  Annahme  einer  Bindung 
zwischen  der  OH-Gruppe  des  Substrats  und  dem  Apoferment. 

3.  Methylaminosauren  werden  nicht  decarboxyliert;  dies  wird  erklart  durch  das  Ausbleiben 
der  Reaktion  mit  der  Aldehydgruppe  des  Pyridoxal-Phosphats  ("Codecarboxylase"). 

4.  Die  Stereospezifitat  der  Aminosauredecarboxylasen  wird  im  Lichte  der  gewonnenen  Resul- 
tate  erlautert. 

REFERENCES 

1  H.  Blaschko,  p.  Holton,  and  G.  H.  Sloane  Stanley,  Brit.  J.  Pharmacol.,  3  (1948)  3i5- 

2  H.  Blaschko,  P.  Holten,  and  G.  H.  Sloane  Stanley,  /.  Physiol.  {London),  108  (1949)  427- 

3  H.  Blaschko,  Biochem.  J.,  44  (1949)  268. 

*  G.  H.  Sloane  Stanley,  Biochem.  J .,  44  (1949a)  373. 

5  G.  H.  Sloane  Stanley,  Biochem.  J.,  44  (1949b)  (in  press). 

8  P.  HoLTZ,  R.  Heise,  and  K.  Ludtke,  Arch,  exptl.  Path.  Pharmakol.,  191  (1938)  87. 

'  H.  M.  R.  Epps,  Biochem.  J.,  38  (1944)  242. 

8  L.  Blum,  Arch,  exptl.  Path.  Pharmakol.,  59  (1908)  269. 

®  A.  Neuberger,  Biochem.  J.,  43  (1948)  599. 

^^  A.  Neuberger,  C.  Rimington,  and  J.  M.  G.  Wilson,  Biochem.  J.,  41  (1947)  438. 
1^  G.  Leaf  and  A.  Neuberger,  Biochem..  J .,  43  (1948)  606. 


VOL.  4  (1950)  SPECIFICITY   OF    DECARBOXYLASES  I37 

^2  H.  Blaschko,  J .  Physiol.  [London),  96  (1939)  50P. 

^^  Z.  M.  Bacq,  Ann.  physiol.  physicochim.  bioL,  10  ^1934)  467. 

"  R.  L.  Stehle  and  H.  C.  Ellsworth,  /.  Pharmacol.  Exptl.  Therap.,  59  (1937)  ii4- 

^^  E.  Friedmann,  Beitr.  chem.  Physiol.  Path.,  8  (1906)  95. 

^®  C.  E.  Dalgliesh  and  F.  G.  Mann,  /.  Chem.  Sac,  (1947)  658. 

^^  H.  Blaschko,  Adrenaline  and  Sympathin  from:  The  Hormones,  Physiology,  Chemistry  and  appli- 
cations. Vol  2  (1949),  New  York,  Academic  Press. 

^*  R.  Danneel,  Z.  Naturforsch.,  1  (1946)  87. 

^®  P.  HoLTZ  AND  G.  Kroneberg,  KUu.  Wochschr.,  26  (1948)  605. 

^"  P.  HoLTON,  Nature,  163  (1949)  217. 

2^  H.  J.  Schumann,  Klin.  Wochenschr.,  26  (1948)  604. 

22  R.  Meier  and  H.  J.  Bein,  Experientia,  4  (1948)  358. 

2^  E.  BiJLBRiNG  AND  J.  H.  BuRN,  Nature,  163  (1949)  363. 

2*  P.  HoLTz  AND  H.  J.  Schumann  (quoted  after  Schumann^I). 

2^  E.  F.  Gale  and  H.  M.  R.  Epps,  Biochem.  J .,  38  (1944)  250. 

2^  I.  C.  Gunsalus,  W.  D.  Bellamy,  and  W.  W.  Umbreit,  /.  Biol.  Chem.,  155  {1944)  685. 

2'  D.  E.  Green,  L.  F.  Leloir,  and  V.  Nocito,  /.  Biol.  Chem.,  161  (1945)  559. 

28  G.  J.  Martin  and  J.  M.  Beiler,  Arch.  Biochem.,  15  (1947)  201. 

2*  T.  S.  Work  and  E.  Work,  The  Basis  of  Chemotherapy ,  London  and  Edinburgh.  Oliver  and  Boyd, 
Ltd.  (1948)  P-  145- 

'"  H.  Blaschko,  C.  W.  Carter,  J.  R.  P.  O'Brien,  and  G.  H.  Sloane  Stanley,  /.  Physiol.,  107 
(1948)  18P,  and  unpublished  observations. 

^^  E.  E.  Snell,  /.  Am.  Chem.  Soc,  167  (1945)  194. 

^2  H.  Blaschko,  J.  Physiol.,  loi  (1942)  337. 

^^  H.  N.  Rydon.  Biochem.  Soc.  Symposia,  I  (1948)  40. 

■'^  E.  F.  Gale,  Advances  in  Enzymol.,  6  (1946)  i. 

^^  H.  Blaschko,  Biochem.  J.,  36  (1942)  571. 

Received  March  21st,  1949 


138  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


GLYCOLYSIS  IN  PHARMACOLOGY^'  ^ 

by 

CHALMERS  L.  GEMMILL 

Department  of  Pharmacology,  Medical  School,  University  of  Virginia, 
Charlottesville,   Virginia  [U.S.A.) 


Classical  pharmacology  deals  with  the  action  of  drugs  on  organ  systems.  If  the 
question  is  raised  as  to  why  a  certain  drug  acts  on  a  particular  organ  system,  the  answer 
may  only  be  obtained  by  searching  for  some  system  inside  the  cell  which  is  sensitive 
to  the  drug  in  question.  The  most  fruitful  line  of  endeavor  has  been  to  test  the  affect 
of  the  drug  on  enzyme  systems  known  to  be  involved  in  cellular  metabolism.  Many 
pharmacological  actions  of  drugs  can  be  explained  in  this  manner.  For  example,  the 
pharmacological  properties  of  vitamins,  physostigmine,  BAL,  and  cyanide  have  been 
explained  to  everyone's  satisfaction  on  an  enzymatic  basis.  During  the  past  war,  a  great 
deal  of  attention  was  paid  to  the  action  of  antimalarial  drugs,  ionizing  radiation  and 
chemical  warfare  agents  on  enzymatic  processes.  In  fact,  there  is  a  growing  school  in 
Pharmacology  which  has  for  its  main  purpose  the  localization  of  drug  action  on  en- 
zymatic processes.  Some  of  this  work  has  been  reviewed  by  Green^,  Bernheim^,  Clark^, 
and  McElroy*.  The  recent  book  by  Work  and  Work^  is  an  excellent  example  of  the 
development  of  this  field  in  chemotherapy. 

Welch  and  Bueding^  have  laid  down  very  severe  criteria  which  should  be  met 
before  the  action  of  a  drug  can  be  attributed  to  its  effects  on  an  enzyme  system.  These 
criteria  involve  concentrations,  organ  and  tissue  specificity  and  close  parallelism  be- 
tween the  activity  of  structurally  related  compounds.  These  criteria  are  very  hard  to 
meet  in  this  field.  It  is  very  difficult  to  determine  how  much  drug  is  acting  on  a  specific 
organ  when  the  drug  is  administered  to  the  whole  animal.  When  working  on  enzyme 
systems,  cell  interfaces  are  destroyed  and  permeability  is  no  longer  a  question,  which 
may  modify  drug  action.  Therefore,  the  criteria  of  Welch  and  Bueding^  should  be 
used  as  an  ultimate  goal  and  not  be  used  to  delay  or  to  give  up  work  and  thinking  in 
this  field. 

It  is  the  purpose  of  this  article  to  give  several  examples  of  drug  action  on  the  gly- 
colytic system  in  order  to  show  how  the  discoveries  of  Meyerhof  are  now  being  used 
in  Pharmacology.  Meyerhof^  used  many  pharmacological  agents  as  chemical  tools  in 
his  work  on  muscle  metabolism.  Narcotics,  methylene  blue,  chloroform,  caffeine,  and 
moniodoacetic  acid  are  a  few  of  many  agents  employed  in  his  work.  More  recently 
Meyerhof  and  his  associates  have  employed  alloxan^  in  their  study  of  glycolysis  of 
brain  preparations  and  have  reported^  the  effects  of  potassium  i,  2-naphthoquinone-4- 
sulfonate  on  the  respiration  and  glycolysis  of  Trypanosoma  equiperdum. 


^  Read  before  a  Seminar  at  the  Army  Chemical  Center,  March  9,  1949. 

2  In  this  paper,  the  term  "glycolysis"  is  used  in  the  general  meaning  for  the  break  down  of 
any  carbohydrate  into  lactic  acid  by  enzymatic  processes. 

References  p.  142  j  143. 


VOL.  4  (1950)  GLYCOLYSIS    IN    PHARMACOLOGY  I39 

Any  abnormal  cell,  invading  organism  or  abnormal  metabolic  event  in  the  body 
involving  or  using  carbohydrate  opens  itself  to  this  mode  of  attack,  namely,  to  find 
a  chemical  substance  which  will  block  or  modify  its  use  of  carbohydrate  but  not  affect 
the  use  of  carbohydrate  by  the  normal  cells  of  the  host.  In  this  manner  the  abnormal 
cells  or  invading  organisms  can  no  longer  use  sugar  for  energy  purposes  and  thus  are 
destroyed.  Abnormal  metabolism  of  carbohydrate  may  also  be  checked  or  diverted  into 
normal  pathways  in  a  similar  manner.  Since  the  carbohydrate  is  generally  oxidized 
by  the  invading  organisms,  two  possibilities  are  available  for  blocking  by  enzymatic 
inhibitors;  a)  in  the  oxidative  chain  and  b)  in  the  glycolytic  system.  In  the  cancer  field, 
for  example,  if  an  agent  could  be  found  which  will  block  the  use  of  glucose  either  by 
oxidation  or  by  glycolysis  in  the  rapidly  growing  cells,  growth  would  cease  since  these 
cells  depend  mainly  on  the  metabolism  of  glucose  for  their  growth.  Therefore,  there 
should  be  a  constant  search  for  compounds  which  inhibit  glycolysis  or  the  oxidation  of 
various  sugars.  Such  a  search  may  some  day  be  rewarded  with  a  differential  inhibitor 
which  will  block  sugar  utilization  in  the  cancerous  cell  and  not  in  the  normal  cell.  Such 
inhibitors  have  been  found  already  for  certain  invading  organisms  and  may  well  be 
found  for  the  cancer  cell.  A  review  of  some  of  the  literature  in  this  field  up  to  1938 
has  been  made  by  Gemmill^". 

Quinine  and  Atabrine:  During  the  war,  Evans  and  his  associates  made  a  very 
intensive  study  of  quinine  and  atabrine  on  glycolysis.  This  group  demonstrated  that 
the  glycolysis  of  the  malarial  parasite  was  similar  to  that  of  the  phosphorylating  gly- 
colysis of  yeast  and  muscle^^.  Following  these  observations  the  effects  of  quinine  and 
atabrine  were  investigated^^  on  this  system  from  malarial  parasites,  yeast  and  mam- 
mahan  muscle.  Atabrine  inhibited  hexokinase  activity  and  the  lactate  dehydrogenase 
in  the  parasite  preparations.  Both  quinine  and  atabrine  inhibited  the  yeast  hexokinase 
while  quinine  was  inhibitory  to  the  phosphorylase  and  the  phosphoglucomutase  from 
rabbit's  muscle.  Lactate  dehydrogenase  from  beef  heart  was  very  susceptible  to  atabrine 
action.  However,  from  the  concentrations  needed  to  inhibit  these  enzymes  in  the  gly- 
colytic systems,  these  authors  concluded  that  the  therapeutic  site  of  inhibition  is 
probably  in  the  oxidative  cycle  unless  there  is  a  possibility  of  a  high  concentration  of 
these  drugs  localizing  inside  the  parasite  cell.  Bovarnick,  Lindsay,  and  Hellerman" 
attribute  the  inhibitory  action  of  atabrine  on  the  oxidation  of  glucose  to  an  interference 
of  phosphorylation  which  is  essential  before  glucose  may  be  oxidized  by  the  malarial 
parasite. 

Naphthoquinones:  There  has  been  considerable  attention  given  to  the  naphtho- 
quinones in  pharmacology  in  recent  years.  In  addition  to  the  discovery  that  vitamin  K 
has  a  naphthoquinone  nucleus,  these  compounds  have  been  investigated  for  their 
antimalarial^*,  fungicidaP^,  antitubercular^^,  and  antibacterial  actions^'.  Some  of  the 
naphthoquinones  have  the  power  to  inhibit  mitosis  which  makes  them  of  interest  from 
the  standpoint  of  tumor  growth^^.  Naphthoquinones  inhibit  acid  formation  in  the  saliva 
which  may  aid  in  the  prevention  of  tooth  decay^^. 

Considerable  work  has  been  done  to  explain  the  action  of  naphthoquinones  on  a 
possible  enzymatic  site.  Wendel^o  has  described  an  inhibition  of  the  oxygen  uptake 
and  the  use  of  carbohydrate  in  red  blood  cells  parasitized  with  a  malarial  parasite. 
Ball,  Anfinsen,  and  Cooper^^  have  made  an  extensive  study  of  the  inhibition  of 
oxygen  uptake  and  have  come  to  the  conclusion  that  the  inhibitory  site  is  between  cyto- 
chrome c  and  b  in  the  chain  of  respiratory  enzymes.  Bueding,  Peters,  and  Waite^^ 
References  p.  142  j  143. 


140 


C.  L.  GEMMILL 


VOL.  4  (1950) 


have  shown  that  2-methyl-i,4-naphthoquinone  inhibits  aerobic  glycolysis  in  Schistosoma 
mansoni,  in  vitro.  Warren^^  has  observed  a  similar  effect  in  bone  marrow.  Meyerhof 
AND  Randall^  have  found  an  inhibition  of  respiration,  glycolysis  and  motility  of 
Trypanosoma  equiperdum,  in  vitro,  using  potassium  i,2-naphthoquinone-4-sulfonate. 
Gemmill^*  has  studied  the  effects  of  various  naphthoquinones  on  anerobic  glycolysis 
of  frog  muscle.  His  results  are  given  in  Table  i. 


TABLE  I 

NAPHTHOQUINONES    WHICH    INHIBITED    GLYCOLYSIS    IN    CONCENTRATIONS    OF    I  •  lO" 


MOLAR    OR    LESS 


1.  Sodium  i,2-naphthoquinone-4-sulfonate 

2.  2-methyl-i,4-naphthoquinone 

3.  Sodium  2-methyl-i,4-naphthohydroquinone  diphosphate 

4.  2-hydroxy-3-methyl-i,4-naphthQquinone  (Phthiocol) 

5.  2-methyl-4-amino-i-naphthol  hydrochloride 

6.  2-hydroxy-i,4-naphthoquinone  (Lawsone) 

7.  1,4-naphthohydroquinone 

8.  2-methyl-3-bromo-i,4-naphthoquinone 

9.  2-chloro-3-N-thiobutyl- 1 ,4-naphthoquinone 

10.  2-methyl-3-thioethyI-i,4-naphthoquinone 

1 1 .  2-hydroxy-3-cyclohexanol- 1 , 4-naphthoquinone 


In  Table  I  may  be  seen  several  napthoquinones  which  are  glycolytic  inhibitors. 
The  relationship  of  concentration  to  inhibition  by  sodium  i,2-naphthoquinone-4- 
sulfonate  may  be  seen  in  Fig.  i.  At  low  concentrations  there  is  a  slight  stimulation  of 
glycolysis.  As  the  concentrations  increase  there  is  a  marked  change  in  glycolysis  with 
practically  complete  inhibition  occurring  with  concentration  of  0.4-10"^  Molar.  Some 

of  the  naphthoquinones  which  have  vitamin 
K  activity  also  are  inhibitors  of  anerobic 
glycolysis:  2-methyl-i,4-napthoquinone,  so- 
dium 2-methyl-i, 4-naphthoquinone  diphos- 
phate and  2-methyl-4-amino-i-naphthol  hy- 
drochloride. Another  interesting  fact  which 
came  out  of  this  work  was  that  the  attach- 
ment of  a  halogen  in  the  2  or  3  position 
increased  the  inhibitory  activity  of  these 
compounds. 

Amidines  and  Related  Compounds:  His- 
torically, the  study  of  the  chemotherapeutic 
properties  of  the  diamidine  compounds  was 
a  direct  result  of  a  search  for  agents  which 
would  block  the  use  of  glucose  by  the  try- 
panosomes-^.  The  early  discovery  that  deca- 
methylene  diguanidine  hydrochloride  (Syn- 
thalin)  was  effective  against  certain  trypan- 
osomes  led  to  a  search  for  less  toxic  substances. 
Out  of  this  search  came  many  guanidines, 
isothioureas,  amidines^^  and  numerous  aro- 
matic diamidines,  among  them  being  stilb- 
amidine    and    pentamidine.     It    was     soon 


% 

120 

-$. 

V 

? 

^^^\ 

100 

\ 

\ 

X 

" 

80 

X 

60 

40 

20 

\ 

\ 

X 

V 

y 

X 

■^^ — 

y 

02 


0.6  1.0 

UlO'^molar 

Fig.  I.  The  effects  of  increasing  concen- 
trations of  sodium  i,2-naphthoquinone-4- 
sulfonate  on  glycolysis.  Abscissae,  i-io— ^ 
Molar  final  concentration:  ordinates,  per 
cent  of  normal  glycolysis. 


References  p.  142I143. 


VOL.  4  {1950)  GLYCOLYSIS   IN    PHARMACOLOGY  I41 

shown  that  doses  of  the  diamidines  which  were  active  against  trypanosomes  did  not 
produce  a  fall  in  blood  sugar  of  the  host.  Therefore,  attention  was  given  to  the  sugar 
metabolism  and  oxygen  utilization  of  these  organisms.  Lourie  and  Yorke"  have 
stated  that  the  diamidines  may  block  the  aerobic  glucose  metabolism  in  the  diamidine- 
sensitive  species.  The  diamidine-insensitive  species  would  be  capable  of  obtaining  their 
energy  from  the  anerobic  glycolysis  in  the  presence  of  the  drug. 

Some  attention  has  been  paid  to  the  possible  enzymatic  site  of  the  action  of  these 
compounds.  Blaschko  and  Duthie^^  have  found  an  inhibitory  action  of  the  various 
amidine  derivatives  on  the  amine  oxidase  activity  of  the  rabbits'  liver.  Bernheim^^ 
has  shown  that  the  oxidation  of  proline  and  alanine  by  E.  coli  is  inhibited  by  prop- 
amidine. However,  the  oxidation  of  glucose,  pyruvate  and  succinate  is  not  affected  by 
this  drug.  Dickens^"  has  demonstrated  that  guanidine  carbonate  increases  the  aerobic 
glycolysis  of  the  rat  brain  cortex.  These  facts  led  to  a  study  of  the  effects  of  diamidines 
and  related  compounds  on  anerobic  glycolysis  of  glycogen  to  lactate  in  muscle  extract 
(Gemmill^i).  The  various  compounds  in  this  series  which  inhibited  glycolysis  are  given 
in  Table  II.  In  the  same  paper  is  given  a  list  of  styryl  and  cyanine  compounds  which 
are  active  inhibitors. 

TABLE  II 

AMIDINES   AND    REL.\TED    COMPOUNDS   WHICH    INHIBITED 
GLYCOLYSIS    IN    CONCENTRATIONS    OF    I  •  lO"^    MOLAR    OR    LESS 


Diamidines :  Diguanidines : 

Ci2-2  HCl  Diguanidine  HCl 

C13.2HCI  C12  HCl 

Monoguanidines:  Diisothioureas: 
Guanidine  HCl  C^^  HBr 

Methylguanidine  sulfate  C^g  HBr 

Arginine  HCl  Stilbamidine 

Cg  HCl  Pentamidine 

Cjo  HCl  Chlorguanidine 


Alloxan:  Since  the  discovery  that  alloxan  may  produce  diabetes  by  destroying 
the  cells  in  the  islets  of  Langerhans,  there  has  been  a  renewed  interest  in  the  effect  of 
alloxan  on  enzyme  systems.  Purr^^  has  demonstrated  that  alloxan  has  the  abihty  to 
inhibit  papain  and  cathepsin  and  Hopkins,  Morgan,  and  Lutwak-Mann^^  have  shown 
the  same  effect  on  the  succinic  dehydrogenase.  Alloxan  may  act  as  a  hydrogen  acceptor 
in  enzyme  solutions^*'  ^^.  Gemmill^^  has  demonstrated  that  alloxan  may  inhibit  gly- 
colysis. The  degree  of  inhibition  was  proportional  to  the  concentration  of  alloxan  and 
the  inhibition  was  partially  reversed  by  cysteine.  Therefore  alloxan  may  be  added  to 
the  group  of  oxidizing  agents  which  can  reversibly  inactivate  glycolysis.  It  would  be  of 
interest  to  show  that  the  cells  in  the  islets  of  Langerhans  have  a  glycolytic  system 
which  was  very  sensitive  to  this  reagent. 

Caffeine:  Considerable  work  has  been  done  on  the  effect  of  caffeine  on  glycolysis 
in  the  intact  muscle.  Meyerhof^^  demonstrated  that  caffeine  increased  lactate  formation. 
Matsuoka^  continued  and  reported  in  detail  this  demonstration.  David^^  has  shoNvn 
a  large  increase  in  lactate  formation  in  caffeine  contracture.  Gemmill*",  in  cell  free 
extracts,  was  able  to  demonstrate  that  caffeine  and  some  theobromine  derivatives 
caused  an  increase  in  the  rate  of  glycolysis  which  was  followed  by  an  inhibition. 
References  p.  142 j  143. 


142  C.  L.  GEMMILL  VOL.  4  (1950) 

Mercury  Compounds:  Gemmill  and  Hellerman*^  studied  the  effects  of  small 
concentrations  of  phenylmercuric  hydroxide,  p-chloromercuric  benzoic  acid  and  mercuric 
chloride  on  glycolysis  in  muscle  extracts.  These  substances  have  the  power  to  inhibit 
glycolysis  and  the  inhibition  is  abolished  by  the  addition  of  cysteine. 

Iodine:  In  the  same  paper  in  which  the  action  of  the  mercury  compounds  on  gly- 
colysis was  described,  Gemmill  and  Hellerman*i  also  demonstrated  that  small 
amounts  of  iodine  inhibited  glycolysis.  This  effect  was  reversed  by  the  addition  of 
cysteine.  Lipmann*-  had  previously  shown  that  iodine  was  an  active  inhibitor  of  gly- 
colysis. Rapkine'*^  traced  the  action  of  oxidizing  agents  to  the  oxidoreduction  between 
phosphoglyceraldehyde  and  pyruvic  acid.  Lipmann**  has  pointed  out  that  there  are 
five  enzymes  in  the  glycolytic  system  which  may  undergo  oxidative  inactivation  and 
reactivation  with  glutathione. 

Anesthetics:  Watts*^,  working  in  this  laboratory,  has  shown  that  methadon  and 
nupercaine  have  the  property  of  maintenance  of  glycolysis  over  and  above  the  normal 
velocity  of  this  process  in  an  activated  homogenate  of  rat  brain.  During  the  first  ten 
minutes,  there  is  no  difference  in  the  rate  of  glycolysis.  However,  after  the  first  ten 
minutes,  the  normal  rate  tends  to  decrease,  while  the  mixture  containing  either  of  these 
two  drugs  maintains  the  same  rate  of  the  original  ten  minute  period.  Using  radioactives 
phosphorous  in  the  form  of  the  phosphate  ion,  Pertzoff  and  Gemmill*^  have  shown 
that  sodium  barbital  and  ether  have  a  retarding  effect  on  the  transfer  of  phosphate 
from  plasma  into  the  red  blood  cell. 


SUMMARY 

Several  examples  of  the  action  of  chemical  compounds  of  therapeutic  interest  on  glycolysis 
have  been  given  in  this  short  review.  In  most  of  these  cases,  the  methods  and  results  of  Professor 
Meverhof  have  been  used  as  a  background  in  this  work.  Many  developments  are  possible  from  this 
type  of  work,  especially  in  the  explanation  of  drug  action  and  the  control  of  disease  through  this 
knowledge.  Therefore,  pharmacology  owes  much  to  the  pioneer  investigations  of  Professor  Meyerhof. 

RfiSUxMfi 

Dans  cette  brfeve  revue  nous  avons  donn^  plusieurs  exemples  de  Taction  sur  la  glycolyse  de 
certains  composes  chimiques  d'interet  pharmaceutique.  Dans  la  plupart  des  cas  les  m^thodes  et  les 
resultats  du  Professeur  Meyerhof  ont  forme  le  point  de  depart  de  ce  travail.  Ce  genre  de  travail 
pent  donner  lieu  a  des  developpements  nombreux,  surtout  pour  expliquer  Taction  des  drogues  et, 
par  ce  fait,  pour  enrayer  la  maladie.  C'est  pourquoi  la  pharmacologic  doit  beaucoup  aux  investiga- 
tions de  pionnier  du  Professeur  Meyerhof. 

ZUSAMMENFASSUNG 

In  dieser  kurzen  tJbcrsicht  wurden  einige  Beispiele  fiir  die  Wirkung  chemischer  Verbindungen 
von  therapeutischem  Interesse  auf  die  Glykolyse  gegeben.  In  den  meisten  Fallen  bildeten  die  Metho- 
den  und  die  Ergebnisse  von  Professor  Meyerhof  den  Hintergrund  dieser  Arbeit.  Vielerlei  Entwick- 
lungen  dieser  Arbeit  sind  moglich,  insbesondere  zur  Erklarung  der  Wirkung  der  Arzneiraittel  und 
dadurch  zur  Eindammung  der  Krankhciten.  Deshalb  hat  die  Pharmakologie  den  Pioniersunter- 
suchungen  von  Professor  Meyerhof  viel  zu  verdanken. 

REFERENCES 

^  D.  E.  Green,  Advances  in  Enzymol.,  i  (1947)  177. 

-  F.  Bernheim,  The  Interaction  of  Drugs  and  Cell  Catalysts,  Burgess  Publ.  Co.,  1942. 


VOL.  4  (1950)  GLYCOLYSIS   IN    PHARMACOLOGY  I43 

3  A.  J.  Clark,  The  Mode  of  Action  of  Drugs  on  Cells,  E.  Arnold  and  Co.,  1933. 

*  W.  D.  McElroy,  Quart.  Rev.  Biol.,  22  (1947)  25. 

5  T.  S.  Work  and  E.  Work,  The  Basis  of  Chemotherapy,  Oliver  and  Boyd,  1948. 

^  A.  D.  Welch  and  E.  Bueding,  Currents  in  Biochemical  Research,  Interscience  Publ.,  1946,  399. 

'  O.  Meyerhof,  Die  Chemischen  Vorgdnge  im  Muskel,  J.  Springer,  1930. 

8  O.  Meyerhof  and  J.  R.  W'Ilson,  Arch.  Biochem.,  17  (1948)  153. 

®  O.  Meyerhof  and  L.  O.  Randall,  Arch.  Biochem.,  17  (1948)  171. 
1°  C.  L.  Gemmill,  Cold  Spring  Harbor  Symposia  Quant.  Biol.,  7  (1939)  216. 
"  J.  F.  Speck  and  E.  A.  Evans  Jr,  J.  Biol.  Chem.,  159  (1945)  71. 
12  J.  F.  Speck  and  E.  A.  Evans  Jr,  J.  Biol.  Chem.,  159  (1945)  83. 

^^  M.  R.  Bovarnick,  a.  Lindsay,  and  L.  Hellerman,  /.  Biol.  Chem.,  163  (1946)  535. 
1*  L.  F.  Fieser  et  al.,  J.  Am.  Chem.  Soc,  70  (1948)  3 151  and/.  Pharmacol  Exptl  Therap.,  94  (1948)  85. 
^^  A.  M.  Kligman  and  W'.  Rosensweig,  Invest.  Dermatol.,  10  (1948)  59. 
1^  J.  B.  Lloyd  and  G.  Middlebrook,  Am.  Rev.  Tuberc,  49  (1944)  539. 
1'  C.  A.  CoLWELL  AND  M.  McCall,  Scicnce,  loi  (1945)  592. 

18  E.  Friedmann,  D.  H.  Marrian,  and  I.  Simon-Reuss,  Brit.  J.  Pharmacol.,  3  (1948)  263. 
1^  L.  S.  Fosdick,  O.  E.  Fancher,  and  J.  C.  Calandra,  Science,  96  (1942)  45. 

20  W.  B.  Wendell,  Federation  Proc,  5  (1946)  406. 

21  E.  G.  Ball,  C.  B.  Anfinsen,  and  O.  Cooper,  /.  Biol.  Chem.,  168  (1947)  257. 

22  E.  Bueding,  D.  Peters,  and  J.  F.  Waite,  Soc.  Exptl  Biol.  Med.,  64  (1947)  m. 
^  C.  O.  Warren,  Am.  J .  Physiol.,  139  (1943)  719. 

2*  C.  L.  Gemmill,  /.  Pharmacol.  Exptl  Therap.  (1949)  (in  press). 
25  E.  B.  Schoenbach  and  E.  M.  Greenspan,  Medicine,  27  (1948)  327. 
28  H.  King,  E.  M.  Lourie,  and  W.  Yorke,  Lancet,  233  (1937)  1360. 

"  E.  M.  Lourie  and  W.  Yorke,  Ann.  Trop.  Med.  Parasitol.,  33  (1939)  305.  (Quoted  from  Schoen- 
bach AND  GrEENSPAN^S). 

28  H.  Blaschko  and  R.  Duthie,  Biochem.  J.,  39  (1945)  347. 

29  F.  Bernheim,  /.  Pharmacol.  Exptl.  Therap.,  80  (1944)  199. 
'"  F.  Dickens,  Biochem.  J .,  33  (1939)  2017. 

^1  C.  L.  Gemmill,  /.  Pharmacol.  Exptl  Therap.  (1949)  (in  press). 

^2  A.  Purr,  Biochem.  J .,  29  (1935)  5. 

^^  F.  G.  Hopkins,  E.  J.  Morgan,  and  C.  Lutwak-Mann,  Biochem.  J.,  32  (1938)  1829. 

^  M.  Dixon  and  L.  G.  Zerfas,  Biochem.  J .,  34  (1940)  371. 

^*  F.  Bernheim,  /.  Biol.  Chem.,  i-Z'i  (1938)  741. 

^*  C.  L.  Gemmill,  Am.  J.  Physiol.,  150  (1947)  613. 

^'  O.  Meyerhof,  Pfliigers  Arch.,  ges.  Physiol.,  188  (1921)  114. 

^8   K.  Matsuoka,  Pfliigers  Arch.  ges.  Physiol.,  204  (1924)  51. 

^*  F.  David,  Pfliigers  Arch.  ges.  Physiol.,  233  (1933)  222. 

*°  C.  L.  Gemmill,  /.  Pharmacol.  Exptl  Therap.,  91  (1947)  292. 

*^  C.  L.  Gemmill  and  L.  Hellerman,  Am.  J.  Physiol.,  120  (1937)  5^2. 

*2  F.  Lipmann,  Biochem.  Z.,  268  (1934)  205. 

*^  L.  Rapkine,  Biochem.  J .,  32  (1938)  1729. 

**  F.  Lipmann,  A  Symposium  on  Respiratory  Enzymes,  Univ.  Wis.  Press.,  1946,  66. 

*5  D.  Watts,  Unpublished  results. 

"  V.  Pertzoff  and  C.  L.  Gemmill,  /.  Pharm.  Exptl  Therap.,  95  (1949)  106. 

Received  March  15th,  1949 


144  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  {1950) 


ZUR  CHARAKTERISIERUNG  DER  SPEZIFITAT 

PHARMAKOLOGISCHER  WIRKUNGEN  UND  DES  SIE  BEDINGENDEN 

REZEPTORSYSTEiyiS  DES  SUBSTRATES 


R.  MEIER  UND  H.  J.  BEIN 

Forschungslaboratorium  der  Ciba,  Basel  {Schweiz) 


Die  Arbeiten  von  Otto  Meyerhof  haben  im  wesentlichen  die  Analyse  physiolo- 
gischer  Reaktionen  zum  Ziele,  besonders  die  quantitative  Feststellung  des  Reaktions- 
ablaufes,  seiner  Bedingungen  und  seiner  Gleichgewichtszustande.  Seine  Auffassung  der 
Dynamik  der  physiologischen  Vorgange  hat  zu  den  —  Grundlage  einer  Arbeitsrichtung 
gewordenen  —  Ergebnissen  gefiihrt.  Auch  fiir  andere  Disciplinen  haben  diese  Unter- 
suchungen  grundsalzliche  Bedeutung  gewonnen,  so  auch  fiir  die  Pharrnakologie,  fiir 
die  gerade  die  quantitative  Analyse  der  physiologischen  Reaktionen  einen  besonderen 
Zugang  zu  ihrem  eigentlichen  Problem  der  Analyse  der  Wirkung  von  x\rzneimitteln 
eroffnet  hat.  Im  allgemeinen  ist  es  allerdings  in  vielen  Fallen  heute  noch  nicht  moglich, 
die  pharmakologische  Wirkung  auf  die  Reaktionsteilnehmer  physiologischer  Reaktionen 
zuriickzufiihren.  In  der  weitaus  grosseren  Zahl  der  Falle  ist  die  Beteiligung  des  zuge- 
setzten  Pharmakons  am  ausgelosten  Prozess  nicht  bekannt,  sodass  sich  die  pharmakolo- 
gische Feststellung  sehr  haufig  zunachst  damit  begniigen  muss,  aus  der  quantitativen 
Ermittlung  des  durch  zugesetzte  Pharmaka  ausgelosten  Reaktionsverlaufes  zu  einer 
praeliminaren  Charakterisierung  des  zugrundeliegenden  Vorganges  zu  kommen.  Be- 
sonders die  Beziehung  zwischen  der  gegebenen  Dosis  und  dem  eintretenden  Effekt 
ist  Gegenstand  der  Analyse  des  Vorganges  geworden.  Die  Forschungen  von  Loewe 
(1928),  Clark  (1933,  1937)  und  Gaddum  (1937)  haben  vor  allem  allgemeingultige 
Folgerungen  an  der  Bewertung  derartiger  Befunde  entwickelt.  In  Parallele  zur  mathe- 
matischen  Behandlung  chemischer  und  physikalisch-chemischer  Reaktionen  war  es 
nahehegend,  die  gleichen  Prinzipien  auch  auf  die  Reaktionen  von  Pharmaka  anzu- 
wenden.  Clark  hat  um  die  Behandlung  biologischer  Daten  in  dieser  Richtung  die 
grossten  Verdienste.  Es  lasst  sich  aber  fiir  diese  Art  der  Analyse  die  Schwierigkeit 
nicht  eliminieren,  inwieweit  die  Dosiswirkungsbeziehung  allein  oder  auch  nur  im 
wesentlichen  durch  die  Reaktion  des  Pharmakons  mit  dem  spezifischen  Rezeptor  be- 
dingt  ist,  da  die  Beteiligung  des  Pharmakons  an  einem  bestimmten  \'organge,  die 
Reaktion  desselben  mit  einem  bestimmten  Reaktionsobjekt  in  der  Zelle  oder  auch  ein 
Reaktionsprodukt  dieses  Vorganges  im  allgemeinen  noch  nicht  exakt  festgestellt  wer- 
den  kann. 

Es  soil  in  dieser  Mitteilung  nicht  zu  den  sich  hier  ergebenden  Problemen  allgemein 
Stellung  genommen  werden,  sondern  nur  eine  Frage  aus  diesem  Zusammenhang  be- 
handelt  werden.  Ein  besonders  wichtiges,  vielleicht  das  wesentlichste  Problem  der 
Literatur  S.  154I155. 


VOL.  4  (1950)  SPEZIFITAT   PHARMAKOLOGISCHER   WIRKUNGEN  I45 

Pharmakologie  ist  die  Erforschung  der  Ursache  der  Spezifitat  pharmakologischer  Wir- 
kungen,  da  bestimmte  differenzierte  Wirkungen  eben  nur  dadurch  moglich  werden,  dass 
eine  Substanz  mit  einer  wesentlich  niedrigeren  Konzentration  an  einem  bevorzugten 
Reaktionsort  zu  wirken  vermag.  Die  zu  behandelnde  Frage  ware  so  zu  umreissen :  Lassen 
sich  quantitative  Beziehungen  des  Reaktionsverhaltens  eines  biologischen  Objektes  auf- 
finden,  welche  mit  der  Spezifitat  der  Wirkung  in  einem  direkten  Zusammenhang  stehen, 
und  welche  Befunde  lassen  Schlussfolgerungen  aaf  die  Art  der  Reaktion  des  spezifischen 
Reaktionssystemes  des  biologischen  Objektes  zu? 

Im  allgemeinen  liegen  nicht  geniigend  Untersuchungen  vor,  welche  das  Reaktions- 
verhalten  von  Substanzen  mit  hoher  Spezifitat  und  anderen  Angehorigen  der  gleichen 
Gruppe  mit  wesentlich  geringerer  Spezifitat  unter  gleichen  Bedingungen  feststellen. 
Ferner  werden  haufig  Befunde  an  verschiedenen  Objekten  untereinander  verglichen. 
Dies  liegt  zum  Teil  in  der  Natur  der  Objekte,  weil  nur  in  Ausnahmefallen  Reaktionen 
verschiedenen  pharmakologischen  Charakters  in  gleicher  Weise  am  gleichen  Objekt 
untersucht  werden  konnen.  Alle  diese  Momente  bieten  Unsicherheiten  fiir  die  Beur- 
teilung.  In  den  letzten  Jahren  wurde  in  unseren  Laboratorien  eine  interessante  Gruppe 
pharmakologischer  Korper  bearbeitet,  welche  fiir  die  Untersuchung  der  genannten 
Fragen  gewisse  Vorteile  bietet,  die  Gruppe  der  aromatischen  Imidazolinderivate.  Diese 
chemische  Struktur  hat  die  besondere  Eigenschaft,  dass  durch  entsprechende  chemische 
Abwandlung  in  dieser  Gruppe  Stof^e  mit  verschiedenartigsten  Wirkungen  sehr  hoher 
Spezifitat  entwickelt  werden  konnen.  Es  finden  sich  in  ihr  neben  Sympathikomimetika 
Sympathikolytika,  neben  Antihistaminen  histaminergische  Stoffe,  ausserdem  Para- 
sympathikolytika  und  Parasympathikomimetika  und  andere  Stoffe  hoher  Spezifitat. 
Es  muss  somit  in  dieser  Struktur  eine  eigenartige  potentielle  MogHchkeit  zur  Reak- 
tion mit  den  verschiedenen  Wirkorten  des  biologischen  Substrates  enthalten  sein,  da 
eine  Gruppe  von  chemischen  Verbindungen  vorliegt,  die  bei  prinzipiell  gleichartiger 
Grundstruktur  sehr  viele  verschiedenartige  Wirkungsqualitaten  aufweist  (Meier,  1947). 
Es  konnen  somit  Dosiswirkungskurven  von  Stoffen  gleichartiger  chemischer  Struktur 
mit  verschiedenem  Spezifitatsgrad  und  verschiedenartigem  Wirkungscharakter  mit  ein- 
ander  verglichen  werden. 

Die  erste  zu  behandelnde  Frage  ist  die:  Bestehen  zwischen  der  Dosiswirkungs- 
beziehung  und  der  Spezifitat  der  Wirkung  Beziehungen  allgemeineren  Charakters?  In 
der  Literatur  werden  eine  Reihe  von  Angaben  liber  diese  MogHchkeit  gegeben  und  zum 
Teil  ziemlich  weitgehende  Aussagen  iiber  die  Bedeutung  bestimmter  Verlaufsformen 
der  Dosiswirkungskurve  fiir  bestimmte  Wirkstoffgruppen  in  Anspruch  genommen 
(Storm  van  Leeuwen,  1919,  Clark,  1937). 

Auf  Grund  unserer  Untersuchungen,  in  denen  verschiedenartigste  Stoffe  in  dieser 
Hinsicht  untersucht  wurden,  geht  nicht  hervor,  dass  sich  in  Veranderungen  der  Dosis- 
wirkungskurven Gesetzmassigkeiten  finden,  welche  direkt  mit  der  Spezifitat  der  Wir- 
kung in  Zusammenhang  stehen.  Man  erkennt  in  Abbildung  i  und  2,  dass  sich  an 
der  isolierten  Samenblase  des  Meerschweinchens  und  an  der  isoliert  durchstromten 
Hinterextremitat  des  Kaninchens  die  Dosiswirkungskurven  verschiedener  Wirkstoffe 
und  von  verschiedener  oder  gleicher  chemischer  Struktur  formal  weitgehend  ahnlich 
verhalten. 

Die  Dosiswirkungsbeziehungen  des  Otrivins  an  der  Meerschweinchen-Samenblase 
und  am  Meerschweinchen-Dunndarm  sind  formal  ebenfalls  praktisch  identisch,  trotz- 
dem  Otrivin  an  der  Samenblase  etwa  die  gleiche  oder  eher  ausgesprochenere  Wirkungs- 
Literatur  S.  154I155. 


146 


R.  MEIER,  H.  J.  BEIN 


VOL.  4  (1950) 


Abb.  I.  Isolierte  Meerschwein- 
chen-Samenblase.  Dosiswir- 
kungskurven  von  Adrenalin, 
Otrivin,  Acetjdcholin  und  Hist- 
amin.      Abszisse :      Konzentra- 

tionen  (logarithmisch). 
Ordinate:  Hubhohe  in  Prozent 
(numerisch)     (Wirsing,    1949). 

o  Adrenalin 


o  Otrivin 

•  Acetylcholin 

•  Histamin 


J  p. 

I  50 

60 
50 
40 
30 
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Abb.  3.  Isolierter  Meerschvveinchen- 
Diinndarm.  Dosiswirkungskurven  von 
Histamin,  Acetylcholin,  Otrivin  und 
Priscol.  Abszisse:  Konzentrationen  (lo- 
garithmisch). Ordinate:  ^Hubhohe  in 
Prozent  (numerisch). 

• Histamin 

• Acetylcholin 

o Otrivin 

o Priscol 


^100 
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1  SO 

60 
50 
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30 
20 
10 
0 


Abb.  2.  Gefassdnrchfluss  der 
isolierten  Kaninchenhinterex- 
tremitat.  Dosiswirkungskurven 
von  6  Imidazolinen  und  3  Phe- 
nylathylaminen.  Abszisse :  Kon- 
zentrationen (logarithmisch) . 
Ordinate :  Durchfluss  in  Prozent 
(numerisch)  (Meier  und 
Pellmont,  1947). 


10-5         10- <> 
Konzeniration 


, 

/ 

/7 

/ 1 

1^ 

/  / 

h 

]\ 

/  / 
/  / 

1 

/ 

/ 

ZJ^^ 

I 

/ 

/ 

w 

/ 

10-^       10-s 


10-7 


10-6        10-s 


IQ-^         10'^ 
Konzentrction 


Literatur  S.  154I155. 


VOL.  4  (1950)  SPEZIFITAT   PHARMAKOLOGISCHER   WIRKUNGEN  I47 

Starke  besitzt  als  Acetylcholin  oder  Histamin,  wahrend  es  am  Diinndarm  rund  500  mal 
schwacher  wirksam  ist  (Abb.  3). 

Naturgemass  bleibt  ein  wichtiges  Moment  bei  diesen  Untersuchungen  verborgen: 
Die  Konzentration  der  Wirkstoffe  an  den  Reaktionsorten.  Es  kann  wohl  ausgeschlossen 
werden,  dass  nicht  geniigend  starke  Konzentrationen  an  die  Reaktionsorte  der  Zelle 
gelangen.  Da  im  allgemeinen  diese  Stoffe  fiir  eine  andere  Reaktion  eine  hohe  Spezifitat 
besitzen  und  diese  ohne  weiteres  ausgelost  werden  kann,  ist  es  nicht  wahrscheinlich, 
dass  ein  wesentlich  kleinerer  Prozentsatz  des  zugesetzten  Stoffes  in  die  Zelle  hinein- 
gelangt.  Immerhin  ist  diese  Moglichkeit  nicht  vollstandig  auszuschliessen.  Vollstandig 
unbekannt  ist  aber,  in  welchem  Umfange  sich  der  Stoff  innerhalb  der  Zelle  zwischen 
spezifischen  und  unspezifischen  Reaktionsorten  verteilt.  Wenn  man  annimmt,  dass  in 
einem  physikalisch-chemischen  Ablauf  der  Reaktion  zwischen  den  spezifischen  und 
unspezifischen  Reaktionsorten  der  Zelle  kein  wesentlicher  Unterschied  besteht  und  die 
Spezifitat  der  Wirkung  ausschliesslich  auf  einer  Verteilung  zwischen  diesen  verschiede- 
nen  Reaktionsorten  beruht,  ist  es  durchaus  moglich,  dass  nur  diese  Unterschiede  der 
Verteilung  die  Ursache  des  Spezifitatsgrades  darstellen.  Es  muss  somit  auf  Grund  dieser 
Untersuchungen  geschlossen  werden,  dass  zwischen  der  Spezifitat  oder  dem  spezifischen 
Charakter  einer  Wirkung  und  der  Dosiswirkungsbeziehung  kein  direkter  Zusammenhang 
besteht.  Wir  glauben  deshalb,  in  der  Interpretation  solcher  Dosiswirkungsbeziehungen 
auf  Grund  unserer  heutigen  Kenntnisse  den  Erklarungsversuchen  von  Gaddum  (1926, 
1937)  folgen  zu  konnen,  der  mit  Shackell  (1925)  und  Fromherz  (1926)  annimmt,  dass 
Konzentrationswirkungskurven  lediglich  die  Wirkung  eines  Giftes  an  einer  Zellpopula- 
tion  zum  Ausdruck  bringen,  d.h.  die  durch  eine  bestimmte  Dosis  hervorgerufene  Wirkung 
ware  eine  Resultante  der  Wirkung  einzelner  aktiver  Elemente,  die  gegeniiber  einem 
einwirkenden  Agens  verschieden  empfindlich  sind,  wobei  unter  den  aktiven  Elementen 
ganze  Zellen  oder  nur  Telle  solcher,  wie  Rezeptoren,  angenommen  werden  konnten. 

Eine  besonders  viel  gebrauchte  Art  der  Charakterisierung  pharmakologischer  Reak- 
tionen  ist  in  den  letzten  Jahren  die  Untersuchung  von  antagonistischen  Wirkungen 
geworden.  "Antagonisten"  besitzen  im  allgemeinen  keine  Eigenwirkung  auf  das  Sub- 
strat,  vermogen  aber  die  durch  einen  bestimmten  Agonisten  hervorgerufene  Reaktion 
eines  Substrates  in  spezifischer  Weise  zu  verhindern.  Es  besteht  somit  die  Moglichkeit, 
dass  bei  diesen  Stoff  en  eine  besonders  giinstige  Situation  gegeben  ist,  um  das  quantita- 
tive Reaktionsverhalten  von  pharmakologischen  Mechanismen  zu  untersuchen.  Es 
wurden  im  wesentlichen  die  gleichen  Untersuchungen  wie  fiir  die  eingangs  besprochenen 
Agonisten  ausgefiihrt.  Es  soil  verzichtet  werden,  auf  die  Befunde  der  Literatur  im 
einzelnen  einzugehen.  Fiir  diese  Gruppe  sind  die  Imidazolinderivate  bezonders  geeignet, 
weil  sich  —  wie  eingangs  erwahnt  —  ausser  primar  wirkenden  Stoffen  wie  Sympathiko- 
mimetika,  histaminergische  Stoffe,  auch  antagonistische  Stoffe  hoher  Spezifitat  in  dieser 
chemischen  Gruppe  finden.  Es  ergibt  sich,  dass  antagonistisch  wirkende  Stoffe,  welche 
einer  im  wesentlichen  gleichen  Grundstruktui  der  aromatischen  Imidazoline  zugehoren, 
aber  von  sehr  verschieden  hohem  Spezifitatsgrad  sind,  im  wesentlichen  einen  gleich- 
artigen  Verlauf  der  Dosiswirkungskurve  zeigen.  Weiterhin  ist  festzustellen,  dass  dieses 
nicht  nur  der  Fall  ist  bei  einem  spezifischen  Vorgang,  wie  z.B.  dem  Antagonismus  der 
Sympathikolytika  gegeniiber  den  Sympathikomimetika,  sondern  dass  auch  bei  den 
iibrigen  Reaktionen  hoher  Spezifitat  wie  dem  Antagonismus  gegen  Histamin  oder  dem 
Antagonismus  gegen  Acetylcholin  ein  weitgehend  uniformes  Verhalten  der  Dosis- 
wirkungsbeziehung antagonistisch  wirkender  Stoffe  vorliegt.  Eine  Sonderstellung  scheint 
Literatur  S.  154 1 155. 


148  R.    MEIER,    H.    J.   BEIN  VOL.  4  (1950) 

unter  den  bisher  untersuchten  antagonistischen  Reaktionen  am  Meerschweinchen- 
Diinndarm  lediglich  dem  Antagonistenpaar  Acetylcholin-Adrenalin  zuzukommen 
(Bein,  1947),  wobei  die  Frage  nach  der  Ursache  dieser  Verschiedenheit  heute  noch  off  en 
gelassen  werden  muss.  Moglicherweise  konnte  dieses  unterschiedliche  Verhalten  dadurch 
bedingt  sein,  dass  es  sich  bei  dieser  Stoffkombination  um  einen  "funktionellen  Antago- 
nismus"  handeln  wiirde. 

Da  nicht  nur  eine  Spezifitatshohe  der  Wirkung  im  Vergleich  verschiedener  chemi- 
scher  Stoffe,  sondern  auch  eine  verschieden  hohe  Spezifitat  der  Wirkung  gegeniiber  einer 
gegebenen  Skala  von  verschiedenen  Reaktionsobjekten  besteht,  sind  auch  die  Dosis- 
wirkungsbeziehungen  an  verschiedenen  Objekten  zu  untersuchen.  Es  sind,  wie  bereits 
erwahnt,  nun  nicht  sehr  viele  Objekte  vorhanden,  an  denen  derartige  Untersuchungen 
fiir  alle  mogHchen  Falle  durchgefiihrt  werden  konnen.  Immerhin  haben  wir  eine  Reihe 
von  Beispielen  aus  diesen  bereits  besprochenen  Stoffgruppen  in  der  Weise  untersucht, 
dass  sowohl  die  Dosiswirkungskurve  von  agonistischen  und  antagonistischen  Wirkungen 
sowohl  an  der  Samenblase  (Abb.  4)  wie  dem  isoherten  Diinndarm  des  Meerschweinchens 
(Abb.  5  und  6)  und  zum  Teil  auch  am  Froschherzen  und  einzelnen  anderen  Objekten 
aufgestellt  wurden.  Das  Untersuchungsmaterial,  welches  in  dieser  Hinsicht  vorhegt,  ist 
nicht  so  vollstandig  wie  es  wiinschenswert  ware.  Es  ergibt  sich,  dass  sowohl  bei  der  Ver- 
wendung  von  Agonisten  verschiedener  chemischer  Struktur  als  auch  verschiedener 
Wirkungsrichtung  Dosiswirkungskurven  erhalten  werden  konnen,  die  fiir  das  eine 
Objekt  einen  etwas  anderen  Wirkungstypus  besitzen  wie  fiir  ein  anderes.  Im  allgemeinen 
sind  die  Dosiswirkungskurven  nicht  bedingt  durch  den  verschiedenen  Spezifitatsgrad 
der  Wirkung  an  diesen  verschiedenen  Objekten,  sondern  die  Dosiswirkungskurven  an 
einem  Objekt  pflegen  im  allgemeinen  einem  bestimmten  Typus  zu  folgen,  wahrend  sie 
an  einem  anderen  Objekt  einen  anderen  Typus  besitzen.  Aus  Abb.  i  geht  hervor,  dass 
beim  Meerschweinchen  die  isolierte  Samenblase  —  ahnlich  dem  isolierten  Uterus 
(Fromherz,  1926)  und  im  Gegensatz  zum  isolierten  Diinndarm  (Abb.  3)  —  die  Tendenz 
zeigt,  bei  verschiedenen  Konzentrationen  von  unterschiedlich  wirksamen  Stoffen  bald 
ein  Maximum  der  Ant  wort  zu  erreichen,  wenn  auch  an  der  Samenblase  wahrscheinlich 
besser  als  beim  Uterus  in  dieser  Hinsicht  noch  eine  gewisse  Differenzierung  zwischen  ein- 
zelnen Pharmaka  durchgefiihrt  werden  kann.  Auch  die  absolute  Wirkungsstarke  von 
antagonistisch  wirkenden  Stoffen  kann  an  verschiedenen  Objekten  stark  wechsehi,  so 
braucht  es  z.B.  am  isolierten  Meerschweinchen-Diinndarm  etwa  fiinfmal  mehr  Antistin, 
um  eine  gegebene  HistamJnkontraktur  zu  unterdriicken,  wahrend  an  der  isolierten 
Meerschweinchen-Samenblase  dieses  Verhaltnis  gerade  umgekehrt  ist.  Es  scheint  somit, 
dass  nicht  die  Spezifitat  der  Wirkung,  sondern  eine  Eigentiimlichkeit  des  Substrates  im 
Verhaltnis  zur  untersuchten  Stoffgruppe  eine  unterschiedliche  Dosiswirkungsbeziehung 
bedingt.  Ganz  ahnlich,  jedenfalls  durchaus  nicht  grundsatzlich  anders  liegen  die  Verhalt- 
nisse  fiir  die  antagonistischen  Reaktionen. 

Die  Feststellung  der  Dosiswirkungsbeziehung  antagonistischer  Reaktionen  bietet 
noch  eine  bcsondere  Moglichkeit  fiir  die  quantitative  Feststellung  des  Reaktionsverhal- 
tens,  die  bisher  von  verschiedenen  Seiten  benutzt  wurde.  Es  kann  festgestellt  werden, 
ob  bei  Steigerung  der  Konzentiation  des  Agonisten  auch  eine  relativ  gleichstarke  Steige- 
rung  des  Antagonisten  zu  erfolgen  hat,  woraus  geschlossen  werden  konnte,  dass  zwischen 
der  Afhnitat  des  Agonisten  imd  des  Antagonisten  unabhangig  von  der  Konzentration  des 
Agonisten  die  gleiche  Reaktionsbereitschaft  besteht.  Auch  ein  derartiges  Verhalten 
konnte  naturgemass  Anhaltspunkte  fiir  die  Spezifitat  einer  Reaktion  geben. 

Liter atur  S.  154 1 155. 


VOL.  4  (1950) 


SPEZIFITAT    PHARMAKOLOGISCHER   WIRKUNGEN 


149 


Abb.  4.  Isolierte  Meerschweinchen- 
Samenblase.  Dosiswirkungskurven  ver- 
schiedener  Stoffkombinationen.  Abs- 
zisse :  Konzentrationen  cler  Antagonis- 
ten  (logarithmisch).  Ordinate:  Hub- 
hohe  in  Prozent  (numerisch)  (Wirsing, 
1949)- 
Spezifische  Aniagonisten: 

Atropin/Acetylcholin 

Antistin/Histamin 

7337n/Adrenalin 

Unspezifische  A  niagonismen: 

Priscol /Adrenalin 

Atropin/.\drenalin 

_. Antistin/Adrenalin 

—  — . .  —  Atropin/Histamin 
7337  n/Histamin 

—  .  ._.  . ._  Antistin/Acetylcholin 


10-^       10-^ 
Konzenfration 


Abb.  5.  Isolierter  Meerschweinchen- 
Diinndarm.  Dosiswirkungskurven  ver- 
schiedener  Histamin-Antagonisten  bei 
einer  gegebenen  Histaminkonzentra- 
tion  von  10-'. 

Abszisse:    Konzentrationen  der  Anta- 
gonisten    (logarithmisch) 
Ordinate:  Hubhohe  in  Prozent  (nume- 
risch) (Meier,  1947) 

• Atropin 

7337; 

_  _  —  Antistin 

Adrenalin 

202o/n 


>100 

'■  90 

\  80 
1 

•  70 
60 
50 
'',0 
30 
20 
10 


1 

~^ 

\ 

A-.     -^ 

\\ 

VWi 

\ 

\  \\\ 

\ 

\  \i 

\ 

\ 

■■■  \ 

y 

\ 

\ 

/O" 


w 


10- 


10- 


10-''         10-^ 
Konzentrotion 


^100 

s  90 
«. 

I  60 

■Q 

60 
50 
40 

30 
20 

to 

0 


\  k 

N.      '•.. 

\ 

\V\ 

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1        '' 

\ 

\ 

\  = 

lA_^ 

\ 

\ 
\ 

i 

\ 

\   I 

\ 

i 

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1 

1        I 

10- 


10- 


10'''         10-^ 
Komentraiion 


Abb.  6.  Isolierter  Meerschweinchen-Diinn- 
darm.  Dosiswirkungskurven  verschiedener 
Acetylcholin-Antagonisten  bei  einer  gege- 
benen Acetjdcholinkonzentration  von  IQ-''. 
Abszisse:  Konzentrationen  der  Antago- 
nisten  (logarithmisch).  Ordinate:  Hubhohe 
in   Prozent    (numerisch)    (Meier,     1947). 


Atropin 
.•\ntiHtin 
Adrenalin 
7337 

2020/n. 


Literatur  S.  154I153. 


150  R.  MEIER,  H.   J.  BEIN  VOL.  4  (1950) 

Bereits  friiheren  Untersuchern  ist  es  aufgefallen,  dass  besonders  mit  relativ  geringen 
Dosen  eines  Agonisten  oder  eines  Antagonisten  eine  solche  Gesetzmassigkeit  dieser  Rela- 
tion nicht  beobachtet  werden  kann.  So  muss,  um  nur  ein  Beispiel  zu  erwahnen,  am  iso- 
lierten  Kaninchendarm  bei  einer  Erhohung  der  Pilocarpinkonzentration  die  fiir  einen 
gleichen  Effekt  notwendige  Atropindosis  um  nur  wenig  mehr  erhoht  werden,  (Magnus, 
1908),  wahrend  umgekehrt  am  isolierten,  elektrisch  gereizten  Ventrikelstreifen  des 
Frosches  in  einem  niedrigen  Dosenbereich  verhaltnismassig  mehr  Atropin  als  Acetyl- 
cholin  fiir  einen  konstanten  Effekt  gegeben  werden  muss  (Clark,  1926).  In  eigenen  Ver- 
suchen,  in  welchen  wir  am  isolierten  Meerschweinchen-Diinndarm  sowohl  die  Konzen- 
tration  von  Agonisten,  Histamin  und  Acetylcholin,  als  auch  diejenige  von  Antagonisten, 
Pyribenzamin,  Neo-Antergan  und  Antistin,  resp.  Atropin  und  Trasentin  steigerten,  ergab 
sich  ebenfalls  ein  inkonstantes  Verhaltnis.  Merkwiirdigerweise  scheint  hier  unter  den  ge- 
wahlten  Versuchsbedingungen  (Einwirkungsdauer  der  Antagonisten  jeweils  2  Minuten) 
bei  hochwirksamen  Antagonisten  (Pyribenzamin,  Neo-Antergan,  Atropin)  eine  relativ 
kleinere  Dosissteigerung  notwendig  zu  sein  als  bei  etwas  weniger  wirksamen  (Antistin, 
Trasentin).  Auf  der  anderen  Seite  muss  bei  den  unspezifischen  antagonistischen  Reak- 
tionen  Neo-Antergan-Acetylcholin  und  Pyribenzamin-Acetylcholin  bei  lo-facher  Stei- 
gerung  der  Acetylcholinkonzentration  die  Konzentration  der  Antagonisten  fiir  einen 
gleichen  Effekt  ebenfalls  nur  um  wenig  mehr  erhoht  werden. 

Wenn  auch  bei  gleichzeitiger  Steigerung  sowohl  einer  Agonisten-  wie  auch  einer 
Antagonistenkonzentration  das  gegenseitige  Mengenverhaltnis,  das  auch  beim  gleichen 
Antagonistenpaar  fiir  verschiedene  Objekte  variiert,  durch  eine  mathematische  Bezie- 
hung  ausgedriickt  werden  kann  (Clark,  1926,  1937;  Gaddum,  1937),  so  bleibt  doch  die 
Schwierigkeit  der  gedanklichen  Vorstellung.  Guzman  Barron  und  Mitarbeiter  (1948) 
haben  kiirzlich  gezeigt,  dass  in  einer  Zelle  zwei  verschiedenartige  Sulfhydrilgruppen 
angenommen  werden  konnen,  die  mit  SH-Gruppen  blockierenden  Giften  je  nach  deren 
Konzentration  reagieren.  Entsprechend  dieser  Vorstellung  konnten  zwei  oder  mehrere 
Rezeptorengruppen  angenommen  werden,  die  sich  gegeniiber  einem  Agonisten  wie  auch 
gegeniiber  einer  antagonistisch  wirkenden  Substanz  verschieden  empfindlich  verhalten. 
Das  gegenseitige  Mengenverhaltnis  Agonist /Antagonist  bei  jeweils  steigenden  Konzen- 
trationen  wiirde  dann  aus  einer  Resultante  der  Wirkung  an  den  verschiedenen  Rezep- 
torengruppen stammen. 

Es  scheint  somit,  so  interessant  diese  Untersuchungen  sind,  und  so  interessant  sie 
fiir  die  Feststellung  der  relativen  Afhnitat  zu  gewissen  Reaktionsorten  der  Zelle  sind, 
dass  sie  offenbar  die  Spezifitatshohe  der  pharmakologischen  Wirkung  nicht  direkt  be- 
griinden  konnen,  wobei  naturgemass  wieder  als  eine  Vermutung  nahegelegt  wird,  dass 
tatsachlich  die  Dosiswirkungskurve  nicht  nur  ein  Ausdruck  der  spezifischen,  sondern 
auch  unspezifischer  Reaktionsorte  der  Zelle  sein  mag. 

Wenn  auch  mit  der  Feststellung  der  spezifischen  Hemmbarkeit  eines  oder  verschie- 
dener  Agonisten  durch  einen  Antagonisten  ein  gemeinsamer  Angriffspunkt  postuliert 
werden  kann,  so  braucht  nun  der  Wirkungsablauf  der  verschiedenen  Agonisten  noch 
nicht  gleich  zu  sein,  da  einerseits  eine  antagonistisch  wirkende  Substanz  eine  Reaktions- 
kette,  von  welcher  wir  meist  nur  die  Endreaktion  beobachten,  an  jeweils  verschiedenen 
Stellen  unterbrechen  kann,  oder  weil  anderseits  die  Reaktionskette  von  einem  primaren 
Ausgangspunkt  an  verschieden  verlauft. 

Ein  weiteres  Vorgehen  besteht  in  der  Feststellung  der  Zeitwirkungskurve,  die  im 
Prinzip  wohl  der  Erreichung  der  Einstellung  eines  Reaktionsgleichgewichtes  zwischen 
Liieratur  S.  154I155. 


VOL.  4  (1950) 


SPEZIFITAT   PHARMAKOLOGISCHER   WIRKUNGEN 


151 


dem  der  Losung  zugesetzten  Wirkstoff  und  den  Reaktionsorten  der  Zelle  angesehen 
werden  kann.  Dass  bei  dieser  Untersuchung  die  Permeabilitatsfrage  eine  wesentliche 
Bedeutung  besitzt,  ist  ohne  weiteres  auf  der  Hand  liegend,  und  es  muss  jedenfalls  diese 
Moglichkeit  bevor  ein  Urteil  iiber  die  Reaktion  mit  spezifischem  Ort  in  der  Zelle  hier 
in  Anspruch  genommen  wird,  im  Auge  behalten  werden.  Immerhin  kann  aber  auch  eine 
solch  "unspezifische"  Reaktion  wie  die  Veranderang  der  Permeabilitat  ebenfalls  ein  fiir 
einen  Wirkstoff  bis  zu  einem  gewissen  Grade  charakteristisches  Verhalten  darstellen. 
Als  besonders  entscheidend  miissen  wieder  diejenigen  Untersuchungen  angesehen  wer- 
den, bei  denen  die  Zeitwirkungskurve  von  Imidazolinvertretern  gleicher  chemischer 
Grundstruktur  aber  verschiedener  Wirkungsspezifitat  angesehen  werden.  Es  ware  an 
und  fiir  sich  moglich,  dass  bei  diesen  die  Eintrittszeiten  verschieden  sind,  weil  natur- 
gemass  bei  Stoffen  verschieden  hoher  Spezifitat  die  Aussenkonzentrationen  verschieden- 
artig  sind,  je  nach  der  Wirkungshohe  des  untersuchten  Stoffes.  Wenn  auch  gewisse 
Unterschiede  bei  Stoffen  verschiedener  chemischer  Struktur  hinsichthch  des  Eintrittes 

Antisiin  3.2-10'^ 
Atropin  7.5-10''' 

if 


Antistin 

Atropin 

32-10'^ 

7.5-10-'' 

-i 

\ 

"i 

r- 

-\ 

64.2% 


-25.5% 


Histamin 


I 


Histamin 
5-10'^ 


-91.5% 


Histamin 
5-10'^ 


Abb. 


Isolierter   Meerschweinchen-Diinndarm.    Einfache  Addition  der  Wirkung  von   zwei  ver- 
schiedenen  antagonistisch  wirkenden  Stoffen  (Mittelwerte  aus  5  Versuchen). 


des  Reaktionsgleichgewichtes  vorhanden  sind,  muss  man  doch  sagen,  dass  bei  den 
Imidazolinderivaten  mit  sehr  unterschiedlicher  Spezifitat  der  Wirkung  keine  typischen 
Unterschiede  zu  beobachten  sind,  die  dafiir  sprechen,  dass  die  Geschwindigkeit  der 
Reaktion  mit  den  fiir  die  Wirkung  verantwortlichen  Reaktionsorten  der  Zelle  in  einem 
direkten  Zusammenhang  mit  der  Spezifitatshohe  der  Wirkung  steht. 

Es  bleiben,  darauf  muss  hingewiesen  werden,  gewisse  Unterschiede  sowohl  der 
Dosiswirkungskurven  wie  der  Zeitwirkungskurven  bestehen.  Die  Abweichungen  dieser 
Befunde  liegen  aber  relativ  so  nahe  in  der  Fehlerbreite  der  Untersuchungsmethoden, 
dass  es  verfriiht  erscheint,  diese  Abweichungen  zum  Gegenstand  allgemeiner  Schluss- 
folgerungen  zu  machen.  Sie  bediirfen  sicher  weiterer  Aufmerksamkeit  und  es  scheint 
moglich,  dass  ihnen  fiir  die  Beurteilung  des  spezifischen  Reaktionsverhaltens  noch  eine 
grossere  Bedeutung  zukommen  wird. 

Da  am  isolierten  Kaninchenuterus  die  voile  Hemmwirkung  z.B.  des  Ergotamins 
gegeniiber  Adrenalin  erst  nach  Stunden  eintritt  (Gaddum,  1926),  am  Kaninchendarm 
jedoch  schon  nach  Minuten  (Rothlin,  1929),  so  konnte  es  auch  sein,  dass  eine  solche 
Zeitmessung  nicht  einen  Vorgang  erfasst,  der  sich  an  den  Rezeptoren  selbst  abspielt, 
sondern  nur  ein  mehr  oder  weniger  rasches  Durchwandern  durch  das  Gewebe  zu  den 
Literatur  S.  154I155. 


152  R.  MEIER,  H.  j.  BEIN  VOL.  4  (1950) 

aktiven  Gruppen  (Gaddum,  1937).  Es  ist  jedoch  mit  dieser  Annahme  schwer  zu  verein- 
baren,  warum  das  Adrenalin,  dessen  Wirkungseintritt,  d.h.  dessen  Verbindung  mit 
seinen  aktiven  Rezeptoren,  innerhalb  von  wenigen  Sekunden  erfolgt,  am  isolierten 
Meerschweinchen-Diinndarm  seine  maximale  antagonistische  Wirkung  gegenuber  Ace- 
tylcholin  auch  nach  10  Minuten  noch  nicht  erreicht  hat  (Vest),  wenn  nicht  angenommen 
wird,  dass  durch  eine  antagonistisch  wirkende  Substanz  nicht  nur  "Rezeptoren 
blockiert",  sondern  moghcherweise  auch  gleichzeitig  andere  Prozesse  wie  z.B.  die  Per- 
meabihtat  oder  Stoffwechselvorgange  und  andeies  mehr,  verandert  werden  miissten. 
Dass  in  diesem  Zusammenhang  auch  der  Frage  der  Haftfestigkeit  eine  Bedeutung  zu- 
kommt,  braucht  wohl  nicht  nahei  ausgefiihrt  zu  werden. 

Hinsichthch  der  eingangs  gestellten  Frage  des  Zusammenhanges  quantitativer 
Reaktionsverhaltnisse  mit  der  Spezifitatshohe  pharmakologischer  Reaktionen  muss 
somit  gesagt  werden,  dass  die  bisher  von  uns  durchgefiihrten  Untersuchungen  keinen 
Anhaltspunkt  dafur  geben,  dass  dieser  quantitative  Reaktionsverlauf  in  irgendeiner 
Weise  zur  Erklarung  der  Spezifitatshohe  herangezogen  werden  kann.  x^n  dem  Substrat, 
an  dem  sich  die  spezifischen  Reaktionen  abspielen,  konnen  sich  die  spezifischen  und 
unspezifischen  Reaktionen  an  sich  nur  dadurch  unterscheiden,  dass  in  der  Verteilung 
innerhalb  verschiedenartiger  Reaktionsorte  in  der  Zelle  die  spezifischen  Wirkstoffe  be- 
vorzugt  die  spezifischen  Reaktionsorte  erreichen,  auch  wenn  in  der  Aussenfliissigkeit 
und  vielleicht  auch  in  der  Zelle  und  an  unspezifischen  Reaktionsorten  eine  hohere  Kon- 
zentration  der  letzteren  vorhanden  ist.  Es  wird  bei  dieser  Sachlage  naturgemass  schwie- 
rig,  hinsichthch  der  spezifischen  Reaktion  allgemein  verbindliche  Schlussfolgerungen  zu 
Ziehen,  da  auch  angenommen  werden  kann,  dass  bei  einem  wesentlichen  Tail  der  Reak- 
tion mit  unspezifischen  Reaktionsorten  der  Zelle  das  gesamte  Verhalten  der  Dosis- 
wirkungsbeziehung  durch  die  unspezifische  Reaktion  mitbedingt  sein  kann.  Es  ist  be- 
sonders  wichtig  zu  entscheiden,  ob  tatsachlich  alle  die  ausgelosten  Reaktionen  eine 
Wirkung  am  gleichen  Reaktionsort  hervorrufen,  oder  ob  nicht  noch  andere  indirekte 
Wirkungsmoglichkeiten  vorhanden  sein  konnen,  welche  den  Eindruck  einer  Wirkung 
am  gleichen  Reaktionsort  besitzen,  trotzdem  sie  eigentlich  nicht  als  "spezifischer" 
Antagonismus  im  eigentlichen  Sinne  aufzufassen  sind.  Fiir  die  Entscheidung  dieser 
Frage  ist  es  von  ausschlaggebender  Bedeutung,  die  Separation  der  spezifischen  Reak- 
tionen nachzuweisen.  Das  hier  meistens  angewandte  Verfahren,  welches  in  einfacher 
Weise  einen  solchen  "Spezifitatsgrad"  der  Wirkung  beweist,  ist  dasjenige,  dass  die  Wir- 
kung eines  Stoffes  an  einem  bestimmten  Reaktionssystem  z.B.  am  histaminergischen 
System,  untersucht  wird,  wahrend  die  Reaktion  des  parasympathischen  Systems  durch 
Atropin  ausgeschaltet  wird.  Auch  dann,  wenn  an  diesem  System  keinerlei  Wirkung 
durch  eine  gegebene  Dosis  Acetylcholin  mehr  ausgelost  wird,  kann  mit  einem  anderen 
Stimulans  z.B.  Histamin,  die  entsprechende  Reaktion  in  gleicher  Weise  ausgelost 
werden.  Dieses  spricht  selbstverstandlich  ohne  weiteres  dafiir,  dass  eine  Differenziertheit 
der  Substrate  vorhanden  ist.  Unterlagen  hinsichthch  der  Dosiswirkungsbeziehung  ver- 
schiedenartiger Stoffe  mit  verschiedenartiger  Spezifitat  unter  derartigen  Bedingungen 
sind  allerdings  nicht  vorhanden.  Eine  weitere  Moghchkeit  besteht  in  der  Verwendung 
der  Addition  verschiedenartiger  spezifischer  Effekte.  Ein  hierfiir  zweckmassiges  Ver- 
fahren ist  die  Auslosung  von  je  50%  des  Maximaleffektes  durch  je  einen  Agonisten,  z.B. 
Histamin  oder  Acetylcholin  am  Meerschweinchendarm.  Bereits  die  additive  Wirkung 
von  derartigen  Dosen  zeigt,  dass  eine  besondere  Differenzierung  zwischen  dem  Reak- 
tionsort und  dem  Kontraktionssubstrat  vorhanden  sein  muss,  der  bewirkt,  dass  der 
Liter atur  S.  154 1 135. 


VOL.  4  (1950)  SPEZIFITAT   PHARMAKOLOGISCHER   WIRKUNGEN  I53 

Effekt  verschiedenartiger  Stimulantien  eine  einfache  Summation  des  Einzeleffektes 
am  Erfolgsorgan  ergibt.  Diese  Feststellung  bietet  gewisse  Schwierigkeiten  fiir  die  Er- 
klarung  mancher  antagonistischer  Wirkungen,  bietet  aber  auch  die  Moglichkeit,  den 
liohen  Spezifitatsgrad  antagonistischer  Wirkungen  nachzuweisen.  Bringt  man  z.B.  einen 
isolierten  Darm  mit  Dosen,  welche  jeweils  eine  50%ige  Kontraktion  der  maximalen 
Histamin-  und  Acetylcholin-Kontraktur  bewirken,  zur  Kontraktion,  so  tritt  eine 
ioo%ige  Kontraktur,  wie  bereits  eben  besprochen  wurde,  ein. Wendet  man  nun  diejenigen 
Konzentrationen  der  Antagonisten,  z.B.  Atropin  und  Antistin,  an,  welche  gerade  50% 
der  Wirkung  zum  Verschwinden  bringen,  so  tritt  auch  in  diesem  Falle  nur  eine  Auf- 
hebung  des  durch  den  entsprechenden  Agonisten  hervorgerufenen  Effektes  auf,  was 
wiederum  beweist,  dass  eine  Separation  der  Angriffspunkte  sowohl  der  agonistischen 
als  auch  der  antagonistischen  Wirkung  vorhanden  ist.  Diese  Befunde  sprechen  wohl 
dafiir,  dass  ein  Verdrangungsvorgang  fiir  die  antagonistische  Reaktion  von  Bedeutung 
ist.  Wenn  nun  eine  Separation  der  Angriffspunkte  der  spezifischen  Agonisten  vorhanden 
ist,  sowohl  untereinander  als  auch  hinsichtlich  des  von  ihnen  bewirkten  Substrates,  so 
lassen  sich  doch  aus  diesen  Befunden  keine  weiteren  Argumente  fiir  die  Struktur  des 
spezifischen  Substrates  erhalten. 

Es  gibt  aber  noch  eine  Moglichkeit,  welche  vielleicht  etwas  weiteren  Aufschluss 
iiber  die  Separation  der  Wirkorte  ergeben  kann.  Es  sind  Stoffe  bekannt  geworden,  welche 
im  gleichen  Molekiil  zwei  spezifische  Wirkungen  besitzen,  z.B.  sympathikolytische  und 
histaminolytische  Wirksamkeit,  atropinartige  und  histaminolytische  und  so  fort.  Nur 
ausnahmsweise  gelingt  es,  Stoffe  zu  erhalten,  bei  denen  die  Wirkungshohe  dieser  beiden 
Wirkungsqualitaten  von  absolut  gleicher  Starke  vorhanden  ist.  Es  lasst  sich  nun  mit 
Hilfe  dieser  Stoffe  folgende  Frage  beantworten.  Bewirkt  ein  derartiger  Stoff  wie  z.B. 
Vertreter  der  Tetrahydrofiuoranthene  eine  antagonistische  Reaktion  z.B.  gegeniiber 
Histamin  und  Acetylcholin,  so  fragt  es  sich,  ob  bei  jeweils  50%iger  Kontraktion  durch 
Histamin  und  50%iger  Kontraktion  durch  Acetylchohn  eine  Konzentration  des  Stoffes 
gebraucht  wird,  welche  die  ioo%ige  Lyse  der  Acetylcholin-  oder  der  Histaminkontrak- 
tur  hervorruft,  oder  ob  fiir  die  Aufhebung  dieses  Effektes  eine  Konzentration  geniigt, 
welche  50%  antagonistisch  beeinfiusst.  Es  stellt  sich  bei  der  Untersuchung  dieser  Frage 
heraus,  dass  in  der  Tat  fiir  die  Aufhebung  einer  summierten  Kontraktion  aus  50% 
Histamin-  und  50%  Acetylcholinkontraktur  nicht  diejenige  Konzentration  gebraucht 
wird,  welche  die  Maximalkontraktion  mit  Histamin,  bzw.  Acetylcholin  lost,  sondern  dass 
nur  diejenige  Konzentration  des  Stoffes  notig  ist,  welche  eine  jeweils  50%ige  Wirkung 
aufzuheben  imstande  ist.  Fiir  dieses  eigenartige  Verhalten  konnten  vor  allem  zwei  ver- 
schiedene  MogHchkeiten  in  Anspruch  genommen  werden,  namlich  dass  die  vorhandene 
Menge  des  antagonistisch  wirkenden  Stoffes,  trotzdem  er  nur  mit  50%  des  Histamin- 
reaktionssubstrates  antagonistisch  reagiert,  auch  gleichzeitig  mit  50%  des  Acetyl- 
cholinsubstrates  reagiert,  wobei  diese  beiden  Substrate  als  separiert  von  gleicher  Em- 
pfindlichkeit  gedacht  sind.  Die  zweite  Moglichkeit  ware  diejenige,  dass  das  gleiche 
Molekiil  des  antagonistisch  wirkenden  Stoffes  gleichzeitig  mit  dem  Acetylcholin-  als 
auch  mit  dem  Histaminrezeptor  reagiert.  Ware  dies  der  Fall,  so  wiirde  daraus  zu  schlies- 
sen  sein,  dass  strukturchemisch  die  Angriffsorte  des  Histamins  und  Acetylcholins  raum- 
lich  so  nahe  beieinander  gelagert  sind,  dass  ein  Molekiil  des  Antagonisten  beide  gleich- 
zeitig beeinflussen  kann.  Es  lasst  sich  zwischen  diesen  beiden  Moglichkeiten  vorlaufig 
nicht  entscheiden ;  es  sind  weitere  Untersuchungen  in  dieser  Richtung  im  Gauge  und  es 
ist  nicht  vollstandig  ausgeschlossen  dass  sich  Argumente  fiir  die  letztere  Moglichkeit 
Liter atur  S.  154I155. 


154  K-  MEIER,  H.  J.  BEIN  VOL.  4  (1950) 

werden  beibringen  lassen.  Der  Nachweis  der  funktionellen  Separation  der  Rezeptions- 
orte  der  Zelle  fiir  die  spezifische  Reaktion  gibt  die  Moglichkeit,  eine  Reihe  von  Eigen- 
schaften  dieses  Reaktionssubstrates  auf  Grand  der  eingangs  besprochenen  Unter- 
suchungen  auf zustellen :  Das  Reaktionssubstrat  muss  in  der  Lage  sein,  mit  hoher  Spezi- 
fitat  mit  Stoffen  verschiedenartiger  chemischer  Grundstruktur  so  zu  reagieren,  dass 
ihnen  der  gleiche  Wirkungscharakter  zukommt.  Das  Substrat  muss  mit  Stoffen  grund- 
satzlich  gleichartiger  chemischer  Struktur  so  reagieren  konnen,  dass  nur  einzelne,  die 
in  bestimmter  Weise  substituiert  sind,  die  hochste  Spezifitat  besitzen,  und  die  Reak- 
tionsorte  verschiedenartigen  Wirkungscharakters  sind  imstande,  Stoffen  gleichartiger 
chemischer  Grundstruktur,  die  sich  nur  durch  bestimmte  Substituenten  voneinander 
unterscheiden,  die  spezifische  Reaktion  zu  erlauben.  Zum  Teil  lassen  sich  diese  Eigen- 
tiimlichkeiten  des  Reaktionssubstrates  durch  die  Wirkung  der  Agonisten  finden,  zum 
Teil  haben  sie  nur  fiir  die  Wirkung  von  Antagonisten  Geltung,  well  nur  mit  Hilfe  dieser 
das  entsprechende  Verhalten  bisher  nachgewiesen  werden  konnte.  Die  Organisation  des 
empfindlichen  Substrates  ist  nicht  dadurch  gekennzeichnet,  dass  quantitative  Einstel- 
lungen  des  Reaktionsgleichgewichtes  die  Ursache  der  unterschiedlichen  Spezifitat  der 
Wirkung  sind.  Ebenso  ist  fiir  die  Spezifitat  der  Reaktion  nicht  die  relative  Empfind- 
lichkeit  gegeniiber  Agonisten  oder  Antagonisten  direkt  verantwortlich.  Diese  verschie- 
denen  Eigentiimlichkeiten  des  Reaktionssubstrates  und  damit  auch  die  Eigenschaften, 
welche  fiir  die  Spezifitat  der  pharmakologischen  Wirkung  verantwortlich  sind,  lassen 
sich  am  einfachsten  so  erklaren,  dass  fiir  die  Spezifitat  der  Wirkung  eine  bestimmte 
chemische  oder  physikalische  Struktur  des  Substrates  verantwortlich  ist.  Da  dieses 
Substrat  ganz  bestimmte  eigentiimliche  Eigenschaften  besitzen  muss,  kann  nur  dann 
eine  Reaktion  an  einem  Substrat  als  Erklarung  oder  als  Analogon  dieses  Reaktions- 
verhaltens  der  Zelle  in  Anspruch  genommen  werden,  wenn  dieses  Substrat  de  facto 
samtliche  Eigenschaften  besitzt,  welche  im  vorstehenden  auf  Grund  der  quantitativen 
Reaktionsverhaltnisse  festgestellt  wurden.  Wenn  somit  diese  Untersuchung  nicht  die 
Frage  der  Zuriickfiihrung  der  Wirkungsspezifitat  auf  allgemeine  physikalische  oder 
chemische  Gesetzmassigkeiten  behandelte,  so  kann  die  quantitative  Analyse  derartiger 
Reaktionsgleichgewichte  doch  dazu  beitragen,  einfachere  Modelle  als  identisch  oder 
nicht  identisch  mit  dem  Substrate  der  pharmakologischen  Wirkung  zu  bezeichnen  oder 
nicht.  Dieses  diirfte  wohl  einer  der  Wege  sein,  auf  dem  versucht  werden  kann,  die 
Komplexitat  des  pharmakologischen  Reaktionsverhaltens  in  seine  einzelnen  Elemente 
aufzulosen. 


LITERATUR 

H.  J.  Bein,  Helv.  Physiol,  el  Pharmacol.  Acta,  5  (1947)  190. 

A.  J.  Clark,  /.  Physiol.,  61  (1926)  547;  The  mode  of  action  of  drugs  on  cells,  Arnold,  London  (1933); 

Hdb.  exp.  Pharmakol.,  4.  Erg.  Bd.  Springer,  Berlin  (1937). 
K.  Fromherz,  Arch,  exptl.  Path.  u.  Pharmakol,  113  (1926)  113. 
J.  H.  Gaddum,  /.  Physiol,  61  (1926)  141;  /.  Physiol,  89  (1937)  7^^  Proc.  Roy.  Soc.  London,  B  121 

(1937)  598. 
E.  S.  Guzman  Barron,  L.  Nelson,  and  M.  J.  Ardao,  /.  Gen.  Physiol.,  32  (1948)  179. 
S.  LoEWE,  Ergeb.  Physiol.,  27  (1928)  47. 
R.  Magnus,  Arch.  ges.  Physiol.,  123  (1908)  95. 
R.  Meier,  Lectures  N.  Y.  Ac.  Sci.  (1947)  (in  press). 

R.  Meier  and  B.  Pellmont,  Helv.  Physiol,  et  Pharmacol.  Acta,  5  (1947)  178. 
E.  RoTHLiN,  /.  Pharmacol.  Exptl  Therap.,  25  (1925)  675. 


VOL.  4  (1950)  SPEZIFITAT   PHARMAKOLOGISCHER   WIRKUNGEN  155 

L.  F.  Shackell  /.  Pharmacol.  Exptl  Therap.,  25  (1925)  275. 

\V.  Storm  van  Leeuwen,  Arch.  ges.  Physiol.,  174  (1919)  120. 

W.  Storm  van  Leeuwen  and  J.  W.  Le  Heux,  Arch.  ges.  Physiol.,  177  (1919)  250. 

M.  Vest,  Dissertation,  Basel  (1948). 

F.  WiRSiNG,  Dissertation,  Basel  (1949). 

Eingegangen  den  i6.  April  1949 


PART  IV 
INTERMEDIATE  METABOLISM 


FREE  RADICALS  DERIVED  FROM  TOCOPHEROL  AND 
RELATED  SUBSTANCES 

L.  MICHAELIS  and  S.  H.  WOLLMAN  * 

Laboratories  of  the  Rockefeller  Institute  for  Medical  Research, 
Neiv  York.N.Y.  {U.S.A.) 


Tocopherol  is  known  to  exhibit  two  properties:  It  serves  as  a  vitamin,  and  also  as 
an  antioxidant  with  respect  to  the  autoxidation  of  unsaturated  fatty  acids.  The  latter 
property  is  shared  with  many  substances  of  phenolic  character.  Although  the  mechanism 
of  the  antioxidant  effect  is  not  fully  understood,  and  the  mechanism  of  its  effect  as 
vitamin  E  is  not  understood  at  all,  the  suggestion  as  to  some  lelationship  of  those  two 
effects  is  almost  inescapable.  The  vitamin  effect  may  be  closely  related  to  the  antioxidant 
effect,  except  of  course  for  the  fact  that  the  more  specific  effect  of  the  vitamin  requires 
a  special  structure  in  addition  to  the  general  feature  of  being  a  substituted  hydroquinone. 
It  may  be  left  undecided  whether  the  specific  structure  is  just  to  make  it  more  fat- 
soluble  or  to  adapt  it  to  any  function  as  a  coenzyme  to  some  enzyme. 

Hydroquinone  is  an  efficient  antioxidant^.  Although  the  mechanism  of  its  action 
is  not  known  in  every  respect,  it  can  scarcely  be  doubted  that  this  effect  is  in  some  way 
connected  with  its  reversible  oxidizability.  However,  also  phenols  with  only  one  (or 
at  least  one  unsubstituted)  hydroxyl  group  are  antioxidants^.  Here  no  reversible 
oxidation  comparable  with  that  of  hydroquinone  can  take  place.  The  reversible  oxida- 
tion of  hydroquinone  leads  to  quinone,  by  a  bivalent  oxidation  passing  through  the 
intermediate  stage  of  a  semiquinone.  For  monophenols,  no  such  bivalent  reversible 
oxidation  is  imaginable.  However,  a  reversible  univalent  oxidation  to  a  free  radical  is 
imaginable  both  for  hydroquinone  and  for  mono-phenols**,  including  tocopherol.  Such 
a  radical  would  be  a  rather  unstable  compound.  Ordinary  oxidizing  agents  may  not  be 
able  to  produce  the  semiquinone  radicals  to  any  readily  recognizable  extent;  yet,  if 
a  free  radical  may  be  produced  only  to  a  slight  extent,  not  recognizable  directly,  the 
high  energy  content  of  the  radical  would  make  it  a  powerful  reactant;  just  as  the  fiee 
OH  radical,  although  never  existing  to  any  directly  recognizable  extent  in  an  aqueous 
solution,  has  been  recognized  as  a  powerful  reagent  in  many  chain  reactions. 

However,  any  speculation  about  such  free  radicals  is  all  too  vague  unless  there  is 
more  direct  evidence  for  their  existence.  It  is  the  purpose  of  this  paper  to  produce  such 
evidence.  It  is  based  on  a  method  devised  by  G.  N.  Lewis^'  *  and  consists  of  the  following 
procedure.  The  substance  to  be  oxidized  is  dissolved  in  an  organic  solvent  such  as,  at 
the  temperature  of  liquid  air,  will  freeze  to  a  homogeneous  glass  without  crystallizing, 


*  Special  Research  Fellow  of  the  National  Cancer  Institute. 
**  At  the  present  time,  it  will  not  be  discussed  whether  even  one  unsubstituted  hydroxyl  grou]) 
is  necessary  at  all  for  the  establishment  of  a  free  radical  of  comparable  structure. 

References  p.  i^g.  156 


VOL.  4  (1950) 


FREE  RADICALS  DERIVED  FROM  TOCOPHEROL 


157 


Fig.  I  shows  the  absorption 
spectrum  of  irradiated  a-toco- 
pherol  at  .iquid  air  tempera- 
ture, photographed  with  a 
spectrograph. 


and  is  irradiated  with  ultraviolet  light  through  quartz  windows  in  a  Dewar  vessel.  Such 
an  irradiation  may  have  two  effects:  one  is,  to  raise  the  energy  of  some  electron  to  a 

higher  level.  The  spontaneous  return  of  this  electron  to  its 
ground  level  will  be  manifested  by  some  luminescence, 
either  fiuoiescence  or  phosphorescence  of  longer  duration, 
according  to  conditions  discussed  by  Lewis.  In  the  second 
place,  if  there  be  an  electron  of  sufficiently  low  ionization 
potential,  the  electron  may  be  knocked  out  altogether,  a 
process  comparable  to  oxidation  by  a  chemical  oxidizing 
agent.  At  the  temperature  of  liquid  air  and  in  the  rigid 
medium  molecular  collisions  are  inhibited.  Free  radicals, 
once  created,  will  accumulate  to  a  concentiation  far  above 
that  permissible  by  thermodynamics,  provided  the  elec- 
trons ejected  are  trapped  in  the  molecules  of  the  solvent 
and  do  not  re-combine  with  the  free  radicals.  In  this  case, 
no  equilibrium  in  which  the  radical  may  be  involved,  can 
be  established.  Reactions  such  as  dismutation,  or  dimeri- 
zation  of  the  radicals  cannot  occur.  If  the  radical  happens  to  be  stable  in  so  far  as  not 
to  suffer  a  decay  by  a  spontaneous  unimolecular  reaction  (such  as  occurs  in  a  radioactive 
atom),  it  will  accumulate  to  a  thermodynamically  impermissible  concentration.  If  the 
radical  should  be  coloured,  it  could  be  seen  in  the  frozen  medium  and  remain  as  long  as 
the  temperature  is  kept  low.  On  slightly 
warming  up  the  solution  the  colour  should 
disappear.  This  may  be  taken  as  evidence 
for  the  that  fact  the  colour  belongs  to  a 
compound  capable  of  existence  to  a  notice- 
able extent  only  under  conditions  where 
the  establishment  of  chemical  equilibria  is 
inhibited*. 

The  colour  produced  in  this  way  can,  in 
suitable  cases,  be  compaied  with  the  colour 
of  free  radicals  produced  by  chemical  oxida- 
tion. In  fact,  the  absorption  spectrum  of 
the  compound  generated  by  either  method 
was  found  to  be  identical^  on  working  with 
such  substances  as  asymmetrical  dimethyl- 
p-phenylene  diamine,  or  tritolylamine^'  ^. 

In  this  paper  we  shall  describe  the  absorp- 
tion spectra  of  several  coloured  substances 
considered  as  free  semiquinone  radicals  pre- 
pared in  this  way  from  substances  related 


- 

a  locopfteryi 

hydroquinone 

- 

/        V 

_- 

-.,. 

\ 

1      \ 

\ 
\ 
\ 
\ 

1        \ 

/          1 

\ 

< 

Tocopherol           \ 

/ 
/ 

1 
1 
1 

, 

1                    1 

!                       1 

1 

39t) 


WO 


if30         «40        ''SO 
^^ovelength  in  mfj 

Fig.    2  shows   tracings,   obtained  w^ith  a  re- 
cording micophotometer,  of  the  spectrum  of 
irradiated    a-tocopherol,    and    of    irradiated 
a-tocopherylhydroquinone     . 


*  According  to  Lewis  and  his  associates,  there  may  be  still  another  effect:  dissociation  of  a 
large  molecule  (such  as  tctraphenylhydrazin)  either  into  two  free  radicals,  or  into  a  positive  and  a 
negative  ion.  Considering  the  structure  of  the  compounds  investigated,  the  possibility  of  such  effects 
may  be  disregarded  here.  The  fact  that  all  the  spectra  obtained  from  the  various  compounds  are 
similar,  is  further  evidence  as  to  the  absence  of  nny  essential  photodecomposition. 

**  The  authors  are  indebted  to  the  Sun  Chemical  Company,  New  York,  for  their  permission 
to  use  their  recording  microphotometer. 

References  p.  i^g. 


158 


L.  MICHAELIS,  S.  H.  WOLLMAN 


VOL.  4  (1950) 


Red 


to  tocopherol.  They  are  all  produced  by  irradiation  of  a  solution  in  a  mixture  of  ether, 
ethanol  and  pentane*,  in  the  volume  proportions  5:2:5,  respectively,  with  an  ultra- 
violet lamp  for  the  duration  of  a  few  minutes  to  about  twenty  minutes.  Although  the 
method  is  not  suitable  in  its  present  form  to  tell  anything  about  the  yield,  it  may  be 
stated,  that  the  radical  of  tocopherol  is  produced  with  ease  to  a  readily  recognizable 
extent. 

Among  the  substances  irradiated  during  this  experimental  study  there  is,  first  of 
all,  hydroquinone.  It  is  irradiated,  then  the  decay  of  the  phosphorescence  is  awaited 
(usually  several  seconds),  without  lifting  the  vessel  out  of  the  liquid  air  environment. 
Now  the  colour  in  transmitted  light  is  observed.  It  is  yellow,  its  absorption  spectrum 
consists  of  several  bands  in  the  visible,  the  maxima  of  which  are  reproduced  in  Fig.  3. 
The  yellov/  substance  is  not  quinone.  Firstly,  its  absorption  spectrum  is  different  from 
that  of  quinone ;  secondly,  this  colour  vanishes 
on  slightly  warming  up  the  frozen  mixture.  In 
addition,  a  spectrum  of  the  same  character  is 
produced  in  this  way  from  hydroquinone-mono- 
methyl  ether.  This,  of  course,  cannot  be  oxidized 
to  the  level  of  a  regular  quinone  but  there  is  no 
reason  why  it  should  not  be  oxidized  to  the  level 
of  d  semiquinone. 

Of  the  various  tocopherols,  samples  of  pure 
a,  8,  and  y  tocopherol**  and  several  samples  of 
commercially  available  a-tocopherol  were  com- 
pared. The  latter  showed  the  same  behaviour  as 
the  pure  a-tocopherol,  whereas  the  S  and  y  com- 
pound showed,  after  irradiation,  absorption 
bands  slightly  different  from  the  a-compound. 
Whereas  the  colour  of  the  radicals  from  hydro- 
quinone and  its  methyl-ether  are  yellow,  that  of 
all  the  tocopherols  is  red,  of  slightly  orange  tint.  This  difference  corresponds  to  the  loca- 
tion of  the  absorption  bands  in  Fig.  3. 

The  problem  arises  whether  this  "oxidation"  by  irradiation  is  a  reversible  one.  Only 
in  this  case  the  substance  could  serve  in  metabolism  as  something  analogous  to  a 
coenzyme  of  an  oxidative  enzyme.  When  tocopherol  is  chemically  oxidized  (say  by 
ferric  chloride),  the  first  oxidation  product  obtainable  is  a  quinone,  tocopherylquinone', 


1 

2 

3                                 1 

^                        1        1 

-VH — 

^"^ 

-j^ 

6 

'      1         1 

^ 

11:: 

360     390     UOO     ^10     ^20     ^30     1>U0     «50  m^ 

Fig.  3  shows  the  location  of  the  absorption 
bands  after  irradiation  as  obtained  ac- 
cording both  to  photographs  such  as 
Fig.   I   and  to  tracings  such  as  Fig.   z. 

1.  (5-tocopherol 

2.  y-tocopherol 

3.  a-tocopherol 

4.  a-tocopheryl  hydroquinone 

5.  hydroquinone  monomethyl  ether 

6.  durohydroquinone 

7.  hydroquinone 


HO. 
H,C' 


CH3  OH 

I        CH2  I 

i^^CH,— C— C,«H, 


^OH 


CH, 


CH, 


a-tocophcrol,  parent  substance  of  radical 
No.  3  in  Fig.  3 


a-tocopherylhydroquinone,    parent    sub- 
stance of  radical  No.  4  in  Fig.  3 


*  G.  N.  Lewis  recommends  isopentane.  We  had  no  trouble  with  ordinary  commercial  pentane. 
If  the  mixed  solvent  shows  any  inclination  to  crystallize  at  liquid  air  temperature,  it  can  be  corrected 
by  adding  slightly  more  ether. 

**  We  owe  these  to  the  courtesy  of  Distillation  Products  Corporation,  Rochester,  N.Y. 

References  p.  i^g. 


VOL.  4  (1950)        FREE  RADICALS  DERIVED  FROM  TOCOPHEROL  I59 

which  cannot  be  re-reduced  directly  to  the  original  tocopherol  because  the  phytol  side- 
ring  is  opened  to  make  the  quinone.  When  this  quinone  is  reduced  to  its  corresponding 
hydroquinone,  and  this  "tocopherylhydroquinone"  is  irradiated  under  proper  condi- 
tions, the  absorption  spectrum  of  the  free  radical  is  different  from  that  produced  by  the 
irradiated  tocopherol  itself.  It  resembles,  with  its  yellow  colour,  more  that  of  the 
hydroquinone-methyl-ether.  Hereby  it  is  shown  that  the  red  radical  produced  from 
tocopherol  does  not  involve  the  opening  of  the  phytol  side-ring.  The  preserva.ion  of  the 
free  radical  will  also  be  aided  by  the  fact  that  the  opening  of  the  phytol  ring  represents 
a  hydrolysis  which  cannot  occur  in  the  absence  of  water.  There  is,  then,  no  reason,  why 
the  univalent  oxidation  of  tocopherol,  especially  in  a  non-aqueous  solvent,  should 
not  be  reversible. 

SUMMARY 

Tocopherol,  dissolved  in  a  suitable  mixture  of  organic  solvents  such  as  will,  at  the  temperature 
of  liquid  air,  form  a  homogeneous  glass,  is  irradiated  with  ultraviolet  light.  A  red  colour  is  developed 
which  disappears  at  slightly  higher  temperature.  Similar  observations  are  made  with  some  other 
substances  related  to  hj'droquinones.  The  coloured  substance  is  interpreted  as  a  free  semiquinone 
radical.  Its  possible  function  for  the  vitamine  and  the  antioxidant  effect  of  tocopherol  is  discussed. 

RESUME 

Le  tocopherol,  dissous  dans  un  melange  approprie  de  solvants  organiques,  melange  qui,  a  la 
temperature  de  I'air  liquide,  forme  un  verre  homogene,  est  irradie  au  moyen  de  lumiere  ultraviolette. 
Une  coloration  rouge  apparait,  qui  redisparait  lorsqu'on  eleve  quelque  peu  la  temperature.  Des 
observations  similaires  ont  ete  faites  avec  quelques  autres  substances  de  nature  hydroquinonique. 
La  substance  coloree  est  consideree  comme  etant  un  radical  semiquinonique  libre.  Son  role  possible 
dans  Taction  vitaminique  et  antioxydante  du  tocopherol  est  discute. 

ZUSAMMENFASSUXG 

Tocopherol,  gelost  in  einer  geeigneten  Mischung  von  organischen  Losungsmitteln,  welche  bei 
der  Temperatur  der  fiiissigen  Luft  zu  einem  homogenen  Glas  erstarren,  w-ird  mit  ultraviolettem  Licht 
bestrahlt.  Es  entsteht  eine  rote  Farbung,  welche  bei  hoherer  Temperatur  wieder  verschwindet. 
Ahnliches  wird  mit  anderen  Hydrochinon-ahnlichen  Verbindungen  beobachtet.  Die  gefarbte  Sub- 
stanz  wird  als  ein  Semichinon  gedeutet  und  ihre  mogliche  Funktion  bei  der  Rolle  des  Tocopherols 
als  Vitamin  und  als  Antioxidant  erortert. 

REFERENCES 

1  J.  L.  BoLL.\ND  AND  P.  TEN  Have,  Traus.  Faraday  Soc,  43  (1947)  201. 

2  J.  L.  BoLLAND  AND  P.  TEN  Have,  in  The  Labile  Molecule,  'Discussions  of  the  Faraday  Soc.,'" 
London  1947. 

3  G.  N.  Lew'is  and  D.  Lipkin,  /.  Am.  Chem.  Soc,  64  (1942)  2801-8. 

*  G.  N.  Lewis  and  Biegeleisen,  /.  Am.  Chem.  Soc,  65  (1944)  2424-6;  65  (1944)  2419. 

^  L.  Michaelis,  M.  p.  Schubert,  and  S.  Granick,  /.  Am.  Chem.  Soc,  61  (1939)  1981-92. 

*  S.  Granick  and  L.  Michaelis,  /.  Am.  Chem.  Soc,  62  (1940)  2241. 
'  Walter  John,  Z.  physiol.  Chem.,  250  (1937)  11;  257  (1939)  173. 

^  L.  T.  Smith,  Chem.  Revs,  27  (1940)  287-320  (Review  of  the  chemistry  of  vitamin  E). 

^  R.  A.  IVIorton,  The  Application  of  Absorption  Spectra  to  the  Study  of  Vitamins,  Hormones  and 

Coenzymes,  2dn  Edition,  Adam  Hilger,  Ltd.,  London  1942. 
^°  Biological  Antioxidants,  Transactions  of  the  first  conference,  Josiah  Macy,  Jr.  Foundation,  X.Y., 
1946.  Second  Conference,  1947;  third  Conference  (in  press)  1948. 

Received  February  14th,  1949' 


l6o  BIOCHIMICA   ET   BIOPHYSICA   ACTA  VOL.  4  (1950) 


THE  COMBINATION  OF  DIPHOSPHOPYRIDINE  NUCLEOTIDE  WITH 
GLYCERALDEHYDE  PHOSPHATE  DEHYDROGENASE 

by 

CARL  F.  CORI,  SIDNEY  F.  VELICK,  and  GERTY  T.  CORI 
Department  of  Biological  Chemistry,  Washington  University,  School  of  Medicine,  St.  Louis, 

Missouri  {U.S.A.) 


It  has  been  shown  in  a  previous  paper^  that  glyceraldehyde  phosphate  dehydrogenase 
from  rabbit  muscle  contains  one  mole  of  diphosphopyridine  nucleotide  (DPN)  per 
50000  g  of  protein.  This  ratio  did  not  change  after  prolonged  dialysis  against  distilled 
water  or  after  repeated  recrystallizations  from  ammonium  sulphate  solutions.  When  an 
aqueous  solution  of  the  enzyme  was  treated  with  activated  charcoal  (norit)  and  filtered, 
DPN  was  removed.  Addition  of  an  excess  of  DPN  and  ammonium  sulphate  to  the  DPN- 
free  enzyme  solution  resulted  in  the  formation  of  crystals  which  contained  the  original 
ratio  of  DPN  to  protein.  From  these  and  other  observations  it  was  concluded  that  the 
enzyme  contained  firmly  bound  DPN.  The  fact  that  DPN  could  be  removed  with  norit 
made  it  clear  that  the  union  between  enzyme  and  coenzyme  was  not  through  a  covalent 
bond. 

Earlier  work^  had  indicated  that  the  dissociation  constant  of  the  enzyme  with 
DPN,  as  estimated  from  the  concentration  of  DPN  at  which  the  reaction  with  glycer- 
aldehyde phosphate  occurred  at  half  maximal  velocity,  was  of  the  order  of  4-  io~^  M/ml. 
This  agreed  with  a  value  obtained  by  Warburg  and  Christian^  with  yeast  enzyme 
and  free  glyceraldehyde  as  substrate.  According  to  existing  criteria  the  constant  so 
obtained  is  sufficiently  large  to  permit  easy  separation  of  enzyme  and  coenzyme  by 
dialysis  or  recrystallization.  The  fact  that  such  a  separation  was  not  observed  suggests 
either  that  the  enzyme  combines  with  DPN  at  two  sites,  one  of  which  binds  DPN  more 
firmly  than  the  other,  or  that  the  conclusions  drawn  from  the  kinetic  measurements  or 
from  dialysis  and  recrystaUization  are  not  valid. 

In  the  present  paper  experiments  are  described  in  which  some  aspects  of  the  two- 
site  hypothesis  are  tested.  In  order  to  make  reactions  of  bound  DPN  measurable  in  a 
I  cm  cell  at  340  m^a  in  the  Beckman  spectrophotometer,  it  is  necessary  to  use  enzyme 
concentrations  of  2  to  4  mg  per  ml  which  are  about  1000  times  greater  than  those 
necessary  to  give  good  rates  with  added  DPN  and  glyceraldehyde  phosphate.  Accord- 
ingly the  reaction  with  glyceraldehyde  phosphate  is  too  rapid  for  convenient  study, 
unless  one  works  at  a  pn  far  from  the  optimum.  When  glyceraldehyde  is  used  as  sub- 
strate, however,  the  reaction  rate  is  conveniently  measurable  over  a  wide  range  of 
conditions,  the  slower  reaction  being  due,  as  will  be  shown,  to  a  low  affinity  of  glycer- 
aldehyde for  the  enzyme. 
References  p.  i6g. 


VOL.  4  (1950)      DPN  AND  GLYCERALDEHYDE  PHOSPHATE  DEHYDROGENASE 


161 


EXPERIMENTAL 

The  enzyme  was  prepared  as  previously  described^  and  recrystallized  four  times.  An  aliquot  of 
the  crystal  suspension  in  ammonium  sulphate  was  centrifuged  at  about  loooo  rpm,  drained,  and 
dissolved  in  0.03  M  sodium  pyTophosphate  —  0.003  ^I  cysteine  buffer  at  pH  8.3.  This  enzyme  solution 
was  prepared  fresh  for  each  experiment.  The  composition  of  reaction  mixtures  is  given  in  the  tables. 


THE    DISSOCIATION    CONSTANT   OF   ENZYME   AND   BOUND    DPN 

The  enzyme  and  bound  DPN  concentrations  cannot  be  varied  independently 
unless  one  resorts  to  partial  removal  of  DPN  with  norit.  The  latter  procedure  introduces 
additional  variables  due  to  the  instability  of  the  DPN-free  enzyme  and  so  a  dilution 
method  was  employed.  It  was  possible  to  follow  the  reactions  in  the  more  dilute  solu- 
tions by  using  cuvettes  with  a  longer  light  path. 

The  experiment  consisted  in  comparing  the  rates  of  reaction  in  two  solutions 
identical  in  all  concentrations  except  that  of  the  enzyme-DPN  complex.  The  results  of 
such  an  experiment  are  described  in  Table  I.  It  may  be  seen  that  the  directly  measured 

TABLE  I 

THE    DISSOCIATION    OF    ENZYME    AND    "BOUND"    DPN 

Two  reaction  mixtures  were  prepared,  one  with  a  total  volume  of  6  ml  and  the  other  of  30  ml.  The 
former  was  in  a  cell  of  2  cm  and  the  latter  in  a  cell  of  10  cm  length.  Both  reaction  mixtures  contained 
in  moles  per  ml,  6-io-®  arsenate,  3-10-^  cysteine,  5-10-^  pyrophosphate  (pn  8)  and  2-10-®  dl- 
glyceraldehyde  (the  latter  added  to  start  the  reaction).  The  two  reaction  mixtures  differed  however 
in  that  the  2  cm  cell  contained  1.77- 10-®  and  the  10  cm  cell  3.54- 10-^  M  per  ml  of  enzyme  -  DPN. 


Time 

2  cm  cell 

10  cm  cell 

(min) 

log  lo/I 

log  lo/I 

I 

0.022 

0.019 

2 

0.037 

0.037 

3 

0.053 

0.053 

4 

0.067 

0.067 

5 

0.079 

0.081 

6 

0.091 

0.092 

7 

0.099 

O.IOI 

00 

0.223 

0.222 

*  After  addition  of  glyceraldehyde  phosphate. 


rates  were  identical.  This  means  that  the  decrease  in  rate  due  to  the  5-fold  dilution 
of  enzyme-DPN  complex  was  exactly  compensated  by  the  5-fold  increase  in  light  path. 
Since  the  observed  rate  was  proportional  to  the  concentration  of  undissociated  enzyme- 
DPN,  it  follows  that  no  measurable  increase  in  dissociation  occurred  on  dilution.  In 
order  for  this  condition  to  hold,  it  would  be  necessary  for  the  dissociation  constant  of 
enzyme-DPN  to  be  of  the  order  of  i-io"^"  M/ml  or  less.  Since  in  fact  no  evidence  of 
dissociation  was  obtained  at  all  in  this  experiment,  the  above  figure  may  be  considered 
only  to  be  an  upper  limit*.  An  analogous  dilution  experiment  with  a  small  amount  of 
enzyme  and  added  DPN  with  glyceraldehyde  phosphate  as  substrate  showed  a  change 


*  In  work  which  will  be  reported  in  detail  at  a  later  date  it  has  been  shown  that  bound  DPN 
equilibrates  rapidly  with  radioactive  DPN  labelled  with  P^^  This  is  in  harmony  with  the  conclusion 
that  the  bond  between  DPN  and  enzyme  is  not  of  the  covalent  type  and  that  the  bound  DPN  ex- 
hibits a  finite  dissociation. 

References  p.  i6g. 

11 


l62 


c.  F.  coRi  et  al. 


VOL.  4  (1950) 


in  rate  between  DPN  concentrations  of  4.4-10"^  and  4.4-10"^  M/ml  that  is  consistent 
with  a  dissociation  constant  of  the  order  of  4-10"^  M/ml. 

The  fact  that  depending  upon  whether  or  not  one  measures  bound  DPN  or  added 
DPN,  one  gets  apparent  dissociation  constants  differing  by  a  factor  of  at  least  100  argues 
for  the  existence  of  two  types  of  catalytic  sites.  We  will  designate  the  still  hypothetical 
site  with  the  higher  DPN  affinity  as  site  I  and  the  site  with  lower  DPN  affinity  as  site 
II  and  proceed  to  examine  the  conditions  that  would  hold  during  the  course  of  a  reaction. 


THE    REACTION    AT   SITE    I 

In  Table  II  is  shown  an  experiment  in  which  the  reduction  of  bound  DPN  is  studied 
as  a  function  of  glyceraldehyde  concentration.  The  glyceraldehyde  concentration  in  all 

TABLE  II 

EFFECT    OF    CONCENTRATION    OF    GLYCERALDEHYDE 

Reaction   mixture   consisted   (in  moles  per  ml)  of  2.4-10-®  enzyme  -  DPN,  e-io-®  arsenate,  3-10-^ 
cysteine,  5-10-*  pyrophosphate  (pn  8.3)  and  varying  amounts  of  DL-glyceraldehyde. 


Time 

Concentration  of  glyceraldehyde  (as  D-form,  moles  per  ml) 

in 
mill 

0.5 -lo-® 

I-IO-® 

2-IO-® 

log  lo/I 

K* 

log  lo/I 

K* 

log  lo/I 

K* 

1-5 
30 

4-5 
6.0 

7-5 

9.0 

10.5 

0.028 

0.054 

0.072 
0.085 
0.096 
0.103 
0.108 

0.14 

0.15 
0.14 
0.14 

0.13 
0.13 

0.051 
0.088 
0.109 
0.123 
0.130 
0.136 
0.140 

0.27 
0.29 
0.29 
0.28 
0.26 

0.087 
0.123 
0.140 
0.147 
0.150 
0.152 
0.152 

0.57 
0.55 
0.57 
0.57 

0.14 

0.28 

0.56 

*  K  =  2.3/t  log  A  (A  —  x),  A  =  initial  concentration  of  DPN. 

cases  was  sufficiently  higher  than  that  of  DPN  so  that  it  was  virtually  constant  during 
the  course  of  the  reaction.  Under  these  conditions  the  rate  is  described  by  a  first  order 
velocity  constant.  The  fact  that  the  first  order  constants  increase  linearly  with  initial 
glyceraldehyde  concentration  means  that  saturation  of  the  enzyme  with  glyceraldehyde 
has  not  been  approached.  The  dissociation  constant  of  enzyme-glyceraldehyde  is  there- 
fore very  large. 

At  the  concentrations  of  enzyme  employed  the  amount  of  free  DPN  in  equilibrium 
with  the  protein  would  be  negligible  if  the  dissociation  constant  at  site  I  is  less  than 
I  •  io~^".  The  above  reaction  is  therefore  first  order  with  respect  to  enzyme-DPN  com- 
plex. This  means  that  each  enzyme  molecule  behaves  as  though  it  reacted  only  once. 

When  DPNH  (in  amounts  equivalent  to  the  bound  DPN  present)  was  added  at  the 
beginning  of  the  reaction,  it  exerted  an  inhibitory  effect.  This  is  indirect  evidence  that 
DPNH  as  well  as  DPN  is  bound  at  site  I.  It  is  also  possible  to  demonstrate  in  a  direct 
manner  that  DPNH  is  bound.  This  was  done  by  reducing  the  bound  DPN  in  a  solution 
containing  10  to  20  mg  of  enzyme  per  ml  with  excess  glyceraldehyde  phosphate  and 
References  p.  i6g. 


VOL.  4  (1950)      DPN  AND  GLYCERALDEHYDE  PHOSPHATE  DEHYDROGENASE 


163 


arsenate  and  then  precipitating  the  enzyme  with  ammonium  sulphate  at  a  final  concen- 
tration of  85%  saturation.  It  was  found  that  90%  or  more  of  the  enzyme  was  precip- 
itated and  that  the  ratio  of  DPNH  to  protein  in  the  precipitate  was  the  same  as  that 
of  DPN  to  protein  in  the  original  solution. 

For  the  interpretation  of  reactions  with  added  DPN  an  additional  consideration 
is  important,  namely,  whether  added  DPN  can  displace  DPNH  at  site  I.  From  the  fact 
that  DPN  at  site  I  is  dissociable  one  would  expect  the  same  to  hold  for  DPNH.  The 
problem  of  displacement  would  then  be  resolved  by, a  determination  of  the  relative 
dissociation  constants  of  enzyme  with  DPN  and  DPNH.  Theoretically  this  could  be 
done  by  determining  the  ratio  of  DPN  to  DPNH  in  the  enzyme  when  enzyme-DPNH 
is  precipitated  in  the  presence  of  added  DPN. 

A  preliminary  experiment  of  this  type  is  presented  in  Table  HI ;  it  gives  qualitative 
evidence  that  displacement  of  DPNH  by  DPN  does  occur  and  that  the  dissociation 

TABLE  III 

COMPETITION  BETWEEN  DPN  AND  DPNH 

DPN  in  enzyme  was  reduced  by  addition  of  arsenate  and  an  equivalent  amount  of  triosephosphate. 
Aliquots  of  the  reduced  enzyme  were  treated  as  follows.  In  (A)  0.5  ml  of  enzyme  containing  12.5  mg 
of  protein,  -f-  o.i  ml  of  HgO,  was  precipitated  with  3  ml  of  saturated  ammonium  sulphate.  In  (B)  0.5  ml 
of  enzyme  +  o.i  ml  of  DPN  solution  (2.4-io— '  M)  was  incubated  for  3  minutes  before  being  pre- 
cipitated with  ammonium  sulphate.  The  precipitates  were  separated  by  centrifugation  at  loooorpm 
and  dissolved  in  cysteine-pyrophosphate  buffer. 


A 

B 

Vol. 
in  ml 

Protein 
mg 

D340 

DPNH 

M-io7 

Vol. 
in  ml 

Protein 
mg 

D340 

DPNH 
M-io' 

Supernatant  fluid 

Precipitate 

Pptate  +  HAsOj-f 

triosephosph.*** 

3-6 
31 

3-25 

II-3 

0.063 
0.304 

0.413 

0.36 

(1-49) 

2.13 

3-6 
3-1 

II. I** 

0.275 
0-175 

1-57 
0.86 

2-49 

2-43 

Calculated  from  optical  density  at  276  m/<. 
Determined  by  biuret  method. 
***  An  excess  of  glyceraldehyde  phosphate  was  added  in  order  to  reduce  DPN  completely. 


constants  of  the  oxidized  and  reduced  forms  with  the  enzyme  are  at  least  of  the  same 
order  of  magnitude.  The  chief  objection  that  might  be  raised  is  that  the  high  concen- 
tration of  ammonium  sulphate  may  change  the  equilibrium. 

An  analysis  of  the  experiment  shows  that  although  a  stoichiometric  amount  of 
glyceraldehyde  phosphate  was  used,  the  reaction  was  only  70%  complete  when  DPN 
was  added.  This  value  is  calculated  from  the  additional  DPNH  which  appeared  when 
excess  triosephosphate  and  arsenate  was  added  to  the  dissolved  precipitate  of  the  enzyme 
in  experiment  A.  Accordingly  there  must  have  been  residual  triosephosphate  in  B  when 
DPN  was  added.  The  excess  DPN  in  B  then  drove  the  reaction  to  completion  as  shown 
by  the  DPNH  recoveries  in  A  and  B. 

Some  of  the  DPNH  in  the  supernatant  fluid  of  B,  therefore,  arose  by  reduction  of 
added  DPN  and  hence  did  not  represent  DPNH  displaced  from  the  enzyme.  A  rough 
estimate  of  the  amount  actually  displaced  is  (by  comparison  with  experiments  A)  equal 
References  p.  i6g. 


164 


c.  F.  CORI  et  al. 


VOL.  4  (1950) 


to  the  total  amount  in  the  supernatant  of  B  (1.57-  io~'  M)  minus  the  amount  arising 
from  residual  triosephosphate,  [(2.13  —  1.49) -lO"'  =  0.64-10"'],  minus  unprecipitated 
protein-DPNH  (0.36-10"').  The  net  displaced  DPNH  is  (1.57  —  0.64  —  0.36) -lO"'  = 
0.57-10"'  M.  A  similar  value  is  arrived  at  by  comparing  DPNH  in  the  precipitated  pro- 
tein in  A  and  B,  namely  (1.49  —  0.86)  •  io~'  =  0.63-  lO"'  M. 

THE    REACTION    AT   SITE    II 

When  reactions  are  studied  with  added  DPN,  site  I  is  saturated,  even  at  low  enzyme 
concentrations  and  site  II  is  saturated  to  an  extent  which  depends  upon  its  dissociation 
constant  and  the  concentration  of  free  DPN.  Reaction  will  be  expected  to  occur  at  both 
sites  but  the  DPNH  formed  at  site  I  will  be  displaced  by  DPN  in  solution  and  site  I 
as  well  as  site  II  will  now  have  a  "turnover".  The  reactions  at  both  sites  will  be  first 
order  provided  that  at  each  site  the  affinity  for  DPN  is  the  same  as  that  for  DPNH. 
Experimentally  it  was  found  that  the  rate  remained  first  order  when  DPN  was  added. 
Table  IV. 

TABLE  IV 

EFFECT  OF  ADDED  DPN  ON  RATE  OF  REACTION 

The  enzyme  concentration  corresponded  to  3.4- lo-*  M  of  bound  DPN  per  ml,  the  pn  was  8.3  and 
the  temperature  26°.  No  DPN  was  added  in  A,  while  in  B  and  C,  3.4  and  7- 10—^  M  per  ml  respectively 
was  added,  giving  the  total  of  concentrations  of  DPN  shown  in  the  table  headings.  The  reaction  was 
started  by  the  addition  of  glyceraldehyde  (final  concentration  as  the  D-form  i.i-io-*  M  per  ml). 
K  =  2.3/t  log  A  (A  —  x),  A  being  the  initial  concentration  of  DPN.  Vq  (initial  velocity)  —  K  times 
the  initial  concentration  of  DPN. 


A 

B 

C 

in 

3.4-10-8  M/ml 

6.8- 10-8  M/ml 

10.4-10-8  M/ml 

mm 

log  lo/I 

K 

log  lo/I                  K 

log  lo/I 

K 

1-5 
30 

4-5 
6.0 

7-5 

9.0 

00* 

0.066 

O.III 

0.142 
0.160 
0.172 
0.183 
0.214 

0.25 
0.24 
0.24 
0.23 
0.22 
0.22 

0.233 

0.105 
0.191 
0.248 
0.290 
0.320 

0.343 

0.428 

0.19 
0.20 
0.19 
0.19 
0.18 
0.18 

0.188 

0.1 12 

0.217 
0.302 

0.371 
0.426 
0.468 
0.658 

0.12 
0.13 
0.14 
0.14 
0.14 
0.14 

0.136 

Vo 

0.79 

1.28 

1.41 

After  addition  of  glyceraldehyde  phosphate. 


By  multiplying  first  order  velocity  constants,  K,  by  the  initial  concentrations  of 
DPN  one  gets  the  initial  velocity  of  the  reaction,  Vq,  in  terms  of  M.  min"^  ml"^.  The 
observed  increase  in  initial  rate  on  addition  of  DPN  can  be  seen  to  be  approaching  a 
maximum  value  which  would  correspond  to  the  saturation  of  both  sites  with  DPN. 
Because  of  the  high  concentration  of  enzyme,  one  cannot  calculate  the  enzyme-coenzyme 
dissociation  constants  by  the  usual  methods  (which  are  based  on  the  assumption  that 
the  concentration  of  free  DPN  is  not  appreciably  diminished  by  combination  with  the 
enzyme).  It  is  furthermore  not  possible  from  this  experiment  to  reach  unambiguous 
conclusions  with  respect  to  the  number  and  type  of  catalytic  sites. 
References  p.  i6g. 


VOL.  4  (1950)      DPN  AND  GLYCERALDEHYDE  PHOSPHATE  DEHYDROGENASE 


165 


EQUILIBRIUM    CONSTANTS 

The  equilibria  of  reactions  with  free  DPN  and  substrate  using  catalytic  amounts 
of  enzyme  and  of  reactions  between  bound  DPN  and  substrate  with  the  protein  present 
in  quantities  equivalent  to  the  DPN  may  be  formulated  respectively  as  follows: 

a)  DPN  +  3-glyceraldehyde  phosphate  +  HPOr  ^^^  DPNH  +  H+  +  1,3" 
diphosphoglyceric  acid 

b)  DPN-enzyme  +  3-glyceraldehyde  phosphate  +  HP04~^=^DPNH-enzyme  + 
H+  +  1.3-diphosphoglyceric  acid. 

In  the  former  case  which  is  a  true  catalytic  reaction,  the  enzyme  forms  transient 
intermediates  with  a  minute  fraction  of  the  substrate  at  any  given  time.  Case  (b)  is  in 
effect  a  different  reaction  in  which  not  free  DPN  and  DPNH  but  the  corresponding 
protein  complexes  are  reactants. 

Meyerhof  and  Oesper*  have  carried  out  a  detailed  study  of  the  reaction  as  re- 
presented by  (a).  Since  one  hydrogen  ion  enters  the  equilibrium,  the  equilibrium  con- 
stant showed  a  dependence  upon  pn-  Equilibrium  measurements  were  made  with  added 
DPN  under  conditions  similar  to  those  employed  by  Meyerhof  and  Oesper.  About 
30  y  of  enzyme  per  ml  were  used  so  that  equilibrium  was  reached  within  one  minute 
after  addition  of  glyceraldehyde  phosphate,  even  at  low  p^  values.  Concentrations  of 
DPN  and  glyceraldehyde  phosphate  in  the  stock  solutions  were  determined  optically 
by  enzyTnatic  methods,  pn  was  measured  with  a  glass  electrode  in  the  reaction  mixture 
at  the  end  of  the  experiment.  The  values  found  for  the  equiHbrium  constants  fall  well 
within  the  range  reported  by  Meyerhof  and  Oesper,  Table  V. 

TABLE  V 

EQUILIBRIUM    OF    REACTION    AT    DIFFERENT    PH 

The  equilibrium  is  compared  for  catalytic  amounts  of  enzyme  (C)  plus  added  DPN,  and  large  amounts 
of  enzyme  (L)  containing  bound  DPN.  The  initial  and  final  concentrations  are  given  in  moles  per 
liter.  GAP  =  glyceraldehyde  phosphate. 


Amount  of 

Initial  Concentrations 

Final  Concentrations 

Present  data 

Data  of  M 

.  and  0.* 

enzyme 

DPN 

GAP 

PO4 

DPN 

GAP 

PO4 

K 

PH 

K 

PH 

L 
C 
L 
C 

•lO^ 

6.23 
7.48 
5-46 
7-53 

•io3 

1-43 
1.42 

1-43 
1-43 

•I03 

82.8 
82.8 

8.66 
8.66 

•io5 

4.08 
4.62 
4-45 
6.35 

•I03 

1-39 
1-37 
1.38 
1.36 

•I03 

82.8 
82.8 
8.62 
8.60 

0.67 
0.65 
16.4 
28.9 

7.09 
7.08 

7.85 
8.10 

0.6-1.4 
19.8* 
21-28 

7-15 
7.85 
8.20 

*  Calculated  from  Meyerhof  and  Oesper's*  data  by  means  of  their  complete  equilibrium 
equation. 


For  equilibrium  measurements  under  the  conditions  of  case  (b)  two  parallel  reaction 
mixtures  were  prepared  which  differed  only  in  that  one  contained  phosphate  and  the 
other  arsenate.  The  former  was  used  for  equilibrium  determination  while  the  latter 
served  for  determination  of  the  amount  of  DPN  present  in  the  enzyme.  The  value  of 
the  equilibrium  constants  that  were  obtained  agree  within  experimental  limits  with  those 
found  with  small  amounts  of  enzyme  and  added  DPN. 

Although  one  cannot  derive  from  these  measurements  evidence  for  the  existence 

References  p.  i6g. 


i66  c.  F.  coRi  et  al.  vol.  4  (1950) 

of  two  catalytic  sites,  the  following  considerations  are  of  interest.  In  case  (a)  the  enzyme 
cannot  contribute  to  the  net  free  energy  change  which  is  lixed  by  the  initial  and  final 
states  of  the  free  reactants.  In  case  (b)  two  of  the  reactants  have  been  altered  by  com- 
plex formation  and  the  initial  and  final  energy  states  are  not  the  same  as  in  case  (a). 
However,  since  only  the  difference  in  initial  and  final  states  determines  the  net  free 
energy  change,  case  (b)  may  or  may  not  have  the  same  equilibrium  constant  as  case  (a). 
These  considerations  apply  irrespective  of  the  physical  nature  of  the  bonding  forces 
involved  and  the  number  and  type  of  binding  sites. 

It  may  be  inferred  from  the  kinetics  that  the  protein  has  the  same  affinity  for 
DPN  as  for  DPNH*.  Conclusions  concerning  the  relative  dissociation  constants  of 
enzyme-DPN  and  enzyme-DPNH  may  also  be  drawn  from  a  comparison  of  the  equi- 
librium constants  in  (a)  and  (b).  If  the  binding  of  the  other  reactants  does  not  alter 
their  energy  differences  then,  from  the  equality  of  equilibrium  constants,  it  follows  that 
the  dissociation  constants  of  enzyme-DPN  and  enzyme-DPNH  are  equal. 

Ph  optimum 

The  rate  of  the  reaction  of  glyceraldehyde  with  enzyme  DPN  was  measured  at  pn 
8.4,  7.5,  and  6.4  in  cysteine-pyrophosphate  buffer.  The  relative  rates  calculated  from 
the  first  order  velocity  constants  were  as  100:30:9.  This  agrees  with  the  pn  activity 
curve  as  determined  previously  with  small  amounts  of  enzyme  (6  y/ml)  and  addition 
of  DPN  and  glyceraldehyde  phosphate  as  substrate^. 

REACTION   WITH    LACTIC   DEHYDROGENASE 

It  has  been  shown  in  a  previous  report^  that  enzyme  DPN,  after  reduction  by 
glyceraldehyde  phosphate,  was  reoxidized  by  addition  of  sodium  pyruvate  and  a  purified 
preparation  of  lactic  dehydrogenase  from  rabbit  muscle.  The  simplest  explanation  of 
this  result  is  that  the  bound  DPNH  has  a  small  but  finite  tendency  to  dissociate  and 
that  it  is  the  dissociated  DPNH  which  reacts  with  the  pyruvate-lactic  dehydrogenase 
system.  In  these  experiments  lactic  dehydrogenase  was  present  in  considerable  excess, 
so  that  the  rate  of  the  reaction  could  not  be  measured. 

The  dissociation  constant  for  lactic  dehydrogenase  and  DPNH  has  been  determined 

by  KuBOWiTZ  AND  Ott^  who  report  a  value  of  5  •  io~^  M/ml.  In  experiment  A,  Table  VI, 

2.3-0.146 
the  initial  concentration  of  bound  DPNH  was =  2.3-10-8  M/ml.  If  the  DPNH- 

1.45 -107 

enzyme  dissociation  constant  were  i  •  io~i"  M/ml,  there  would  not  be  enough  free  DPNH 
in  solution  to  give  25%  saturation  of  lactic  dehydrogenase  and  the  rate  of  reaction 
would  be  much  slower  than  in  experiment  C,  where  the  concentration  of  added  DPNH 
was  3.i-io~8M/ml  or  enough  to  saturate  the  enzyme.  The  fact  that  such  a  difference** 

*  This  inference  arises  from  the  fact  that  in  the  presence  of  a  large  excess  of  glyceraldehyde 
and  arsenate  the  reduction  of  bound  and  of  added  DPN  may  be  described  by  a  first  order  velocity 
constant.  If  one  assumes  that  DPNH  has  the  same  affinity  for  the  catalytic  site  as  does  DPN,  then 
the  first  order  kinetics  may  be  shown  to  be  due  to  the  formation  of  DPNH  which  acts  as  a  com- 
petitive inhibitor^. 

**  Actually  the  rate  was  faster  in  A  than  in  C.  One  possible  explanation  was  that  lactic  dehydro- 
genase in  C  was  acting  in  the  absence  of  "protective"  protein.  In  order  to  compensate  for  this  differ- 
ence, lactic  dehydrogenase  was  added  in  other  experiments  to  a  solution  containing  the  same  amount 
of  triosephosphate  dehydrogenase  the  DPN  of  which  had  not  been  reduced.  The  rate  of  reaction  of 
lactic  dehydrogenase  with  "bound"  and  with  added  DPNH  was  then  approximately  the  same. 

References  p.  169. 


VOL.  4  (1950)      DPN  AND  GLYCERALDEHYDE  PHOSPHATE  DEHYDROGENASE 


167 


TABLE  VI 

REACTION    OF    "BOUND"    DPNH    WITH    LACTIC    DEHYDROGENASE    SYSTEM 

The  DPN  in  24  mg  of  glyceraldehyde  phosphate  dehydrogenase  was  first  reduced  by  addition  of 
glyceraldehyde  phosphate  and  arsenate.  One  ahquot  (A)  was  precipitated  directly  with  ammonium 
sulphate,  while  another  aliquot  (B)  was  first  exposed  to  0.024  M  iodoacetate  before  being  precipitated 
with  ammonium  sulphate.  The  precipitates  were  separated  by  centrifugation,  dissolved  in  cysteine- 
pyrophosphate  buffer,  pH  8.3,  and  pyruvate  (i-io-^  M/ml)  was  added.  The  reaction  was  started  by 
the  addition  of  a  catalytic  amount  of  lactic  dehydrogenase.  To  reaction  mixture  (C)  free  DPNH  was 
added  in  place  of  glyceraldehyde  phosphate  dehydrogenase  containing  bound  DPNH. 


Time 

A 

B 

C 

in  min 

log  lo/I 

A 

log  lo/I 

A 

log  lo/I 

A 

0 

0.146 

0.146 

0.195 

I 

0.112 

0.034 

0.098 

0.048 

0.169 

0.026 

2 

0.084 

0.062 

0.071 

0.075 

0.153 

0.042 

3 

0.068 

0.078 

0.050 

0.096 

0.140 

0.055 

4 

0.050 

0.096 

0.037 

0.109 

0.127 

0.068 

5 

0.045 

O.IOI 

0.029 

0.117 

0.113 

0.082 

in  rate  was  not  observed  leaves  one  with  two  alternatives.  Either  DPNH  is  more  highly- 
dissociated  than  has  been  assumed  or  lactic  dehydrogenase  can  react  with  bound 
DPNH.  The  latter  alternative  would  involve  collisions  between  protein  molecules  which, 
from  a  kinetic  standpoint,  is  not  incompatible  with  a  rapid  rate  of  reaction. 

In  a  final  experiment  we  tried  to  see  whether  the  lactic  dehydrogenase  system  could 
reduce  the  DPN  bound  to  the  enzyme.  The  reaction  mixture  contained  4  mg  of  glycer- 
aldehyde phosphate  dehydrogenase  per  ml  as  a  source  of  DPN,  sodium  lactate,  cyanide 
(to  trap  the  pyruvate  formed)  and  a  catalytic  amount  of  lactic  dehydrogenase.  The 
bound  DPN  was  reduced  at  a  good  rate  as  soon  as  the  lactic  dehydrogenase  was  added. 
The  considerations  mentioned  above  when  the  reverse  reaction  was  discussed  apply 
here  as  well. 


IODOACETATE 

Iodoacetate  (0.004  M)  completely  inhibited  the  reduction  of  enzyme  DPN  by  sub- 
strate. An  enzyme  solution  of  about  8  mg  of  protein  per  ml  was  prepared  with  pyro- 
phosphate buffer  at  pn  8.4  containing  no  cysteine.  Five  minutes  at  25°  was  allowed  for 
reaction  with  iodoacetate  before  glyceraldehyde  was  added.  A  suitable  control  without 
iodoacetate  was  run  simultaneously.  This  was  necessary  because  the  enzyme  loses 
activity  quite  rapidly  in  the  absence  of  cysteine.  No  enzymatic  activity  could  be  detected 
in  the  presence  of  iodoacetate.  Whether  or  not  a  differentiation  of  two  catalytic  sites 
is  possible  by  means  of  addition  of  smaller  concentrations  of  iodiacetate  has  not  been 
tried. 

In  experiment  B,  Table  V,  iodoacetate  was  added  after  the  DPN  bound  to  the 
enzyme  had  been  reduced.  The  object  was  to  see  whether  the  inactivation  of  the  enzyme 
by  iodoacetate  would  influence  the  rate  of  reaction  of  bound  DPNH  with  the  lactic 
dehydrogenase  system.  As  shown  in  Table  V  no  difference  could  be  detected. 

This  paper  is  presented  as  a  token  of  esteem  for  the  numerous  scientific  contributions 
of  Otto  Meyerhof. 

References  p.  i6g. 


i68  c.  F.  coRi  d  al.  vol.  4  (1950) 

SUMMARY 

The  theory  has  been  examined  that  glyceraldehyde  phosphate  dehydrogenase  from  rabbit 
muscle  contains  two  catalytic  sites,  having  dissociation  constants  with  DPN  which  differ  by  a  factor 
of  100  or  more.  The  facts  in  favour  of  a  very  slightly  dissociated  site  are  that  the  enzyme  retains 
on  recrystallization  or  dialysis  a  stoichiometric  amount  of  DPN.  From  observations  made  in  kinetic 
measurements  this  DPN  does  not  measurably  dissociate  on  five  fold  dilution  of  the  enzyme.  Further- 
more, evidence  is  presented  that  DPNH  is  also  bound  to  the  enzyme  and  that  it  can  be  displaced 
by  added  DPN  to  an  extent  which  indicates  relative  affinities  of  the  protein  for  the  oxidized  and 
reduced  forms  of  at  least  the  same  order  of  magnitude.  The  fact  that  bound  DPN  can  be  removed 
from  the  enzyme  by  adsorption  on  charcoal  and  that  it  exchanges  rapidly  with  DPN  labelled  with 
P32  allows  the  conclusion  (a)  that  the  binding  is  not  of  the  covalent  type  and  (b)  that  bound  DPN 
has  a  measurable  dissociation. 

Other  approaches  to  the  problem  did  not  reveal  differences  between  the  reaction  with  enzyme- 
DPN  and  the  reaction  with  a  catalytic  amount  of  enzyme  plus  added  DPN.  In  both  cases,  in  the 
presence  of  an  excess  of  substrate,  the  reaction  was  first  order  with  respect  to  the  total  DPN  concen- 
tration, and  the  pn  optimum  was  the  same.  The  equilibrium  constants  with  bound  and  with  added 
DPN  were  also  the  same.  lodoacetate  inhibited  the  reaction  at  the  bound  site.  Kinetic  studies  in- 
volving simultaneous  reaction  of  bound  and  added  DPN  showed  that  with  increasing  concentrations 
of  the  latter  a  saturation  value  was  approached,  but  the  data  could  not  be  resolved  to  give  an  une- 
quivocal answer  in  terms  of  two  catalytic  sites. 

Enzyme  DPNH  was  shown  to  react  rapidly  with  lactic  dehydrogenase  plus  pyruvate,  or  in  the 
reverse  reaction,  bound  DPN  was  found  to  react  with  lactic  dehydrogenase  plus  lactate.  On  the  basis 
of  the  assumption  that  bound  DPNH  has  a  very  low  dissociation,  the  observed  rate  of  reaction  with 
lactic  dehydrogenase  would  have  to  be  attributed  to  collisions  between  protein  molecules. 

In  the  light  of  available  evidence  the  hypothesis  that  glyceraldehyde  phosphate  dehydrogenase 
has  two  catalytic  sites  which  differ  in  their  affinity  for  DPN  requires  further  examination. 

RfiSUMfi 

Un  examen  a  ^te  fait  de  la  theorie  selon  laquelle  la  deshydrog6nase  de  I'aldehyde  phospho- 
glycerique  du  muscle  de  lapin  possederait  deux  positions  catalytiques  dont  les  constantes  de  dissocia- 
tion avec  le  DPN  difif^reraient  par  un  facteur  de  loo  ou  davantage.  Les  faits  en  faveur  d'une  position 
ou  la  dissociation  est  tres  faible  sont  que  I'enzyme,  lors  de  la  recristallisation  ou  de  la  dialyse,  retient 
une  quantity  sto^chiometrique  de  DPN.  D 'observations  faites  au  cours  de  mesures  cin^tiques,  il 
d^coule  que  ce  DPN  ne  dissocie  pas  d'une  fa9on  appreciable  lorsqu'on  dilue  I'enzyme  au  cinquieme. 
En  outre,  des  preuves  sont  apport^es  que  le  DPNH  est  lui  aussi  lie  a  I'enzyme  et  peut  etre  deplace 
de  cette  combinaison  par  I'addition  de  DPN,  jusqu'a  une  limite  qui  indique  que  les  affinites  relatives 
de  la  prot^ine  pour  la  forme  oxyd^e  et  pour  la  forme  r^duite  sont  en  tout  cas  du  meme  ordre  de 
grandeur.  Le  fait  que  le  DPN  li^  peut  etre  ^limind  de  I'enzyme  par  adsorption  a  du  charbon  actif,  et 
qu'il  s'^tablit  un  ^change  rapide  avec  du  DPN  marque  au  P^^  permet  de  conclure:  a)  que  le  mode  de 
liaison  n'est  pas  du  type  covalent  et  b)  que  le  DPN  possede  une  dissociation  mesurable. 

D'autres  m^thodes  d'approche  du  probleme  pos6  n'ont  pas  r^vele  de  differences  entre  la  reaction 
de  la  combinaison  enzyme-DPN  et  celle  d'une  quantite  catalytique  d'enzyme  plus  du  DPN  additionn^. 
Dans  les  deux  cas,  en  presence  d'un  exces  de  substratum,  la  reaction  6tait  du  premier  ordre  par 
rapport  a  la  concentration  totale  en  DPN,  et  le  pjj  optimum  6tait  le  meme.  Les  constantes  d'6quilibre 
avec  du  DPN  116  ou  additionn^  ^talent  ^galement  identiques.  L'acide  iodac^tique  inhibe  la  reaction 
au  point  de  liaison.  Des  etudes  cin6tiques  impliquant  la  reaction  simultan^e  de  DPN  116  et  de  DPN 
additionn6  ont  montr6  que  lorsque  les  concentrations  de  ce  dernier  augmentent,  on  tend  vers  une 
valeur  de  saturation,  mais  il  n'a  pas  ete  possible  d'ordonner  les  r^sultats  de  fa9on  a  donner  une 
r^ponse  non  Equivoque  a  la  question  de  I'existence  de  deux  positions  catalytiques. 

II  a  et6  montr6  que  le  DPNH  lie  r6agit  rapidement  avec  la  d6shydrog6nase  lactique  plus  pyru- 
vate, ou,  en  sens  inverse,  le  DPN  116  avec  la  d6shydrog6nase  lactique  -f-  lactate.  Si  Ton  assume  que 
le  DPNH  116  dissocie  tres  faiblement,  la  vitesse  observ6e  de  la  r6action  avec  la  d6shydrog6nase  lactique 
devrait  etre  attribu6e  a  des  collisions  entre  des  mol6cules  de  prot6ine  ou  a  la  formation  de  complexes 
enzymatiques  organis6s.  A  la  lumiere  des  faits  6tablis,  I'hypothese  que  la  d6shydrog6nase  de  I'alde- 
hyde phospho-glyc6rique  possede  deux  positions  catalytiques  diff6rant  par  leur  affinit6  pour  le  DPN 
demande  de  nouvelles  6tudes. 

ZUSAMMENFASSUNG 

Es  wurde  die  Theorie  untersucht,  welche  besagt  dass  Glycerinaldehydphosphat-Dehydrogenase 
aus  Kaninchenmuskel  zwei  katalytische  Stellen  besitzt,  deren  Dissoziationskonstanten  mit  DPN  um 

References  p.  i6g. 


VOL.  4  (195O)      DPN  AND  GLYCERALDEHYDE  PHOSPHATE  DEHYDROGENASE  169 

mehr  als  das  Hundertfache  von  einander  abweichen.  Die  Tatsachen,  die  fiir  eine  sehr  wenig  disso- 
ziierte  Haftstelle  sprechen,  sind  die,  dass  das  Enzym  beim  Umkristallisieren  oder  bei  der  Dialyse 
eine  stochiometrische  Menge  DPN  zuriickhalt.  Aus  Beobachtungen  bei  kinetischen  Messungen  geht 
hervor,  dass  dieses  DPN  bei  fiinffacher  Verdiinnung  des  Enzyms  nicht  messbar  dissoziiert.  Obendrein 
werden  Belege  dafiir  erbracht,  dass  auch  DPNH  an  das  Enzym  gebunden  ist,  und  aus  dieser  Verbin- 
dung  durch  zugesetztes  DPN  verdrangt  werden  kann  bis  zu  einem  Grade,  welcher  relative  Affinitaten 
des  Proteins  zur  oxydierten  und  zur  reduzierten  Form  von  mindestens  gleicher  Grossenordnung 
anzeigt.  Die  Tatsache,  dass  gebundenes  DPN  durch  Adsorption  an  Kohle  aus  dem  Enzym  entfernt 
werden  kann,  und  dass  die  Austauschreaktion  mit  DPN  welches  mit  P^-  markiert  ist  eine  rasche  ist, 
erlaubt  den  Schluss:  a)  dass  die  Bindung  nicht  covalenter  Art  ist  und  b)  dass  gebundenes  DPN 
messbar  dissoziiert. 

Andere  Angriffsarten  auf  das  gestellte  Problem  zeigten  keine  Unterschiede  auf  zwischen  der 
Reaktion  mit  Enzym-DPN  und  der  Reaktion  mit  einer  katalytischen  Menge  Enzym  plus  zugesetztem 
DPN.  In  beiden  Fallen  war,  in  Gegenwart  eines  Uberschusses  an  Substrat,  die  Reaktion  erster  Ord- 
nung  mit  Bezug  auf  die  gesamte  DPN-Konzentration  und  das  pn-Optimum  war  das  Gleiche.  Die 
Gleichgewicntskonstante  mit  gebundenem  und  mit  zugesetztem  DPN  war  ebenfalls  dieselbe.  Jod- 
acetat  hinderte  die  Reaktion  an  der  Bindungsstelle.  Kinetische  Untersuchungen,  bei  welchen  gleich- 
zeitig  gebundenes  und  zugesetztes  DPN  reagierte,  zeigten  an,  dass  man  sich  mit  wachsender  Konzen- 
tration  des  Letzteren  einem  Sattigungswert  naherte;  jedoch  konnten  die  Ergebnisse  nicht  so  darge- 
stellt  werden,  dass  sie  eine  unzweideutige  Antwort  auf  die  Frage  gegeben  hatten,  ob  zwei  katalytische 
Stellen  bestehen. 

Es  wurde  gezeigt,  dass  gebundenes  DPNH  rasch  mit  Milchsaure-Dehydrogenase  plus  Pyruvat 
reagierte,  oder  in  umgekehrter  Richtung  gebundenes  DPN  mit  Milchsaure-Dehydrogenase  plus 
Lactat.  Auf  der  Grundlage  der  Annahme,  dass  gebundenes  DPNH  sehr  wenig  dissoziiert,  miisste 
die  beobachtete  Reaktionsgeschwindigkeit  mit  Milchsaure-Dehydrogenase  durch  Zusammenstosse 
zwischen  Proteinmolekeln  erklart  werden,  oder  durch  die  Bildung  von  geordneten  Enzym-Kom- 
plexen.  Im  Lichte  der  vorhandenen  Belege  gesehen  bedarf  die  Hypothese,  dass  Glycerinaldehyd- 
phosphat-Dehydrogenase  zwei  katalytische  Stellen  besitzt,  welche  sich  in  ihrer  Afhnitat  fiir  DPN 
unterscheiden,  weiterer  Untersuchung. 

REFERENCES 

^  J.  F.  Taylor,  S.  F.  Velick,  G.  T.  Cori,  C.  F.  Cori,  and  M.  W.  Slein,/.  Biol.  Chem.,  173  (1948)  619. 

2  G.  T.  Cori,  M.  W.  Slein,  and  C.  F.  Cori,  /.  Biol.  Chem.,  iji  (1948)  605. 

3  O.  Warburg  and  W.  Christian,  Biochem.  Z.,  303  (1939)  40. 
*  O.  Meyerhof  and  p.  Oesper,  /.  Biol.  Chem.,  170  (1947)  i. 

^  F.  KuBowiTZ  AND  P.  Ott,  BiocHcm.  Z.,  314  (1943)  94. 

Received  April  19th,  1949 


170  BIOCHIMICA   ET   BIOPHYSICA   ACTA  VOL.  4  (1950) 


GARUNG  und  phytochemische  reduktion 


CARL  NEUBERG 

Polytechnic  Institute  of  Brooklyn,  New  York,  N.Y.  {U.S.A.) 


Rein  chemische  Erfahrungen  haben  vor  Decennien  den  Gedanken  nahe  gelegt,  dass 
der  Abbau  der  Hexosen  in  der  Natur  iiber  Stoffe  der  3-Kohlenstoffreihe  erfolge.  Es  ist 
namentlich  die  1871  von  Hoppe-Seyler  aufgefundene  Entstehung  von  rf,/-Milchsaure 
aus  Traubenzu'cker^  gewesen,  die  auf  diesen  Gedanken  gefiihrt  hat.  Er  wurde  befestigt 
durch  bestatigende  und  erweiternde  Beobachtungen^  von  Schutzenberger  (1876), 
Nencki  und  Sieber  (1881)  und  Kiliani  (1882).  Eine  Umwandlung  etwa  von  ^-Glucose 
zu  einer  der  optisch  aktiven*  Raumformen  der  Milchsaure  oder  auch  nur  eine  einfache 
Depolymerisation  zu  Triosen,  zu  optisch  aktivem  Glycerinaldehyd  oder  zu  Dioxyaceton, 
war  nicht  ausgefiihrt.  Der  umgekehrte  Vorgang,  die  Condensation  von  Triosen  zu  race- 
mischen  Hexosen,  war  in  den  Jahren  1887-1890  verwirkhcht;  er  bildet  eine  der  Grund- 
lagen  der  Zuckersynthesen  von  Emil  Fischer.  In  friihe  Zeit  (1904)  fallen  die  ersten 
physiologischen  Versuche  mit  Glycerose,  dem  Gemisch  von  Dioxyaceton  und  ^,/-Gly- 
cerinaldehyd,  das  schon  damals*  in  ziemlich  reinem  Zustande  erhaltlich  war.  Mit  diesem 
Material  haben  Neuberg  und  Blumenthal^  den  ersten  experimentellen  Beweis  dafiir 
geliefert,  dass  Triosen  im  Tierkorper  zu  optisch  aktiven  Hexosen  condensiert  werden, 
und  Glycogenbildner  sind.  Diese  Feststellung  ist  dann  vielfach  bestatigt  worden,  so  von 
MosTOWSKY,  Parnas,  Embden  und  Mitarbeitern,  Ringer  und  Frankel,  Stohr^  u.a. 
Diese  und  eine  Reihe  ahnlicher  Befunde,  d.h.  Biosynthesen  von  Hexosen  mittels  niederer 
Zucker,  waren  als  Beispiele  einer  Aldolcondensation  verstandlich.  Der  stereochemische 
gerichtete  Verlauf  war  mit  den  Prinzipien  der  asymmetrischen  Synthese  erklarlich. 
Dagegen  war  der  Mechanismus  des  biochemischen  Zuckerabbaus  unerforscht.  Es  fehlten 
z.B.  alle  Grundlagen  fiir  die  Herleitung  der  Methylgruppe,  wie  sie  fiir  die  typischen 
Produkte  der  Glycolyse,  fiir  Milchsaure  und  Weingeist,  charakteristisch  ist.  Dieses 
Problem  ist  der  Losung  zugefuhrt  mit  der  1911  begriindeten  Lehre  von  der  Rolle  der 
Brenztraubensaure  fiir  den  Umsatz  der  Zucker.  Damit  war  die  Aera  eingeleitet,  in  der 
die  biochemische  Zerreissung**  der  6-Kohlenstoff  kette,  die  Desmolyse  der  Zymohexosen, 
zu  Substanzen  der  3-Kohlenstoffreihe  experimentell  bewiesen  wurde.  Mit  der  halftigen 
Aufteilung  der  Hexose  in  2  Mol  Methylglyoxal-hydrat  {C^Ri^Pq  =  2C3H6O3),  die  1928- 
1929  Neuberg  und  Kobel^  mit  verschiedenen  Enz3^mpraparaten  pflanzlicher  und 
tierischer  Provenienz  herbeifiihrten,  schien  das  Problem  gelost.  Meyerhof  und  Loh- 
mann^  zeigten   1934,   dass  unmittelbare  Vorlaufer  des  isolierten  Methylglyoxals  die 

*  Die  Behauptung  Duclaux's,  dass  im  Sonnenlicht  aus  einer  alkalischen  Glucoselosung  d- 
Milchsaure  in  grosser  Ausbeute  entstehe,  ist  nach  Jacobsohn*  auf  eine  Verwechslung  mit  optisch 
aktiven  Saccharinsauren  zuriickzufiihren. 

**  Fiir  den  Vorgang  der  enzymatischen  Trennung  von  -C-C-  Bindungen  hat  sich  die  1925  von 
Neuberg  und  Oppenheimer^  eingefiihrte  Bezeichnung  Desmolyse  eingebiirgert. 

Liieratur  S.  lyyIiyS. 


VOL.  4  (1950)  GARUNG    UND    PHYTOCHEMISCHE    REDUKTION  I7I 

Trioseii  sind,  und  zwar  in  Form  ihrer  unter  den  Versuchsbedingungen  zum  Zerfall  in 
Methylglyoxal  neigenden  Phosphorsaureester^".  Das  Ausgangsmaterial  fiir  das  des- 
molytisch  gebildete  Methylglyoxal  ist  das  Fructosediphosphat  gewesen.  Schon  diese 
Tatsache  sprach  fiir  eine  hierbei  massgebliche  Rolle  der  Phosphorylierung.  Eine  solche 
war  niemals  abgelehnt.  Es  ist  jedoch  in  manchen  Darstellungen  nicht  beachtet,  dass  in 
den  damaligen  Foimulierungen  die  Beteiligung  der  Phosphorsaure  der  Vereinfachung 
wegen  fortgelassen  wurde  und  ausdriicklich  bemerkt  ist,  dass  phosphorylierte  Zwischen- 
stufen  und  die  Triosen  dem  gegebenen  Abbild  ungezwungen  eingefiigt  werden  konnen, 
sobald  sie  nachgewiesen  sein  wiirden^^. 

Gleichfalls  in  die  3-KohIenstoffreihe  fiihrten  drei  andere  biochemische  Reaktionen 
der  Zymohexosen :  die  1917  bekannt  gegebene  Spaltung  in  Glycerin,  Kohlendioxyd  und 
Acetaldehyd  C^Kn^e  =  CH^OH.CHOH.CHpH  +  CO,  +  CH3.CHO  (II.  Vergarungs- 
form),  die  1919  beschriebene  Zerlegung  in  Glycerin,  Kohlendioxyd,  Aethanol  und  Essig- 
saure  2C6H12O6  +  H^O  =  2CH.PH.CHOH.CH2OH  +  2CO2  +  C2H5OH  +  CH3.COOH 
(III.  Vergarungsform) ,  und  schliesslich  die  Aufteilung  der  Hexose  in  aequimole- 
kulare  Mengen  Glycerin  und  Brenztraubensaure  CgHiaOg  =  CH2OH.CHOH.CH2OH  + 
+  CH3.CO.COOH  (IV.  Vergarungsform).  Die  letzte  ist  experimentell  am  spatesten 
(1929)  begriindet.  Infolge  der  Ausschaltung  des  Carboxylase-Systems  findet  man  hier 
Primarprodukte.  Unter  den  Bedingungen  des  Abfangverfahrens  ist  dagegen  noch 
carboxylatische  Spaltung  der  Brenztraubensaure  moglich,  und  es  entstehen  die  Erzeug- 
nisse  der  2.  Vergarungsform,  wahrend  bei  schwach  alkalischer  Reaktion,  welche  die 
biochemische  Dismutation*  des  Acetaldehyds  begiinstigt,  die  Stoffe  der  3.  Vergarungs- 
form auftreten.  Meyerhof,  Lohmann  und  Kiessling^^  haben  gelehrt,  dass  Glycerin 
wie  Brenztraubensaure  phosphorylierte  Vorstufen  haben,  /-Glycerin-  i-phosphorsaure 
einerseits,  Enol-phosphobrenztraubensaure,  bzw.  in  Position  2  und  3  phosphorylierte 
^-Glycerinsaure  anderseits.  Phosphoglycerinsaure,  die  schon  1928  synthetisiert  war^^, 
ist  1930  in  einer  denkwiirdigen  Arbeit  Nilsson's^*  als  Produkt  einer  von  Fluorid 
beeinflussten  Zuckerspaltung  durch  Hefe  entdeckt  worden.  Die  urspriinglich  schwer 
erhalthche  Substanz  konnten  Neuberg  und  Kobel^^  mit  biochemischer  Methodik  als 
schon  kristallisierendes  saures  Bariumsalz  leicht  zuganglich  machen.  Sie  zogen,  wie  auch 
NiLSSON^^,  die  Schlussfolgerung,  dass  die  Verbindung  als  normales  Zwischenprodukt  der 
Glycolyse  fungieren  moge,  da  sie  diese  Substanz  mittels  Hefen  und  Milchsaurebakterien 
in  Brenztraubensaure  iiberfiihren  und  im  Gegensatz  zu  freier  Glycerinsaure**  vergaren 
konnten.  In  Wiirdigung  ihrer  sich  immer  mehr  offenbarenden  Bedeutung  ist  sie  von 
Meyerhof  und  Embden  als  Glied  in  die  Kette  der  obligatorischen  Zwischenprodukte 
der  Desmolyse  eingereiht  worden.  Substrate  der  Carboxylase  und  Ketonaldehydmutase 
sind  Brenztraubensaure  und  Methylglyoxal,  und  die  spater  nachgewiesenen  umbauenden 
Enzyme  Isomerase,  Phosphoglyceromutase,  Enolase  u.s.w.greifen  ebenfalls  an  3-Kohlen- 
stoffverbindungen  an.  Somit  ist  es  selbstverstandlich,  dass  diese  Substanzen  in  jedem 


*  Wenn  Hefe  Acetaldehyd  statt  zur  Dismutation  zu  carboligatischer  Erzeugung  von  Acyloin 
verwendet,  so  ist  nach  L.  Elion  [Biochem.  Z.,  169  (1926)  471)  auch  unter  diesen  Bedingungen,  wie 
bei  der  2.,  3.  und  4.  Vergarungsform,  Glycerin  das  Reduktionsaequivalent  zur  Oxydationsstufe 
Acetaldehyd. 

**  Freie  Glycerinsaure  wird  unter  keiner  Bedingung  von  Hefe  vergoren.  Das  ist  schon  von 
C.  Neuberg  und  J.  Kerb  [Ber.,  47  (1914)  1308)  und  unter  kritischer  Beriicksichtigung  der  Literatur 
spater  wieder  von  O.  v.  Schonebeck  [Biochem.  Z.,  276  (1935)  421)  dargetan.  Dagegen  greifen  Bak- 
terien,  die  Hefe  evtl.  verunreinigen,  nach  C.  Antoniani  {Biochem.  Z.,  267  (1933)  380)  freie  Glycerin- 
saure an.  Siehe  auch  A.  I.  Virtanen,  [Biochem.  Z  .,279  (1935)  262)  und  I.  Tikka  [Biochem.  Z.,  279 
(1935)  264). 

Literatur  S.  lyjiiyS. 


172  C.  NEUBERG  VOL.  4  (1950) 

Schema  der  glycolytischen  Processe  zentrale  Platze  einnehmen.  Das  Kernstiick  bleibt 
immer  die  primare  Desmolyse  zur  Stufe  der  3-Kohlenstoffverbindungen.  Das  kommt  in 
dem  Schema  von  Neuberg^^  zum  Ausdruck,  das  die  bis  zum  Jahre  1933  festgestellten 
Tatsachen  zu  erklaren  versucht,  und  in  den  fortentwickelten  Paradigmen  von 
Embden,  Deuticke  und  Kraft^^,  sowie  von  Meyerhof^^  und  Cori^",  wo  die  vor  der 
eigentlichen  Desmolyse  liegenden  Umformungen  und  die  generelle  Rolle  der  Phosphory- 
lierung  und  Dephosphorylierung  ausfiihrlich  beriicksichtigt  sind.  Erhebliche  Fort- 
schritte  sind  zu  verzeichnen,  namentlich  ist  die  Beteiligung  der  Cofermente  und  an- 
organischen  Erganzungsstoffe,  sowie  die  Reversibilitat  der  meisten  Reaktionsfolgen 
erkannt.  Was  die  primare  Desmolyse  als  den  charakteristischen  Ausdruck  der  Glycolyse 
anbelangt,  so  ist  der  Ubergang  der  Hexosen  zur  Wertigkeitsstufe  der  Triosen  die  inte- 
grierende  Reaktion  geblieben.  Auch  die  Massnahmen,  die  zur  Abfangung,  Anhaufung 
und  Isolierung  von  Intermediargebilden  oder  zu  Stabilisierungsprodukten  (Essigsaure, 
Glycerin)  fiihren,  sind  prinzipiell  von  gleicher  Art.  Durch  kiinstliche  Eingriffe  wird 
irgendwie  die  normale  Korrelation  der  Biokatalysatoren  gestort  und  die  Weiterver- 
arbeitung  unterbunden,  mag  dies  durch  Fixierung  eines  Zwischenproduktes,  Abschwa- 
chung  eines  der  Partialagentien,  durch  Zusatze  oder  Verdiinnung* ,  durch  Ferment-oder 
Coferment-  ausschaltung  oder  spezielle  Begiinstigung  einer  der  Enzymreaktionen  ge- 
schehen. 

Wir  sind  liber  die  biochemische  Bildungsweise  diverser  3-Kohlenstoff  korper  unter- 
richtet.  Ungeklart  ist  bis  heute,  wie  das  Glycerin  entsteht,  das  in  kleinen  Mengen  bei  der 
normalen  alkoholischen  Garung  auftritt.  Die  Herleitung  aus  den  Triosen  lage  nahe,  da  die 
rein  chemische  Reduktion  des  Dioxyacetons^^  wie  des  Glycerinaldehyds^^  zum  Glycerin 
keine  Schwierigkeiten  bietet.  Schon  be  vor  Meyerhof  die  bedeutsame  Isolierung  der 
phosphorytierten  Triosen  in  Substanz  gegliickt  war,  hat  man  mehrfach  inGarfliissigkeiten 
und  Zellelementen  kleine  Mengen  eines  Materials  beobachtet**  (Iwanoff,  v.  Euler, 
Warkany,  Kluyver,  Struyk,  Boyland,  Dische  u.a.) ,  das  bei  der  Destination  mit  H2SO4 
von  20%  das  leicht  nachweisbare  Methylglyoxal  liefert.  Wahrscheinlich  handelt  es  sich 
um  gebundene,  nicht  um  freie  Triosen.  Dass  erstere  durch  Dismutationsreaktionen  Gly- 
cerophosphat  liefern  konnen,  haben  Meyerhof  und  Kiessling^*  dargetan.  Die  erste  in 
der  Hefe  aufgefundene  Phospho-monoesterase  ist  die  Glycerophosphatase.  Sie  spaltet,  wie 
friihzeitig^^  dargetan  ist,  leicht  die  Salze  der  Glycerinphosphorsaure.  So  erscheint  es 
moglich,  dass  die  bei  der  2.  und  3.  Vergarungsform  gebildeten  Stoffe,  insbesondere  das 
Glycerin,  iiber  phosphorylierte  Vorstufen  entstehen.  Es  ware  bei  den  jetzt  erkannten 
Beziehungen  zwischen  enzymatischer  Zuckerspaltung  und  Bioreduktion^®  auch  denkbar, 
dass  die  Triosenphosphate  zunachst  der  Dephosphorylierung  anheimfallen  und  dann  der 
phytochemischen  Reduktion  zu  Glycerin  unterliegen.  Die  nachstehend  beschriebenen 
Versuche  mit  monomolekularem  Dioxyaceton  und  ^,Z-Glycerinaldehyd  lehren,  dass 
keiner  dieser  Stoffe  durch  garende  Ober-  und  Unterhefe  in  Glycerin  iibergefiihrt  wird. 
Da  beide  Triosen  quantitativ  iibrigbleiben,  scheidet  auch  die  Eventualitat  aus,  dass 

Zur  Theorie  des  Verdiinnungseffekts,  siehe  F.  Lynen^^. 
**  Lit.  s.  bei  M.  Kobel  und  C.  Neuberg,  35.  Meeting  of  the  Soc.  of  American  Bacteriologists, 
Philadelphia  1933;  Biochem.  Z.,  269  (1934)  41^  und  273  (1934)  445-  Sie  konnten  durch  zweckmassige 
Versuchsanordnung  die  bis  dahin  nur  als  minimal  befundene  Quantitat  <  1%  auf  31%  steigern. 
Hinzuzufiigen  ist  als  allem  Anschein  nach  alteste  einschlagige  Angabe  cine  Notiz  von  F.  Bordas 
UND  De  Razkowski  (Compt.  rend.,  126  (1898)  1050).  Ihr  zufolge  sollen  in  umgeschlagenen  (turned) 
franzosischen  W^einen  3  Bakterienarten  vorkommen,  die  Glucose  spurenhaft  in  Dioxyaceton  um- 
wandeln.  Experimentell  ist  diese  Behauptung  nicht  hinreichend  gestiitzt,  vielleicht  hat  es  sich  um 
Acetylmethylcarbinol  gehandelt. 

Literatur  S.  lyyjiyS. 


VOL.  4  (1950)  GARUNG    UND    PHYTOCHEMISCHE    REDUKTION  I73 

eine  Componente  des  raceniischen  Glycerinaldehyds  in  Reaktion  trate.  Im  Gegensatz 
zu  den  Triosenphosphaten  sind  somit  die  freien  Triosen  fiir  gewohnliche  Hefe  (s.  S. 
174)  unter  den  obwaltenden  anaeroben  Bedingungen  keine  angreifbaren  Substrate. 
Dass  Phosphorylierung  die  biologische  Dignitat  einer  Substanz  vollig  verandert,  ist 
ausser  an  dem  erwahnten  Beispiel  der  Glycerinsaure  (s.  S.  172)  auch  sonst  beobachtet,  so 
von  Pringsheim"  bei  ^-Galactose-phosphat  und  namentlich  von  Warburg^  und 
DiCKENS^^  fiir  die  Oxydasen  der  GIucose-6-phosphorsaure  bezw.  6-Phosphogluconsaure. 
Die  P-freien  Stoffe  sind  keine  Substrate  fiir  diese  Enzyme. 

Die  Resistenz  der  Triosen  beruht  nicht  auf  einer  Schadigung  der  benutzten  Hefe 
durch  die  3-Kohlenstoffzucker.  Die  abzentrifugierte  Hefe  erweist  sich  als  ungeschwacht. 
In  Gegenwart  beider  Triosen  werden  zugesetzte  Zymohexosen  glatt  vergoren*.  Der  von 
Lehmann  und  Needham^"  angegebene  Einfluss  des  Glycerinaldehyds  auf  die  glycoly- 
tischen  Vorgange  macht  sich  nicht  geltend,  er  ist  auch  in  den  Versuchen  von  Neuberg 
UND  HoFMANN^^  nicht  zu  Tage  getreten. 

Das  Verhalten  der  Triosen  ist  insofern  unerwartet,  als  die  nahestehenden  Sub- 
stanzen  Milchsaurealdehyd,  CH3.CHOH.CHO,  und  Acetol,  CH3.CO.CH2OH,  die  als 
Desoxyderivate  vom  Glycerinaldehyd  und  Dioxyaceton  aufgefasst  werden  konnen,  und 
das  Anhydrid  der  Triosen,  das  Methylglyoxal,  CH2:CH(0H).CH0,  der  Bioreduktion  zu 
dem  mit  Glycerol  nahe  verwandten  Propylenglycol  zuganglich  slnd^^. 

Die  normale  Funktion  der  benutzten  Hefe  offenbart  sich  ferner  in  Versuchen,  die 
im  Anschluss  beschrieben  seien,  obzwar  sie  mit  der  Glycerinfrage  als  solcher  nichts  zu 
tun  haben.  Die  phytochemische  Reduktion  des  Cyclopentanons  zum  Cyclopentanol  sowie 
die  des  d-  und  d,l-Campherchinons  {2,j-Dioxycaniphans)  gelingt  ohne  Schwierigkeiten. 
Sie  wird  im  letzten  Falle  halbseitig  vollzogen,  indem  in  der  Hauptsache  3-Oxy-campher 
entsteht.  Die  Bioreduktion  des  ^,/-Campherchinons  verlauft  partiell  asymmetrisch. 
Dasselbe  trifft  fiir  die  phytochemische  Reduktion  des  d,l-Methyl-n-propylacetaldehyds 
(Isocapronaldehyds)  zu,  die  2-Methyl*pentanol-i  mit  einem  Uberschuss  an  hnks- 
drehender  Form  liefert. 

Auf  Kosten  vergarender  Zucker  ist  somit  die  Bioreduktion  in  der  Cyclopentanreihe 
und  bei  einem  o-Chinon  der  hydroaromatischen  Reihe  moglich.  Selbst  ein  so  ober- 
flachenaktiver  Stoff  wie  der  erwahnte  Hexylalkohol  verhindert  den  Eintritt  der  Bio- 
reduktion nicht. 

Der  Beginn  der  hier  mitgeteilten  Versuche  reicht  langer  zuriick.  Zu  verschiedenen 
Zeiten  haben  daran  mitgearbeitet  Prof.  Dr  N.  N.  Iwanoff,  Leningrad,  Dr  Hilda 
LusTiG,  New  York,  und  Dr  Elisabeth  Peiser,  BerHn.  Ihnen  alien  schulde  ich  Dank. 
Ich  statte  ihn  in  trauernder  Erinnerung  ab,  alle  drei  weilen  nicht  mehr  unter  den 
Lebenden. 

A.    VERSUCHE    MIT   GLYCERINALDEHYD 

Kristallisierter  «f, /-Glycerinaldehyd  ist  jetzt  unschwer  zuganglich'^  wird  er  in  wassriger  Losung 
24h  bei  Zimmertemperatur  aufbewahrt,  so  vollzieht  sich  nach  Wohl  und  Neuberg^'  der  tlbergang 
in  die  monomolekulare  Form.  Er  wurde  in  i.o,  0.5  und  0.25%  Concentration  verwendet. 

In  je  100  ml  der  Glycerinaldehydlosung  wurden  10  g  Rohrzucker  oder  Glucose 
gelost.  Auf  Zugabe  von  2-3  g  obergariger  Brennereihefe  trat  bei  25°  schnelle  Garung  ein, 

*  Glycerinaldehyd  kann  sogar  als  Aktivator  der  alkoholischen  Zuckerspaltung  f  ungieren : 
C.  Neuberg  und  M.  Ehrlich,  Biochem.  Z.,  loi  (1920)  242. 

Liieratur  S.  lyyjiyS. 


174  C.  NEUBERG  VOL.  4  (1950) 

die  2-3  Tage  anhielt.  Dann  war  alle  Hexose  verschwunden.  Mit  mehr  Hefe  wurde  keine 
neue  Garung  entfacht.  Die  schon  in  der  Kalte  eintretende  Reduktion  von  FEHLiNG'scher 
Mischung  lehrte,  dass  unveranderte  Triose  vorhanden  war. 

Obgleich  Methoden  zur  Bestimmung  von  Triose  neben  Hexose  ausgearbeitet  sind^*, 
eriibrigte  sich  deren  Anwendung,  da  keine  Hexosen  mehr  zugegen  waren.  Die  zentrifu- 
gierten  Fliissigkeiten,  die  kein  Drehungsvermogen  aufwiesen,  zeigten  gegen  FEHLiNG'sche 
Mischung  dasselbe  Reduktionsvermogen,  wie  die  urspriingHche  Glycerinaldehydlosung; 
die  Reduktionskraft  der  Triose  ist  schon  von  Wohl^^  ermittelt. 

Verdoppelung  der  Mengen  von  Hexose  und  Hefe  sowie  erneuter  Zusatz  von  Glucose 
und  Hefe  nach  beendeter  Garung  (in  toto  3  Mai)  anderte  nichts  an  dem  Ergebnis,  so 
wenig  wie  die  Heranziehung  einer  anderen  Hefesorte  (untergariger  Bierhefe).  Eine 
phytochemische  Reduktion  des  (f,/-GlycerinaIdehyds  war  nicht  nachweisbar. 

B.    VERSUCHE    MIT   DIOXYACETON 

Die  Versuche  mit  monomolekularem  Dioxyaceton  wurden  wie  die  mit  Glycerin- 
aldehyd  ausgefiihrt.  Das  Ergebnis  war  gleich,  alle  Ketotriose  blieb  unverandert. 

Kristallisiertes  Dioxyaceton  ist  nach  Neuberg  und  Hofmann^^  in  einfacher  Weise  erhaltlich. 
Bei  richtiger  Arbeitsweise  kristallisiert  die  Ketotriose  direkt  in  einer  Ausbeute  von  77%,  berechnet 
auf  das  in  Arbeit  genommene  Glycerin,  praktisch  rein  aus*.  Durch  Aufarbeitung  der  Mutterlauge 
Uber  das  2.4-Dinitrophenylhydrazon^2  kann  man  noch  10-14%  3-"  kristallisiertem  Dioxyaceton,  in 
toto  also  90%,  gewinnen.  Der  Rest  diirfte  das  von  Levene  und  Walti^*  beschriebene  polymere 
Condensationsprodukt  enthalten. 

Man  kann  sich  von  den  mitgeteilten  Tatsachen  auch  durch  Anstellung  der  Versuche 
in  kleinstem  Umfange  iiberzeugen.  Statt  eines  titrimetrischen  Verfahrens  wahlt  man 
dann  die  Methode  der  Destination  mit  H2S04^^.  Es  entsteht  dabei  quantitativ  Methyl- 
glyoxal,  und  dieses  kann  jetzt  in  y-Bereichen  bestimmt  werden*".  Voraussetzung  ist 
natiirlich,  dass  keine  Spezialhefen  in  Anwendung  kommen,  die  Triosen  angreifen,  sei  es 
durch  Condensation  zu  Hexosederivaten*^,  sei  es  durch  wirkliche  Vergarung^^. 


C.    VERSUCHE    MIT   CYCLOPENTANON 

Die  Anstellung  kann  in  der  friiher^"  fiir  2-Methylcyclohexanon  angegebenen  Weise 
geschehen.  Zur  Trennung  von  unverandertem  Keton  schaltet  man  zweckmassig  eine 
Rektification  iiber  ^-Nitrophenylhydrazin  oder  2.4-Dinitropenylhydrazin  ein.  Das 
Cyclopentanol  vom  Siedepunkt  141°  wurde  in  einer  Ausbeute  von  42%  isoliert. 

D.  VERSUCHE    MIT   d-   UND   d,  /-CAMPHERCHINON 

Die  phytochemische  Reduzierbarkeit  der  Diketone  ist  am  Beispiel  des  Diacetyls 
aufgefunden^^.  Auch  andere  Polyketone  sind  der  Hydrierung  durch  garende  Hefe 
zuganghch,  solche  der  aliphatischen,  aromatischen  und  heterocyclischen  Reihe^^.  Im 


*  Mit  sehr  ahnlicher  Methodik  haben  auch  Underkofler  und  Fulmer"**  gute  Resultate 
erzielt.  Die  von  ihnen  erhaltene  Ausbeute  ist  etwas  geringer  gewesen.  Die  von  ihnen  angegebenen 
80%  beziehen  sich  namlich  nicht  auf  eingesetztes  Glycerin,  sondern  auf  Prozente  von  reduzierender 
Substanz.  Diese  besteht  ausserdem  nicht  nur  aus  Triose,  vielmehr  ist  nach  Bousfield,  Wright  und 
Walker^'*  ein  starker  reduzierender  Korper  beigemengt. 

Literatur  S.  lyyliyS. 


VOL.  4  (1950)  GARUNG    UND   PHYTOCHEMISCHE    REDUKTION  I75 

Campherchinon  liegt  ein  bequem  zuganglicher  Vertretcr  von  Diketonen  der  hydro- 
aromatischen  Reihe  vor. 

(^-Campherchinon  und  (/, /-Campherchinon  warden  von  garender  Hefe  unschwer  und 
in  erhebhchem  Ausmasse  reduziert.  Die  Hydrierung  konnte  zum  2,3-Dioxycamphan 
fiihren,  aber  auch  zu  einem  Oxy-oxo-campher.  Das  angewendete  Campherchinon  ging 
in  einen  Oxycampher  iiber.  Drehungen  und  Schmelzpunkte  der  Derivate  hegen  denen 
des  3-Oxy-camphers  (2-Oxo-3-oxy-camphans)  am  nachsten.  Die  physikaHschen  Daten 
stimmten  nicht  genau  damit  iiberein,  sondern  sind  ganz  ahnhch  wie  bei  dem  Material, 
das  durch  Verfiitterung  von  2,3-Dioxocamphan  an  Hunde  entsteht.  Hier  tritt  neben 
2-Oxy-3-oxo-campher  ein  nicht  naher  charakterisierter  3-Oxycampher  auf*^.  Auch  die 
rein  chemische  Reduktion  des  Campherchinons  liefert  ein  Isomerengemisch^^. 

Auf  alle  Falle  findet  eine  partielle  Bioreduktion  statt.  Sie  ergreift  nur  eine  der  beiden 
Carbonylgruppen.  Dass  die  phytochemische  Reduktion  in.  Stufen  erfolgt,  ist  fiir  die 
Umwandlungen  des  Diacetyls,  des  Furils  und  auch  sonst  nachgewiesen^^.  Oxyketone 
sind  Zwischenglieder  bei  der  Entstehung  der  Glycole.  Beim  Benzil  ist  bislang  iiberhaupt 
nur  die  biochemische  Bildung  von  Benzoin  zu  erzielen  gewesen^^.  Ob  unter  den  Be- 
dingungen  einer  forcierten  langanhaltenden  phytochemischen  Reduktion,  die  nach 
F.  G.  Fischer  auch  Doppelbindungen  erfasst^®,  die  zweite  Carbonylgruppe  betroffen 
werden  kann,  bleibe  dahingestellt. 

Das  6?-Campherchinon  wurde  nach  der  Vorschrift  von  Evans,  Ridgion  und  Simonsen^*  be- 
reitet;  aus  Ligroin  umkristallisiert  schmolz  es  bei  198°.  [ajo  =  — 92°. 

Fine  Losung  von  10  g  (i-Campherchinon  in  50  ml  Alkohol  lasst  man  zu  dem  garenden 
Gemisch  von  250  g  Backerhefe  und  2.5  Litem  10%  Rohrzuckerlosung  fliessen.  Bei  lang- 
samem  Zusatz  wird  die  COg-Entwicklung  nicht  unterbunden.  Der  Eintritt  der  Umwand- 
lung  ist  ohne  weiteres  daran  zu  erkennen,  dass  die  vom  Chinon  herriihrende  gelbe  Farbe 
verschwindet.  Nach  2-tagiger  Digestion  bei  Zimmertemperatur  saugt  man  die  Hefe 
ab  und  schiittelt  das  Filtrat  mit  Aether  aus.  Nach  Trocknen  des  Aetherextraktes  iiber 
Natriumsulfat  wurde  das  Losungsmittel  abdestilliert.  Es  hinterblieb  ein  farbloser 
kristallinischer  Riickstand,  der,  aus  Petrolather  umkristallisiert,  bei  200-202°  schmolz. 
a  in  ii%iger  alkoholischer  Losung  im  i  dm  -  Rohr  =  +  3.8^°.  [a]p  =  -f  34.9°.  Aus- 
beute  6.3  g.  Aus  dem  Hefeschlamm  liess  sich  mit  Wasserdampf  nur  eine  ganz  geringe 
Menge  einer  fliichtigen  Substanz  abtreiben,  die  vernachlassigt  werden  kann. 

Zur  Identifizierung  wurde  das  Semicarbazon  dargestellt. 

Nach  der  Vorschrift  von  Bredt  und  Ahrens*^  wurden  0.42  g  Semicarbazid-chlorhydrat  und 
0.35  g  KaUumacetat  in  Wasser  gelost,  0.5  g  Substanz  und  soviel  Methylalkohol  hinzugegeben,  dass 
eine  klare  Losung  entstand.  Nach  eintagigem  Stehen  schied  sich  ein  Ol  ab,  das  nach  starker  Ab- 
kiihlung  und  Reiben  mit  einem  Glasstabe  kristallinisch  erstarrte.  Es  wurde  auf  Ton  abgepresst  und 
aus  Petrolather  umkristalhsiert.  Die  Verbindung  schmolz  bei  189°.  a  in  6%  alkoholischer  Losung 
im  I  dm  -  Rohr  =  -\-  0.26°  [a]D  =  4-  4-4°- 

CioHigOiN.NH.CO.NHg.Ber.N  =  18.7%;  gef.  N  =  18.9% 

8  g  J,/-Campherchinon  (aus  synthetischem  ^,/-Campher  bereitet)  wurden  mit  200  g 
Zucker  und  200  g  Hefe  in  2  1  Wasser  vergoren.  Das  Filtrat  wurde  mit  Aether  extrahiert 
und  der  Aetherriickstand  aus  Petrolather  umkristallisiert.  Er  schmolz  bei  200-203°. 
Ausbeute  5  g  an  "3-Oxycampher".  a  im  i  dm  -  Rohr  in  10%  alkoholischer  Losung  = 
-|-  0.47°.  [a]p  =  -f-  4.7°.  Die  phytochemische  Reduktion  verlauft  also  partiell  asym- 
metrisch. 

Literatur  S.  ijyjiyS. 


176  C.  NEUBERG  VOL.  4  (1950) 

E.    VERSUCHE   MIT   ISOCAPRONALDEHYD    (i, /-METHYL-W-PROPYL-ACETALDEHYD) 

Die  Arbeitsweise  fiir  die  phytochemische  Reduktion  des  verwendeten  Isocapron- 
aldehyds  schloss  sich  an  diejenige  an,  welche  fiir  die  entsprechende  Umwandlung  des 
Isovaleraldehyds  angegeben  ist^®. 

10  g  des  racemischen  Ausgangsmaterials  (Kp  115-116°)  lieferten  6.5  g  2-Methyl- 
pentanol-i  (Kp  147-149°).  Dieser  Hexylalkohol  zeigte  (unverdiinnt)  im  2  dm  -  Rohr 
eine  Linksdrehung  von  a  =  —  0.9°.  Fiir  ein  synthetisch  gewonnenes  Produkt,  das 
vielleicht  keine  maximale  Drehung  besessen  hat,  ist  in  der  Literatur^^  [aj^  =  —  1.25° 
angegeben. 

ZUSAMMENFASSUNG 

Im  Anschluss  an  Betrachtungen  iiber  Entstehung,  Verhalten  und  Bedeutung  der  3-Kohlen- 
stoffkorper,  insbesondere  der  freien  wie  phosphorylierten  Triosen,  wird  folgendes  gezeigt:  Gewohn- 
liche  obergarige  und  untergarige  Hefen,  die  Dioxyaceton  und  Glycerinaldehyd  nicht  vergaren,  be- 
vvirken  keine  phytochemische  Reduktion  der  beiden  Triosen  zu  Glycerin.  Die  3-Kohlenstoffzucker 
werden  nicht  verandert.  Sie  sind  in  Konzentrationen  von  i  %  fiir  Hefe  ungiftig  und  verhindern  die 
glatte  Vergarung  zugefiigter  Zymohexosen  nicht.  Die  Resistenz  der  Triosen  gegen  phytochemische 
Reduktion  ist  insofern  bemerkenswert,  als  die  Desoxytriosen,  Acetol  und  Milchsaurealdehj'd,  ebenso 
wie  das  Anhydrid  der  Triosen,  das  Methylglyoxal,  unter  vergleichbaren  Bedingungen  zu  dem  mit 
Glycerol  nahe  verwandten  Propylenglykol  reduziert  werden. 

Die  verwendeten  Hefen  sind  zu  Bioreduktionen  durchaus  geeignet  befunden  worden.  Sie  fiihren 
Cyclopentanon  in  Cyclopentanol,  d-  und  ^./-Campherchinon  durch  Bioreduktion  einer  Carbonylgruppe 
in  Oxycampher  und  Isocapronaldehyd  in  2-Methylpentanol-i  iiber.  Sobald  dazu  die  Moglichkeit 
besteht,  verlauft  die  phytochemische  Reduktion  asymmetrisch.  Diese  selber  ist  nunmehr  auch  in  der 
Cyclopentanreihe  und  bei  einem  Dike  ton  der  hydroaromatischen  Reihe  verwirklicht  worden. 

SUMMARY 

In  connection  with  considerations  about  the  origin,  behaviour,  and  significance  of  C3-substances, 
particularly  free  as  well  as  phosphorylated  trioses,  it  has  been  shown  that :  Ordinary  top  and  bottom 
fermentation  yeasts,  which  do  not  ferment  dihydroxyacetone  or  glyceraldehyde,  effect  no  phyto- 
chemical  reduction  of  the  two  trioses  to  glycerol.  The  Cg-sugars  are  unchanged.  They  are  not  toxic 
to  yeast  in  concentrations  of  1%,  nor  do  they  inhibit  the  smooth  fermentation  of  added  zymohexoses. 
The  resistance  of  the  trioses  to  phytochemical  reduction  is  noteworthy  insofar  as  the  desoxytrioses, 
monohydroxyacetone  and  lactic  aldehyde,  just  like  the  triose  anhydride,  methylglyoxal,  are  reduced 
to  propylene  glycol  (which  is  closely  related  to  glycerol)  under  comparable  conditions. 

The  yeasts  used  have  been  found  to  be  entirely  suitable  for  bioreductions.  They  convert  cyclo- 
pentanone  into  cyclopentanol,  d-  and  rf,/-camphorquinone  by  bioreduction  of  a  carbonyl  group  into 
hydroxycamphor,  and  isocaproic  aldehyde  into  2-methylpentanol-i.  As  soon  as  the  possibihty  exists, 
the  phytochemical  reduction  takes  an  asymmetric  course.  This  has  now  been  carried  out  in  the 
cyclopentane  series  and  with  a  diketone  of  the  hydroaromatic  series. 


RfiSUMfi 

A  la  suite  de  considerations  sur  la  formation,  le  comportement  et  I'importance  des  corps  a  trois 
atomes  de  carbone,  sp^cialement  des  trioses,  tant  libres  que  phosphorylees,  on  montre  ce  qui  suit: 

Des  levures  hautes  ou  basses  ordinaires,  qui  ne  font  pas  fermenter  la  dioxj'ac^tone  et  I'ald^hyde 
glycerique,  ne  provoquent  pas  davantage  de  reduction  phytochimique  de  ces  deux  trioses  en  glycerine. 
Les  deux  corps  ne  sont  pas  transformes.  A  la  concentration  de  1%,  ils  ne  sont  pas  toxiques  pour  la 
levure  et  n'inhibent  pas  la  fermentation  reguliere  de  zymohexoses  additionn^s.  La  resistance  des 
trioses  a  la  reduction  phytochimique  est  d'autant  plus  remarquable  que  les  desoxytrioses,  Tac^tol  et 
I'aldehyde  lactique,  de  meme  que  I'anhydride  des  trioses,  le  methylglyoxal,  sont  r^duits  en  propylfene- 
glycol,  proche  parent  de  la  glycerine,  dans  des  conditions  comparables. 

Les  levures  utilis6es  ont  ete  trouvees  parfaitement  aptes  a  effectuer  des  reductions  biochimiques. 

Literatur  S.  lyjjiyS. 


VOL.  4  {1950)  GARUNG    UND    PHYTOCHEMISCHE    REDUKTION  I77 

Elles  transforment  la  cyclopentanone  en  cyclopentanol;  la  d-  et  la  ci,/-camphoquinone  donnent,  par 
reduction  de  I'un  des  deux  groupes  carbcnyle,  de  I'oxycamphre;  I'ald^hyde  isocaproique  fournit  le 
2-methvlpentanol-i.  Des  que  la  possibilite  en  est  donnee,  la  phytor^duction  prend  un  cours  asyin6- 
trique.  Cette  phytoreduction  a  maintenant  et^  realis^e  aussi  dans  la  sdrie  cyclopentanique  et  chez 
une  dicetone  de  la  serie  hydroaromatique. 

LITERATUR 
1  F.  Hoppe-Seyler,  Ber.,  4  (1S71)  346. 

-    P.    SCHUETZENBERGER,    Bull.    SOC.   chim.    [2]    25    (1876)    289;    M.    NENCKI    UND    N.    SlEBER,  /.    pTakt. 

Chem.  [N.F.]  24  (1881)  498;  H.  Kiliani,  Ber.,  15  (1882)  136  u.  699. 
3  K.  P.  Jacobsohn,  Biochem.  Z.,  215  (1929)  216. 

*  E.  Fischer  und  J.  Tafel,  Ber.,  21  (1888)  2634;  22  (1889)  106;  H.  J.  H.  Fenton  und  H.  Jackson, 
/.  Chem.  Soc,  75  (1899)  4. 

'  C.  Neuberg  und  F.  Blumenthal,  Verhandl.  Berliner  Physiolog.  Ges.  Sitzung  vom  25.  Marz  1904; 
Arch.  Anat.  u.  Physiol.,  Physiol.  Abt.  (1904)  571. 

*  St.  Mostowsky,  Compt.  rend.,  152  (191 1)  1276;  J.  K.  Parnas,  Cenir.  Physiol.,  26  (191 2)  671; 
G.  Embden,  K.  Baldes  und  E.  Schmitz,  Biochem.  Z.,  45  (1912)  108;  A.  J.  Ringer  und  E.  M. 
Frankel,/.  Biol.  Chem.,  15  (1914)  233;  R.  Stohr.Z.  physiol.  Chem.,  20b  [ig^z)  211,  u.  212  (1932)  85. 

■  C.  Neuberg  und  C.  Oppenheimer,  Biochem.  Z.,  166  (1925)  451 ;  C.  Oppenheimer,  Die  Fermente, 
5.  Aufi.  II,  1213  (1926);  C.  H.  Werkman  und  H.  G.  Wood  in  Bamann-Myrbaeck,  Methoden  der 
Fermeniforschuiig  194 1,  1191;  J.  B.  Sumner  und  G.  F.  Somers,  Chetnistry  and  Methods  of  Enzymes, 
2nd.  Ed.  1947,  317. 

8  C.  Neuberg  und  M.  Kobel,  Biochem.  Z.,  203  (1928)  463;  207  (1929)  232;  210  (1929)  466.  Vergl. 
auch  C.  Neuberg  und  M.  Scheuer,  Monatsh.,  53,  54  (1929)  103 1  (Wegscheider-Festschrift). 

9  O.  Meyerhof  UND  K  LoHMANN,  Biochem.  Z.,  271  (1934)  89;  273  (1934)  73  u.  413;  O.  Meyer- 
HOF,  Bull.  soc.  chim.  biol.,  20  (193S)  1033  u.  1345;  21  (1938)  965;  O.  Meyerhof  und  R.  Junowicz- 
KocHOLATY,  /   Biol.  Chem.,  149  (1943)  71. 

1"  Siehe  r.euerdings  auch  E.  Baer  und  H.  O.  L.  Fischer,/.  Biol.  Chem.,  150  (1943)  223;  J.  A.  Sibley 

UND  A.  L.  Lehninger,  /.  Biol.  Chem.,  ijj  (1949)  859. 
11  C.  Neuberg  in  C.  Oppenheimers  Handbuch  der  Biochemie,  II.  Aufl.,  2  (1925)  442;  M.  Kobel  und 

C.  Neuberg,  Klein's  Handbuch  der  Pjlanzenanalyse,  4  (1933)  1253. 
1-  K.  Lohmann  und  O.  Meyerhof,  Biochem.  Z.,  273  (1934)  60;  O.  Meyerhof  und  W.  Kiessling, 

Biochem.  Z.,  283  (1935)  83.  Siehe  namentlich  auch  H.  O.  L.  Fischer,  Naturujissenschajtcn,  25 

(1937)  589;  W.  Kiessling  und  Ph.  Schuster,  Ber.,  yi  (1938)  123. 
1^  C.  Neuberg,  F.  Weinmann  und  M.  Vogt,  Biochem.  Z.,  199  (1928)  248;  M.  Vogt,  Biochem.  Z., 

211  (1929)  I. 
"  R.  Nilsson,  Arkiv  Kemi,  Mineral.  Geol.  10  A  No.  7  (1930)  121. 
"  C.  Neuberg  und  M.  Kobel,  Biochem.  Z.,  263  (1933)  219;  264  (1933)  456.  Siehe  auch  Arch.  Biochem. 

I  (1942)  311- 
^'  R.  Nilsson,  Svensk.  Kem.  Tid.,  45  (1933)  129. 
^'  C.  Neuberg  und  E.  Simon,  Ergeb.  Enzymforsch.,  2  (1933)  118. 
^^  G.  Embden,  H.  J.  Deuticke  und  G.  Kraft,  Klin.  Wochschr.,  12  (1933)  213. 
1*  O.  Meyerhof,  Ergcb.  Physiol.,  39  (1937)  '^°'>  Symposium  on  Respiratory  Enzymes,  The  University 

of  Wisconsin  Press,  Madison,  1942,  9;  Experientia,  4  {1948)  169. 
^°  C.  F.  Cori,  Symposium  on  Respiratory  Enzymes,  The  University  of  Wisconsin  Press,  Madison, 

1942,  188. 
2^  F.  Lynen,  Ann.,  539  (1939)  i. 
22  O.  PiLOTY,  Ber.,  30  (1898)  3161. 
2'  I.  St.  Neuberg,  Biochem.  Z.,  255  (1932)  i. 

2*  O.  Meyerhof  und  W.  Kiessling,  Biochem.  Z.,  267  (1933)  313. 
25  C.  Neuberg  und  L.  Karczag,  Biochem.  Z.,  36  (191 1)  64. 
2S  C.  Neuberg,  Advances  in  Carbohydrate  Chem.,  4  (1949)  75. 
2'   H.  Pringsheim,  Biochem.  Z.,  156  (1925)  109. 
28  O.  Warburg  und  W.  Christian,  Biochem.  Z.,  242  (1931)  206;  O.  Warburg,  W.  Christian  und 

A.  Griese,  Biochem.  Z.,  282  (1935)  167. 
-^  F.  Dickens,  Biochem.  J.,  32  (1938)  1626,  1645. 
^°  H.  Lehmann  und  J.  Needham,  Enzymologia,  5  (1938)  98. 
^1  C.  Neuberg  und  E.  Hofmann,  Biochem.  Z.,  280  (1935)  167. 
^2  H.  Collatz  und  I.  St.  Neuberg,  Biochem.  Z.,  255  (1932)  27. 
^^  A.  WoHL  und  C.  Neuberg,  Ber.,  33  (1900)  3095. 
^*  Beilstein,  Handb.,  E.  I.  429,  E.  II.  892;  A.  I.  Virtanen  und  B.  Baerlund,  Biochem.  Z.,  169 

(1926)  169. 


178  C.  NEUBERG  VOL.  4  (1950) 

■'^  A.  WoHL,  VON  LiPPMANN,  Chcmie  der  Zuckerarten,  Braunschweig  1904. 

36  C.  Neuberg  und  E.  Hofmann,  Biochem.  Z.,  279  (1935)  318.  Vergl.  audi  dieselben  Biochem.  Z., 

224  (1930)  496- 
3^"  L.  A.  Underkofler  und  E.  J.  Fulmer,  /.  Am.  Chem.  Soc,  59  (1937)  3°i- 
^''^  E.  G.  BousFiELD,  G.  G.  H.  Wright  und  T.  K.  Walker,  /.  Instit.  Brewing,  53  (1947)  258. 

38  P.  A.  Levene  und  a.  Walti,  /.  Biol.  Chem.,  78  (1928)  23.  ,, 

39  C.  Neuberg,  E.  Faerber,  A.  Levite  und  E.  Schwenk,  Biochem.  Z.,  83  (1917)  263.  Auch  fiir 
Triosephosphate  anwendbar  siehe  bei  M.  Kobel  und  C.  Neuberg,  Biochem.  Z.,  269  (1934)  44i' 

'"  C.  Neuberg  und  E.  Strauss,  Arch.  Biochem..,  7  (1945)  21 1. 

*^  C.  Neuberg  und  M.  Kobel,  Ann.  brass,  dist.,  27  (1928)  65;  Biochem.  Z.,  203  (1928)  452.  Siehe 

ebenfalls  O.  Meyerhof,  Ann.  brass,  dist.,  27  (1928)  81;  K.  Iwasaki,  Biochem.  Z.,  203  (1928)  237. 
*2  Y.  Ashahino  und  M.  Ishidate,  Ber.,  67  (1934)  71;  F.  Reinartz  und  W.  Zanke,  Ber.,  67  (1934) 

548. 
*^  H.  RuPE  und  W.  Thommen,  Helv.  Chim.  Ada,  30  (1947)  939. 
■•*  W.  C.  Evans,  J.  M.  Ridgion  und  J.  L.  Simonsen,  /.  Chem.  Soc.  (1934)  137. 

**  J.  Bredt  und  H.  Ahrens,  /.  prakt.  Chem.  [2],  112  (1926)  297;  J.  Bredt,  ebenda,  121  (1929)  165. 
*5  P.  A.  Levene  und  L.  A.  Mikeska,  /.  Biol.  Chem.,  84  (1929)  571. 

Eingegangen  den  12.  Mai  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  I79 


ESSAIS  DE  BILANS  DE  LA  FERMENTATION  ALCOOLTOUE 
DUE  AUX  CELLULES  DE  LEVURES 

par 

L.  GENEVOIS 

Faculte  des  Sciences  de  Bordeaux  [France) 


I.  LES    DIVERS    PRODUITS   DE    LA    FERMENTATION    ANAEROBIE 

I.  Essai  d'un  hilan  des  produits  secondaires  de  la  fermentation 

Tres  peu  d'auteurs  se  sont  attaches  a  etablir  un  bilan  complet  de  la  fermentation 
alcoolique.  Recemment,  E.  Peynaud,  au  laboratoire  de  Bordeaux,  s'est  attache  a  suivre 
les  divers  produits  formes  par  la  levure  vivante  a  cote  de  I'alcool;  les  premiers  resultats 
relatifs  a  I'acide  acetique  ont  paru  deja  en  1939^;  les  resultats  principaux  ont  ete  publics 
en  1946,  1947^  et  1948.  lis  verifient  une  hypothese  sur  I'origine  des  produits  formes 
publics  par  I'auteur  de  ces  lignes  des  1936^.  Le  travail  analytique  considerable  fourni 
par  Peynaud  n'a  pas  eu  pour  seul  resultat  d'accumuler  des  chiffres,  ou  meme  de  verifier 
des  hypotheses ;  il  a  apporte  quelques  notions  nouvelles  plus  ou  moins  inattendues. 

L'hypothese  publiee  en  1936^  et  veriiiee  depuis,  etait  la  suivante :  le  glycerol  prend 
naissance  dans  une  fermentation  glyceropyruvique : 

1.  CfiHiA  =  CH2OH.CHOH.CH2OH  +  CH3COCO2H 

L'acide  pyruvique  forme  est  decarboxyle  en  acetaldehyde ;  I'acetaldehyde  est 
dirige  vers  3  voies  differentes : 

2.  a)  elle  est  dismutee  en  alcool  et  acide  acetique 

2CH3CHO  +  H2O  =  CH3CH2OH  +  CH3CO2H 

b)  elle  est  condensee  en  acetylmethylcarbinol,  reduit  ensuite  en  2-3  butyleneglycol 

3.  CH3CHO  +  CH3CHO  +  AH2  -  CH3CHOH  CHOH  CH3  +  A 

c)  elle  est  condensee  en  acide  succinique,  avec  formation  correlative  de  3  molecules 
d'alcool 

4.  5CH3CHO  +  2H2O  -  CO2HCH2CH2CO2H  +  3CH3CH2OH 

La  voie  a  avait  ete  decrite  par  C.  Neuberg  sous  le  nom  de  fermentation  alcaline ; 
la  voie  b  avait  ete  decrite  par  le  meme  auteur  pour  levures  fermentant  en  presence 
d'acetaldehyde;  la  voie  c  n'avait  a  ma  connaissance  pas  ete  envisagee.  Pasteur  avait 
deja  en  1861  affirme  que  l'acide  succinique  ne  pouvait  provenir  que  du  sucre,  car  la 
masse  d' acide  succinique  formee  pouvait  atteindre  2  fois  la  masse  de  levure  formee; 
Ehrlich  avait  fait  en  191 1  une  autre  hypothese,  qui  a  ete  depuis  generalement  admise 
sans  preuve  experimentale  serieuse,  a  savoir  que  I'acide  succinique  proviendrait  de 

Bibliographic  p.  igifigQ. 


l80  L.  GENEVOIS  VOL.  4  (1950) 

I'acide  glutamique.  Malheureusement,  les  levures  sont  pauvres  en  acide  glutamique,  et 
la  quantite  d'acide  succinique  trouvee  est  20  fois  au  moins  la  quantite  d'acide  glutamique 
de  la  levure  qui  I'a  engendree;  les  huiles  de  fusel  apparaissent  effectivement  en  quantites 
10  a  20  fois  plus  faibles  que  I'acide  succinique,  ce  que  montrent  par  exemple  les  travaux 
de  Claudon  et  Morin  en  1887. 

L'hypothese  de  1936  conduit  a  une  equation  que  voici:  entre  le  nombre  g  de  mole- 
cules de  glycerol,  a  d'acide  acetique,  b  de  butyleneglycol,  m  d'acetylmethylcarbinol, 
s  d'acide  succinique,  h  d'acetaldehyde  presentes  cote  a  cote  dans  le  milieu  a  un  moment 
quelconque  de  la  fermentation,  doit  exister  la  relation: 

5-  g  =  2a  +  b  +  2m  +  5s  +  h. 

L'analyse  de  plus  de  60  fermentations  conduites  dans  des  milieux  divers,  et  avec 
diverses  levures,  a  permis  a  E.  Peynaud  d'etablir  I'equation  empirique*'  ^' ': 

6.  2  ==  2a  +  b  +  2m  +  5s  +  h  =  0.9  g 

ce  qui  signifie  que  10%  de  I'acetaldehyde  donne  des  produits  qui  echappent  pour  le 
moment  a  l'analyse.  Peynaud  a  trouve  qu'il  se  formait  un  peu  d'acide  citrique,ou 
du  moins  d'un  acide  en  ayant  tons  les  caracteres  analytiques  (insolubilite  du  sel  de 
baryum  dans  I'alcool  a  30°,  formation  d'acetone  par  oxydation  permanganique  a 
I'ebullition  a  p^  4^"'  ^^).  Si  Ton  admet  que  I'acide  citrique  se  forme  suivant  I'equation 
de  bilan  suivante  (qui  n'a  nullsment  I'ambition  de  representer  la  marche  reelle  de  la 
formation  de  I'acide  citrique) : 

7.  9CH3CHO  +  4H2O  =  CO2H.CH2COH  CH2  CO2H 

CO2H 
+  6CH3CH2OH 

I'introduction  de  I'acide  citrique  c  avec  le  coefficient  9  dans  I'equation  6  aboutit  a  un 
bilan  se  bouclant  aux  erreurs  experimentales  pres: 

8.  2a  +  b  +  2m  +  5s  +  h  +  9c  =  g 

2.  Relation  entre  le  CO^  et  Valcool 

Des  equations  2,  3,  et  4,  on  pent  deduire  de  meme  une  relation  entre  le  COg  d^gage 
au  cours  de  la  fermentation  et  I'alcool  forme;  comme  un  certain  nombre  de  molecules 
d'acetaldehyde  donnent  autre  chose  que  de  I'alcool,  on  doit  trouver  plus  de  molecules 
K  de  CO2  que  de  molecules  d'alcool  A,  et  la  difference  K — A  est  donnee  par: 

9.  K — A  =  a  +  2b  +  2m  4-  28  +  3c  +  h 

Cette  derniere  equation  est  particulierement  difficile  a  verifier,  car  la  difference 
K  —  A  est  de  I'ordre  de  2  %  de  K  ou  A ;  pour  mesurer  cette  difference  d'une  fagon 
utile,  il  faudrait  doser  K  et  A  a  i/ioooo^™®  pres,  ce  qui  presente  des  difficultes  techni- 
ques considerables,  aussi  bien  pour  le  CO2  (qui  est  tres  soluble  dans  I'eau)  que  pour 
I'alcool  (qui  est  souille  d'huiles  de  fusel). 

II  faut  remarquer  qu'au  debut  de  la  fermentation,  la  difference  K  —  A  est  bien 
superieure  a  2%.  II  est  commode  d'exprimer  K  —  A  en  fonction  du  glycerol  g,  en  partant 
de  I'equation  8. 
Bibliographic  p.  igilig2. 


VOL.  4  {1950)  BILANS    DE    FERMENTATION    ALCOOLIQUE  18I 

10.  K  —  A  =  g  +  b  —  (a  +  3s  +  6c) 

L'ordre  de  grandeur  du  phenomene  est  donne  par  la  valeur  de  g;  or,  comme  nous 
le  verrons  plus  loin,  au  debut  d'une  fermentation,  g  represente  20  ou  30%,  voire  plus, 
si  Ton  se  rapproche  du  debut  de  la  fermentation,  des  molecules  de  sucre  fermentees; 
on  doit  done  mesurer  aisement  K  —  A  au  debut  de  la  fermentation.  La  solubilite  du 
CO2  introduit  une  cause  d'erreur  grave,  qui  a  fait  croire  a  certains  experimentateurs 
qu'il  se  formait  au  debut  de  la  fermentation  plus  d'alcool  que  de  CO2.  En  realite, 
il  ne  pent  pas  se  former  une  molecule  d'alcool  sans  decarboxylation,  et  liberation 
de  CO2;  I'inverse  n'est  pas  vrai;  il  peut  apparaitre  du  COg,  sans  liberation  d'alcool, 
par  exemple,  lorsqu'il  se  fait  de  I'acctaldehyde,  de  I'acetylmethylcarbinol  ou  du 
butyleneglycol. 

3.  Methodes  d'adiition  ou  de  soustraction  d' acetaldehyde 

Les  hypotheses  faites  precedemment  ont  ete  demontrees  par  Peynaud,  non  seule- 
ment  par  Tanalyse  d'un  grand  nombre  de  fermentations  produites  en  milieu  sterile 
par  des  levures  pures  et  selectionnees,  mais  encore  par  I'analyse  de  fermentations  en 
milieux  modifies,  et  modifies  de  deux  fa9ons: 

a)  par  addition  progressive  d'acetaldehyde  au  milieu^,  on  augmente  les  quantites 
d'acide  acetique,  d'acide  succinique,  de  butyleneglycol,  qui  se  forment;  on  double  ces 
quantites;  les  3  corps  se  comportent  de  la  meme  fagon. 

b)  par  addition  progressive  de  dimedon  au  milieu,  on  diminue  la  quantite  d'acet- 
aldehyde libre,  on  "capture"  I'acetaldehyde,  et  on  diminue  dans  des  proportions  con- 
siderables les  trois  corps  qui  en  derivent ;  on  peut  reduire  I'acide  acetique  forme  au 
^/g  de  sa  valeur  dans  le  temoin. 

A  cote  de  ces  resultats  prevus,  des  notions  nouvelles,  les  unes  attendues,  les  autres 
completement  inattendues,  ont  apparu. 

4.  Role  du  milieu  et  de  la  race 

Deux  notions  nouvelles  et  non  surprenantes  ont  ete  apportees: 

1.  Les  proportions  d'acides  acetique  et  succinique,  et  de  butyleneglycol,  par  rapport 
au  glycerol  forme,  varient  beaucoup  en  fonction  du  milieu,  non  seulement  du  pjj,  mais 
encore  de  beaucoup  d'autres  facteurs  (Genevois,  Peynaud,  Ribereau-Gayon'.) 

2.  Dans  un  meme  milieu  (jus  de  raisin  filtre  et  sterilise)  les  diverses  races  de  levure 

se  comportent  tres  differemment ;  il  est  commode  de  considerer  le  rapport  -  de  I'acide 

b 
acetique  a  I'acide  succinique,  et  le  rapport  -  du  butyleneglycol  au  glycerol.  Le  rapport 

a  b  g 

-  varie  de  0.5  a  3,  le  rapport  -  de  0.04  a  0.12  (Peynaud,  Ribereau-Gayon^). 

s  g 

Ainsi  la  fermentation  alcoolique,  qui,  d'apres  des  dosages  simples  d'alcool  et  de 
CO2,  varie  tres  peu  en  fonction  du  milieu  et  de  la  race  de  levure,  est  au  contraire  une 
fonction  tres  sensible  du  milieu  et  de  la  race,  si  Ton  considere  les  produits  accessoires 
issus  de  la  dismutation  de  I'acetaldehyde. 

Acide  acetique,  butyleneglycol,  acetylmethylcarbinol  sont  des  elements  tres  impor- 
tants  de  I'appreciation  des  vins,  de  sorte  que  nous  saisissons  comment  des  levures  diffe- 
rentes  peuvent  donner  des  vins  differents  a  partir  d'un  meme  mout. 
Bibliographic  p.  igilig2. 


l82 


L.  GENEVOIS 


VOL.  4  (1950) 


5.  La  reduction  do  Vacide  acetique 

Une  notion  nouvelle  et  tout  a  fait  inattendue  a  ete  apportee  par  Peynaud^  :  I'acide 
acetique  pent  a  la  fois  apparaitre  et  disparaitre  au  cours  d'une  meme  fermentation ;  il 
se  forme  en  quantites  relativement  grandes  au  debut,  puis  sa  formation  s'arrete,  et  on 
assiste  a  la  disparition  de  proportions  importantes  (parfois  les  2/3)  de  I'acide  acetique 
forme  (Fig.  2).  L'acide  acetique  est  peut-etre  reduit  en  alcool,  car  les  acides  propionique 
et  butyrique  donnent  un  pen  d'alcools  propylique  et  butylique.  Correlativement,  il 
apparait  dans  le  milieu  de  I'acide  succinique.  Tout  se  passe,  au  point  de  vue  du  bilan, 
comme  si  Ton  avait  la  reaction. 

II.  3CH3CHO  +  CH3CO2H  +  H2O  =  COaH.CHaCHgCOoH  +  2CH3CH2OH 


On  pent  encore  supposer  la  reaction 


12. 


CH3CHO 


2CH3CO2H 


CHXHoOH  +  CO.HCHoCHoCOoH 


J 


Comme  i  molecule  d'acide  acetique  correspond  a  2  molecules  d'acetaldehyde, 
d'apres  (2),  rien  n'est  change  au  bilan  5,  quelle  que  soit  I'hypothese  adoptee. 

L'equation  (ii)  laisse  prevoir  que  la  quantite  d'acide  acetique  qui  sera  reduite  sera 
toujours  inferieure  a  la  quantite  d'acide  succinique  formee.  Dans  la  fermentation  d'une 
molecule  de  glucose  (180  g)  il  se  forme  de  4  a  6  millimolecules  d'acide  succinique; 
effectivement,  la  reduction  de  I'acide  acetique  ne  depasse  pas  5  millimolecules  par  litre, 
et  cela  lorsque  Ton  ajoute  un  exces  (12.4  millimolecules)  d'acide  acetique  au  debut  de 
la  fermentation.  Lorsque  la  levure  reduit  son  propre  acide  acetique,  la  quantite  reduite 
ne  depasse  pas  3  millimolecules. 

Ce  phenomene  de  reduction  depend  du  milieu;  si  I'on  ajoute  un  sel  de  cuivre  au 
milieu,  20  mg  par  exemple,  la  reduction  de  I'acide  acetique  est  empechee;  le  cuivre 
forme  des  complexes  avec  la  cysteine  et  la  glutathion,  et  c'est  peut-etre  la  le  mecanisme 
de  son  action.  Si  Ton  ajoute  au  contraire  de  la  cysteine  au  milieu,  le  maximum  d'acide 
acetique  et  la  teneur  finale  en  acide  acetique  sont  nettement  plus  faibles  (Fig.  3). 

Ce  phenomene  depend  de  la  levure :  il  est  des  levures  reductrices,  qui  font  dispa- 
raitre les  2/3  de  I'acide  acetique  qu'elles  produisent ;  on  observe  en  fin  de  fermentation 
un  Ph  bas,  de  I'ordre  de  9.  II  est  au  contraire  des  levures  sans  action  sur  I'acide  acetique 
qu'elles  forment;  ces  levures  donnent  au  miheu  oil  elles  fermentent,  un  pn  relativemen^ 


TABLEAU 

PRODUITS  J-ORMtS    AU    COURS     d'UNE 


Temps 

Sucres 
g 

Alcool 
degres 

Glycerol 
Mill. 

Cations 

Jours 

Acid. 

Alcal. 

NH3 

Somme 

0 
I 
2 

3 

8 

Bilan 

166 

134 
86 

56.5 
6.5 

2°.2 

5°-o 
6°.8 
9°-9 

0 

53 

58 
67 

82 

95 
104 
107 
107 
112 

+  17 

61 

61.7 
62 
62 
62.4 

+  1-4 

1-9 
0.4 
0.4 

0.3 
0-3 

—  1.6 

158 
166 
169 
169 
175 

+  16.8 

Cation  et  anions  sont  exprim^s  en  milliequivalents  par  litre. 
Aux  cations:  Acid,  signifie  Acidite  de  titration  (a  pH  7-5) • 
Alcal.  signifie  Alcalinite  des  cendres. 

Bibliographie  p.  igiligz. 


VOL.  4  (1950) 


BILANS   DE   FERMENTATION    ALCOOLIQUE 


183 


eleve  de  12  a  16.  II  est  des  levures  ayant  une  action  moderee  sur  I'acide  acetique;  on 
observe  alors  des  pjj  de  10  a  11.  Des  etudes  ulterieures  preciseront  les  substances  qui 
sont  a  I'origine  de  ces  differences. 

II,  ETUDE  EXPERIMENTAIE  DES  PRODUITS  FORMES  AU  COURS  DE  LA  FERMENTATION 

I .  Le  depart  de  la  fermentation 

Le  Tableau  I,  emprunte  a  E.  Peynaud^  (i947)  montre  revolution  d'une  fermen- 
tation d'un  mout  du  cepage  de  raison  rouge  petit  Verdot,  a  25°;  Le  mout  a  ete  sterilise 
au  moment  de  la  recolte,  conserve  en  bouteilles,  ensemence  ensuite  au  laboratoire  d'une 
levure  pure  particulierement  reductrice.  On  a  suppose  que  le  bitartrate  ne  precipitait 
pas,  et  le  bilan  a  ete  calcule  en  rajoutant  le  bitartrate  precipite  a  celui  subsistant  dans 
le  milieu.  Les  equilibres  de  precipitation  de  bitartrate  sont  en  effet  fort  longs  a  atteindre. 
Des  echantillons  de  chaque  stade  de  fei mentation  ayant  ete  preleves  et  conserves,  un 
tableau  a  pu  etre  dresse  en  tenant  compte  du  bitartrate  precipite,  I'equilibre  de  solu- 
bilite  ayant  ete  realise  (Peynaud^). 

La  fermentation  est  partie  rapidement,  de  sorte  que,  au  bout  de  24  heures,  32  g 
de  glucose  et  de  levulose  avaient  deja  fermente,  ce  sucre  represente  178  millimolecules; 
il  a  engendre  53  millimolecules  de  glycerol;  30%  du  sucre  ont  done  suivi  la  voie  de  la 
fermentation  glyceropyruvique.  Si  Ton  pouvait  suivre  le  sort  des  20  premieres  milli- 
molecules de  sucre  fermentees,  il  est  probable  que  la  fermentation  glyceropyruvique 
predominerait. 

En  meme  temps  que  le  glycerol,  on  voit  apparaitre  2  millimolecules  d'acide  lactique, 
ce  qui  montre  qu'au  depart  de  la  fermentation  alcoolique,  1%  au  moins  du  sucre  em- 
prunte la  voie  de  la  fermentation  lactique.  Cette  proportion  s'elevera  sensiblement  a  la 
fin:  les  50  demiers  grammes  —  280  millimolecules  —  engendreront  5  millimolecules 
d'acide  lactique,  ce  qui  represente  1.8%  du  sucre  consomme.  Si  I'acide  lactique  etait 
du  a  des  bacteries,  on  n'observerait  pas  ce  phenomene:  les  bacteries  lactiques  sont 
toujours  inhibees  par  I'alcool,  en  sorte  que  Ton  verrait  se  former  plus  d'acide  lactique 
au  debut  qu'a  la  fin  de  la  fermentation. 

Outre  I'acide  lactique,  il  apparait  au  debut  de  la  fermentation  de  I'acide  acetique, 
5.5  milliequivalents,   de  I'acide  succinique,   2.9  milliequivalents,   de  I'acide  citrique 


FERMENTATION    ALCOOLIQUE 


Anions 

Tart. 

Mai. 

Citr. 

Ac6t. 

Succin. 

Lact. 

Phosph. 

Somme 

lOI 

46.5 

3-5 

I.O 

0 

1-5 

153 

lOI 

45 

4.0 

6.5 

2.9 

2.0 

1-5 

163 

lOI 

45 

4.2 

5-5 

7-1 

3-2 

1-5 

167 

lOI 

43-5 

4-5 

50 

8.7 

4.0 

1-5 

167 

lOI 

41.4 

4-7 

3-8 

II. 2 

9.0 

1.0 

172 

—  51 

+  1.2 

+  2.8 

-f  II. 2 

+  9 

+  I9-I 

Bibliographie  p.  igijigs. 


i84 


L.  GENEVOIS 


VOL.  4  (1950) 


0.5  milliequivalents.  Si  Ton  suit  ces  acides  au  cours  de  la  fermentation,  on  observe  I'aug- 
mentation  progressive  des  acides  succinique  et  citrique,  mais  par  contre  on  voit  dispa- 
raitre  progressivement  I'acide  acetique,  sur  6.5  milliequivalents  au  bout  de  24  heures  de 
fermentation,  2.7  disparaissent,  et  il  ne  reste  finalement  que  3.8  milliequivalents. 

En  fin  de  fermentation,  il  est  apparu  11.2  milliequivalents  d'acide  succinique, 
9  d'acide  lactique,  2.8  d'acide  acetique,  1.2  d'acide  citrique.  Considerer  I'acidite  formee 
au  cours  de  la  fermentation  comme  due  au  seul  acide  succinique  est  done  ignorerla 
complexite  du  phenomene. 

2.  V acide  lactique 

Peynaud^  a  dose  I'acide  lactique  forme  au  cours  de  la  fermentation  d'un  movit 
de  raisin  a  156  g  de  sucre  au  litre,  de  pjj  3.26,  par  15  levures  pures,  retirees  presque 
toutes  de  vins  de  la  Gironde;  il  a  trouve  de  5  a  7  milliequivalents  d'acide  lactique 
forme,  ce  qui,  ramene  a  180  g  de  sucre,  represente  de  6.0  a  8.5  milliequivalents. 

II  a  fait  fermenter  un  mout  de  raisin  a  180  g  de  sucre  du  litre,  qu'il  a  ajuste  k  des 
Pji  allant  de  2.7  a  7.0;  une  levure  de  Fronsac  a  donne  des  quantites  d'acide  lactique 
allant  de  5.4  a  6.7  milliequivalents,  sans  relation  avec  le  p^;  une  levure  de  Saint- 
Emilion  a  donne  de  5.1  a  6.3  milliequivalents,  egalement  sans  relation  avec  le  p^- 
L'acide  lactique  est  done  un  produit  tres  constant  de  la  fermentation  par  les  levures, 
qui  ne  varie  pratiquement  pas  entre  de  larges  limites  de  p^. 

3.  Formation  et  disparition  de  V acide  acetique 

La  formation  d'acide  acetique  varie  enormement : 

1.  Avec  la  race  de  levure. 

2.  Avec  les  conditions  de  milieu. 

Avec  la  plupart  des  levures,  I'acide  acetique  forme  passe  par  un  maximum,  parfois 
tout  au  debut  de  la  fermentation,  le  plus  sou  vent  lorsque  la  moitie  ou  les  deux  tiers  du 
sucre  ont  fermente.  Ce  maximum  est  compris  entre  2  et  9  milliequivalents  par  litre; 

I'acide  acetique  en  fin  de  fermenta- 
tion est  compris  entre  i  et  8  milliequi- 
valents, tout  cela  pour  des  fermenta- 
tions suivies  dans  des  jus  de  raisin 
filtres  et  steriles  (Peynaud^'  ^) 
(Fig.  I). 

Dans  les  conditions  de  la  vini- 
fication  normale,  la  proportion  d'acide 
acetique  formee  est  beaucoup  plus 
elevee,  les  vins  contiennent  normale- 
ment  de  10  a  20  milliequivalents 
d'acide  acetique  que  Ton  pent  attri- 
buer  a  la  fermentation  alcoolique, 
independamment  de  la  piqure  aceti- 
que, ou  des  traces  d'acide  ac^tiques 
formees  dans  la  fermentation  malo- 
lactique  (Genevois,  Peynaud, 
Ribereau-Gayon^)  . 

La  formation  d'acide  acetique  en 
fonction  du  p^  presente  toujours  un 


50 


100  150 

Sucre  fermente  en  g.  par  litre 


Fig.  I 
Bibliographic  p.  igijigs. 


VOL.  4  (1950) 


BILANS   DE    FERMENTATION    ALCOOLIQUE 


185 


100  150 

Sucre  ferment^  en  g.  par  Ufr« 


minimum,  minimum  dont  la  valeur  abso- 
lute est  tres  variable  selon  la  race,  de  2  a 
10  milliequivalents,  et  qui  se  produit 
pour  des  pn  allant  de  3.5  a  5  (Peynaud^) 
(Fig.  2). 

En  milieu  neutre  (p^  7),  la  produc- 
tion d'acide  acetique  va  de  15  a  25  millie- 
quivalents par  litre,  selon  la  race  et  aug- 
mente  rapidement  avec  le  pn-  On  tend 
vers  la  "fermentation  alcaline"  de  C. 
Neuberg.  Cu  empeche  la  reduction,  la 
cysteine  favorise  la  reduction  de  I'acide 
acetique  (Fig.  3). 

4.  Formation  d'acide  citrique  au  cours  de 
la  fermentation 

La  levure  forme,  en  anaerobiose,  une 

petite  quantite  d'un  acide  ayant  tous  les 

caracteres  analytiques  de  I'acide  citrique. 

Cet  acide  a  ete  recherche  dans  la  fermen- 
tation d'un  mout  de  raison  a  166  g  de 

sucres,  contenant  deja  4  milliequivalents 

d'acide  citrique  au  litre.  7  levures  diffe- 

rentes  ont   donne   des   quantites  d'acide 

citrique  supplementaires  allant  de  i  a  2 

milliequivalents  par  litre.  On  pent  se  demander  si  cet  acide  citrique  ne  provient  pas 

de  I'acide  malique  present  normale- 
ment  dans  le  mout.  La  levure  detruit 
en  effet  de  10  a  20%  de  I'acide  mali- 
que present,  en  passant  par  le  stade 
d'acide  oxalacetique ;  or  I'acide  oxal- 
acetique  reagit  biochimiquement  avec 
I'acide  pyruvique  pour  donner  de 
I'acide  citrique.  Mais  I'experience 
montre  que  les  quantites  d'acide  citri- 
que formees  sont  independantes  des 
quantites  d'acide  malique  presentes, 
ou  transformees. 

Si  Ton  fait  fermenter  5  fois  un 
meme  milieu,  auquel  on  ajoute  apres 
chaque  fermentation  du  sucre  apres 
elimination  de  I'alcoGl,  on  observe 
la  formation  de  quantites  reguliere- 
ment  croissantes  d'acide  citrique,  de 
1.5  milliequivalents  a  chaque  opera- 
tion (Peynaud^'  ^"j. 


Fig. 


100  150 

Sucre  ferments  en  g.  per  litre 


Fig.  3 
Bibliographic  p.  igijigs. 


i86 


L.  GENEVOIS 


VOL.  4  (1950) 


III.   INFLUENCE   DES  ADDITIONS   OU   SOUSTRACTIONS   D'ACETALDEHYDE   SUR  LA 
FERMENTATION   ALCOOLIQUE 

I.  Addition  d'acetaldehyde 

II  est  impossible  d'aj outer  brutalement  do  I'acetaldehyde  au  milieu  de  fermenta- 
tion, car  I'ethanal  est  toxique  pour  la  levure  a  des  doses  superieures  a  M/ioo.  II  faut 
ajouter  I'ethanal  lentement,  a  raison  de  0.2  g  par  jour  et  par  litre,  par  exemple,  pendant 
10  jours;  la  fermentation  est  simplement  un  peu  ralentie.  La  levure  arrive  ainsi  a  trans- 
former en  10  jours  2  g  d'acetaldehyde,  pour  190  g  de  sucre  fermente,  ce  qui  represente 
5%  du  nombre  des  molecules  de  sucre  fermentees.  Cela  suffit  pour  modifier  profonde- 
ment  les  quantites  de  produits  secondaires  de  la  fermentation  (Genevois,  Peynaud, 
Ribereau-Gayon')  . 

L'experience  a  ete  repetee  avec  deux  levures:  une  levure  de  vin  rouge  typique, 
levure  de  Pomerol,  une  levure  industrielle  de  boulangerie. 

Les  resultats  sont  portes  sur  le  Tableau  II,  dans  les  deux  cas,  les  trois  produits 
secondaires  de  la  fermentation,  acides  acetique  et  succinique,  et  butyleneglycol,  aug- 
mentent  massivement. 


tableau  II 

ADDITION    d'acetaldehyde    A    DEUX    FERMENTATIONS    (jUS    DE    RAISON    k    IQO    g    DE    SUCRE) 
MILLIMOLECULES    POUR    lO    LITRES    DE    MILIEU    FERMENTE    (SAUF    POUR   L'aLCOOL) 


Aldehyde 
ajoutee 

H 

Alcool 

Glycerol 
g 

Acides 

Acetyl 
methyl 
carbinol 

m 

Butylene 
glycol 

b 

Aldehyde 
restant 

h 

E 

g 

Levure 

Acetique 
a 

Succin. 
s 

Pomerol 

0 
502 

ii°o 

II°2 

570 
500 

92 

144 

33 

73 

0.2 
0.3 

34                  8 
93                69 

363 

-l-  0.2 

—  70 

+  52 

+  40 

+  0.1 

+  59 

+  61 

83 

Boulangerie 


502 


io°8 
ii°o 


580 
510 


-|-  0.2         —  70 


37 
109 


+  72 


51 
0.6 


+  33 


0.6 
10.3 


+  9-7 


42 
119 


77 


8 
28 


4-  20 


406 


L'experience  consiste  a  comparer  une  fermentation  de  jus  de  raison  (190  g  de  sucre 
reducteur  par  litre,  p^  =  3- 17)  recevant  0.2  g  d'acetaldehyde  par  jour,  a  une  fermen- 
tation temoin,  marchant  parallelement,  avec  la  meme  levure. 

La  levure  de  Pomerol  fait  passer  I'acide  succinique  de  33  a  73  millimolecules  pour 
10  litres,  I'acide  acetique  de  92  a  144,  le  butyleneglycol  de  34  a  93,  I'acide  succinique 
augmente  de  120%,  le  butyleneglycol  de  160%,  45%  de  I'acetaldehyde  ajoutee  sont 
passes  a  former  de  I'acide  succinique  et  de  I'alcool  selon  I'equation  4.  La  levure  de  bou- 
langerie "travaille"  moins  I'acetaldehyde  a  I'etat  d'acide  succinique,  neanmoins  34% 
de  I'aldehyde  se  retrouve  encore  dans  I'acide  succinique  et  I'alcool  correspondant. 
Chaque  levure  possede  sa  fagon  personelle  de  distribuer  I'acetaldehyde  entre  les  acides 
acetique  et  succinique,  le  butyleneglycol  et  I'acetylmethylcarbinol. 
Bibliographic  p.  igilig2. 


VOL.  4  (1950) 


BILANS    DE    FERMENTATION   ALCOOLIQUE 


187 


2.  Addition  de  dimedon 

Le  moyen  le  moins  brutal  de  soustraire  de  I'acetaldehyde  au  milieu  de  fermentation, 
consiste  a  aj  outer  du  dimedon  (Peynaud^).  Le  dimedon  etant  tres  peu  soluble  dans 
I'eau,  il  faut  I'ajouter  en  solution  alcoolique ;  pour  qu'il  reste  convenablement  en  solution, 
il  faut  que  la  teneur  initiale  du  milieu  en  alcool  soit  de  4°  environ;  pour  qu'il  reagisse 
assez  vite  avec  I'aldehyde,  il  faut  que  le  pn  du  milieu  soit  d'au  moins  4  et  de  preference  6. 
Le  Tableau  III  porte  deux  fermentations,  realisees  dans  du  jus  de  raisin  a  4°  d'alcool, 
a  Ph  4-0  et  6.5,  chaque  fois  avec  et  sans  dimedon.  Sans  dimedon,  I'acide  acetique  appa- 
rait  des  les  premiers  jours  de  fermentation,  puis  n'augmente  plus  que  lentement;  avec 
dimedon,  la  formation  d'acide  acetique  est  tres  faible  au  debut,  et  se  poursuit  lentement, 
au  cours  de  toute  la  fermentation. 

TABLEAU  III 

ACTION    DU    DIMEDON    SUR    LA    FORMATION    D'ACIDE    ACETIQUE 
JUS    DE    RAISIN    A    4°    d'alcool    ET    A    1%    DE    DIMEDON 


Temoin 

+  1%  dim6don 

Ph  du  milieu 

Temps 
Jours 

Sucre 

fermente 

g 

Acide 
Acetique 
Milliequ. 

Temps 
Jours 

Sucre 

fermente 

g 

Acide 
Acetique 
Milliequ. 

4.0 
6.5 

3 

4 
5 

3 
5 
6 

56 

96 

125 

48 
105 
125 

3-6 
4.1 
4-3 

9-3 
17-3 
20.3 

5 

7 

12 

3 
5 
6 

36 
68 

133 

26 

89 

105 

0.9 
1.2 

2.2 

1.0 
2.9 
4-3 

A  Ph  4,  le  dimedon  reagissant  lentement,  la  formation  d'acide  acetique  est  reduite 
a  la  moitie  de  sa  valeur  normale.  A  p^  6.5,  le  dimedon  reagissant  mieux,  I'acide  acetique 
est  reduit  a  21%  de  la  valeur  du  temoin. 


IV.  BILAN    des    PRODUITS    SECONDAIRES    de    la   FERMENTATION 

I.  Milieu  constitue  par  du  jus  de  raison  sterilise 

Sur  un  meme  jus  de  raisin  a  190  g  de  sucre  au  litre,  de  pn  =  3-i7.  ont  fermente 
29  levures  differentes  de  provenances  tres  varices:  16  levures  de  vins  rouges  de  la 
Gironde,  2  levures  ayant  pousse  spontanement  sur  des  jus  de  raisins  concentres,  i  levure 
de  vin  blanc,  i  levure  de  boulangerie,  et  9  levures  de  vins  suisses  (Peynaud^^).  Les 
dosages  ont  porte  sur  les  substances  figurant  dans  les  bilans  (5)  et  (6),  savoir:  glycerol 
(g),  acides  acetique  (a)  et  succinique  (s),  acetylmethylcarbinol  (m),  2-3  butyleneglycol 
(b),  acetaldehyde  (h).  La  production  de  glycerol  a  relativement  peu  varie  d'une  levure 
a  I'autre:  52  a  75  millimolecules  par  litre;  I'acide  acetique  a  varie  de  3  a  12,  I'acide  succi- 
nique de  4.8  a  9,  I'acetylmethylcarbinol  de  0.02  a  o.ii,  le  butyleneglycol  de  3  a  6, 
I'acetaldeh^^de  de  1,5  a  4,  le  tout  en  millimolecules.  Malgre  ces  grandes  variations,  dues 
a  la  diversite  des  races  physiologiques  de  levure,  le  bilan  6  se  verifie  aux  erreurs  d'ex- 
periences  pres  (Tableau  IV)  (Genevois,  Peynaud,  Ribereau-Gayon^).  Si  Ton  appelle 

T  la  somme:  v       .-     1   ^^    1   ^^    1   k    1   k 

^  2,  =  5s  +  2a  +  2m  +  b  +  ri 

Bibliographic  p.  igijigB. 


i88 


L.  GENEVOIS 


VOL.  4  (1950) 


y 

on  observe  que  le  rapport  —  est  compris  entre  0.82  et  0.96,  les  chiffres  les  plus  frequents 

g 
etant  voisins  de  0.90. 

TABLEAU  IV 

BILAN    DES    PRODUITS    SECONDAIRES    DE    LA    FERMENTATION 
FERMENTATIONS    SUR    JUS    DE    RAISIN    STERILE    (J)    ET    SUR    SOLUTION    DE    SACCHAROSE    (S) 

MILLIMOLECULES    POUR    ID    LITRES 


Levure 

Type 

Milieu 

g 

a 

s 

m 

b 

h 

E 

100— 
g 

a 

s 

b 

1000- 

g 

Margaux 

Succinogene 

J 

S 

650 

840 

43 
153 

77 
68 

0.2 
0.4 

36 

85 

37 
10 

544 
741 

84 
88 

0.56 

2.2 

55 

lOI 

Boulanger 

Id.  glycol. 

J 

s 

640 

820 

37 
153 

80 
65 

0.7 
0.5 

55 
83 

26 
10 

555 
784 

86 
89 

0.7 
2.4 

84 

lOI 

La  Tresne 

Equilibre 

J 

s 

610 

780 

78 
220 

70 
40 

0.3 
0.4 

33 

84 

33 

7 

572 
731 

93 
92 

I.I 
5-5 

55 
106 

Malvoisie 

Id.  glycol. 

J 

s 

600 

750 

59 
160 

60 
59 

I.I 
0.9 

57 
71 

27 
4 

502 
640 

83 
85 

I.O 

2.7 

95 
94 

Pau  iliac 

Acetogene 

J 

s 

630 
750 

84 
218 

58 
38 

0.4 
0.6 

34 

77 

31 
5 

523 

708 

83 
94 

1.4 

5-7 

54 
102 

Bonarda 

Id.  glycol. 

J 

s 

610 

790 

100 
233 

48 
36 

0.7 
0-5 

56 
59 

31 

8 

527 
713 

86 
90 

2.0 
6.5 

92 
75 

Glycol.  =  abreviation  pour  "butyleneglycologene" 

II  reste  done  un  ou  plusieurs  constituants,  derivant  de  I'acetaldehyde,  qui  restent 

a  determiner,  mais  ils  ne  representent  pas  plus  de  10  a  15%  de  I'acetaldehyde  derivant 

de  la  fermentation  glyceropyruvique. 

a 
II  est  commode  de  considerer  le  rapport  de  I'acide  acetique  a  I'acide  succinique  -  et 

b  a 

le  rapport  du  butyleneglycol  au  glycerol,  -;  le  rapport  -  varie  de  0.4  a  2.1,  selon  les 

b  g  s 

levures,  le  rapport  -  varie  de  0.048  a  0.095,  ces  rapports  permettent  un  classement 

g  /a  \  /a  \ 

physiologique  des  levures,  en  levures  succinogenes    -  <  0.75    acetogenes  I  —  >   1.25  1 

a  b  ^^  I  .       ^        . J,  , 

et  equilibrees  (0.75  <  -  <  1.25);  les  levures  ou  -  >  0.070  pourront  etre  considerees 

s  g 

comme  glycologenes  (Peynaud  et  Ribereau-Gayon^). 

On  peut  repartir  les  levures  dans  six  categories  physiologiques  differentes  (Tabl.  IV). 

Ainsi  ce  travail  analytique  considerable  aboutit  a  deu.x  resultats:  verifier  les  hypo- 
theses faites  sur  I'origine  des  acides  acetique  et  succinique,  et  du  butyleneglycol,  donner 
une  description  logique  des  diverses  races  de  levures. 

2.  Milieu  constitue  par  une  solution  de  saccharose  et  d'eau  de  levure 

L'experience  precedente,  portant  sur  29  levures  differentes,  a  ete  repetee  sur  une 
solution  de  saccharose  a  180  g  au  litre,  contenant  10%  d'extrait  de  levure.  Le  p^  de  cc 
milieu  se  stabilisait  aux  environs  de  5.  Les  memes  produits  que  precedemment  ont  etc 
Bibliographic  p.  igilig2. 


VOL.  4  (1950)  BILANS   DE   FERMENTATION    ALCOOLIQUE  189 

doses;  ils  se  sont  trouves  systematiquement  differents  (Gevenois,  Peynaud,  Ribereau- 
Gayon'). 

1.  le  glycerol  g  augmente  de  10  a  40% 

2.  I'acide  acetique  a  est  en  moyenne  3  fois  plus  elevc 

3.  I'acide  succinique  s  diminue  de  10  a  20% 

4.  le  butyleneglycol  b  double  generalement 

5.  racetylmethylcarbinol  m  est  en  quantites  du  meme  ordre 

6.  I'acetaldehyde  h  tombe  au  quart  de  sa  concentration. 

Par  contre,  I'equation  (6)  se  verifie  comme  prucedemment ;   le  mecanisme  de  la 

fermentation  est  le  meme,  mais  la  distribution  de  I'acetaldehyde  entre  les  divers  pro- 

a 
duits  de  fermentation  est  differente.  Le  rapport  -,  au  lieu  de  varier  de  0.4  a  2.1  varie  de 

b  ^ 

2.0  a  6.5;  le  rapport  -  varie  de  0.07  a  o.ii  (Tableau  IV). 
g 
Les  differentes  categories  de  levures,  caracterisees  par  leur  fermentation  sur  jus 

de  raisin  filtre  et  sterile,  presentent  sur  milieu  au  saccharose  d'autres  constantes,  comme 

a 
il  est  normal;  chez   toutes  les  levures,  le  rapport  -  augmente  considerablement,  les 

s 
a  , 

levures  succinogenes  presentent  des  rapports  -  de  2  a  3,  au  lieu  de  0.4  a  0.75 ;  les  levures 

s 

acetogenes  presentent  des  rapports  -  de  5  a  7  au  lieu  de  1.25  a  2.  Les  levures  dites 

a  a 

"equilibrees",  au  lieu  d'un  -  voisin  de  i,  donnent  pour  -  des  valeurs  echelonnees  de 

s  s 

3  a  5.5.  Ces  trois  categories  de  levures  se  retrouvent  done  sans  difficulte. 

b 
Les  levures  qui  presentaient  des  rapports  -  faibles,  de  0.04  a  0.07,  presentent  des 

b  § 

rapports  -  voisin  de  o.io.  Les  levures  caracterisees  comme  glycologenes  precedem- 

§        b      .,        .  . 
ment,  avec  un  —  deja  voisin  de  o.io,  gardent  sensiblement  la  meme  valeur  pour  le 

b  g 

rapport  -,  comme  s'il  y  avait  un  "plafond"  pour  la  formation  de  butyleneglycol. 

^      y  '         - 

Le  rapport  -=  oscille,  comme  precedemment,  entre  0.82  et  0.95.  II  a  done  la  une 
g 
veritable  "constante"  biologique,  independante  dans  une  large  mesure  de  la  race  de  la 
levure  et  de  la  nature  du  milieu  fermente. 

3.  Fermentation  dans  les  vins 

La  fermentation  dans  les  vins  est  rarement  une  fermentation  alcoolique  pure ;  dans 
a  peu  pres  tons  les  vins  non  sulfites  ni  additionnes  d'alcool,  I'acide  malique  est  trans- 
forme  en  acide  lactique,  par  fermentation  malolactique  due  a  des  bacteries  speciales; 
d'apres  Peynaud^,  qui  a  soigneusement  etudie  ce  type  de  fermentation  a  Bordeaux, 
il  apparait,  non  seulement  de  I'acide  lactique,  a  raison  d'une  molecule  par  molecule 
d'acide  malique  detruit,  mais  encore  un  peu  d'acide  acetique,  de  i  a  7  milliequivalents 
par  litre,  qui  semble  provenir  d'une  autre  source.  Les  chiffres  les  plus  frequents  pour 
I'acide  acetique  ainsi  forme  vont  de  2  a  4  milliequivalents.  II  semblerait  done  que  le 
bilan  indique  par  I'equation  (6)  ne  doive  plus  se  verifier.  Cependant,  si  Ton  considere 
Bibliographie  p.  igilig2. 


igo 


L.  GENEVOIS 


VOL.  4  (1950) 


TABLEAU  V 

BILAN   DES   PRODUITS  SECONDAIRES   DE  LA  FERMENTATION   DANS   LES  VINS  ROUGES   ET  BLANCS 


E 

a 

b 

Annee 

Type 

Vin 

g 

a 

s 

m 

b 

h 

E 

100— 

g 

s 

1000- 
g 

1945 

Rouge 

Pomerol 

850 

112 

90 

0.9 

81 

757 

89 

1.24 

95 

1945 

Blave 

830 

163 

71 

0.5 

79 

761 

90 

2.16 

93 

1944 

Moulis 

804 

122 

85 

I.I 

65 

736 

91 

1-4 

81 

1944 

St.  Emilion 

654 

122 

51 

2.2 

48 

551 

«5 

2.4 

73 

1943 

Listrac 

862 

174 

84 

I.I 

70 

840 

98 

2.1 

81 

1943 

,, 

Bourg 

890 

200 

71 

0-5 

71 

827 

93 

2.8 

80 

1946 

Blanc 

Tuchan 

590 

66 

65 

64 

16 

537 

91 

I.O 

108 

1946 

,, 

Tautavel 

730 

90 

74 

92 

36 

678 

93 

1.2 

126 

1945 

Tautavel 

770 

99 

71 

94 

35 

682 

88 

1.4 

122 

1946 

" 

Frontignan 

250 

53 

16 

32 

17 

235 

94 

3-3 

128 

des  vins  jeunes,  de  un  a  deux  ans  d'age,  des  vins  pasteurises,  des  vins  "vines"  c'est-a-dire 
additionnes  d'alcool  au  cours  de  la  fermentation,  pour  garder  du  sucre,  le  bilan  (6)  se 
verifie  presque  tou jours. 

Par  exemple,  sur  20  echantillons  de  vins  rouges  de  la  Gironde  analyses  par  Peynaud* 

I 
au  printemps  1946,  18  presentent  des  rapports  -=  normaux,  allant  de  0.82  a  0.98  et  2 

g 

seulement  des  rapports  superieurs  a  i,  par  suite  d'un  exces  d'acide  acetique. 

a 
L'experience  montre  que  le  rapport  -  est  dans  un  vin  rouge  issu  de  la  fermentation 

s 

de  la  maceration  de  la  totalite  de  la  bale  de  raison  fraiche,  tres  different  de  ce  qu'il 

est  dans  la  fermentation  d'un  jus  de  raison  filtre  et  sterilise. 

a 
Dans  les  vins  rouges,  le  rapport  -  s'est  toujours  trouve  compris  entre  i  et  3,  en 

s 

eliminant  les  echantillons  contenant  visiblement  de  I'acide  acetique  du  a  une  fermen- 
tation acetique.  La  valeur  absolue  de  a  va  de  10  a  20  milliequivalents,  alors  que  sur 
jus  de  raison  sterile  il  va  de  3  a  12;  les  levures  frangaises  donnent  meme  pour  a  des 
valeurs  comprises  entre  3  et  8.  Meme  en  tenant  compte  de  I'acide  acetique  de  la  fermen- 
tation malolactique,  2  a  4,  il  est  clair  que  la  fermentation  due  aux  levures  se  fait  dans 
le  mout  naturel  autrement  que  dans  nos  fiacons,  et  que  le  rendement  en  acide  acetique 
est  au  moins  double. 

Dans  le  cas  des  vins  blancs  du  midi^,  tres  riches  en  sucre,  mutes  a  I'alcool  en  cours 

y 

de  fermentation,  ce  qui  empeche  Taction  des  bacteries  malolactiques,  le  rapport  —  s'est 

g 
trouve  compris  entre  0.88  et  0.94.  Le  cas  du  muscat  de  Frontignan,  mute  apres  fermen- 

a 
tation  du  quart  a  peine  de  son  sucre,  est  tres  interessant ;  le  rapport  —  est  eleve,  3.3, 

b  ^ 

comme  il  est  normal  au  depart  de  la  fermentation ;  le  rapport  -  est  aussi  remarquable- 

ment  eleve,  0.13;  le  rapport  ■=-  est  normal,  0.94. 


Bibliographic  p.  igijig2. 


VOL.  4  (1950)  BILANS    DE    FERMENTATION   ALCOOLIQUE  I9I 

CONCLUSIONS 

La  determination  des  produits  secondaires  de  la  fermentation,  glycerol,  acides  acetique  et  succi- 
nique,  acetylmethylcarbinol,  butyleneglycol  et  acetaldehyde    presente  done  un  grand  interet: 

1.  Toutes  ces  substances  sont  des  produits  normaux  de  la  fermentation  alcoolique. 

2.  EUes  proviennent  d'une  fermentation  glyceropyruvique,  qui  pr^domine  au  depart  de  la 
fermentation,  mais  se  poursuit  durant  toute  la  destruction  du  Sucre. 

3.  Elles  obeissent  a  I'equation  (6). 

4.  Les  rapports  de  I'acide  acetique  a  I'acide  succinique,  du  butyleneglycol  au  glycerol,  varient 
en  fonction  du  moment  de  la  fermentation,  de  la  race  de  levure,  enfin  de  la  nature  du  milieu  fer- 
mente  (pn.  etc.  .  .). 

5.  Ces  rapports  peuvent,  dans  un  milieu  donne,  servir  a  caracteriser  des  races  de  levures. 

6.  L'acide  acetique  suit  au  cours  de  la  fermentation  une  evolution  compliquee,  qui  le  fait 
apparaitre,  puis  disparaitre. 

7.  Dans  les  fermentations  naturelles  (vin),  la  consideration  de  I'equation  (6)  permet  de  carac- 
tdriser  certaines  alterations  bacteriennes  graves. 

CONCLUSIONS 

Determination  of  the  secondary  products  of  alcoholic  fermentation :  glycerol,  acetic  acid,  suc- 
cinic acid,  acetyl  methyl  carbinol,  butyleneglycol  and  acetaldehyde,  is  of  great  interest,  for: 

1.  All  these  compounds  are  normal  products  of  alcohohc  fermentation. 

2.  They  arise  from  a  glycero-pyruvic  fermentation,  which  dominates  in  the  beginning  of  the 
fermentation,  but  perseveres  during  the  destruction  of  all  the  sugar. 

3.  They  agree  with  equation  (6). 

4.  The  relation  between  acetic  acid  and  succinic  acid,  as  between  butyleneglycol  and  glycerol, 
depends  upon  the  phase  of  the  fermentation,  the  strain  of  yeast,  and  finally  also  upon  the  nature  of 
the  medium  in  which  fermentation  takes  place  (pn,  etc.). 

5.  In  a  given  medium  these  relations  can  serve  to  characterize  the  strains  of  yeasts. 

6.  During  the  fermentation  acetic  acid  is  subject  to  a  complicated  evolution,  which  causes  it 
to  appear  and  then  to  disappear  again. 

7.  In  natural  fermentations  (wine)  a  consideration  of  equation  (6)  enables  the  characterization 
of  certain  serious  bacterial  changes. 


SCHLUSSFOLGERUNGEN 

Die  Bestimmung  der  Nebenprodukte  der  alkoholischen  Garung:  Glycerin,  Essigsaure,  Bernstein- 
saure,  Acetylmethylcarbinol,  Butylenglykol  und  Acetaldehj^^d  ist  aus  folgenden  Griinden  wichtig: 

1.  Alle  diese  Verbindungen  sind  normale  Produkte  der  alkoholischen  Garung. 

2.  Sie  stammen  aus  einer  Glycero-Brenztraubensaure-Garung,  die  zu  Beginn  der  Garung  vor- 
herrscht,  aber  wahrend  der  ganzen  Zersetzung  des  Zuckers  fortdauert. 

3.  Sie  sind  im  Einklang  mit  Gleichung  (6). 

4.  Das  Verhaltnis  Essigsaure/Bernsteinsaure  und  Butylenglykol/Glycerin  hangt  von  der  Phase 
der  Garung,  von  dem  benutzten  Hefestamm  und  endlich  von  der  Natur  des  Milieus  ab,  in  dem  die 
Garung  stattfindet  (pn,  usw.). 

5.  In  einem  bestimmten  Milieu  konnen  diese  Verhaltnisse  zur  Charakterisierung  der  Heferasse 
dienen. 

6.  Die  Essigsaure  ist  wahrend  der  Garung  einem  komplizierten  Prozess  unterworfen,  durch  den 
sie  zuerst  auftritt  und  dann  wieder  verschwindet. 

7.  Bei  natiirlichen  Garungen  (Wein)  kann  man  durch  Betrachtung  der  Gleichung  (6)  gewisse 
ernste  bakterielle  Veranderungen  charakterisieren. 


BIBLIOGRAPHIE 

1  Peynaud,  Annates  des  fermentations,  5  (1939)  321,  385. 

2  Peynaud,  These,  Bordeaux  1946;  Industries  agricoles  et  alimentaires,  64  (1947)  ^7-  ^^7>  3°i<  399- 
^  Genevois,  Bull.  soc.  chim.  biol.,  18  (1936)  295. 

*■  Genevois,  Peynaud,  RiB]d;REAU-GAYON,  Compt.  rend.  acad.  sci.,  223  (1946)  693. 

^  Peynaud  et  Rib6reau-Gayon,  Ibidem,  224  (1947)  1388. 

®  Genevois,  Peynaud,  Ribereau-Gayon,  Ibidem,  224  (1947)  766. 


ig2  L.  GENE VO IS  VOL.  4  (1950) 

•  Genevois,  Peynaud,  Ribereau-Gayon,  Ibidem,  226  (1948)  126. 
**  Genevois,  Peynaud,  Ribereau-Gayon,  Ibidem,  226  (1948)  439. 
9  Genevois,  Peynaud,  Ribereau-Gayon,  Ibidem,  227  (1948)  227. 

10  Peynaud,  Bull,  intern,  du  vin,  118  (1938)  33- 

11  Peynaud,  Ann.  chim.  anal.,  28  (1946)  m- 

12  Peynaud,  Industries  agricoles  et  alimentaires  (1948)- 

Re9u  le  5  avril  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  I93 


TRIOSEPHOSPHORSAURE    ALS    INTERMEDIARPRODUKT    BEI    DER 
ZUCKERGARUNG  MIT  INTAKTER  HEFE 


WILHELM  KIESSLING 

Biochemisches  Laboratorium  der  wissenschaftlichen  Abteilung  von  C.  H.  Boehringer  Sohn, 

IngelheimjRhein  [Deutschland) 


Der  heute  allgemein  anerkannte  Abbauweg  bei  der  Vergarung  von  Zucker  zu  Alkohol 
iiber  phosphorylierte  Intermediarprodukte  wurde  fiir  den  zellfreien  Fermentextrakt 
(Macerationssaft)  als  Schema  im  Jahre  1933  von  O.  Meyerhof^  im  Princip  das  erstemal 
aufgestellt  und  im  Jahre  1935^  und  1937^  erganzend  erweitert.  Bei  diesem  Schema  wird 
zwischen  Initialphase  und  stationarer  Phase  unterschieden.  In  der  Erganzung  von  1937 
findet  sich  auch  eine  Erklarung  fiir  die  Entstehung  der  HARDEN-YouNG'schen  Ganings- 
phase  im  zellfreien  Macerationssaft ;  aber  gerade  die  Bildung  von  Hexoseestern  ist  fiir 
die  Gegner  dieses  Schemas  immer  ein  Punkt  der  Kritik,  nach  der  der  Abbau  des  Zuckers 
in  der  lebenden  Zelle  anders  verlaufen  sollte,  weil  bei  ihr  diese  Ester  als  Garungs- 
zwischenprodukte  nicht  nennenswert  sich  anhaufen  und  nachzuweisen  sind.  In  einem 
wirklichen  stationaren  Zustand  ist  nun  die  Anreicherung  eines  Intermediarproduktes 
nicht  zu  erwarten,  da  jeder  Fall  dieser  Art  eine  Zustandsanderung  voraussetzt,  sei  es 
Alterung,  Hemmung  durch  Gifte  oder  Erschopfung  von  Nahrstoffen  u.s.w.  Auch  die 
Initialphase  der  Garung  ist  eine  solche  Zustandsanderung.  Im  folgenden  soil  die  vor- 
iibergehende  Anreicherung  einer  Triosephosphorsaure  als  Intermediarprodukt  bei  der 
Angarung  von  intakter  Hefe  beschrieben  werden. 

METHODIK 

Triosephosphorsaure  wurde  nachgewiesen  als  Milchsaure,  welche  durch  alkaUsche  Verseifung 
bei  Zimmertemperatur  nach  der  von  O.  Meyerhof  und  K.  LoHMANN^a  beschriebenen  Reaktion 
entsteht:  Triosephosphat  ->  Milchsaure  +  Phosphat.  Die  so  gebildete  Milchsaure  in  den  Garansatzen 
wurde  folgendermassen  bestimmt:  20  ml  Garlosung  wurden  filtriert,  schwach  Phenolphtalein-alkalisch 
zur  Trockne  verdampft,  2  mal  mit  Wasser  aufgenommen  und  nochmals  verdampft,  im  Schwefelsaure- 
exsiccator  iiber  Nacht  getrocknet;  dann  in  20  ml  Wasser  gelost,  und  zur  Verseifung  der  Triosephos- 
phorsaure mit  I  ml  25%  Natronlauge  versetzt.  Nach  10  Minuten  Stehen  bei  Zimmertemperatur 
wurde  mit  i  ml  25%  Salzsaure  neutralisiert,  mit  CuS04-Ca(OH)2  gefallt,  ein  aliquoter  Teil  abge- 
nommen  und  in  der  iiblichen  Weise  nach  Friedemann,  Contonio  und  Shaffer*  die  Milchsaure 
bestimmt.  Als  Hefe  wurde  Weinhefe  Steinberg  aus  Geisenheim  am  Rhein  oder  Weinhefe  Oppenheimer 
Kreuz  aus  Oppenheim  am  Rhein  verwandt. 

Natiirliche  Nahrsubstrate  waren  Traubenmoste  oder  Moste  aus  anderen  Friichten.  Als  synthe- 
tische  Nahrlosung  diente  ein  modifizierter  Garansatz  nach  Henneberg^,  bestehend  aus  15%  Rohr- 
zucker,  0.2%  Pepton,  0.5%  KHjPO^,  0.2%  MgSO^.  pH  4-8. 

Um  die  Triosephosphorsaure  zu  isolieren  und  fernerhin  die  bereits  vorgebildete  Milchsaure  von 
derjenigen  zu  unterscheiden,  die  erst  durch  alkahsche  Verseifung  entsteht,  wurden  die  Garansatze 
durch  Fallung  mit  Bariumacetat  und  Alkohol  fraktioniert.  Beim  Fraktionieren  wurden  20-50  ml 
der  mit  Trichloressigsaure  enteiweissten  Garansatze  mit  Bariumacetat  versetzt  und  bei  schwach 
lackmussaurer  Reaktion  mit  3  Teilen  Alkohol  gefallt.  Der  Niederschlag  wurde  mit  Alkohol- Aether  ge- 

Literatur  S.  198. 

13 


194 


W.  KIESSLING 


VOL.  4  (1950) 


waschen  und  im  Schwefelsaureexsiccator  iiber  Nacht  getrocknet.  Darauf  wurde  im  urspriinglichen 
Volumen  Wasser  mit  einigen  Tropfen  2  n  Salzsaure  gelost,  Ba  mit  Na2S04  ausgefallt,  mit  i  ml  25%iger 
Natronlauge  bei  Zimmertemperatur  verseift;  dann  mit  i  ml  25%iger  Salzsaure  neutralisiert  und  nach 
CuS04-Ca(OH)2-Behandlung  in  einem  aliquoten  Teil  die  Milchsaure  bestimmt.  Ein  entsprechender 
Anteil  wurde  vor  der  CuS04-Ca(OH)2-Behandlung  zur  PjO^-Bestimmung  der  Triosephosphorsaure 
abgenommen  und  als  anorganisches  Phosphat  nach  der  Verseifung  mit  normaler  Natronlauge  be- 
stimmt. Die  kolorimetrische  Phosphorsaurebestimmung  nach  Lohmann  und  Jendrassik^  wurde 
in  einem  lichtelektrischen  Kolorimeter  nach  Havemann  mittels  einer  Eichkurve  vorgenommen. 
Zunahme  des  anorganischen  Phosphats  nach  alkalischer  Verseifung  entspricht  der  Triosephosphor- 
saure. 

In  der  Mutterlauge  der  Bariumfallung  konnte  die  wirkliche  Milchsaure,  d.h.  diejenige,  die  nicht 
erst  durch  alkalische  Verseifung  entstanden  ist,  nach  Ba-Fallung  mit  Natriumsulfat  in  der  oben 
angegebenen  Weise  ebenfalls  bestimmt  werden. 


^160 

o 
o 

«> 

^120 

;§ 

o> 
6 

80 
60 
40- 
20- 


(\ 

Oar 

ungsr 

■nilchsc 

■jure 

'^  \ 

i 

Normalgarung 

Nach  Zusatz  von  Aspergillus  -Extrakt 

V 

A 

V 

=^ 

Ti— 



X 

--^ 

W        i      -_y- 

! 

' 

' 

Jl 

Maxin 

tale  0 

irgesc 

)windig 

keif 

10       15      20      25       30 


Abb.  I 


35       40      45 
Versuchsiag 


und  Forschungsanstalt  fiir  Wein-  und  Obstbau  zu 

gezeichnet.   Das  pn  dieses  Mostes  betrug  3.3  und 

9-11°.  Kurve  I  (Abb.  i)   zeigt  in    -^  70 

der  Angarungsphase  zwischen  dem    § 

2.  und  8.  Tag  einen  Anstieg  der    .c 

Milchsaure  bis  zu  65  mg/%,  der 

dann   bei  einsetzender  maximaler 

Gargeschwindigkeit  auf  46  mg/% 

absinkt    und   wahrend   und   nach 

beendeter    Garung    bei    ungefahr 

40  mg  bleibt  bis  zum   Einsetzen 

der  bakteriellen  Sauregarung.   Es 

wurde  nun  weiter  gefunden,  dass 

sich  dieser  Milchsaureanstieg  durch 

Zusatz  eines  wassrigen  Extraktes 

aus  getrockneten   Schimmelpilzen 

[Aspergillus  niger)  bedeutend  stei- 

gern  lasst.   Nach    v.  Euler  und 

Nielsen'  enthalt  A  spergillus  niger 

einen   wasserlcisHchen    Wuchsstoff 

fiir  Hefe,  der  wahrscheinhch   zur 

Biosgruppe  gehort.  Bei  Zusatz  von 

Literatur  S.  198. 


VERSUCHE 

Verfolgt  man  die  Milchsaure- 
bildung  eines  garenden  Trauben- 
saftes  wahrend  des  ganzen  Garver- 
laufes,  so  ist  zunachst  in  der 
Angarungsphase  ein  Ansteigen  der 
Milchsaure  festzustellen,  die  bei 
Beginn  der  stationaren  Phase  auf 
einen  gleichbleibenden  Gehalt  bis 
zum  Ende  der  Garung  abfallt.  In 
der  Abb.  i  ist  eine  derartige  Milch- 
saurebildung  bei  der  normalen  Ver- 
garung  von  1200  1  Traubenmost 
aus  Sylvanertrauben  in  der  Versuchs- 
Geisenheim  aus  dem  Jahre  1944  auf- 
die  Kellertemperatur  Ende  Oktober 


J,  60 

3 
O 

« 

5  50 

o 

6 

30 


20 


10 


^ 

/ 

N 

/ 

/ 

\ 
\ 

\ 

/ 

/ 

; 

\ 

\ 
\ 

/ 

\ 

1 

/ 

\ 

\ 

Oarungsmilchsaure 

\ 

Normalgarung 

\ 

\ 

Aspergillus  -  Exfraht 

\ 

I                 1                 1 

\ 

\ 

i    / 

\ 

'           / 

\ 

N 

1          / 

\ 

V 

/ 

\ 

\ 

■..--'''' 

■  —  -; 

'       / 

1  / 

>^ 



— ' — 



6  7 

Versuchsfag 


Abb.  2 


VOL.  4  (1950) 


TRIOSEPHOSPHORSAURE    IN    DER   ZUCKERGARUNG 


195 


2%  eines  derartigen  io%igen,  wassrigen  Extraktes  stieg  die  Milchsaure  nach  5  Tagen 
bis  auf  159  mg  in  100  ml  Garlosung  an  (Kurve  II). 

Da  das  pn  eines  Traubenmostes  zwischen  3  und  3.5  relativ  ungiinstig  fiir  die  Hefe 
liegt,  erstreckt  sich  diese  Angarungsphase  bei  einer  verhaltnismassig  niedrigen  Tempe- 
ratur  zwischen  9  und  12''  auf  etwa  6  Tage;  bei  Zimmertemperatur  und  bei  einem  fiir  die 
Hefe  giinstigen  pjj  von  4.8  beschrankt  sich  diese  Phase  auf  2  Tage,  wie  in  Laboratoriums- 
versuchen,  z.B.  mit  einem  Moste  von  Hagebuttenfriichten,  der  mit  Zucker  auf  etwa 
20%  versetzt  war,  zeigt  (Abb.  2).  Auch  hier  ist  der  Milchsaureanstieg  (Kurve  I)  deuthch 
zu  erkennen  und  betragt  bei  Zusatz  von  Aspergillusextrakt  mehr  als  das  Doppelte 
(Kurve  II). 

Auch  mit  kiinstlicher  Nahrlosung,  wie  sie  oben  beschrieben  wurde,  bei  einem  pjj 
von  4.8,  ist  dieser  Anstieg  und  seine  wesenthche  Steigerung  durch  einen  Aspergillus- 
Extrakt  als  Garungsaktivator  klar  ersichtlich,  wie  die  Tabelle  I  zeigt.  Die  Angarungs- 
phase dauerte  hier  ebenfalls  nur  2  Tage. 

TABELLE   I 


Zeit  in  Stunden 

mg  Milchsaure  in  100  ml 
ohne  Zusatz 

mgMilchsaure  in  100  ml 
mit  Aspergilluszusatz 

23 
27 
44 
52 

6.7 
33-8 
22.5 
15.0 

9 
67.5 
33-8 
16.65 

Die  Wirkung  des  Aspergillusaktivators  ist  mengenmassig  begrenzt  und  erreicht 
zwischen  0.2  und  0.5%,  auf  Pilztrockengewicht  berechnet,  das  Maximum.  In  einem 
Garansatz  mit  synthetischer  Nahrlosung  wurden  nach  17  Stunden  die  Milchsaurewerte 
der  Tabelle  II  erhalten.  Konzentrationen  iiber  0.5%  wirkten  hemmend. 


TABELLE  II 

Ansatz  Aspergillus  % 

mg  Milchsaure/ioo  ml  Nahrlosung 

0.05 

0.1 

0.2 

0.5 
1.0 

15.0 
27-3 
31.6 

34-1 
21.0 

Fraktioniert  man  die  in  den  beschriebenen  Versuchen  gebildete  Milchsaure  derart, 
dass  man  mit  Bariumacetat  und  Alkohol  bei  schwach  lackmussaurer  Reaktion  eine 
Fallung  vornimmt,  dann  findet  man  in  dieser  BariumfaUung  nach  alkahscher  Verseifung 
ebenfalls  Milchsaure.  Diese  Fallung  kann  aber  keine  vorgebildete  Milchsaure  enthalten, 
da  das  Ba-Salz  der  Milchsaure  noch  in  75%  Alkohol  spielend  losHch  ist.  Fallbar  mit 
Barium  und  Alkohol  und  zur  Milchsaure  umgesetzt  mit  Alkali  werden  aber  von  alien 
Intermediarprodukten  bis  jetzt  nur  die  Triosephosphorsauren.  Tatsachlich  lassen  sich 
auch  die  annahernden  Mengen  anorganisches  Phosphat  nach  der  Verseifung  mit  Alkali, 
herriihrend  aus  einer  alkali-empfind lichen  Phosphorsaureverbindung,  nachweisen.  Es  ist 
also  sicher  anzunehmen,  dass  in  dieser  fallbaren  Substanz  eine  Triosephosphorsaure 
Literatur  S.  ig8. 


196 


W.  KIESSLING 


VOL.  4  (1950) 


%70 


.60 


I 

■^50 


Odrungsmilchsaure         '  I 

Aus  der  Ba-  faltung ;  Normatansafz 

„     ,,     ^,        „      jZusatz  von 

Aspergillus-Exfrakt 

Mit  Ba  nicht  fallbar;  Normalansafz 

„     „     „        ^,       ;Zusatz  von  I 

Asperg.-Exfrakf  ,y^ 

/ 


vorliegt.  In  Abb.  3  ist  ein  derartiger  Versuch 
aufgezeichnet.  Kurve  I  ist  die  gebildete  Triose- 
phosphorsaure  bei  der  Angarung  ohne  Zusatz, 
Kurve  II  nach  Zusatz  von  0.2%  getrocknetem 
Aspergillusmicel.  Sie  erreicht  ihr  Maximum  bei 
40  mg  Milchsaure  pro  100  ml  Ansatz  und  ist 
um  das  1.75  fache  gegeniiber  dem  Normalansatz 
gesteigert.  Beide  Werte  fallen  bei  beginnender, 
sichtbarer  Garung  ab  und  betragen  bei  voller 
Garung,  also  im  stationaren  Zustand,  kaum  noch 
bestimmbare  Mengen.  Die  der  Milchsaure  ent- 
sprechende  Phosphat-Menge  ist,  soweit  sie  be- 
stimmbar  war,  in  Tabelle  III  aufgezeichnet. 

Die  gefundenen  Milchsaurewerte  in  der 
Mutterlauge  der  Ba-FaUungen,  die  der  prafor- 
mierten  Milchsaure  entsprechen,  sind  in  Kurve 
III  bzw.  Kurve  IV  aufgezeichnet.  Aus  ihrem 
Verlauf  sieht  man  deutlich,  dass  sie  im  Laufe 
der  Garung  bis  zu  Werten  von  ca.  50-60  mg  in 
100  ml  ansteigen.  Dieser  ungefahre  Wert  wird 

bei  alien  Garungen  mit  lebender  Hefe  gefunden.  Der  grosste  Teil  wird  in  der  Angarungs- 

phase  gebildet. 


Abb.  3 


TABELLE  III 


Zeit  in 

mg  Milchsaure/ 1 00  ml 

mg  PjOg  gefunden/ 1 00  ml 

mg  PjOg  berechnet/ioo  ml 

Tagen 

ohne  Zusatz 

mit  Aspergillus 

ohne  Zusatz 

mit  Aspergillus 

ohne  Zusatz 

mit  Aspergillus 

2 
3 

14.8 
234 
17-4 

31.2 

38.5 
25.2 

10.6 

17-3 
10.4 

22.3 
26.8 
16.9 

II. 7 
18.4 
13-7 

24.8 

30-4 
19.9 

ZUSAMMENFASSUNG 


Die  Angarungsphase  mit  intakter  Hefe  zeigt  das  Ansteigen  einer  scheinbaren  Milchsaurebildung, 
die  durch  Zusatz  eines  wassrigen  Aspergillus  Mtg-fy-Extraktes  wesentlich  gesteigert  werden  kann. 
Bei  Ubergang  zur  stationaren  Phase  fallt  diese  Milchsaure  bis  zu  einem  wahrend  der  ganzen  Garung 
gleichbleibenden  Wert  ab.  Durch  Ba-Salzbildung  kann  man  diese  Milchsaure  in  2  Fraktionen  zer- 
legen,  von  denen  die  alkoholunlosliche  Ba-Fallung  nach  ihren  Eigenschaften  als  eine  Triosephosphor- 
saure  angesprochen  werden  muss.  Damit  scheint  bewiesen,  dass  die  Spaltung  des  Zuckers  zu  Alkohol 
auch  mit  intakter,  lebender  Hefe  ebenso  wie  beim  zellfreien  Macerationssaft  iiber  Triosephosphor- 
saure  als  Zvvischcnprodukt  verlauft.  Es  ist  beliebig  unwahrscheinlich,  dass  die  darauffolgenden  Reak- 
tionen  einen  anderen  Weg  als  den  in  dem  Garungsschema  angegebenen  einschlagen  werden.  Die  Bil- 
dung  dieser  Triosephosphorsaure  ist  in  der  Angarungsphase  mit  den  darauffolgenden  Reaktionen 
nicht  synchronisiert,  sodass  es  moglich  ist,  die  vorauseilende  Bildung  dieser  Triosephosphorsaure 
analytisch  zu  erfassen.  In  der  stationaren  Phase  jedoch  liegt,  wie  zu  erwarten  war,  dieses  Zwischen- 
produkt  nicht  angereichert  vor. 

Weiter  zeigen  diese  Versuche  auch  den  Ursprung  der  bei  jeder  Hefegarung  entstehenden,  gerin- 
gen  Mengen  Milchsaure.  Er  liegt  hauptsachlich  in  der  Initialphase  und  steigt  langsam  bis  zu  einem 
konstant  bleibenden  Wert  wahrend  des  Garverlaufes  an,  der  f  iir  die  untersuchten  Weinhefen  zwischen 

Literatur  S.  ig8. 


VOL.  4  (1950)  TRIOSEPHOSPHORSAURE    IN    DER   ZUCKERGARUNG  I97 

30  und  60  mg/ioo  ml  Most  liegt.  Es  ist  noch  unklar,  ob  diese  Milchsaure  aus  einer  Anderung  des 
Stoffwechsels  von  der  ruhenden  zur  sprossenden  Hefe  stammt,  oder  ob  diese  Milchsaure  als  Produkt 
der  vorauseilenden  Triosephosphorsaurebildung  aufgefasst  werden  muss.  Im  letzteren  Fall  wiirde  sie 
natiirlich  nicht  durch  alkalische  Verseifung  entstanden  sein.  Sie  konnte  aber  aus  dem  spontanen  Zer- 
fall  von  Triosephosphat  zu  Methylglyoxal  herriihren.  Das  Methylglyoxal  wiirde  dann  durch  die 
Methylglyoxalase  in  Milchsaure  umgewandelt.  Damit  wiirde  zum  ersten  Mai  diesem  Enzym  in  der 
Hefe  eine  Funktion  zugewiesen  (Siehe  hierzu  auch  K.  Lohmann^). 


SUMMARY 

The  initial  phase  of  fermentation  with  whole  yeast  shows  the  onset  of  an  apparent  formation 
of  lactic  acid,  which  can  be  markedly  increased  by  watery  extracts  of  Aspergillus  niger.  On  transition 
to  the  stationary  phase  this  lactic  acid  decreases  to  an  amount  which  remains  constant  throughout 
the  fermentation.  By  forming  baryum  salts  this  lactic  acid  can  be  separated  into  two  fractions.  One 
of  these,  the  precipitate  which  is  insoluble  in  alcohol,  is  to  be  regarded  as  a  triose-phosphoric  acid, 
according  to  its  properties.  This  seems  to  prove  that  the  decomposition  of  sugar  to  alcohol  by  intact 
living  yeast  also  proceeds  by  way  of  triose  phosphoric  acid  as  intermediate,  just  as  in  the  case  of 
cell-free  maceration  juice.  It  is  rather  improbable  that  the  subsequent  reactions  would  follow  another 
route  than  has  been  indicated  in  the  scheme  of  alcoholic  fermentation. 

The  formation  of  this  triose  phosphoric  acid  has  not  yet  been  "synchronized"  with  the  following 
reactions  during  the  initial  phase  of  fermentation,  so  the  preceding  formation  of  this  triose  phosphoric 
acid  can  be  demonstrated  analytically.  In  the  stationary  phase,  however,  this  intermediate  is  not 
present  in  larger  amount,  as  is  to  be  expected. 

These  experiments  also  reveal  the  origin  of  the  small  amounts  of  lactic  acid  which  are  formed 
during  each  yeast  fermentation.  This  origin  is  to  be  found  in  the  initial  phase  and  the  amount  of 
lactic  acid  gradually  increases  when  the  fermentation  proceeds  until  a  constant  value  is  attained  which 
is  mostly  30-60  mg/ioo  ml  wort  for  the  wine  yeasts  investigated. 

It  is  not  yet  clear  whether  this  lactic  acid  originates  from  a  conversion  of  the  metabolisni  of 
resting  yeast  to  that  of  budding  yeast,  or  whether  it  must  be  regarded  to  be  a  product  of  the  preceding 
formation  of  triose  phosphoric  acid.  In  the  latter  case  it  would  of  course  not  have  been  formed  by 
alkaline  saponification.  It  could  however  arise  from  the  spontaneous  decomposition  of  triose  phosphate 
to  methylglyoxal.  The  latter  would  then  be  converted  into  lactic  acid  by  methylglyoxalase.  This 
would  be  the  first  time  that  a  function  is  appointed  to  this  enzyme  in  the  yeast  (See  also  K.  Lohmann*). 

(■ 
RfiSUMfi 

La  phase  initiale  de  la  fermentation  avec  de  la  levure  intacte  montre  une  augmentation  de  la 
formation  apparente  d'acide  lactique  qui  peut  etre  considerablement  accrue  par  I'adjonction  d'un 
extrait  aqueux  d' Aspergillus  niger.  Lors  du  passage  a  la  phase  stationnaire  la  quantity  d'acide  lactique 
d6croit  jusqu'a  une  valeur  qui  reste  constante  pendant  toute  la  duree  de  la  fermentation.  Par  trans- 
formation en  sels  de  barium  cet  acide  lactique  apparent  peut  etre  separe  en  deux  fractions;  le  pr^cipite; 
de  barium  insoluble  dans  I'alcool  doit  etre  considere,  d'apres  ses  propri^t^s,  comme  provenant  d'un 
acide  triose-phosphorique.  Ceci  semble  d^montrer  que  la  transformation  du  sucre  en  alcool  se  produit 
sous  Taction  de  la  levure  intacte  vivante,  de  meme  que  sous  Taction  d'un  extrait  exempt  de  cellures 
en  passant  par  Tacide  triose-phosphorique  comme  intermediaire.  II  est  assez  peu  probable  que  les 
reactions  suivantes  passent  par  un  autre  chemin  que  celui  indique  dans  le  schema  de  la  fermentation 
alcoolique. 

Dans  la  phase  initiale  cette  formation  d'acide  triose-phosphorique  n'est  pas  "synchronisee"  avec 
les  reactions  suivantes  et  c'est  pourquoi  il  est  possible  de  demontrer  son  existence  analytiquement 
Cependant,  ainsi  que  Ton  pouvait  s'y  attendre,  ce  produit  intermediaire  n'est  pas  accumule  pendant 
la  phase  stationnaire . 

Ces  experiences  revelent  de  plus  Torigine  des  faibles  quantites  d'acide  lactique  rencontr^es  dans 
toute  fermentation  produite  par  la  levure.  La  formation  d'acide  lactique  commence  dans  la  phase 
initiale  at  augmente  pendant  la  fermentation  jusqu'a  une  valeur  constante  qui  est  de  30  a  60  mg/ioo 
ml  de  moiit  pour  les  levures  de  vin  examinees. 

Cet  acide  lactique  provient-il  du  passage  du  metabolisme  de  la  levure  au  repos  a  celui  de  la 
levure  bourgeonnante  ou  bien  est-il  un  produit  de  la  formation  prec^dente  d'acide  triose-phospho- 
rique ?^  Detoutes  fa9ons,  dans  ce  dernier  cas,  il  ne  pourrait  pas  provenir  d'une  saponification  alcaline 
mais  bien  d'une  decomposition  spontanee  du  triose-phosphate  en  m^thylglyoxale.  Ce  dernier  serait 
ensuite  transforme  en  acide  lactique  par  le  methylglyoxalase.  Ce  serait  la  premiere  fois  qu'une 
onction  fiit  attribuee  a  cet  enzyme  dans  la  levure  (voir  aussi  K.  Lohmann^). 

Literatur  S.  198. 


igS  W.  KIESSLING  VOL.  4  (1950) 

LITERATUR 

^  O.  Meyerhof  und  W.  Kiessling,  Biochem.  Z.,  267  (1933)  313. 

2  O.  Meyerhof  und  W.  Kiessling,  Biochem.  Z.,  281  (1935)  249. 

^  O.  Meyerhof,  W.  Kiessling  und  W.  Schulz,  Biochem.  Z.,  292  (1937)  25. 

*aO.  Meyerhof  und  K.  Lohmann,  Biochem.  Z.,  271  (1934)  ^9- 

*  Friedemann,  Contonio  und  Shaffer,  /.  Biol.  Chem.,  73  (1927)  355. 

^  Henneberg,  Handbuch  d.  Gdrungsbakteriologie,  II.  Auflage  (1926)  Seite  50. 

^  K.  Lohmann  und  Jendrassik,  Biochem.  Z.,  178  (1926)  419. 

^  H.  V.  Euler  und  Nielsen,  Zentr.  Bakt.  Parasitenk.  Abt.  II,  100  (1939)  435. 

®  K.  Lohmann,  Biochem.  Z.,  254  (1932)  332. 

Eingegangen  den  14.  Marz  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  I99 


CONFIGURATIONAL  RELATIONSHIPS  BETWEEN  NATURALLY 
OCCURRING  CYCLIC  PLANT  ACIDS  AND  GLUCOSE 

TRANSFORMATION  OF  QUINIC  ACID  INTO  SHIKIMIC  ACID 

by 

GERDA  DANGSCHAT  and  HERMANN  O.  L.  FISCHER 

Department  of  Biochemistry,  University  of  California  Berkeley  4, 

California  {U.S.  A .) 


The  old  idea  that  meso-inositol  could  be  formed  by  cyclization  of  D-glucose  gained 
considerable  strength  by  the  determination  of  the  configuration  of  meso-inositol^  by 
Gerda  Dangschat,  which  later  was  confirmed  by  Th.  Posternak.  Similar  circum- 
stances could  be  demonstrated  in  the  field  of  cyclic  plant  acids,  for  instance  Quinic  Acid 
and  Shikimic  Acid.  We  were  able  to  prove  their  constitution^  and  their  planar  con- 
figuration^. 

In  1937  we  succeeded*  by  the  degradation  of  shikimic  acid  into  2-desoxygluconic 
acid,  IX^  in  demonstrating  the  same  configuration  for  carbon  atoms  3,  4  and  5  of 
shikimic  acid  as  is  found  for  carbon  atoms  3,  4  and  5  of  D-glucose. 

An  analogous  relationship  between  quinic  acid^,  which  is  more  commonly  found 
in  the  plant  kingdom,  and  D-glucose,  seemed  very  probable  at  that  time.  This  phy- 
siologically important  relationship  could  be  established  with  certainty  by  transforming 
quinic  acid  into  shikimic  acid.  In  this  communication  we  describe  the  successful  trans- 
formation of  derivatives  of  quinic  acid  into  those  of  shikimic  acid'. 

The  use  of  the  acetone  compounds  of  quinic  acid,  which  in  previous  work  with 
these  substances  had  proven  highly  satisfactory,  met  with  unexpected  difficulties.  We 
therefore  employed  the  formaldehyde  derivatives  which  are  described  in  the  preceding 
paper*,  after  having  determined  that  the  methylene  group  blocked  the  hydroxyls  of 
carbon  atoms  4  and  5  of  the  quinic  acid  as  did  the  acetone. 

We  used  a-toluene  sulphonyl  derivatives  of  quinic  acid  and  found  that  the  formation 
of  a  double  bond  by  the  splitting  off  of  the  toluene  sulphonic  acid  by  alkali  only  pro- 
gressed smoothly  after  conversion  to  the  nitrile,  thus  considerably  weakening  the  stabi- 
lizing influence  of  the  carboxyl  group.  By  prolonged  treatment  of  the  3-acetyl-4,5-formal 
quinic  amide,  P  with  excess  of  ^-toluene  sulphonyl  chloride  and  pyridine  we  performed 
three  reactions  in  one  operation:  toluene  sulphonylation  of  the  amide,  nitrilization  of 
the  amide,  and  finally  the  splitting  off  of  the  toluene  sulphonyl  group  from  the  nitrile, 
with  the  result  that  the  nitrile  of  the  expected  3-acetyl-4,5-methylene  shikimic  acid,  II, 
could  be  isolated.  This  could  be  converted  by  means  of  alkali  into  the  methylene  deriv- 
ative of  shikimic  acid,  IIP"  which  was  transformed  into  free  shikimic  acid,  V,  in  acid 
solution.  The  identification  of  shikimic  acid  was  made  by  melting  points,  mixed  melting 
points,  and  optical  determinations. 
References  p.  203I204. 


200 


G.  DANGSCHAT,  H.  O.  L.  FISCHER 


VOL.  4  (1950) 


This  shows  conchisively  that  quinic  acid  bears  the  same  steric  relationship  to 
D-glucose  as  that  which  has  already  been  demonstrated  for  shikimic  acid^^. 

Furthermore,  since  the  structural  connection  between  quinic  acid,  IV,  -»  shikimic 
acid,  V,  ->  gallic  acid,  VI,  is  obvious,  it  seems  to  us  that  in  this  chemical  relationship 
we  have  an  indication  that  many  hydroaromatic  and  aromatic  plant  products  aie 
actually  formed  biologically  from  carbohydrates.  In  addition,  it  might  be  mentioned 
that  our  transformation  in  vitro  of  quinic  acid  to  citric  acid,  VII,  by  means  of  periodic 
acid^2  has  perhaps  its  biological  counterpart  in  the  work  of  But^ewitsch"  who  suc- 
ceeded in  establishing  a  connection  between  the  fermentability  of  quinic  acid  and  the 
formation  of  citric  acid  in  the  hfe  of  bacteria  and  fungi. 


H      H 


H      H 


H       H 


-CN 


NaOH 


CH, 


COOH 


H        H 

Formal  shikimic  Acid 
III 


H 


HO 


H       H 
'OH    H^ 


XOH    HX 

H       H 

Quinic  Acid 
IV 


H       H 


COOH 


OH 


H      /OH 


HO    XoH    H> 
H       H 

Shikimic  Acid 
V 


HO 


H 


-COOH 


HO- 


ho: 


HO-C-COOH 


C-COOH 


HOOC-HjC        CH2-COOH 

Citric  Acid 

VII 


HOOC-HC        CH2-COOH 

Aconitic  Acid 

VIII 


COOH 


H 
Gallic  Acid 

vi 


COOH 
H-C-H 
HO-C-H 
H-C-OH 
H-C-OH 
CH2OH 
2-Desoxygluconic  Acid 
IX 


SUPPLEMENT 


Our  experiments  described  in  this  paper  on  the  transformation  of  quinic  acid  into 
shikimic  acid  by  splitting  out  water  clearly  show  how  strongly  the  carboxyl  of  the 
quinic  acid  influences  its  tertiary  hydroxyl  in  the  a  position,  and  probably  also  the 
remainder  of  the  molecule. 


References  p.  203I204. 


VOL.  4  (1950) 


CYCLIC   PLANT   ACIDS   AND    GLUCOSE 


201 


Our  previous  papers  on  the  oxidation  of  quinic  acid,  shikimic  acid  and  dihydro- 
shikimic  acid  by  means  of  periodic  acid  have  made  available  a  series  of  1.5-dialdehydes, 
which,  depending  on  their  origin,  possess  either  a  free  or  blocked  hydroxyl  and  carboxyl 
group,  or  a  carboxyl  group  alone.  The  possession  of  these  aldehydes  led  us  to  an  alkaloid 
synthesis,  along  the  lines  of  the  lobelanine  synthesis  of  Schopf^^.  The  condensation, 
however,  was  successful  only  after  the  elimination  of  the  electro-negative  groups  and 
the  choice  of  a  1.5-dialdehyde  which  no  longer  contained  any  hydroxyl  groups  and  only 
a  carboxyl  group  in  the  form  of  its  nifrile.  This  was  the  dialdehyde,  XI,  which  is  obtained 
by  treating  the  nitrile  of  the  dihydro-shikimic  acid  with  2  molecules  of  periodic  acid^'^-^-. 


C=N 

H-C 
HjC/        jCHg        NaOCH^ 

AcOChI       JcHOAc 
CHOAc 


2HI04 


/ 

H-C 


C=N 


H,C 


CH, 


H-C=0  C=0 


H-C 

2C,HiCO-CH;-COOH        HgCj^        1CH2 
NHj-CH,  ^  I 

0=C-CH2-H  Cv      /'CH  •  CH2-C=0 


C«H, 


X 


XI 


I 

CH3 

XII 


C«H, 


Experimentally  the  synthesis  was  carried  out  in  the  following  way:  Triacetyl 
dihydro-shikimic  acid  amide,  was  transformed  into  the  corresponding  nitrile  X,  by 
heating  with  acetic  anhydride.  The  nitrile  was  de-acetylated  with  a  minimum  amount 
of  sodium  methylate  according  to  Zemplen.  and  the  free  nitrile  was  transformed  into 
the  dialdehyde,  XI,  by  the  action  of  2  molecules  of  periodic  acid.  The  dialdehyde  was 
not  isolated,  but  was  condensed  directly  in  aqueous  solution  with  2  molecules  of  benzoyl 
acetic  acid  ester  and  i  molecule  of  monomethylamine  at  a  p^  of  4. 

The  3-cyano-lobelanine,  XII,  was  isolated  in  a  yield  of  30%  (calculated  on  the 
amount  of  triacetyl  dihydro-shikimic  acid  nitrile),  and  showed  the  usual  precipitation 
reaction  of  alkaloids,  e.g.  with  perchloric  acid,  picric  acid  and  picrolonic  acid.  It  crystal- 
lized in  long  shining  silklike  needles  similar  to  those  of  caffein,  and  showed  a  melting 
point  of  143°. 

experimental 

Preparation  of  the  acetyl-methylene-shikimic  acid  nitrile  from  monacetyl-methylene-quinic  acid  amide 
5  g  monacetyl-methylene-quinic  acid^^  were  shaken  with  10  g  (2^4  molecules)  ^-toluene  sul- 
phonyl chloride  in  15  ml  dry  pyridine  for  a  short  time  until  dissolved.  The  brown  coloured  solution 
was  kept  for  seven  days  at  37°.  The  solution  was  then  diluted  with  20  ml  of  water,  and  an  oily  sub- 
stance separated.  It  was  allowed  to  stand  with  occasional  shaking  for  15  minutes  at  room  tempera- 
ture in  order  to  destroy  any  unused  toluene  sulphonyl  chloride.  The  solution  was  then  extracted 
twice  with  a  large  volume  of  chloroform.  The  united  chloroform  fractions  were  next  shaken  up  with 
small  portions  of  dilute  sulphuric  acid  until  all  the  pyridine  was  neutralized,  and  no  more  acid  was 
used  up.  The  solution  was  washed  with  a  little  water  and  then  dried  with  sodium  sulphate.  The  mix- 
ture was  next  filtered  and  the  filtrate  was  evaporated  in  the  vacuum  of  a  water  pump  to  remove  all 
solvent.  The  light-brown  oil  (4.5  g)  remaining  was  distilled  under  high  vacuum.  A  light  yellow  oil 
(2.3-2.8  g,  i.e.,  54-65%  of  the  theoretical  yield)  distilled  over  at  0.2  mm  and  a  bath  temperature 
of  150-165°.  It  had  a  boiling  point  of  128°.  After  a  second  distillation  it  was  almost  colourless,  but 
had  a  slight  odour  of  toluene  sulphonic  acid  and  a  minimum  content  of  sulphur. 

Preparation  of  unsaturated  nitrile  from  monacetyl-isopropylidenequinic  acid  amide^^ 

Reaction  and  processing  follow  exactly  as  described  for  the  corresponding  methylene  compound. 
4.3  g  {i.e.,  77%  of  the  theoretical  yield)  unsaturated  acetyl-isopropylidene  nitrile  were  obtained 

from  6.5  g  monacetyl-isopropylidene-quinic  acid  amide.  The  compound  had  a  light  yellow  colour 

and  a  boihng  point  of  i25°/o.i5  mm. 

References  p.  203I204. 


202  G.  DANGSCHAT,  H.  O.  L.  FISCHER  VOL.  4  (1950) 

A  sample  twice  redistilled  was  used  for  analysis: 

5.068  mg  gave  11.195  rng  COj  and  2.860  mg  HjO; 
3.169  mg  gave  0.151  ml  Nj  (26°  and  741  mm). 
C12H15O4N  (237.1):  Calc.     C60.7     H  6.4     N  5.9 
Found  C  60.8     H  6.3     N  5.4 

Hydrolysis  of  acetyl-methylene-shikimic  acid  nitrile  to  methylene-shikimic  acid 

3.3  g  distilled  acetyl-methylene-shikimic  acid  nitrile  were  boiled  for  two  and  a  half  hours  with 
45  ml  N  sodium  hydroxide  (about  3  molecules).  A  condenser  was  attached  to  take  off  the  water 
vapours  and  the  ammonia.  Water  was  added  to  the  distillation  flask  during  boiling  so  that  the  volume 
was  not  reduced  below  one-half  the  original.  The  condensate  was  caught  in  an  ice-cooled  receiver 
and  at  the  end  of  the  time  the  ammonia  could  be  determined  almost  quantitatively.  No  formaldehyde 
was  found  in  the  distillate  even  after  acid  hydrolysis.  The  reaction  liquid,  which  was  coloured  dark 
brown,  was  cooled  and  the  alkali  was  neutralized  by  addition  of  41  ml  N  sulphuric  acid  and  4  ml 
N  hydrochloric  acid.  The  weak  acetic  acid  solution  was  reduced  to  dryness  in  the  best  vacuum  ob- 
tainable by  a  water  pump,  during  which  time  the  bath  temperature  was  not  allowed  to  rise  above  35°. 
The  residue  was  extracted  thoroughly  several  times  with  ethyl  acetate,  and  the  united  filtered  extrac- 
tions were  evaporated  under  reduced  pressure.  If  crystals  are  at  hand  for  inoculation,  the  yellow 
syrup  remaining  will  begin  to  crystallize  on  inoculating.  33-38%  of  the  theoretical  yield  of  crystal- 
lized methylene-shikimic  acid  was  obtained  from  the  concentrated  ethyl  acetate  solution,  but  these 
crystals  still  had  a  yellow  colour.  Using  animal  charcoal,  a  recrystallization  from  ethyl  acetate  was 
made  for  further  purification. 

The  substance,  well  crystallized  in  rhombic  plates,  had  a  m.p.  of  138°  and  showed  no  depression 
of  the  melting  point  on  addition  of  an  equal  quantity  of  a  preparation  made  from  shikimic  acid.  The 
preparation  twice  recrystallized  gave  in  aqueous  solution  the  following  rotation: 

[a]Jf°  =  _88.7°i7  (I  dm  tube,  c  =  2.17,  a^  =  —1.93°). 

Further  quantities  of  the  acid  could  be  obtained  from  the  motherliquor  of  the  isolated  methylene- 
shikimic  acid  in  the  following  manner:  The  methylene-shikimic  acid  methyl  ester  was  formed  by 
esterification  with  diazomethane  and  was  distilled  under  a  high  vacuum  at  a  bath  temperature  of 
170-190°.  It  was  then  kept  for  two  to  three  days  at  37°  together  with  pyridine  and  toluene  sulphonyl 
chloride.  The  toluene  sulphonyl-methylene-shikimic  acid  ester  (m.p.  and  m.p.  of  the  mixture  1 18-1 19°) 
crystallized  out  readily  on  gradual  addition  of  water  and  trituration.  This  isolated  quantity  corres- 
ponds to  a  further  15-20%  of  the  theoretical  yield  of  methylene-shikimic  acid,  so  that  together 
about  52%  of  the  acid  obtained  from  the  nitrile  can  be  definitely  identified  as  a  derivative  of  the 
shikimic  acid.  The  methylene-shikimic  acid  is  easily  isolated  and  identified  by  preparing  its  toluene 
sulphonyl-methyl  ester,  which  readily  crystallizes.  This  process  is  to  be  recommended,  if  no  inocula- 
tion crystals  of  the  free  methylene-shikimic  acid  are  at  hand  or  if  difficulties  appear  during  the  isola- 
tion of  the  free  acid.  After  washing  with  50%  alcohol  the  ester  is  at  once  obtained  in  the  pure  state. 
For  analysis  and  optical  determinations  it  has  to  be  recrystallized  once  more  from  alcohol: 

5071  mg  substance  gave  10.090  mg  COj  and  2.350  mg  HgO 
7921  mg  substance  gave    5170  mg  BaSO^ 
CisHigO^S  (354.2):  Calc.      C  54.2     H  5.1     S  9.1 
Found  C  54.2     H  5.2     S  9.0 
[a]  ff°  =  —42.5°  (in  chloroform)i8  (i  dm  tube,  c  =  3.25,  a^  =  —1.38°). 

Hydrolysis  of  the  unsaturated  acetyl-isopropylidene  nitrile 

The  hydrolysis  of  the  unsaturated  isopropylidene  nitrile  can  be  carried  out  under  the  same  mild 
conditions  as  the  corresponding  methylene  compound.  In  this  reaction  the  ammonia  can  also  be 
determined  nearly  quantitatively  after  about  two  hours  boiling  with  dilute  alkali.  Furthermore,  it 
was  found  that  25%  of  the  theoretically  possible  amount  of  acetone  was  split  off  by  the  alkali.  The 
acetone  could  be  determined  in  the  distillate  by  titration  with  alkaline  hypoiodite  solution  and 
identified  as  the  ^-nitro-  or  dinitrophenylhydrazone.  The  further  processing  parallels  the  procedure 
used  for  the  methylene  nitrile.  From  the  acetonated  compound,  however,  it  was  not  possible  to  isolate 
the  free  acetonated  acid,  nor  to  crystallize  a  derivative  of  the  acetonated  unsaturated  ester,  which 
had  been  obtained  by  esterification  with  diazomethane  and  subsequent  distillation  in  a  high  vacuum. 
If,  however,  the  unsaturated  ester,  of  which  27%  of  the  theoretical  yield  was  obtained  by  distillation, 
is  hydrolysed  by  acetic  acid,  about  4.5%  of  the  theoretical  amount  (based  on  the  amount  of  nitrile 
used)  is  obtained  in  crystallized  form^®  as  shikimic  acid  methyl  ester.  After  two  recrystallizations 
from  ethyl  acetate  and  ligroin,  the  m.p.  was  112-114°  ^.nd  there  was  no  depression  of  the  melting 
point  when  the  substance  was  mixed  with  equal  amounts  of  a  compound  prepared  from  shikimic 
acid  for  comparison. 

References  p.  203/204. 


VOL.  4  (1950)  CYCLIC   PLANT   ACIDS   AND   GLUCOSE  2O3 

SUMMARY 

The  transformation  of  quinic  acid  into  shikimic  acid  by  means  of  the  methylene  derivatives 
of  these  acids  has  been  described.  Thus  the  configuration  of  the  carbon  atoms  3,4  and  5  of  quinic 
acid  has  been  shown  to  be  the  same  as  in  shikimic  acid,  which  had  previously  been  configurationally 
related  to  D-glucose. 

3-cyano-lobelanine  has  been  synthesized  from  dihydroshikimic  acid  nitrile,  benzoyl  acetic  acid, 
and  monomethyl  amine  under  conditions  sufficiently  mild  so  that  they  might  exist  in  plant  or  animal 
organisms. 

RfiSUMfi 

Nous  avons  decrit  la  transformation  de  I'acide  quinique  en  acide  shikimique  a  I'aide  des  derives 
methyMniques  de  ces  acides.  Nous  avons  montre  ainsi  que  la  configuration  des  atomes  de  carbone 
3,  4  et  5  dans  I'acide  quinique  est  la  meme  que  dans  I'acide  shikimique,  dont  la  configuration  avait 
6t6  pr^cedemment  reliee  a  celle  du  D-glucose. 

La  3-cyano-lobelanine  a  6te  synthetisee  a  partir  du  nitrile  de  I'acide  dihydro-shikimique,  de 
I'acide  benzoylacetique  et  de  la  monomethylamine  sous  des  conditions  suffisamment  douces  pour 
exister  dans  I'organisme  vegetal  ou  animal. 

ZUSAMMENFASSUNG 

Wir  beschreiben  die  Umwandlung  der  Chinasaure  in  die  Shikimasaure  iiber  die  entsprechenden 
Methylenderivate.  Es  wurde  also  gezeigt,  dass  die  Konfiguration  der  Kohlenstoffatome  3,  4  und  5 
in  der  Chinasaure  dieselbe  ist  wie  in  der  Shikimasaure,  deren  Konfiguration  schon  friiher  auf  die  der 
D-Glucose  zuriickgefiihrt  wurde. 

3-Cyanolobelanin  wurde  aus  Dihydroshikimisaure-nitril,  Benzoylessigsaure  und  Monomethyl- 
amin  unter  milden  Bedingungen  synthetisiert,  wie  sie  auch  im  pflanzlichen  oder  tierischen  Organismus 
vorkommen  konnen. 


REFERENCES 

^  G.  Dangschat,  Naturwisenschaften  ,30  (1942)  146;  C.  A.,  37  (1943)  3408®. 

Th.  Posternak,  Helv.  Chini.  Acta,  25  (1942)  746. 

Confer  also  H.  O.  L.  Fischer,  Harvey  Lectures,  Ser.  40  (1945)  156-178. 

H.  G.  Fletcher  Jr,  Advances  in  Carbohydrate  Chem.,  Vol.  3,   Academic   Press,   Inc.,   New  York 

1948. 
2  H.  O.  L.  Fischer  and  G.  Dangschat,  Ber.,  65  (1932)  1009  and  Helv.  Chim.  Acta,  17  (1934)  1200. 

*  H.  O.  L.  Fischer  and  G.  Dangschat,  Helv.  Chim.  Acta,  18  (1935)  1206. 
^  H.  O.  L.  Fischer  and  G.  Dangschat,  Helv.  Chim.  Acta,  20  (1937)  705- 

*  Max  Bergmann  et  al.,  Ber.,  55  (1922)  158;  Ber.,  56  (1922)  1052. 
P.  A.  Levene  and  G.  Mikeska,  /.  Biol.  Chem.,  88  (1930)  791. 

®  Quinic  acid  occurs  not  only  in  the  free  state  in  the  plant  kingdom  but  also  for  example  in  chloro- 
genic  acid  as  a  depside  with  caffeic  acid.  For  the  constitution  of  chlorogenic  acid  cf.  H.  O.  L.  Fischer 
and  G.  Dangschat,  Ber.,  65  (1932)  1037. 

^  A  preliminary  notice  on  the  same  subject  has  been  published  in  Die  Naturwissenschaften,  26 
(1938)  562. 

**   gth  Communication  on  Quinic  Acid  and  derivatives,  J.A.C.S.,  in  press. 

*  gth  Communication  on  Quinic  Acid  and  derivatives,  J.A.C.S.,  in  press. 
'"  gth  Communication  on  Quinic  Acid  and  derivatives,  J.A.C.S.,  in  press. 

^1  This  relationship  is  also  a  confirmation  of  the  assumption  of  the  cis  position  of  the  hydroxyls 
4  and  5  of  quinic  acid  and  shikimic  acid  which  we  have  always  made  on  the  basic  of  the  work  of 
Boeseken  (cf.  also  Huckel,  Theoretische  Grundlagen  der  Chemie,  i  (65-66). 

^^  H.  O.  L.  Fischer  and  G.  Dangschat,  Helv.  Chim.  Acta,  17  (1934)  1196.  Cf.  also  shikimic  acid 
-^  aconitic  acid,  VIII,  H.  O.  L.  Fischer  and  G.  Dangschat,  Helv.  Chim.  Acta,  18  (1935)  1204. 

1^  Wl.  Butkewitsch,  Biochem.  Z.,  145  (1924)  442. 

!■*  C.  ScHOPF  and  G.  Lehmann,  Liebig's  Ann.,  518  (1935)  1-37. 

^*  See  gth  Communication  on  Quinic  Acid  and  derivatives,  J.A.C.S.,  in  press. 

^®  H.  O.  L.  Fischer  and  G.  Dangschat,  Ber.,  65  (1932)  1020.  The  yield  is  increased  if  the  processing 
is  performed  two  hours  after  action  of  the  acetylation  reagent. 

^'  See  gth  Communication  on  Quinic  Acid  and  derivatives,  J.A.C.S.,  in  press. 


204 


G.  DANGSCHAT,  H.  O.  L.  FISCHER  VOL.  4  (1950) 


18  After  acid  hydrolysis  following  the  prescription  given  in  the  "gth  Communication  on  Quinic  Acid 
and  derivatives"  free  shikimic  acid  is  obtained: 

its  m.p.  and  m.p.  of  a  50%  mixture  184-185°;  [a]^°  —  — 183° 
(in  water,  i  dm  tube,  c  =  1.23,  ajf  =  — 2.25°). 
"  The  small  yield  of  crystallized  substance  suggests  that  the  acetyl-isopropylidene-shikimic  acid 
nitrile  contains,  unlike  the  corresponding  methylene  compound,  a  considerable  quantity  of  a 
1,2  unsaturated  product. 

Received  June  23rd,  1949 


VOL.  4  (1950)  BIOCHIMICA   ET   BIOPHYSICA   ACTA  205 


PARTIAL  PURIFICATION  OF  ISOCITRIC  DEHYDROGENASE  AND 
OXALOSUCCINIC  CARBOXYLASE* 

by 

ALLAN  L.  GRAFFLIN**  and  SEVERO  OCHOA 

Department  of  Pharmacology,  New  York  University  College  of  Medicine, 
New  York  {U.S.A.) 


It  has  been  shown^'^  that  the  over-all  reversible  Reaction  i,  catalysed  by  enzymes 
present  in  a  number  of  tissues,  involves  two  steps  (Reactions  2  and  3). 

1.  rf-Isocitrate  +  TPN^^  ^^^^  a-ketoglutarate  +  CO2  +  TPN,ed 

2.  (^-Isocitrate  +  TPN^^  v  "*  oxalosuccinate  +  TPN^ed 

3.  Oxalosuccinate  "*  a-ketoglutarate  +  COg 

Crude  enzyme  solutions  from  heart  muscle^,  liver^  and  higher  plants'*  catalyse 
Reaction  i  in  either  direction,  as  well  as  the  decarboxylation  of  oxalosuccinate  (Reac- 
tion 3),  in  the  presence  of  added  manganous  ions.  Reaction  2  can  be  shown  to  occur  in 
either  direction  with  the  same  enzyme  solutions  when  Mn++  is  excluded^. 

Partial  purification  of  isocitric  dehydrogenase,  as  tested  by  Reaction  i,  was  pre- 
viously reported^.  A  four-fold  purification  of  the  activity  exhibited  by  extracts  of  ace- 
tone-dried pig  heart,  with  very  low  yield,  was  obtained  at  that  time.  Lynen  and 
ScHERER^  have  recently  reported  the  synthesis  of  oxalosuccinic  acid  and  the  catalysis 
of  the  decarboxylation  of  this  compound  by  enzymes  from  various  sources.  Their  work, 
carried  out  without  knowledge  of  the  work  of  this  laboratory,  led  essentially  to  the  same 
results.  They  also  reported  partial  purification  of  the  oxalosuccinic  carboxylase  activity 
(Reaction  3)  of  horse  liver. 

A  somewhat  improved  method  of  purification  of  the  isocitric  dehydrogenase  and 
oxalosuccinic  carboxylase  activities  of  pig  heart,  as  determined  according  to  Reactions 
I  and  3,  is  described  in  this  paper.  A  six-fold  purification  of  the  activity  of  the  extracts 
with  a  yield  of  about  15%  has  been  obtained.  There  was  no  separation  of  activities  as 
tested  by  Reactions  i  and  3,  but  both  these  activities  were  increased  with  respect  to 
malic  dehydrogenase.  Thus,  the  question  whether  Reactions  2  and  3  are  catalysed  by 
distinct  enzymes  (isocitric  dehydrogenase  and  oxalosuccinic  carboxylase  respectively), 
or  by  a  single  enzyme,  still  remains  unsettled. 


*  Aided  by  grants  from  the  United  States  Public  Health  Service,  the  American  Cancer  Society  (re- 
commended by  the  Committee  on  Growth  of  the  National  Research  Council),  the  Office  of  Naval  Research 
and  the  Lederle  Laboratories  Division,  American  Cyanamid  Company. 

Senior  Fellow  in  Cancer  Research,  American  Cancer  Society,  upon  recommendation  of  the 
Committee  on  Growth  of  the  National  Research  Council.  Present  address,  Department  of  Anatomy, 
School  of  Medicine,  The  Johns  Hopkins  University,  Baltimore,  Maryland. 

References  p.  210. 


206 


A.  L.  GRAFFLIN,  S.  OCHOA 


VOL.  4  (1950) 


OPTICAL   TESTS   AND   ENZYME    UNITS 

Over-all  Reaction.  —  The  activity  determination  is  based  on  Reaction  i.  The  early  rate  of 
reduction  of  triphosphopyridine  nucleotide  (TPN)  in  the  presence  of  enzyme,  Mn+"'",  and  an  excess  of 
isocitric  acid,  is  proportional  to  the  concentration  of  the  enzyme  within  certain  limits.  The  measure- 
ment is  carried  out  in  the  Beckman  spectrophotometer  at  wave-length  340  m^  using  either  glass  or 
silica  cells  of  i.o  cm  light  path.  One  enzyme  unit  was  defined  as  the  amount  of  enzyme  causing  an 
increase  in  optical  densit}'  of  0.0 1  per  minute  calculated  for  the  third  15  second  period  after  the  start 
of  the  reaction. 

The  reaction  mixture,  in  a  final  volume  of  3.0  ml  contained  0.025  ^  glycyl-glycine  buffer  pH 
7.4,  0.6- 10-^  M  MnClg,  0.45  •  10-*  M  TPNqx,  enzyme,  and  0.175  •  1°"^  M  rf,/-isocitrate.  The  volume  was 
made  up  with  water  adjusted  to  a  temperature  of  22-23°.  The  blank  cell,  for  setting  at  100%  light 
transmission,  contained  all  the  above  components  except  TPN.  The  reaction  was  started,  after  taking 
a  zero  time  reading,  by  addition  of  either  enzyme  or  isocitrate.  The  presence  of  phosphate  in  concen- 
trations higher  than  0.0003  M  should  be  avoided  because  turbidity,  due  to  precipitation  of  manganous 
phosphate,  may  develop.  Typical  results  obtained  with  an  extract  of  washed  acetone-dried  pig 
heart  containing  6.0  mg  of  protein  per  ml  are  shown  in  Table  I. 

TABLE  I 

OPTICAL    TEST    FOR    REACTION    I 
PROPORTIONALITY    OF    RATE    TO    ENZYME    CONCENTRATION 


/llog  (lo/I) 

Enzyme 
concentration 

between  30  and 

45  seconds,  at 

340  mij, 

Units 

Specific  activity 

mg  protein  in 

unitsjm.g  protein 

3.0  ml 

0.012 

-\-  0.005 

2.0 

166 

0.024 

+  0.012 

4.8 

198 

0.036 

-1-  0.017 

6.8 

188 

0.048 

+  0.023 

9.2 

192 

0.060 

-j-  0.026 

10.4 

173 
Average  183 

The  protein  content  of  the  enzyme  solutions  was  determined  spectrophotometrically  by  mea- 
suring the  absorption  of  light  at  wave-lengths  280  and  260  m//.  The  protein  concentration  was  cal- 
culated from  the  absorption  at  280  m/^  with  a  correction  for  the  nucleic  acid  content  from  the  data 
given  by  Warburg  and  Christian®. 

Oxalosuccinic  Carboxylase.  —  The  oxalosuccinic  carboxylase  activity  (Reaction  3)  was  deter- 
mined by  means  of  a  rapid  and  sensitive  optical  test.  The  test  is  based  on  the  fact  that,  in  the  presence 
of  Mn"*""^  and  oxalosuccinate,  the  enzyme  causes  a  pronounced  increase  in  the  absorption  of  light  at 
the  wave-length  240  m/i,  presumably  as  a  result  of  increased  formation  of  an  intermediate  oxalo- 
succinate-manganese  complex ;  this  increase  is  followed  by  a  rapid  drop  indicating  decarboxylation^. 
The  early  rate  of  increase  of  light  absorption  is,  within  certain  limits,  proportional  to  the  concentra- 
tion of  enzj^me.  The  measurement  is  carried  out  in  the  Beckman  spectrophotometer  using  silica  cells 
of  1.0  cm  light  path.  One  enzyme  unit  was  defined  as  the  amount  of  enzyme  causing  an  increase 
in  optical  density  of  o.oi  per  minute  calculated  for  the  first  15  second  period  after  the  start  of  the 
reaction. 

The  reaction  mixture,  in  a  final  volume  of  3.0  ml,  contained  0.134  ^^  potassium  chloride,  enzyme, 
0.167-10-^  M  MnClg,  and  approximately  0.167-10-^  M  oxalosuccinate*.  The  volume  was  made  up 
with  water  adjusted  to  a  temperature  of  15°.  The  blank  cell  contained  no  oxalosuccinate.  The  reaction 
was  started  by  addition  of  oxalosuccinate,  which  was  blown  into  the  mixture  from  a  Lang-Levy 
micropipette^,  and  readings  of  the  optical  density  were  made  at  15  second  intervals  thereafter  for  i 
or  2  minutes.  The  optical  density  of  the  oxalosuccinate  was  determined  separately  and  furnished  the 
zero  time  value.  The  amount  of  enzyme  was  so  adjusted  that  an  increase  in  optical  density  not  below 
0.07  nor  above  0.20  was  obtained  in  the  first  15  seconds.  The  reason  for  the  presence  of  potassium 
chloride  is  that  it  was  found  to  increase  the  activity  of  the  enzyme.  This  effect  appears  to  be  a  non- 
specific one  caused  by  the  increased  ionic  strength'.  The  presence  of  phosphate  in  concentrations  higher 
than  0.0003  M  should  be  avoided  for  the  reasons  already  stated.  Typical  results  obtained  with  the 
acetone  powder  extract  of  pig  heart  are  shown  in  Fig.  i.  , 

*  Prepared  as  previously  described*. 

References  p.  210. 


VOL.  4  (1950) 


ENZYMES    FROM   PIG    HEART 


207 


Malic  Dehydrogenase. —  The  optical  test  for  malic  dehydrogenase  activity  is  based  on  Reaction  4. 

(4)  Oxalacetate  +  DPNred  ^  /-malate  +  DPNqx 

The  test  is  carried  out  in  the  Beckman  spectrophotometer,  at  wave-length  340  m/z,  using  cells  of 
i.o  cm  light  path.  It  is  based  on  the  fact  that  the  early  rate  of  oxidation  of  reduced  diphosphopyridine 
nucleotide  (DPNred)  by  oxalacetate  is  proportional  to  the  enzyme  concentration  within  certain  limits. 
One  enzyme  unit  was  defined  as  the  amount  of  enzyme  causing  a  decrease  in  optical  density  of  o.oi 
per  minute  calculated  for  the  third  15  second  period  after  the  start  of  the  reaction.  The  reaction 
mixture,  in  a  final  volume  of  3.0  ml,  contained  0.025  M  glycylglycine  buffer  pfj  7.4,  0.4-10"*  M 
DPNj-ed.  enzyme,  and  0.25-10-^  M  oxalacetate.  The  volume  was  made  up  with  water  adjusted  to  a 
temperature  of  22-23°.  The  blank  cell  contained  no  DPN.  The  reaction  was  started,  after  taking  a 
zero  time  reading  of  the  optical  density,  by  addition  of  either  oxalacetate  or  enzyme. 


ir>    0.20 


0.15 


0.10 


0-05 


0  0.01  0.02  0.03 

CC  Pig  heart  extract 

Fig.  I.   Optical  test  for  oxalosuccinic  carboxylase  (Reaction  3).  Proportionality  of  rate  to  enzyme 

concentration. 


II 

y^  j 

II    ^^ 


PREPARATION   OF   ENZYME 

Extraction.  —  Acetone-dried  pig  heart  was  prepared  by  the  method  described  by 
Straub^°.  The  dry  material  was  ground  to  a  fine  powder  in  a  mechanical  mortar.  The 
powder  was  extracted  with  o.i  M  phosphate  buffer  pn  7.4  at  room  temperature  following 
the  method  of  Straub^". 

Ammonium  Sulphate  Fractionation.  ■ — -  The  clear  extract  was  cooled  to  0°,  brought 
to  50%  saturation  with  solid  ammonium  sulphate,  and  the  mixture  was  filtered  with 
suction  in  the  cold  room  using  filter-aid  (Hyflo-Supercel)  to  facilitate  filtration.  The 
precipitate  was  discarded  and  the  supernatant  was  brought  to  60%  saturation  with 
solid  ammonium  sulphate.  The  mixture  was  filtered  as  before.  The  supernatant  was 
discarded  and  the  precipitate  was  dissolved  in  cold  0.04  M  phosphate  buffer  pfj  7.4  to 
give  a  concentration  of  about  3%  protein.  The  solution  was  clarified  by  filtration  and 
dialysed  against  0.04  M  phosphate  buffer  pjj  7.4  at  2-^"  for  4-5  hours. 

Ethanol  Fractionation.  —  The  dialysed  solution  was  fractionated  with  ethanol  at 
low  temperature.  Details  of  the  procedure  have  been  described  elsewhere^^.  The  most 
active  fraction  was  usually  obtained  between  20  and  30%  ethanol  by  volume  at  -5°. 
The  precipitate  was  collected  by  centrifugation  at  -5°,  dissolved  in  cold  o.oi  M  phosphate 
buffer  Ph  7.4,  and  dialysed  for  a  few  hours  at  2-3°  against  the  same  buffer. 
References  p.  210. 


208 


A.  L.  GRAFFLIN,  S.  OCHOA 


VOL.  4  (1950) 


TABLE  II 

PARTIAL    PURIFICATION    OF    ISOCITRIC    DEHYDROGENASE    AND    OXALOSUCCINIC    CARBOXYLASE 
800  gm  OF  POWDER  OF  WASHED,  ACETONE-DRIED,  PIG  HEART 


Step 

Volume 
of  so- 
lution 
ml 

Protein 
mg 

Oxalosuccinic 
carboxylase 

Isocitric 
dehydrogenase* 

Ratio 

(a) /(b) 

Yield 
(OS  car- 
boxylase) 
% 

Malic  dehy- 
drogenase 

Units 

S.A.** 
(a) 

Units 

S.A.** 
(b) 

Units 

S.A.** 

Extract 

(NHJjSO^ 

fractionation 

(0.5-0.6  sat.) 

Ethanol 

fractionation 

(20-25'%) 

8300 

134 

37 

48200 
7210 
1254 

22244000 

12542400 

3596400 

462 
1740 
2860 

7968000 
1 108  150 

165 

885 

2.8 

3-2 

100 

55 
16 

17928000 
I 116956 

373 
890 

*  Over-all  reaction  isocitrate  -f  TPN,, 
**  Specific  activity  (units/mg  protein) 


a-ketoglutarate  -f  COj  +  TPNjed 


These  preparations  are  very  unstable  and  lose  activity  rather  rapidly  even  when 
stored  at  0°.  If  dried  from  the  frozen  state,  30  to  40%  of  the  activity  is  lost  but,  on  the 
other  hand,  the  remaining  activity  persists  unchanged  for  many  months  when  the  dry 
powder  is  stored  in  the  cold  over  calcium  chloride.  The  preparations  contain  no  aconitase 
and  only  traces  of  lactic  dehydrogenase. 

The  results  of  a  typical  fractionation  are  summarized  in  Table  II. 

Occasionally  the  purification  obtained  after  ammonium  sulphate  and  ethanol 
fractionation  may  be  lower  than  that  reported  in  Table  II.  The  purity  of  these  prepa- 
rations can  be  increased  about  1.5  times,  with  a  yield  of  60%  or  better,  by  adsorption 
on  calcium  phosphate  gel.  For  this  purpose  the  enzyme  solution  is  diluted  with  o.oi  M 
phosphate  buffer  pjj  7.4  to  give  a  protein  concentration  of  about  1%.  The  adsorption 
is  carried  out  successively  with  small  amounts  of  the  gel,  until  all  the  activity  has  been 
removed  from  solution,  and  the  sediments  are  separately  eluted  with  o.i  M  phosphate 
buffer  Ph  7.4.  The  eluates  are  tested  separately  and  the  best  ones  are  combined.  The 
calcium  phosphate  gel  was  prepared  following  the  directions  of  Keilin  and  Hartree^^. 


COMPARISON   OF   MANOMETRIC   AND   OPTICAL   DETERMINATION   OF   OXALOSUCCINIC 

CARBOXYLASE    ACTIVITY 

The  specific  oxalosuccinic  carboxylase  activity  of  the  extract  of  acetone-dried  pig 
heart,  as  determined  manometrically,  has  been  previously  reported^.  The  determinations 
were  carried  out  at  pn  5-6  and  15°,  in  the  presence  of  0.0014  M  MnClg  and  0.0065  M 
oxalosuccinate,  and  the  COg  evolution  due  to  spontaneous  decarboxylation  was  sub- 
tracted from  the  total  to  obtain  the  enzyme-catalysed  decarboxylation  rate.  Pig  heart 
extract  catalysed  the  evolution  of  70  /ul  of  COg  during  the  first  5  minutes  per  mg  of 
protein.  The  activity  of  pig  liver  extract  was  about  one  tenth  of  this  value. 

The  manometric  specific  activity  of  70  corresponds  to  an  optical  specific  activity 
of  462  (cf.  Table  II).  Thus,  the  activity  of  the  ethanol  fraction  of  Table  II  is  2,860-70/462 
or  435  1^^  of  CO2  in  5  minutes  per  mg  of  protein  (at  15°).  The  best  fraction  of  Lynen 
AND  Scherer^  had  a  specific  activity  of  100  jul  CO2  (corrected  for  spontaneous  decarboxy- 
References  p.  210. 


VOL.  4  (1950) 


ENZYMES    FROM   PIG   HEART 


209 


lation)  in  the  Jfirst  2  minutes  per  mg  of  protein,  tested  at  30^^*.  Allowing  for  the  difference 
in  temperature  in  the  manometric  tests  of  the  two  laboratories,  it  would  appear  that 
the  specific  oxalosuccinic  carboxylase  activity  of  Lynen  and  Scherer's  preparation 
from  horse  liver  was  only  about  one  fourth  of  that  obtained  by  us  starting  with  pig  heart. 

INHIBITION    OF   OXALOSUCCINIC   CARBOXYLASE   BY   ISOCITRIC   ACID 

It  has  been  reported  that  isocitric  acid  strongly  inhibits  the  enzymatic  decarboxy- 
lation of  oxalosuccinic  acid  as  followed  manometrically^.  As  shown  in  Fig.  2,  this 
inhibition  can  also  be  observed  under  the  conditions  of  the  optical  test.  The  test  system 


Fig.  2.  Inhibition  of  oxalosuccinic  carboxylase  activity  by  isocitric  acid;  optical  test. 

(Description  in  text). 

was  as  indicated  in  a  previous  section.  Curves  i  and  2  were  obtained  with  0.02  and  0.04 
ml  respectively  of  the  acetone  powder  extract  of  pig  heart  (about  0.12  and  0.24  mg  of 
protein).  Oxalosuccinate  (final  concentration,  0.167-  io~^  M)  was  added  at  zero  time  in  all 
cases.  Curves  3  (-0-0-)  and  4  {-A-A-)  both  with  0.04  ml  of  extract  and  either  0.35-  io~^ 
M  (curve  3)  or  0.35-  ic"*  (curve  4)  fl',/-isocitrate.  Curve  5  (-3-3-)  with  0.02  ml  of  extract 
and  0.35-  io~^  M  (^,^-isocitrate. 

A  cknowledgement 

We  are  indebted  to  Mr  Morton  C.  Schneider  for  technical  assistance. 


SUMMARY 

Partial  purification  of  the  isocitric  dehydrogenase  and  oxalosuccinic  carboxylase  activities  of 
pig  heart  has  been  obtained  by  means  of  ammonium  sulphate  and  ethanol  fractionation  of  an  acetone 

*  Manometric  test  with  o.ooi  M  MnSO^  and  0.002  oxalosuccinate,  pn  6.0.  The  purification  proce- 
dure involved  water  extraction  of  the  fresh  liver,  precipitation  with  acetone,  fractionation  with 
nucleic  acid  between  pH  5-i8  and  4.6,  and  precipitation  with  ethanol.  The  average  specific  activity 
of  solutions  of  the  acetone  precipitate  was  3.8.  Yields  were  not  reported  and  the  fractions  were  not 
tested  for  isocitric  dehydrogenase. 

References  p.  210. 


210 


A.  L.  GRAFFLIN,  S.  OCHOA  VOL.  4  (1950) 


powder  extract.  The  purification  reached  was  about  six-fold  with  a  yield  of  about  15%.  No  separation 
of  the  two  activities  has  thus  far  been  accomplished.  The  strong  inhibition  of  oxalosuccinic  carboxylase 
activity  by  isocitric  acid  has  been  confirmed  using  an  optical  test  system. 

r£sum£ 

Nous  avons  reussi  une  purification  partielle  des  principes  actifs  de  I'isocitrate-dehydrogenase 
et  de  I'oxalosuccinate-carboxylase  par  fractionnement  au  sulfate  d'ammonium  et  a  I'ethanol  d'un 
extrait  acetonique  de  poudre  de  coeurs  de  Pigeon.  Apres  purification  I'activite  etait  environ  six  fois 
plus  grande,  tandisque  le  rendement  6tait  de  15%  environ.  Les  deux  activites  n'ont  pas  encore  pu 
etre  separees.  Nous  avons  confirme  par  test  optique  que  I'activite  de  I'oxalosuccinate-carboxylase 
est  fortement  inhibee  par  I'acide  isocitrique. 

ZUSAMMENFASSUNG 

Die  Isocitrat-Dehydrogenase  und  die  Oxalosuccinat-Carboxylase  aus  einem  Acetonextrakt  von 
getrocknetem  pulverisierten  Taubenherz  wurden  durch  fraktionierte  Fallung  mit  Ammoniumsulphat 
und  Athanol  teilweise  gereinigt.  Die  Aktivitat  wurde  ungefahr  sechsmal  angereichert,  wobei  die 
Ausbeute  etwa  15%  betrug.  Es  wurde  keinerlei  Trennung  der  beiden  Aktivitaten  beobachtet.  Die 
Starke  Hemmung  der  Oxalosuccinat-Carboxylase  durch  Isozitronensaure  wurde  durch  einen  optischen 
Test  bestatigt. 

REFERENCES 

1  S.  OcHOA,  J.  Biol.  Cheni.,  159  (1945)  243;  174  (1948)  i33- 

2  S.  OcHOA  AND  E.  Weisz-Tabori,  /.  Biol.  Chem.,  159  (1945)  245;  i74  (1948)  123. 
^  S.  Grisolia  and  B.  Vennesland,  J.  Biol.  Chem.,  170  (1947)  461. 

*  J.  Ceithalm  and  B.  Vennesland,  J.  Biol.  Chem.,  178  (1949)  i33- 
5  F.  Lynen  and  H.  Scherer,  Ann.  Chem.,  560  (1948)  163. 
•*  O.  Warburg  and  W.  Christian,  Biochem.  Z.,  310  (1941-42)  384. 
'  A.  Kornberg,  S.  Ochoa,  and  A.  H.  Mehler,  /.  Biol.  Chem.,  174  (1948)  159. 
®  S.  Ochoa,  /.  Biol.  Chem.,  174  (1948)  115. 

'  M.  Levy,  Compt.  rend.  trav.  lab.  Carlsberg,  Serie  chim.,  21  (1936)  loi. 
'0  F.  B.  Straub,  Z.  physiol.  Chem.,  275  (1942)  63. 

11  S.  Ochoa,  A.  H.  Mehler,  and  A.  Kornberg,  /.  Biol.  Chem.,  174  (1948)  979- 

12  D.  Keilin  and  E.  F.  Hartree,  Proc.  Roy.  Sac.  (B),  124  (1938)  397- 

Received  April  13th,  1949 


VOL.  4  (1950) 


BIOCHIMICA   ET  BIOPHYSICA   ACTA 


211 


SPECTROPHOTOMETRIC  MEASUREMENTS  OF  THE  ENZYMATIC 
FORMATION  OF  FUMARIC  AND  C75-ACONITIC  ACIDS 

by 

E.  RACKER 

Department  of  Microbiology,  New  York  University  College  of  Medicine  and  College  of  Dentistry, 

New  York  {U.S.A.) 


Fumaric  and  czs-aconitic  acids  are  intermediates  in  the  main  pathway  of  substances 
oxidized  through  the  tricarboxylic  acid  cycle.  With  the  exception  of  the  keto-acid 
oxidases,  the  enzymes  participating  in  the  cycle  have  been  obtained  in  solution  and 
after  purification  can  be  studied  in  isolated  and  defined  systems.  Compounds  such  as 
fumaric  and  cw-aconitic  acid  with  an  unsaturated  C  =  C  linkage  have  a  marked  absorption 
in  the  ultraviolet.  This  property  can  be  utilized  in  a  spectrophotometric  test,  measuring 
appearance  and  disappearance  of  these  substances  in  the  course  of  enzymatic  reactions. 

A  rapid  and  convenient  test  for  the  ^i  i.ooOr 
enzymes  catalysing  the  formation  of  fuma- 
ric acid  from  malic  acid  or  aspartic  acid  and 
the  formation  of  czs-aconitic  acid  from 
citric  acid  or  isocitric  acid  will  be  described 
in  this  paper. 


0.800 


EXPERIMENTAL 


0.600 


0.400 


0.200 


Ultraviolet  Absorption  Spectrum  of 

Fumaric  Acid  and  Cis-Aconitic  Acid 

The  ultraviolet  absorption  spectrum 
of  the  sodium  salts  of  these  two  acids  is  re- 
corded in  Fig.  I.  The  fumaric  acid  used  in 
this  experiment  was  a  recrystalHzed  com- 
mercial preparation;  the  czs-aconitic  acid 
was  kindly  supplied  by  Dr  S.  Ochoa.  As 
can  be  seen  from  Fig.  i,  the  absorption  of 
these  compounds  shows  a  steady  rise  to- 
ward the  short  wave  lengths.  Because  pro- 
teins and  nucleic  acids  absorb  considerable 
amounts  of  ultraviolet  light  in  this  region, 
enzymes  used  for  spectrophotometric  stu- 
dies must  have  a  fairly  high  turnover  num- 
ber so  that  activity  measurements  can  be 

carried  out  at  high  enzyme  dilutions.  The  activity  of  enzymes  with  a  low  turnover 
number  can  be  tested  spectrophotometrically  only  after  considerable  purification,  with 

References  p.  214. 


Fig. 


Ultraviolet  absorption  spectrum  of  sodium 
fumarate  and  sodium  c/s-aconitate. 


212  E.  RACKER  VOL.  4  (195OJ 

removal  of  interfering  absorbing  substances,  particularly  proteins  and  nucleic  acid. 
Of  the  enzymes  catalyzing  the  formation  of  unsaturated  intermediates  of  meta- 
bolism, fumarase,  aconitase  and  aspartase  were  selected  for  study. 

PREPARATION    OF   ENZYMES 

a)  Fumarase.  Fumarase  was  prepared  according  to  the  method  of  Laki  and  Laki^  and  fumarase 
activity  was  measured  at  each  stage  of  the  purification^.  It  was  found  that  the  preparation  at  the 
final  stage  still  contained  contaminating  proteins.  The  crystalline  precipitate  obtained  was  found  to 
have  lost  most  of  the  fumarase  activity  after  four  subsequent  recrystallizations  while  the  supernatant 
retained  the  fumarase  activity^.  These  findings  confirm  the  report  by  Scott'  who  observed  that  the 
crystalline  fraction  lost  fumarase  activity  on  recrj'stallization  while  the  amorphous  fraction  had  a 
specific  activity  equal  to  that  ascribed  to  the  crystals  by  Laki  and  Laki^.  Furthermore,  the  purified 
fumarase  preparations  of  Laki  and  Laki  still  contain  considerable  quantities  of  contaminating 
proteins.  Appreciable  aconitase  activity  has  been  found  in  these  preparations  as  will  be  described 
below,  as  well  as  very  active  lactic  acid  dehydrogenase  which  represents  about  20%  of  the  protein 
present^. 

b)  Aconitase.  Fumarase  prepared  by  the  method  of  Laki  and  Laki^,  and  kindly  supplied  by 
Dr  J.  B.  V.  Salles,  was  found  to  contain  an  active  aconitase  as  noted  above.  This  preparation  of 
fumarase  had  been  kept  at  0°  for  several  weeks  and  retained  considerable  aconitase  activity.  Because 
of  the  known  lability  of  purified  aconitase,  it  was  decided  to  investigate  this  preparation  further. 

Fumarase  was  prepared,  therefore,  according  to  the  method  of  Laki  and  Laki^  and  fumarase 
and  aconitase  activity  were  measured  in  all  fractions^.  A  large  proportion  of  the  aconitase  activity 
was  retained  by  the  heart  muscle  pulp  after  thorough  washing  with  water;  the  pulp  was  then  ex- 
tracted by  the  phosphate  buffer  treatment  used  for  obtaining  the  fumarase  activity  ^.  Both  aconitase 
and  fumarase  were  purified.  Aconitase  showed  a  somewhat  greater  sensitivity  to  the  acid  pn  used 
in  the  course  of  the  purification.  On  fractionation  with  ammonium  sulphate,  the  fumarase  precipitated 
at  lower  salt  concentrations,  so  that  partial  separation  of  the  two  enzymes  was  accomplished. 

An  aconitase  preparation  was  also  made  from  Fleischmann's  baker's  yeast.  Maceration  juice 
was  obtained  by  extracting  dried  yeast  with  M/15  disodium  phosphate  for  3  hours  at  37°.  The  macera- 
tion juice  was  fractionated  at  -5°  with  acetone.  An  active  fraction  was  obtained  which  precipitated 
between  30  and  50%  acetone  concentration.  This  was  dissolved  in  cold  water  and  dialysed  for  two 
hours  against  running  tap  water.  Following  centrifugation,  the  supernatant  was  further  fractionated 
by  the  addition  of  solid  ammonium  sulphate.  The  precipitate  obtained  at  50%  saturation  was  col- 
lected. Solid  ammonium  sulphate  was  added  to  the  supernatant  and  the  fractions  precipitated  up  to 
80%  saturation  were  also  collected.  The  aconitase  activity  of  these  fractions  will  be  described  later 
in  this  paper. 

c)  Aspartase.  This  enzyme  was  prepared  from  E.  coli  (strain  B).  The  bacteria  were  grown  in 
neopeptone  broth  for  18  hours  at  37°  with  vigorous  aeration,  then  centrifuged  and  washed  once 
distilled  water.  They  were  then  suspended  in  a  small  volume  of  distilled  water  and  disintegrated  by 
sonic  vibration^  for  five  minutes.  After  centrifugation  for  20  minutes  at  18000  rpm  in  a  refrigerated 
centrifuge,  the  supernatant  was  fractionated  by  means  of  ammonium  sulphate.  The  precipitate  ob- 
tained at  50%  saturation  was  dissolved  and  dialysed  against  distilled  water  at  0°  for  24  hours.  This 
preparation  of  aspartase  was  used  for  the  studies  described  in  this  paper  and  was  found  to  be  quite 
stable  if  kept  at  0°. 

SPECTROPHOTOMETRIC   MEASUREMENTS 

a)  Fumarase.  The  enzymatic  activity  of  fumarase  was  determined  in  a  Beckman  DU 
quartz  spectrophotometer.  The  final  volume  was  3  ml  including  0.05  M  potassium- 
phosphate  buffer  at  Ph  74  and  0.05  M  sodium  L-malate.  After  addition  of  the  enzyme, 
the  changes  in  absorption  at  240  m/<  were  recorded  at  intervals  of  15  seconds.  The  control 
cell  contained  all  the  solutions  except  the  substrate.  The  enzymatic  reaction  follows  a 
zero  order  course  for  several  minutes  and  is  measured  during  this  period.  One  unit  is 

defined  as  a  change  of  log  -5-  of  o.ooi  per  minute.  The  increments  in  optical  density  at 
240  m/u.  are  proportional  to  the  amount  of  enzyme  added  (Fig.  2). 


*  Sonic  oscillator  manufactured  by  Ratheon  Corp.,  Waltham,  Massachusetts,  U.  S.  A. 
References  p.  214. 


VOL.  4  (1950) 


TEST   FOR    FUMARASE    AND   ACONITASE 


213 


■0.20 


bl- 


under these  experimental  conditions  the  Michaelis  constant  for  fumarase  as 
determined  by  the  method  of  Lineweaver  and  Burk*  was  4.1-10-=^  (moles  x  Hter-i) 
with  sodium  L-malate  as  substrate. 

The  enzymatic  activity  of  fumarase  can  also  be  followed  with  sodium  fumarate  as 
the  substrate.  Due  to  the  high  specific  absorption  of  fumaric  acid,  only  limited  amounts 
of  this  substrate,  which  are  not  sufficient  to  saturate  the  enzyme,  can  be  used  in  the 
spectrophotometric  test.  The  rates,  therefore,  are  slower  and  fall  off  more  rapidly  than 
with  L-malic  acid  as  the  substrate.  However,  with  an  active  enzyme  preparation  the 
equilibrium  is  quite  rapidly  established  from 
either  direction. 

b)  Aconitase.  For  the  measurement  of 
aconitase  activity  the  test  system  was  the 
same  as  that  for  fumarase  except  that  the 
substrate  used  was  either  0.03  M  sodium  cit- 
rate or  o.oi  M  sodium  D,L-isocitrate  (kindly 
supplied  by  Dr  S.  Ocho.\).  Since  the  enzyme 
is  unstable  in  dilute  solutions,  all  estimations 
were  carried  out  immediately  following  di- 
lution in  0.1  M  phosphate  buffer.  The  en- 
zymatic acti\'ity  followed  a  zero  order  course 
for  several  minutes  and  was  proportional  to 
the  amount  of  enzyme  added  (Fig.  2). 

The  specific  activity  (units/mg  protein) 
of  aconitase  preparations  when  tested  with 
isocitrate  was  always  found  to  be  greater 
than  with  citrate.  Considerable  variation  in 
the  relative  activities  was  found  in  different 
fractions  during  purification.  Although  no 
evidence  was  obtained  of  a  separation  of  the 
enzyme  activity  for  the  two  substrates,  the 
respective  activities,  for  the  sake  of  conve- 
nience, are  referred  to  as  citrase  and  isoci- 
trase.  Thus,  in  a  crude  heart  extract,  a  ratio 
isocitrase/citrase  activity  of  2.1  was  found, 

while  the  purified  preparation^  had  a  ratio  of  7.5.  Similarly,  the  fractions  obtained 
from  yeast  by  acetone  and  ammonium  sulphate  precipitation,  showed  considerable 
variation  in  the  relative  citrase  and  isocitrase  activities.  The  ammonium  sulphate 
precipitate  obtained  at  50%  saturation  showed  an  isocitrase/citrase  ratio  of  2.0,  while 
the  fractions  obtained  between  60  and  80%  saturation  showed  a  ratio  of  about  7.0. 

The  Michaelis  constant  of  aconitase  measured  with  sodium  citrate  as  substrate 
was  found  to  be  i.i-  lO"^  and  for  D-isocitrate  4-10"*  M. 

c)  Aspartase.  This  enzyme  was  measured  in  the  same  manner  as  the  other  hydrases 
with  0.15  M  sodium  aspartase  as  the  substrate.  A  high  concentration  of  substrate  i.s 
required  for  maximal  activity  of  this  enzyme.  With  substrate  concentration  sufficient 
for  enzyme  saturation,  proportionaUty  between  enzyme  concentration  and  increments 
in  optical  density  was  found  (Fig.  2). 

The  Michaelis  constant  of  aspartase  was  found  to  be  in  the  neighbourhood  of 

References  p.  214. 


0.16 


0.12 


0.08 


0.04 


Fig.  2.  Quantitative  determination  of  fumarase, 

aconitase  and  aspartase.   Relation  of  enzyme 

concentration  to  activity  per  minute. 


214  ^-  R-^CKER  VOL.  4  (1950) 

3-  io~2  M.  Some  variation  around  this  value  was  found  with  different  preparations.  This 
variation  might  be  explained  by  the  presence  of  two  different  aspartases  reported  by 
Gale^. 

discussion 

Rapid  and  convenient  spectrophotometric  methods  for  the  determination  of  gly- 
colytic enzymes  of  the  Meyerhof-Embden  scheme  have  been  developed  by  Warburg 
and  his  school.  These  methods  have  been  valuable  in  following  purification  and  also 
for  kinetic  studies  of  these  enzymes.  The  high  absorption  coefficients  in  the  ultraviolet 
of  unsaturated  compounds  such  as  fumaric  and  aconitic  acid  have  been  made  the  basis 
for  a  method  of  measuring  their  enzymatic  formation.  Other  compounds  such  as  crotonic 
and  vinyl-acetic  acid  were  also  found  to  show  a  high  absorption  in  the  ultraviolet.  These 
latter  compounds  are  known  to  be  metabolized  by  animal  tissues  and  by  bacteria  and 
may  be  intermediates  of  fatty  acid  metabolism.  In  view  of  their  high  specific  light 
absorption,  their  enzymatic  formation  and  breakdown  could  be  followed  by  spectro- 
photometric tests  similar  to  those  described  in  this  paper. 

The  occurrence  of  unsaturated  compounds  as  intermediates  of  metabolism  of 
amino  acids  such  as  serine  and  threonine  has  been  postulated^.  The  probably  high 
absorption  in  the  ultraviolet  of  such  intermediates  may  help  in  the  elucidation  of  the 
pathway  of  the  metabolic  breakdown  of  these  amino  acids.  Advantage  has  been  taken 
of  the  high  absorption  coefficients  of  reduced  coenzymes  I  and  II,  keto  acids,  dehydro- 
peptides,  and  am.ino  acids,  such  as  tyrosine  for  enzymatic  studies  with  these  compounds. 
The  present  study  shows  that  the  metabolism  of  unsaturated  organic  substances  may 
be  followed  by  2.  similar  technique. 

SUMMARY 

A  spectrophotometric  method  of  measuring  the  enzymatic  formation  and    disappearance  of 
umaric  and  cis-aconitic  acids  is  reported. 

RESUMfi 

Nous  decrivons  une  methode  spectrophotometrique  qui  permet  de  mesurer  la  formation  et  la 
disparation  enzymatique  de  I'acide  fumarique  et  de  I'acide  cis-aconitique. 

ZUSAMMENFASSUNG 

Eine  spektrophotometrische  Methode  zur  Messung  der  enzymatischen  Bildung  und  Zferstorung 
von  Fumarsaure  und  czs-Akonitsaure  wird  beschrieben. 

REFERENCES 

1  E.  Laki  and  K.  Laki,  Enzymologia,  g  (1941)  139. 

2  S.  OcHOA  AND  E.  Racker,  Unpublished  experiments. 

3  E.  M.  Scott,  Arch.  Biochem.,  18  (1948)  131. 

*  H.  LiNEWEAVER  AND  D.  BuRK,  /.  Ar}t.  Chem.  Soc,  56  (1934)  658. 

''  E.  F.  Gale,  Biochem.  J.,  32  (1938)  1583. 

^  E.  Chargaff  and  D.  B.  Sprinson,  /.  Biol.  Chem.,  151  (1943)  273. 

Received  April  28th,  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  215 


THE  INTERCONVERSION  OF  THE  RETINENES 
AND  VITAMINS  A  IN  VITRO 

by 

GEORGE  WALD* 
Biological  Laboratories  of  Harvard  University,  Cambridge,  Mass.  (U.S.A.) 


In  the  summer  of  1933  I  was  woiking  as  a  National  Research  Council  Fellow  in 
Otto  Meyerhof's  Institute  in  Heidelberg,  measuring  the  distribution  of  phosphates  in 
the  frog  retina  in  light  and  darkness.  I  had  noticed  that  the  trichloracetic  acid  used 
to  extract  the  phosphates  turned  the  red  colour  of  the  dark  adapted  retina  to  bright 
orange,  and  that  thereafter  the  retina  behaved  as  a  pu  indicator,  orange  in  acid  and 
colourless  in  alkaline  solution.  Light  adapted  retinas  were  colourless  under  all  circum- 
stances. 

All  about  us  the  Third  Reich  was  coming  into  flower,  and  the  laboratory'  remained 
an  island  of  sanity  in  a  world  increasingly  committed  to  unreason  and  repression.  Under 
the  urging  of  the  Society  of  Animal  Friends,  led  by  a  retired  general,  the  government 
of  Baden  had  forbidden  the  killing  of  frogs  —  that  is,  Geiman  frogs;  there  seemed 
to  be  no  objection  to  importing  foreign  frogs  for  laboratory  use. 

In  August,  just  after  Professor  Meyerhof  and  his  assistants  left  on  their  vacations, 
and  I  had  all  but  terminated  my  phosphate  experiments,  a  large  sh'pment  of  frogs 
arrived  from  Hungary.  The  Diener  was  prepared  to  throw  them  into  the  Neckar,  but 
it  seemed  a  pity  to  waste  them,  and  I  decided  to  use  them  to  try  to  learn  something 
of  the  orange  p^  indicator  which  results  from  the  destruction  of  rhodopsin  in  the  retina. 
It  was  under  these  circumstances  that  I  found  retinenci,  and  had  a  first  view  of  its 
interplay  with  vitamin  Aj  in  the  rhodopsin  cycle. 

It  is  only  within  the  past  few  months  that  the  chemistry  of  these  relationships 
has  been  clarified.  At  a  key  point  in  this  investigation  it  fell  in  with  the  pattern  of 
Meyerhof's  classic  experiments  on  the  role  of  cozymase  in  the  lactic  fermentation. 
For  cozymase  is  also  the  substance  which  reduces  the  retinenes  to  the  vitamins  A;  and 
to  learn  this  we  entered  on  a  line  of  experiment  developed  by  Meyerhof  many  years 
before. 

It  is  therefore  in  a  double  sense  that  I  offer  this  essay  to  Otto  Meyerhof:  first, 
for  his  personal  connection  with  its  beginnings ;  and  again,  for  the  debt  to  him  and  to 
his  work  which  I  share  with  all  who  do  biochemistry. 

retinenEj  and  vitamin  Ai 

Vision  in  dim  light  is  mediated  in  all  vertebrates  through  the  retinal  receptors 
known  as  rods.  In  land  and  sea  vertebrates,  these  organs  contain  the  red,  light-sensitive 

*  The  recent  investigations  described  in  this  paper  have  been  supported  in  part  by  the  Medical 
Sciences  Division  of  the  Office  of  Naval  Research. 

References  p.  228. 


2l6  G.  WALD  VOL.  4  (1950) 

pigment  rhodopsin.  This  substance  takes  part  with  the  carotenoids  retinenCi  and 
vitamin  Aj  in  a  cycle  of  reactions  of  the  following  form* : 

Rhodopsin 
(500  m^i) 

^\ 
(3)/  \Light 

(i)r\        Orange  intermediates 

\  \ 
(2)       \    ^ 

Vitamin  A^  +  protein    < Retinene^  +  protein 

(325  m/i  in  petroleum  ether)                             (365  m^  in  petroleum  ether) 
(4-  SbClg  — >  615-620  m/j.)  (-}-  SbCl3 >   664  m/n) 

Rhodopsin  bleaches  in  the  light  over  unstable  orange  intermediates  to  a  mixture 
of  yellow  retinenej  and  colourless  protein ;  the  retinenej,  is  then  transformed  to  colourless 
vitamin  Aj)  and  both  vitamin  Aj  and  retinenej — ^or  its  orange  precursors — recombine 
with  protein  to  form  new  rhodopsin  (Wald,  1935-36  a,  b). 

One  has  only  to  separate  the  retina  from  contact  with  the  underlying  tissues  which 
line  the  optic  cup  to  abolish  the  synthesis  of  rhodopsin  from  vitamin  A^  (reaction  3). 
According  to  KOhne  this  process  requires  the  cooperation  of  a  living  pigment  epithelium 
(EwALD  AND  KiJHNE,  1878,  page  255;  KiJHNE,  1879). 

When  the  system  is  further  reduced  by  bringing  rhodopsin  into  aqueous  solution, 
processes  (i)  and  (2)  are  usually  also  eliminated.  Nothing  then  remains  but  the  succes- 
sion of  light  and  "dark"  reactions  which  transform  rhodopsin  into  retinenej  and  protein. 

The  present  paper  is  concerned  primarily  with  reaction  (2),  the  conversion  of  reti- 
nene^  to  vitamin  A^.  This  is  a  slow,  irreversible  process  which  goes  to  completion  in 
the  isolated  retina  in  about  an  hour  at  room  temperature**.  In  1942-43  we  succeeded 
in  biinging  this  process  into  a  cell-free  brei  prepared  from  cattle  retinas;  and  recently 
Bliss  (1948)  has  shown  that  it  occurs  under  certain  conditions  in  freshly  prepared 
rhodopsin  solutions.  These  demonstrations  that  it  can  proceed  in  vitro  form  a  prelude 
to  the  present  experiments.  Their  other  antecedent  is  the  clarification  of  chemical  rela- 
tions between  retinenC]  and  vitamin  A^,  due  primarily  to  the  work  of  Morton  and  his 
colleagues  in  Liverpool. 

Vitamin  Aj  is  the  primary  alcohol  C19H27CH2OH.  Ball,  Goodwin,  and  Morton 
(1948)  found  that  on  mild  oxidation  this  is  transformed  to  a  product  which  agrees  in 
spectrum  and  antimony  chloride  reaction  with  retinenei.  They  have  crystallized  this 
product  and  shown  it  to  be  an  aldehyde,  which  they  believe  to  be  simply  vitamin  A^ 
aldehyde,  C19H27CHO.  Their  analytic  data  do  not  establish  this  formulation  unequi- 
vocally as  yet;  but  all  that  is  now  known  of  retinenci  from  the  work  of  Morton's 
laboratory  and  our  own  is  consistent  with  the  view  that  it  is  vitamin  A^  aldehyde.  We 
shall  accept  this  as  its  structure  in  what  follows. 

The  wavelength  values  written  below  components  of  this  cycle  represent  maxima  in  the  ab- 
sorption spectra  of  these  substances  in  solution,  or,  when  so  indicated,  of  the  products  which  these 
substances  yield  when  treated  with  antimony  trichloride. 

Designating  this  as  an  irreversible  process  is  not  intended  to  exclude  the  possibility  that 
it  is  in  fact  reversible,  but  with  the  equilibrium  far  over  toward  vitamin  A  formation.  It  might  for 
example  be  possible  by  greatly  increasing  the  concentration  of  vitamin  Aj  in  the  system  to  demon- 
strate a  small  reversion  to  retinenCj. 

References  p.  228. 


VOL.  4  (1950) 


RETINENES   AND   VITAMINS    A 


217 


THE   OXIDATION    OF   VITAMIN    Aj    TO    RETINENEi 

In  their  simplest  procedure  for  oxidizing  vitamin  A,  to  retinenej,  Ball  et  al.  (1946) 
added  a  little  manganese  dioxide  powder  to  a  solution  of  vitamin  Aj  in  petroleum  ether, 
and  placed  this  mixture  in  a  refrigerator.  After  3-4  days  they  found  that  retinene,  had 
replaced  vitamin  Aj  in  the  supernatant  solution. 

On  examining  this  process  we  found  its  mechanism  to  be  as  follows.  Vitamin  Aj 
is  strongly  adsorbed  on  manganese  dioxide,  and  is  oxidized  to  retinene  ^  in  the  adsorbed 
condition.  Retinene,  is  much  less  strongly  adsorbed  and  so  is  displaced  from  the  manga- 
nese dioxide  by  new  vitamin  A^  as  fast  as  it  is  formed.  In  this  way  all  the  vitamin  A, 
passes  over  the  manganese  dioxide  surface,  and  is  replaced  by  retinene j  in  the  super- 
natant solution.  At  the  close  of  the  process,  the  final  charge  of  vitamin  Aj  on  the 
adsorbent  is  oxidized  to  retinenci,  and  then,  wdth  no  vitamin  A^  remaining  to  displace 
it,  is  oxidized  further  to  what  I  have  called  the  545  m/^-chromogen.  This  can  be  recoveied 
from  the  manganese  dioxide  by  elution  with  a  polar  organic  solvent  such  as  ethanol. 


300 


uoo 


600  700 

Wavelength-ma 

Fig.  I.  Comparison  of  natural  and  synthetic  retinene^.  Absorption  spectra  of  cattle  retinene^  in 
chloroform  and  of  the  blue  product  which  squid  retinene^  yields  with  antimony  chloride,  compared 
with  similar  preparations  of  synthetic  retinencj.  The  absorption  is  plotted  as  extinction  or  optical 
density,  log  I^/I,  in  which  I^  is  the  incident  and  I  the  transmitted  intensity  (From  Wald,  1947-48). 


For  this  reason  the  proportions  of  vitamin  Aj  and  manganese  dioxide  used  in  the  pro- 
cedure are  important.  If  too  much  manganese  dioxide  is  used,  it  adsorbs  all  the  vitamin 
Al  at  once,  and  oxidizes  all  of  it  to  the  545  m^u-chromogen  (Wald,  1947-48). 

Once  the  nature  of  this  reaction  was  appreciated,  we  recast  it  in  more  convenient 
form.  The  manganese  dioxide  powder  is  packed  into  a  short  column  such  as  is  used  in 
chromatography.  To  oxidize  10  mg  of  vitamin  A^,  about  0.6  g  of  manganese  dioxide 
is  employed.  A  solution  of  crystaUine  vitamin  A^  in  peti oleum  ether  is  poured  in  at  the 
top  of  the  column,  and  a  solution  of  nearly  pure  retinene^  is  drawn  off  under  light  suction 
in  the  filtrate. 

On  washing  through  the  column  for  a  time  with  more  petroleum  ether,  a  high  yield 
of  retinene^  is  obtained.  This  can  be  freed  of  traces  of  contaminating  substances  by 
chromatographic  adsorption  on  a  column  of  calcium  carbonate.  It  is  adsorbed  as  a 
diffuse  yellow  zone,  which  travels  slowly  down  the  column  on  washing  with  petroleum 
ether,  and  is  collected  as  an  isolated  fraction  of  high  purity  in  the  filtrate.  The  properties 
of  this  product  are  virtually  identical  with  those  of  purified  natural  retinene^  (Fig.  i). 
References  p.  228. 


2l8  G.  WALD  VOL.  4  (1950) 

I  have  referred  to  this  procedure  as  a  chromatographic  oxidation.  The  founder  of 
chromatography,  Michael  Tswett,  looked  forward  to  the  discovery  of  an  entire  class 
of  such  reactions,  in  which  dry  powders  act  at  once  as  adsorbents  and  reagents,  and  I 
have  no  doubt  that  this  is  a  correct  view.  Such  reactions  probably  possess  a  degree  of 
specificity  and  orientation  not  commonly  realized  in  free  solution,  mimicking  on  occasion 
the  character  of  enzymic  processes.  The  range  and  properties  of  such  chromatographic 
procedures  deserve  careful  sj'stematic  examination. 

THE   COENZYME    OF   RETINENE    REDUCTION* 

A  simple  procedure  has  been  described  for  oxidizing  vitamin  Aj  to  retinenci.  In 
the  retina  just  the  reverse  process  occurs :  retinene^  is  leduced  irreversibly  to  vitamin  Aj. 

Several  years  ago,  as  noted  above,  we  brought  this  reaction  into  a  cell-free  prepara- 
tion from  cattle  retinas.  The  retinas  were  frozen-dried,  ground  to  a  fine  powder,  and 
were  extracted  exhaustively  with  petroleum  ether,  all  in  darkness.  The  residue  was 
stirred  into  a  brei  with  neutral  phosphate  buffer.  On  exposing  this  to  light,  its  rhodopsin 
was  bleached,  and  the  retinenCi  so  formed  was  converted  almost  completely  to  vita- 
min Aj. 

In  a  study  of  the  bleaching  of  rhodopsin  in  aqueous  solution  some  years  ago,  we 
found  that  freshly  prepared  solutions  undergo  a  special  type  of  bleaching,  which  con- 
tinues further  than  the  bleaching  of  the  same  solutions  after  a  period  of  aging  (Wald, 
1937-38).  Bliss  (1948)  has  lately  reported  that  the  basis  of  this  extra  bleaching  in 
fresh  rhodopsin  solutions  is  the  conversion  of  retinencj  to  vitamin  A^.  We  have  con- 
firmed this  observation.  A  fresh  rhodopsin  solution,  however,  is  not  a  satisfactory  pre- 
paration in  which  to  study  the  reduction  of  retinenCj,  for  while  this  reaction  is  in 
progress,  the  enzyme  system  which  accompHshes  it  is  being  rapidly  inactivated,  the 
vitamin  A^  formed  is  being  destroyed,  and  the  intrusion  of  intermediates  between 
rhodopsin  and  retinenCi  leaves  equivocal  the  actual  substrate  in  the  process. 

In  order  to  analyse  such  systems  further  one  would  ordinarily  attempt  to  fractionate 
them  into  their  components.  We  had  already  begun  such  experiments  when  the  investi- 
gation took  a  new  turn  with  the  discovery  that  the  enzyme  system  can  be  fractionated 
anatomically  through  the  structure  of  the  retinal  rods. 

The  vertebrate  rod  is  composed  of  two  sections,  the  inner  and  outer  limbs  or  seg- 
ments. The  inner  limb  contains  the  nucleus,  and  is  the  principal  seat  of  the  ordinary 
cellulai  functions.  The  outer  limb  is  a  specialized  outgrowth,  which  contains  all  the 
rhodopsin  of  the  retina,  and  includes  within  its  small  compass  the  whole  photoreceptor 
process. 

When  a  retina  is  removed  from  the  eye  into  Ringer  solution  with  all  possible  care, 
the  solution  is  found  to  contain  large  numbers  of  rod  outer  limbs  which  had  broken 
off  in  the  course  of  the  dissection,  just  at  their  junctures  with  the  inner  segments.  By 
scraping,  one  can  break  away  about  half  the  outer  limbs  from  the  surface  of  the  retina, 
and  collect  them  in  a  dense  suspension,  free  from  other  retinal  tissues,  by  filtration  or 
differential  centrifugation  (Fig.  2). 

When  this  procedure  is  cariied  out  in  dim  red  light,  the  outer  limbs  contain  a 
large  quantity  of  rhodopsin.  On  exposure  to  white  light  this  bleaches;  but  in  the  isolated 

*  A  detailed  account  of  the  experiments  reviewed  in  this  section  will  be  found  in  the  paper  of 
Wald  and  Hubbard  (1948-49). 

References  p.  228. 


VOL.  4  (1950) 


RETINENES   AND   VITAMINS   A 


219 


outer  segment,  unlike  the  whole  retina,  the 
retinene^  which  results  is  not  converted  to 
vitamin  Aj.  The  isolated  outer  limb  lacks 
some  component  of  the  system  which  per- 
forms this  conversion. 

It  does  not  help  this  situation  to  sus- 
pend outer  limbs  in  the  presence  of  intact 
retinas.  But  if  whole  retinas  are  ground  up 
in  Ringer  solution  or  phosphate  buffer, 
though  in  the  process  almost  all  the  outer 
segments  are  detached  from  other  struc- 
tures, the  suspension  which  results  does 
convert  its  retinenci  efficiently  to  vitamin 
Aj.  The  crushing  of  the  retinal  cells  relea- 
ses substances  which  promote  this  process 
in  the  outer  limbs. 

If  such  a  retinal  brei  is  centrifuged  at 
high  speed  and  the  clear,  colourless  superna- 
tant solution  is  poured  off,  the  solid  residue 
—  which  retains  all  the  rhodopsin  —  has  lost 
the  power  to  reduce  retinenci.  It  regains  this 
capacity  on  re-adding  to  it  the  supernatant. 
Fuithermore,  if  one  suspends  isolated  rod 
outer  segments  in  such  a  water  extract  of 
crushed  retinas,  they  now  reduce  their 
retinenci  to  vitamin  A^.  The  retinal  extract 
supplies  whatever  the  isolated  outer  limb 


Fig.  2.  Rod  outer  segments  of  the  frog,  sus- 
pended in  Ringer  solution.  Magnification 
about  500  diameters.  The  longitudinal  stria- 
tions  which  can  be  seen  in  most  of  the  outer 
limbs  are  characteristic  of  fresh  preparations, 
and  probably  are  evidence  of  a  fibrillar  struc- 
ture. Later,  cross-striations  appear,  and  even- 
tually dominate  the  structure;  the  first  of 
these  also  are  visible  in  the  photograph  (From 

W.\LD  .\ND   HUBB.\RD,    I948-49). 


lacks  for  performing  this  conversion  (Fig.  3). 

The  water-soluble  factor  concerned  with  this  process  did  not  seem  to  involve  a  pro- 
tein. It  was  relatively  heat-stable,  retaining  most  of  its  activity  after  boiling  for  as 
long  as  seven  minutes.  Also  the  ease  and  completeness  with  which  it  left  the  retinal 
tissue  in  a  single  extraction  suggested  that  it  was  made  up  of  small  and  relatively 
simple  molecules  —  perhaps  a  coenzyme,  01  a  hydrogen-donating  substrate. 

Now  one  would  expect  an  enzyme  protein  to  be  rela- 
tively specific ;  and  since  retinenci  is  found  only  in  retinas, 


Fig.  3.  Rod  outer  limbs  suspended  in  a  water  extract  of  retina 
convert  retinene^  to  vitamin  A^;  washed  retinal  tissue  is  inactive. 
Isolated  rod  outer  limbs  were  frozen-dried  and  preextracted  with 
petroleum  ether  in  the  dark.  Whole  retinal  tissue  was  ground, 
extracted  with  neutral  phosphate  buffer,  and  the  outer  limb 
material  was  suspended  in  the  extract.  Both  this  suspension  and 
the  washed  retinal  tissue  were  irradiated,  incubated,  and  extracted 
with  hexane.  Spectra  of  the  antimony  chloride  tests  of  these 
extracts  are  shown.  That  from  the  washed  retinal  tissue  displays 
the  band  of  unchanged  retinenej  (curve  b);  while  the  outer  limb 
preparation  suspended  in  retinal  washings  has  converted  its 
retinenei   entirely  to  vitamin   A^    (curve  a).   (From  W.\ld   and 

HUBB.^RD,   1948-49). 


40 

b 

/'N' 

c 

\ 

/ 

\ 

V 

30 

/ 

\ 

sC 

\ 

/ 

/ 

\ 

\ 

20 

/ 

/ 

\ 

/ 

/ 

10 

/ 

r 

/ 

' 

n 

700 


600 
Wavelength- mu. 


References  p.  228. 


220 


G.  WALD 


VOL.  4  (1950) 


-.50 


AO 


30 


20 


10 


700 


600 
Wavelength -mu. 


its  reductase  might  well  be  restricted  to  this  tissue.  A  coen- 
zyme or  substrate,  however,  would  ordinarily  be  unspecilic, 
and  one  would  expect  to  find  it  widely  distributed  among 
the  tissues.  This  thought  led  us  to  try  an  extract  of  frog 
muscle  as  a  suspension  medium  for  rod  outer  limbs. 

The  preparation  we  used  was  the  Muskelkochsaft  —  the 

Fig.  4.  Boiled  muscle  juice  activates  isolated  rod  outer  limbs.  Equal 
numbers  of  rod  outer  segments  were  suspended  in  phosphate  buffer 
and  in  a  boiled  juice  of  frog  muscle.  The  suspensions  were  exposed 
to  light,  left  at  room  temperature  for  i  hour,  and  the  residues  extracted 
with  petroleum  ether.  The  spectra  of  the  antimony  chloride  tests 
with  these  extracts  are  shown.  The  outer  limbs  in  buffer  had  failed 
to  convert  their  retinene^  to  vitamin  A^  (curve  a) ;  those  suspended 
in  boiled  muscle  juice  had  done  so  completely  (curve  b).  The  relatively 
low  content  of  vitamin  Aj  shown  in  curve  b  is  due  to  its  destruction  in 
preparations  of  this  type.  (From  Wald  and  Hubbard,  1948-49). 


boiled  muscle  juice  —  of  Meyerhof  (1918).   Rod  outer  segments  suspended  in  this 
medium  converted  their  retinene^  quantitatively  to  vitamin  A^  (Fig.  4). 

Boiled  muscle  juice  contains  a  number  of  substances  which  could  donate  hydrogen 
for  the  reduction  of  retinencj.  It  also  contains  a  major  coenzyme  of  hydrogen  transfer, 
cozymase,  Coenzyme  I,  or  DPN. 

When  rod  outer  limbs  were  suspended  in  a  buffer  solution  to  which  DPN  had  been 
added,  they  failed  to  transform  theii  retinenci  to  vitamin  A^.  But  if  they — or  an  inactive 
preparation  of  washed  retinal  tissue — ^were  provided  with  reduced  cozymase,  DPN-Hg, 
they  performed  this  conversion  quantitatively  (Fig.  5). 

Given  a  proper  substrate,  rod  outer  limbs  can  themselves  reduce  cozymase.  We 
have  found  a  first  such  substrate  in  fructose  diphosphate.  Rod  outer  segments  suspended 
in  a  solution  to  which  both  DPN  and  fructose  diphosphate  were  added  converted  their 
retinencj  completely  to  vitamin  A^.  The  outer  segments  must  therefore  contain  an 
enzyme  system  for  reducing  DPN  when  a  suitable  hydrogen  donor  is  made  available. 
It  is  highly  improbable  that  fructose  diphosphate  itself  is  the  source  of  hydrogen  in 
this  reaction.  More  probably  the  outer  limbs  also  possess  the  enzyme  aldolase,  which 
cleaves  fructose  diphosphate  to  yield  3-glyceraldehyde  phosphate,  the  normal  substrate 
for  the  reduction  of  DPN  in  the  lactic  acid  fermentation. 

The  convex  sion  of  retinenej  to  vitamin  A^  is  there- 
fore a  coupled  reduction  in  which  DPN-Hg  acts  as 
coenzyme.  The  essential  process  is  the  transfer  of  two 


S30 


Fig.  5.  The  action  of  reduced  cozymase  on  washed  retina. 
Equal  portions  of  a  preparation  of  water-extracted  frog  retina 
were  suspended  in  a  solution  containing  reduced  DPN,  and  in 
an  otherwise  identical  solution  lacking  only  the  DPN-Hj.  Both 
suspensions  were  bleached  in  the  light,  incubated,  and  the 
residues  extracted  with  petroleum  ether.  Spectra  of  the  anti- 
mony chloride  tests  with  these  extracts  are  shown.  The  control 
preparation  yielded  retincncj  alone  (curve  a) ;  while  in  the 
washed  retina  to  which  reduced  DPN  had  been  added  this  had 
been  converted  almost  completely  to  vitamin  Aj  (curve  b). 
(From  Wald  and  Hubbard,  1948-49). 

References  p.  228. 


20 


I            \b 
a,'  X.  ^ 


700 


600 
Wavelength-miL 


VOL.  4  (1950)  RETINENES    AND   VITAMINS   A  221 

hydrogen  atoms  from  DPN-H^  to  retinene,,  reducing  its  aldehyde  group  to  the  primary 
alcohol  group  of  vitamin  A^.  We  may  assume  that  in  this  process  an  apoenzyme,  retinene 
reductase,  still  to  be  revealed,  takes  part.  The  reaction  may  be  written: 

CijHj^CHO  +  DPX-H,         retinene  reductase  ^    Q^^U^-CH^OU  +  DPN 
retinene^  vitamin  Aj 

In  the  rod  outer  limb  this  system  works  in  conjunction  with  a  second  dehydrogenase 
system  which  reduces  DPN,  using  a  derivative  of  fructose  diphosphate  as  hydiogen 
donor.  The  total  process  may  be  formulated: 

Rhodopsin 

71  \ 

\light 

Orange  intermediates 
\ 

- , . .         .       .        ,             .    .                  retinene  reductase         t-.    ,  •  ,  .    ■ 

\  itamm  Aj  +  protem  < Retmenci  +  protem 

DPN-H2  <-- 

fructose 
diphosphate 

+ 

dehydrogenase 

system 

^  DPN 


THE    RETINENE    REDUCTASE    SYSTEM 

With  the  coenzyme,  the  first  component  of  the  retinene  reductase  system  was 
defined.  Up  to  this  point  the  apoenzyme  had  remained  a  matter  of  surmise,  buried  in 
the  structure  of  the  rod  outer  limb.  The  substrate  had  been  obtained  by  bleaching 
rhodopsin,  and  was  both  equivocal  in  character  and  very  limited  in  quantity. 

The  nature  of  the  substrate  was  resolved  with  the  observation  that  for  this  one 
could  use  pure  synthetic  retinene j  prepared  as  described  above  by  the  chromatographic 
oxidation  of  crystalline  vitamin  Aj  on  manganese  dioxide.  Retinenej  is  fat-soluble, 
and  was  originally  introduced  into  the  system  with  the  aid  of  digitonin,  with  which  it 
forms  a  water-soluble  complex.  Later  the  digitonin  proved  to  be  unnecessary,  for  reasons 
to  be  discussed  below. 

The  apoenzyme  was  found  to  be  readily  extracted  with  dilute  salt  solutions  from 
homogenates  of  frog  or  cattle  retinas,  forming  clear,  almost  colourless  solutions.  Though 
the  apoenzyme  has  not  yet  been  isolated  as  a  pure  substance,  it  has  been  separated 
from  the  other  components  of  the  system  and  some  of  its  properties  have  been  deter- 
mined. It  is  precipitated  by  half-saturated  ammonium  sulphate  and  re-dissolves  without 
losing  its  activity.  It  is  retained  by  a  Visking  membrane,  and  survives  dialysis  for  18 
hours  at  5°  C  against  neutral  phosphate  buffer.  It  is  destroyed  by  heating  at  100° 
within  30  seconds.  Its  pn  optimum  lies  at  about  6.5. 

The  retinene  reductase  system  can  therefore  now  be  assembled  from  its  separate 
components,  all  in  true  solution:  the  coenzyme,  DPN-Hj,  prepared  by  the  method 

*  A  short  account  has  been  published  of  the  experiments  which  follow  (Wald,  1949).  A  more 
complete  description  of  these  experiments  will  appear  in  the  Journal  of  General  Physiology. 

References  p.  228. 


222 


G.  WALD 


VOL.  4  (1950) 


0.3 


0.1 


Vitamin  Ai-^—Retinenef 

Ar\ 

Frog 
apormyine 

- 1 

/v 

-  ■ 

^  \ 

\ 

K 

■ 

Vitamin  Ai—Rftin 

'flpj 

■ 

/x 

\ 

/\ 

\ 

■ 

^  \ 

\ 

■ 

VJ^ 

1      1      J       1             1       1       1       1 

300  20  40   60   80  400  20  40   60    00 

Wavelength- mu. 


of  Ohlmeyer  (1938);  the  substrate,  synthetic  retinenci; 
and  the  apoenzyme,  contained  in  a  clear,  almost  colourless 
extract  of  homogenized  frog  or  cattle  retinas.  When  these 
three  components  are  mixed  and  incubated  for  1-2  hours 
at  room  temperature,  the  retinencj  is  quantitatively 
reduced  to  vitamin  Aj  (Fig.  6,  upper  half). 


Fig.  6.  The  action  of  frog  retinene  reductase  on  synthetic  retinene^ 
and  retinencj.  Each  of  the  experimental  mixtures  included  a 
synthetic  retinene  dissolved  in  1%  digitonin,  0.7  mg  of  reduced 
cozymase  per  ml,  5.5  mg  of  nicotinamide  per  ml,  and  extracts  of 
homogenized  frog  retinas  in  m/30  phosphate  buffer,  pn  6.81.  The 
controls  differed  only  in  that  the  retinal  extracts  were  replaced 
with  either  the  same  extract  which  had  been  boiled  for  Yo  minute 
(upper  figure)  or  with  the  phosphate  buffer  alone  (lower  figure). 
The  enzyme  and  control  mixtures  were  incubated  together  for 
2  hours  at  23°  C.  Methanol  was  added  to  each  to  a  concentration 
of  60%,  and  they  were  extracted  with  hexane.  The  spectra  of  the 
hexane  extracts  are  shown.  Those  from  the  controls  (solid  circles) 
show  the  spectra  of  the  unaltered  retinenes;  those  from  the  enzyme 
mixtures  (open  circles)  show  complete  conversion  to  the  corre- 
sponding vitamins  A. 


RETINENE2   AND   VITAMIN    A.;    SPECIFICITY   OF    RETINENE    REDUCTASE 

In  the  rods  of  freshwater  fishes,  cyclostomes  and  certain  amphibia,  rhodopsin  is 
replaced  by  the  purple,  light-sensitive  porphyropsin.  This  takes  part  in  a  retinal  cycle 
identical  in  form  with  the  rhodopsin  system,  but  based  upon  the  new  carotenoids, 
retinenCo  and  vitamin  A_^  (Wald,  1937;  1945-46): 

Porphyropsin 
(522  m^) 

\ 

\light 

\Russet  intermediates 

\         \ 
\         ^ 
Retinencj  -|-  protein 


Vitamin  A^  -\-  protein 

I 
(345-350  m/n  in  petroleum  ether) 
(-f  SbClg >  692-696  m/u) 


(384  m/t  in  petroleum  ether) 
{+  SbCl3 >  705  m//) 


The  structure  of  vitamin  Ag  is  still  uncertain.  It  seems  clear,  however,  that  like  A, 
it  is  a  primary  alcohol;  and  that  retinene.j,  as  emerges  from  experiments  of  Morton  et  al. 
and  from  those  discussed  below,  is  in  all  probability  its  aldehyde. 

Morton,  Sal  ah,  and  Stubbs  (1946)  reported  that  when  solutions  of  vitamin  Ao 
in  petroleum  ether  are  let  stand  in  the  cold  over  solid  manganese  dioxide,  the  vitamin 
is  replaced  by  a  product  resembling  retinene^  in  spectrum  and  antimony  chloride  reac- 
tion. They  found  that  this  product  forms,  as  does  retinenCi,  a  2-4-dinitrophenyl- 
hydrazone,  indicating  the  presence  of  a  carbonyl  group.  That  this  substance  possesses 
a  conjugated  carbonyl  group  is  shown  also  b}^  a  large  displacement  of  its  spectrum  in 
References  p.  228. 


VOL.  4  (1950) 


RETINENES   AND   VITAMINS   A 


223 


ethanol  as  compared  with  hexane  (cf.  Wald,  1947-48).  That  the  carbonyl  group  replaces 
the  primary  alcohol  group  of  vitamin  A,  is  shown  by  the  fact  that  though  the  vitamin 
is  hypophasic,  its  oxidation  product  is  epiphasic  in  partition  between  hexane  and  90% 
methanol.  This  information,  together  with  what  follows,  leaves  little  doubt  that  this 
product  is  retinene.,,  and  that  it  is  the  aldehyde  of  vitamin  A2. 

As  in  the  manufacture  of  retinene  1,  we  have  found  that  the  oxidation  of  vitamin  A2 
to  retinene.j  can  be  carried  out  conveniently  in  chromatographic  form.  The  procedure 
is  identical  with  that  used  in  making  retinencj ;  but  in  this  case  only  about  half  as  much 
manganese  dioxide  is  employed  —  0.3  g  to  oxidize  10  mg  of  vitamin  A,.  The  yield  of 
retinenca  is  in  the  neighbourhood  of  50% ;  and  it  can  be  brought  to  a  state  of  high  purity 
by  chromatographic  adsorption  on  a  column  of  calcium  carbonate. 

In  our  past  experience  one  of  the  most  remarkable  properties  of  the  porphyropsin 
system  has  been  its  detailed  parallelism  in  chemical  behaviour  with  the  rhodopsin 
cycle.  In  the  present  instance  this  parallelism  is  maintained,  for  retinencj  is  reduced  to 
vitamin  A.,  by  an  enzyme  system  entirely  similar  to  that  which  reduces  retinene^. 

This  system  can  be  assembled  from  the  following  components:  the  coenzyme, 
DPN-Ho;  the  substrate,  sjmthetic  retinene^,  prepared  by  the  chromatographic  oxidation 
of  vitamin  A;,  on  manganese  dioxide;  and  the  apoenzyme,  contained  in  a  clear,  almost 
colourless  saline  extract  of  homogenized  freshwater  fish  retinas  (yellow  perch,  sunfish). 
When  these  three  components  are  mixed  and  left  at  room  temperature  for  two  hours, 
the  retinene.,  is  reduced  almost  entirely  to  vitamin  A.^  (Fig.  7,  upper  half). 

Since  the  coenzyme  of  retinene  reduction  is  common  to  the  rhodopsin  and  por- 
phyropsin cycles,  one  may  inquire  into  the  specificity  of  the  apoenzyme.  To  test  this, 
experiments  were  performed  in  which  the  frog  apoenzyme  was  allowed  to  act  on  retinene.^ 
and  the  fish  apoenzyme  on  retinene j .  It  emerged  that  the  reduction  proceeded  as  smooth- 
ly and  completely  with  the  crossed  as  with  the  normal  substrates  (Figs  6  and  7). 

There  is  no  reason  therefore  to  designate  the  apoen- 
zyme differently  in  the  rhodopsin  and  porphyropsin     § 
systems.  We  have  to  deal  with  a  single  apoenzyme,    |o.j 
retinene  reductase,  which  with  the  single  coenzyme,    i 
dihydrocozymase,  reduces  either  of  the  retinenes  to  the       o.z 
corresponding  vitamin  A. 

This  enzyme  system  introduces  a  new  vitamin  into       a, 
the  chemistry  of  rod  vision,  for  the  central  component 


Fig.  7.  Action  of  retinene  reductase  from  a  freshwater  fish  on 
synthetic  retinencj  and  retinenej.  The  experimental  mixtures 
included  solutions  of  the  retinenes  in  1%  digitonin,  2.4  mg 
reduced  cozymase  per  ml,  6-7  mg  nicotinamide  and  i  mg 
a-tocopheryl  phosphate  per  ml  to  stabilize  the  system;  and 
extracts  of  homogenized  yellow  perch  retinas  in  m/30  phosphate 
buffer,  ph  6.81.  The  controls  differed  only  in  that  the  retinal 
extracts  were  replaced  by  the  phosphate  buffer  alone.  All  the 
mixtures  were  left  for  2  hours  at  22°  C;  then  methanol  was 
added  to  a  concentration  of  60%,  and  they  were  extracted 
vWth  hexane.  The  spectra  of  the  hexane  extracts  are  shown. 
Those  from  the  controls  (solid  circles)  show  the  unaltered 
retinenes;  those  from  the  enzyme  mixtures  (open  circles)  show 
almost  complete  conversion  to  the  corresponding  vitamins  A. 
In  each  figure  a  short  vertical  line  shows  the  position  of  the 
absorption  maximum  of  vitamin  Aj  or  .\j  in  hexane. 

References  p.  228. 


0.3 


VHamin  A^- — Rttii 

1 

ene2 

- 

.^ 

\ 

- 

/>                   \ 

A 

- 

^ 

■ 

Vitamin-^ —  ftftintne 

yellow 

- 

/7\\ 

apoenzyme 

- 

y    \\ 

■ 

\ 

■ 

X 

■      III 

•           <           i 

300  20    40    60    eO  MO  70    M    60    80 
Wavelength -mji 


224  ^-  ^VALD  VOL.  4  (1950) 

of  cozymase  is  nicotinamide,  the  anti-pellagra  factor  of  the  vitamin  B  complex.  It 
presents  also  the  novel  phenomenon  of  widely  distinct  vitamins  not  only  interacting  in 
vitro,  but  of  one  of  them  paiticipating  directly  in  the  regeneration  of  the  others.  I  do  not 
know  a  comparable  i  elation  in  the  whole  of  biochemistry. 

STABILITY 

It  has  been  known  for  some  time  that  animal  and  certain  plant  tissues  contain  a 
nucleotidase  which  cleaves  cozymase  and  dihydrocozymase,  and  which  is  released  into 
homogenates  and  tissue  breis  by  the  breaking  of  the  cells.  Measurements  made  on  various 
tissues  of  the  rat  have  shown  this  enzyme  to  be  particularly  active  in  brain,  to  which 
of  course  retina  is  closely  related  (Mann  and  Quastel,  1941 ;  Handler  and  Klein, 
1942).  The  action  of  this  enzyme  makes  a  number  of  the  preparations  which  we  have 
described  unstable. 

It  was  noted  above  that  solutions  of  rhodopsin,  prepared  by  extracting  fresh  retinal 
tissue  with  water  solutions  of  digitonin,  rapidly  lose  the  power  to  reduce  retinenCi. 
Within  3-4  hours  their  ability  to  perform  this  process  usually  falls  to  very  low  levels. 
The  principal  cause  of  this  failure  is  the  loss  of  cozymase. 

This  is  shown  by  the  following  experiment.  A  freshly  prepared  rhodopsin  solution 
was  kept  at  about  23°  C  for  18  hours.  At  the  end  of  this  period  it  was  divided  into  halves, 
and  to  one  half  reduced  cozymase  was  added  (1.5  mg  per  ml).  Both  portions  were 
bleached  in  the  light  and  were  incubated  for  i  hour.  The  control  portion  converted  no 
more  than  a  trace  of  its  retinenci  to  vitamin  A^;  that  to  which  DPN-Ho  was  added 
had  completed  this  conversion.  It  is  clear  that  the  apoenzyme  in  such  preparations  is 
relatively  stable;  their  loss  of  activity  is  caused  by  the  destruction  of  the  coenzyme. 

Cozymase  and  reduced  cozymase  are  protected  from  the  action  of  the  nucleotidase 
by  the  presence  of  free  nicotinamide  (2-20  mg  per  ml)  (Mann  and  Quastel,  1941; 
Handler  and  Klein,  1942).  It  has  recently  been  reported  also  that  a-tocopheryl  phos- 
phate (about  I  mg  per  ml)  similarly  protects  cozymase  (Spaulding  and  Graham,  1947). 

The  nucleotidase  has  been  reported  to  be  in  general  insoluble  in  water  or  dilute 
salt  solutions.  Our  experiments  show  that  it  does  go  into  solution  in  the  2%  digitonin 
with  which  we  extract  rhodopsin.  It  also  is  active  in  all  our  retinal  homogenates  and 
particulate  preparations.  Whether  it  enters  the  saline  extracts  which  contain  our 
apoenzyme  we  have  not  yet  determined.  A  number  of  our  fish  enzyme  preparations 
have  definitely  been  unstable,  but  they  also  tend  to  be  slightly  turbid,  and  may  contain 
small  amounts  of  very  fine  particles. 

In  any  case  we  have  taken  the  precaution  ordinarily  to  add  nicotinamide  to  our 
enzyme  preparations;  and  to  those  from  freshwater  fish  retinas,  in  which  the  nucleoti- 
dase appears  to  be  particularly  active,  we  have  added  also  a-tocopheryl  phosphate. 

These  adjustments  extend  still  further  the  participation  of  vitamins  in  the  retinene 
reductase  system.  Nicotinamide  acts  not  only  as  the  key  component  of  the  cozymase 
molecule,  but  in  the  free  condition  protects  cozymase  fiom  destruction.  In  this  action 
it  is  aided  by  vitamin  E  phosphate.  As  many  as  three  vitamins  therefore  interact  with 
one  another  in  this  single  system. 

the  state  of  the  retinenes 
With  the  first  use  of  the  synthetic  retinenes  as  substrates  there  arose  the  problem 
how,  as  typically  fat-soluble  substances,  they  were  to  be  introduced  into  the  aqueous 
References  p.  228. 


VOL.  4  (1950)  RETINENES    AND   VITAMINS   A  225 

enzyme  system.  This  was  solved  initially  by  bringing  the  retinenes  into  water  solution 
with  the  aid  of  digitonin,  with  which  they  form  water-sduble  complexes. 

The  use  of  digitonin,  however,  proved  to  be  unnecessary.  The  retinal  extracts  which 
contain  the  apoenzyme  take  up  the  retinenes  directly.  If  either  of  the  retinenes  is 
concentrated  in  a  few  drops  of  petroleum  ether,  and  is  agitated  together  with  a  water 
extract  of  retinas  while  the  last  of  the  petroleum  ether  is  drawn  off  under  suction,  the 
retinenes  gradually  are  taken  up  to  yield  clear  yellow  solutions.  This  is  one  indication 
that  the  retinenes  couple  with  water-soluble  substances  from  the  retina.  Primarily  in 
these  preparations  they  attach  to  protein,  for  they  are  precipitated  from  such  solutions 
with  the  protein  fraction. 

It  has  been  known  for  some  time  that  in  the  product  of  bleaching  rhodopsin  in 
solution,  most  of  the  retinenej  is  found  loosely  coupled  with  protein  (Wald,  1937-38, 
pp.  812-813).  In  this  condition  it  behaves  as  a  pn  indicator,  deep  yellow  in  acid  and 
almost  colourless  in  alkaline  solution;  hence  Lythgoe's  proposal  that  it  be  called 
"indicator  yellow".  Synthetic  retinenei  does  not  change  its  spectrum  at  all  with  pn; 
nor  does  natural  letinene^  after  partial  purification  by  adsorption  and  elution  (Wald, 
1947-48).  Ball  et  al.  have  now  shown  that  the  pn  indicator  property  is  characteristic 
of  retinenei  in  the  coupled  condition  (Ball,  Collins,  Morton,  and  Stubbs,  1948). 
Retinenei  condenses  spontaneously,  as  do  aldehydes  generall}^  with  a  variety  of  amino 
compounds  —  proteins,  amino  acids,  aromatic  amines  —  and  in  this  state  acts  as  a  pn 
indicator.  Indeed  a  second  evidence  that  the  synthetic  retinenes  added  directly  or  in 
digitonin  solution  to  our  apoenzyme  extracts  couple  with  other  molecules  is  that  they 
have  acquired  this  property.  They  have  in  fact  come  to  resemble  closely  the  natural 
products  of  bleaching  rhodopsin  and  porphjn-opsin  in  solution. 

A  third  evidence  that  synthetic  retinene^  couples  with  other  molecules  in  our 
enzyme  system  is  that  it  becomes  more  and  more  difficult  to  extract  with  fat  solvents 
as  the  mixture  is  made  more  alkaline.  If  to  a  solution  of  retinenci  in  digitonin  one  adds 
methanol  in  a  final  concentration  of  60  %  and  shakes  vigorously  with  petroleum  ether, 
almost  all  the  retinene  enters  the  petroleum  ether  regardless  of  the  pn-  But  if  retinenej 
in  digitonin  is  mixed  with  a  water  extract  of  the  retina  prior  to  carrying  out  this  pro- 
cedure, smaller  and  smaller  fractions  of  the  retinene  enter  the  petroleum  ether  as  the 
alkalinity  is  increased.  At  pn  4  about  2/3  of  the  retinene  is  extracted  with  petroleum 
ether  in  one  partition;  at  pn  9  only  about  1/6  of  the  retinene  is  extracted.  What  this 
probably  means  is  that  since  retinene^  is  coupled  by  the  condensation  of  its  carbonyl 
group  with  the  amino  groups  of  other  molecules,  alkalinity  favours  this  process  by 
increasing  the  proportion  of  free  amino  groups,  while  acidity  hinders  it  by  converting 
amino  groups  to  ammonium  ions*. 

The  net  result  of  these  considerations  is  that  we  must  regard  the  normal  state  of 
the  retinenes  in  retinas  and  retinal  extracts  as  a  labile  equilibrium  between  free  mole- 
cules and  those  loosely  coupled  to  other  substances.  There  is  no  unique  retinal  molecule, 
however,  with  which  the  retinenes  couple  and  which  therefore  should  be  designated 
"visual  yellow"  or  "indicator  yellow".  On  the  contrary,  the  retinenes  regularly  condense 
with  a  variety  of  molecules,  some  protein,  some  forming  fat-soluble  complexes.  So,  for 
example,  when  the  retinenes  have  been  extracted  from  retinas  with  petroleum  ether. 


*  On  observing  that  retinenej  is  not  readily  extracted  with  petroleum  ether  from  alkaline  solu- 
tions of  bleached  rhodopsin,  Bliss  (194S)  concluded  that  it  had  not  been  formed.  It  is  formed,  but 
like  added  retinencj  it  is  retained  by  coupling  with  other  retinal  molecules. 

References  p.  228. 


15 


226  G.  WALD  VOL.  4  (1950) 

and  are  hence  protein-free,  they  still  behave  as  pn  indicators,  and  are  therefore  still 
in  the  coupled  condition. 

Not  only  do  the  retinenes  form  a  variety  of  retinal  complexes,  but  normally  they 
migrate  from  one  such  association  to  another.  One  such  migration  is  established  by  the 
present  experiments.  Rhodopsin  and  retinene  reductase  are  different  proteins.  Retinenes 
originates  on  rhodopsin  protein,  but  it  must  transfer  to  the  reductase  protein  preparatory 
to  its  reduction.  RetinenCa  is  involved  in  a  like  situation.  Such  changes  of  the  molecules 
with  which  the  retinenes  are  coupled  must  play  an  important  part  in  retinal  metabolism. 


SUMMARY 

The  retinenej  which  results  from  the  bleaching  of  rhodopsin  now  appears  to  be  vitamin  Aj 
aldehyde.  Morton  et  al.  have  given  the  best  evidence  for  this,  and  have  shown  that  retinene^  can 
be  prepared  by  the  mild  oxidation  of  vitamin  A^.  A  simple  procedure  is  described  for  performing 
this  process  chromatographically  on  a  column  of  manganese  dioxide. 

In  the  retina,  retinencj  is  converted  irreversibly  to  vitamin  Aj^  by  an  enzyme  system  in  which 
reduced  cozymase  (reduced  Coenzyme  I,  DPN-Hj)  serves  as  coenzyme.  The  essential  process  is  the 
transfer  of  two  hydrogen  atoms  from  DPN-Hj  to  retinencj,  reducing  its  aldehyde  group  to  the  primary 
alcohol  group  of  vitamin  A^. 

The  enzyme  system  which  performs  this  reduction  can  be  assembled  in  solution  from  the  fol- 
lowing components:  the  coenzyme,  DPN-Hjl  as  substrate,  synthetic  retinene^;  and  the  apoenzyme 
extracted  with  dilute  salt  solutions  from  homogenized  frog  or  cattle  retinas.  The  apoenzyme  is 
non-dialysable,  is  precipitated  by  half-saturated  ammonium  sulphate,  and  is  destroyed  by  heating 
at  100°  C  within  30  seconds.  Its  pn  optimum  lies  at  about  6.5. 

In  the  rods  of  freshwater  fishes,  a  parallel  enzyme  system  reduces  retinenej  to  vitamin  Aj. 
This  can  be  assembled  from  the  following  components,  all  in  true  solution:  the  coenzyme,  DPN-Hgl 
as  substrate,  synthetic  retinenej,  prepared  by  the  chromatographic  oxidation  of  vitamin  Ag  on 
manganese  dioxide;  and  the  apoenzyme  extracted  with  dilute  salt  solutions  from  freshwater  fish 
retinas  (sunfish,  yellow  perch). 

The  apoenzyme  from  frog  retinas  reduces  retinene2  as  effectively  as  retinenej.  Similarly  the 
fish  apoenzyme  acts  equally  well  upon  both  retinenes.  One  need  consider  only  one  apoenzyme,  retinene 
reductase,  which  together  with  one  coenzyme,  DPN-Hj,  reduces  either  of  the  retinenes  to  the  cor- 
responding vitamin  A. 

The  retinene  reductase  system  brings  a  second  vitamin  into  the  chemistry  of  rod  vision.  It 
presents  the  novel  phenomenon  of  one  vitamin  regenerating  another,  for  the  central  component  of 
DPN-H,  is  nicotinamide,  the  anti-pellagra  factor  of  the  vitamin  B  complex. 

Rhodopsin  solutions  and  retinal  homogenates  rapidly  lose  their  power  to  reduce  the  retinenes, 
through  destruction  of  their  DPN  by  a  nucleotidase.  Rhodopsin  solutions  which  have  lost  their 
activity  in  this  way  are  re-activated  by  the  addition  of  new  DPN-Hj.  The  coenzyme  can  also  be 
protected  by  the  presence  of  free  nicotinamide  and  of  a-tocopheryl  phosphate. 

On  addition  to  the  enzyme  system,  the  synthetic  retinenes  rapidly  couple  with  other  molecules, 
and  primarily  with  protein.  The  normal  state  of  the  retinenes  in  retinas  and  retinal  extracts  is  a  labile 
equilibrium  between  the  free  and  the  coupled  conditioft.  The  retinenes  couple  with  a  variety  of  retinal 
molecules,  and  migrate  freely  from  one  to  the  other. 

RfiSUMfi 

Le  retinene^,  qui  resulte  du  blanchissement  de  la  rhodopsine,  apparait  maintenant  comme  6tant 
I'aldehyde  de  la  vitamine  A^.  Morton  et  collab.  en  ont  donne  la  meilleure  preuve  en  montrant  que 
le  retinenci  pent  etre  prepare  par  une  oxydation  menagee  de  la  vitamine  Aj.  Un  proc6d6  simple 
est  decrit,  qui  permet  d'effectuer  cette  operation  par  chromatographic  sur  une  colonne  de  bioxyde 
de  manganese. 

Dans  la  rdtine,  le  retinenej  est  converti  irr6versiblement  en  vitamine  Aj  par  un  systeme  enzy- 
matique  dans  lequel  la  cozymase  I  r^duite  (DPN-H2)  sert  de  coenzyme.  Le  processus  consiste  essen- 
tiellement  en  un  transfert  de  deux  atomes  d'hydrogene  du  DPN-Hg  sur  le  r^tinenej,  r^duisant  sa 
fonction  aldehydique  en  fonction  alcoolique  primaire  de  la  vitamine  Aj. 

Le  systeme  enzymatique  qui  cffectue  cette  r6duction  pent  etre  constitu6  en  solution  a  partir 

References  p.  228. 


VOL.  4  (1950)  RETINENES   AND   VITAMINS   A  227 

des  composantes  suivantes:  la  coenzyme,  DPN-Hj;  comme  substratum  du  r^tinenej  synth6tique; 
et  I'apoenzyme,  extraite  de  retines  homogeneisees  de  grenouilles  ou  de  bceufs  au  moyen  de  solutions 
salines  diluees.  L'apoenzyme  n'est  pas  dialysable ;  elle  est  precipitee  par  le  sulfate  d'ammonium  a  demi- 
saturation  et  detruite  par  chauffage  a  100°  pendant  30  secondes.  Son  pn  optimum  est  d'environ  6.5. 

Dans  les  batonnets  de  la  retine  de  poissons  d'eau  douce,  il  existe  un  systeme  enzymatique 
parallele,  qui  reduit  le  retinenej  en  vitamine  A2.  Ce  systeme  pent  etre  constitue  a  partir  des  compo- 
santes suivantes,  toutes  en  vraie  solution:  la  coenzyme,  DPN-Hj;  comme  substratum,  du  r^tinencg 
synthetique,  prepare  par  oxydation  chromatographique  de  la  vitamine  A,  au  bioxyde  de  manganese; 
et  l'apoenzyme,  extraite  au  moyen  de  solutions  salines  diluees  a  partir  de  retines  homogeneisees  de 
poissons  d'eau  douce  (poisson-soleil,  perche  jaune). 

L'apoenzyme  de  la  retine  de  grenouille  reduit  le  r6tinene2  aussi  bien  que  le  retinencj.  De  meme, 
l'apoenzyme  de  poissons  d'eau  douce  agit  egalement  bien  sur  les  deux  retinenes.  II  n'est  done  besoin 
de  considerer  qu'une  seule  apoenzyme,  la  reductase  du  r^tinene,  qui,  en  presence  d'une  coenzyme, 
le  DPN-Hg,  reduit  I'un  ou  I'autre  des  deux  retinenes  en  la  vitamine  A  correspondante. 

Le  systeme  de  la  reductase  du  r^tinene  introduit  une  seconde  vitamine  dans  la  chimie  de  la 
vision  par  batonnets.  II  pr^sente  le  phenomene  nouveau  d'une  vitamine  qui  en  regenere  une  autre, 
attendu  que  la  composante  essentielle  du  DPN-Ho  est  la  nicotamide,  le  facteur  antipellagreux  du 
complexe  vitamique  B. 

Des  solutions  de  rhodopsine  et  d'extraits  homogen^is^s  de  ratines  perdent  rapidement  leur 
pouvoir  de  r6duire  les  retinenes,  de  par  la  destruction  de  leur  DPN  par  une  nucleotidase.  Des  solu- 
tions de  rhodopsine  ayant  ainsi  perdu  leur  pouvoir  r^ducteur  sont  reactivees  par  I'addition  d'une 
quantite  fraiche  de  DPN-Hjj.  La  coenzyme  pent  Egalement  etre  protegee  par  la  presence  de  nicota- 
mide libre  et  de  phosphate  d'a-tocopheryle. 

En  plus  du  systeme  enzymatique  etudie,  les  retinenes  synthetiques  forment  des  produits 
d'addition  avec  d'autres  molecules,  et  specialement  avec  les  proteines.  L'etat  normal  des  retinenes 
dans  les  retines  et  leurs  extraits  est  un  equilibre  labile  entre  la  forme  libre  et  la  forme  associ^e.  Les 
retinenes  s'associent  avec  une  varidt6  de  molecules  r^tinales  et  migrent  librement  de  I'une  a  I'autre. 


ZUSAMMENFASSUNG 

Das  Retinen^,  welches  bei  der  Bleichung  des  Rhodopsins  entsteht,  entpuppt  sich  jetzt  als  Vita- 
min A^-Aldehyd.  Morton  und  Mitarb.  haben  dafiir  den  besten  Beweis  geliefert,  dadurch  dass  sie 
gezeigt  haben  dass  Retinenj  durch  milde  Oxydation  von  Vitamin  Aj  gebildet  werden  kann.  Es  wird 
eine  einfache  Prozedur  beschrieben,  um  diesen  Vorgang  chromatographisch  mittels  einer  Mangan- 
dioxyd-Saule  zu  bewerkstelligen. 

In  der  Netzhaut  wird  Retinenj  irreversibel  in  Vitamin  A^  verwandelt  durch  ein  Enzymsystem 
in  welchem  reduzierte  Cozj^mase  I  (DPN-H,)  als  Coenzym  dient.  Die  Hauptreaktion  besteht  dabei 
in  der  Ubertragung  von  zwei  Wasserstoflfatomen  vom  DPN-Hj  auf  das  Retinen^,  dessen  Aldehyd- 
gruppe  zur  primaren  Alkoholgruppe  des  Vitamins  Aj  reduziert  wird. 

Das  Enzymsystem  welches  diese  Reduktion  vollfiihrt,  kann  in  Losung  aus  folgenden  Kompo- 
nenten  zusammengestellt  werden:  das  Coenzym,  DPN-H2;  ^^^  Substrat,  sjmthetisches  Retinen^; 
und  das  Apoenzym,  welches  durch  verdiinnte  Salzlosungen  aus  homogenisierten  Frosch-  oder  Rinder- 
Netzhiiuten  ausgezogen  wird.  Das  Apoenzym  ist  nicht  dialysierbar ;  es  wird  durch  halbgesattigte 
Ammoniumsulfat-Losung  gefallt  und  durch  Erhitzen  auf  100°  innerhalb  30  Sek.  zerstort.  Sein  pn 
Optimum  liegt  bei  ca  6.5. 

In  den  Stabchen  von  Siisswasserfischen  besteht  ein  paralleles  Enzymsystem,  welches  Retinenj 
zu  Vitamin  Ag  reduziert.  Es  kann  aus  folgenden,  alle  in  wahrer  Losung  befindlichen  Komponenten 
zusammengestellt  werden:  das  Coenzym,  DPN-Hg;  als  Substrat,  synthetisches  RetineUj,  durch 
chromatographische  Oxydation  von  Vitamin  A2  an  Mangandioxyd  dargestellt;  und  das  Apoenzym, 
welches  durch  verdiinnte  Salzlosungen  aus  den  Netzhauten  von  Siisswasserfischen  (Sonnenfisch, 
gelber  Barsch)  ausgezogen  wird. 

Das  Apoenzym  aus  Froschnetzhauten  reduziert  RetineUg  so  wirksam  wie  Retinen^.  Desgleichen 
wirkt  das  Fisch-Apoenzym  gleich  gut  an  beiden  Retinenen.  Man  hat  also  nur  ein  einziges  Apoenzym 
zu  betrachten,  die  Retinen-Reduktase,  welche  zusammen  mit  einem  Coenzym,  dem  DPN-Hj,  beide 
Retinene  zu  den  entsprechenden  A-Vitaminen  reduziert. 

Das  System  der  Retinen-Reduktase  fiihrt  ein  zweites  Vitamin  in  die  Chemie  des  Stabchen- 
Sehens  ein.  Es  zeigt  das  neuartige  Phanomen  eines  Vitamins  welches  ein  anderes  regeneriert,  denn 
die  wichtigste  Komponente  vom  DPN-Hj  ist  das  Nikotinamid,  der  Antipellagra-Faktor  des  Vitamin 
B-Komplexes. 

Losungen  von  Rhodopsin  und  homogenisierten  Netzhautextrakten  verlieren  rasch  ihr  Ver- 
mogen,  Retinene  zu  reduzieren;  ihr  DPN  wird  namlich  von  einer  Nukleotidase  zerstort.  Auf  solche 
Art  inaktivierte  Rhodopsin-Losungen  konnen  durch  Zugabe  von  DPN-H,  reaktiviert  werden.  Das 

References  p.  228. 


228  G.  WALD  VOL.  4  (1950) 

Coenzym  kann  auch  diirch  die  Gegenwart  von  freiem  Nikotinamid  oder  von  a-Tokopherylphosphat 
geschiitzt  werden. 

Ausser  mit  dem  Enzymsystem.verbinden  sich  die  synthetischen  Retinene  auchraschmit  anderen 
Molekiilarten,  besonders  mit  Proteinen.  Der  Normalzustand  der  Retinene  in  der  Netzhaut  und  in 
Netzhautextrakten  ist  ein  labiles  Gleichgewicht  zwischen  freier  und  gebundener  Substanz.  Die 
Retinene  wandern  leicht  von  ciner  zur  anderen  der  verschiedenen  in  der  Netzhaut  befindlichen 
Molekeln  mit  denen  sie  lose  Vcrbindungen  eingehen. 

REFERENCES 

S.  Ball,  F.  D.  Collins,  R.  A.  Morton,  and  A.  L.  Stubbs,  Nature  161  (194S)  424. 

S.  Ball,  T.  W.  Goodwin,  and  R.  A.  Morton,  Biochem.  J.,  40  (1946)  Proc.  lix. 

S.  Ball,  T.  W.  Goodwin,  and  R.  A.  Morton,  Biochem.  J.,  42  (1948)  516. 

A.  F.  Bliss,  /.  Biol.  Chem.,  172  (1948)  165. 

A.  EwALD  and  W.  KiJHNE,  Untersiich.  physiol.  Inst.  Univ.  Heidelberg,  i  (187S)  248. 

P.  Handler  and  J.  R.  Klein,  /.  Biol.  Chem.,  143  (1942)  49. 

\V.  KiJHNE,  Chemische   Vorgange  in  der  Netzhaut,  Handbuch  der  Physiologie,  L.  Hermann,  editor, 

Leipzig,  F.  C.  W.  Vogel,  3  (1879)  pt.  i,  312. 
P.  J.  G.  Mann  and  J.  H.  Quastel,  Biochem.  J.,  35  (1941)  502. 
O.  Meyerhof,  Z.  physiol.  Chemie.,  102  (1918)  i. 

R.  A.  Morton,  M.  K.  Salah,  and  A.  L.  Stubbs,  Biochem.  J.,  40  (1946)  Proc.  lix. 
P.  Ohlmeyer,  Biochem.  Z.,  297  (1938)  66. 

M.  E.  Spaulding  and  W.  D.  Graham,  /.  Biol.  Chem.,  170  (1947)  71 1- 
G.  Wald,  /.  Gen.  Physiol.,  19  (i935-36a)  351. 
G.  Wald,/.  Gen.  Physiol.,  19  (i935-36b)  781. 
G.  Wald,  Nature,  139  (1937)  1017. 
G.  Wald,  /.  Gen.  Physiol.,  21  (1937-38)  795. 
G.  Wald,  /.  Gen.  Physiol.,  22  (1938-39)  775- 
G.  Wald,  Harvey  Lectures,  41  (1945-46)  117. 
G.  Wald,  /.  Gen.  Physiol.,  31  (1947-48)  489. 
G.  Wald,  Science,  109  (1949)  482. 
G.  Wald  and  R.  Hubbard,  /.  Gen.  Physiol.,  32  (1948-49)  367. 

Received  May  24th,  1949 


VOL.  4  (1950) 


BIOCHIMICA   ET  BIOPHYSICA   ACTA 


229 


EXPERIMENTELLE  BINDUNG  VON  EIWEISSKORPERN  AN 
ZELLKERNE  UND  NUKLEINSAUREN 

(kurze  mitteilung) 

von 

PAUL  OHLMEYER 
Physiologisch-Chemisches  Institut  der  U  iversitat  Tubingen  (Deiitschland) 


Zu  einem  Reaktionsansatz  der  Prostataphosphatase  mit  Glycerinphosphat  bei 
Pjj  =  3.7  haben  wir  isolierte  Zellkerne  der  Thymusdriise  zugesetzt  und  eine  Hemmung 
des  Ferments  auf  etwa  die  halbe  Wirkung  beobachtet.  Bei  Pn  =  5  und  ebenso  in  Gegen- 
wart  gewisser  (verdrangender)  Eiweisskorper  bleibt  die  Hemmung  aus  (Tab.  I). 

TABELLE  I 

ZELLKERNE    HEMMEN    DAS    FERMENT 
(protein    E    1ST    EINE    FRAKTION    AUS    MUSKULATUR) 


PH  = 

=  3-7 

PH  = 

=  5-0 

Kerne  (y) 

— 

35 

140 

280 

(  + 

350 
157  Protein  E) 

— 

3500 

Fermentwirku  ng 
(rel.  Zahlen) 

100 

85 

61 

49 

102 

130 

135 

Die  Hemmung  beruht  auf  einer  Bindung  des  Ferments  an  den  Zellkern ;  die  schiit- 
zende  Wirkung  des  Proteins  auf  einer  Verdrangung.  Die  Verbindung  lasst  sich  durch 
Zentrifugieren  abtrennen;  unter  passenden  Bedingungen  wird  dabei  ein  fermentfreier 
Uberstand  erhalten.  Der  Niederschlag  hat  dann  eine  der  Hemmung  entsprechende  Fer- 
mentwirkung,  die  durch  Zugabe  von  Eiweiss  auf  etwa  die  urspriingHche  Hohe  gebracht 
warden  kann. 


II 

Die  Wirkung  der  Zellkerne  beruht  auf  ihrem  Gehalt  an  Nukleinsaure.  Auch  Thymo- 
nukleinsaure  bildet  eine  Verbindung  mit  dem  Ferment,  die  pn-abhangig  ist  und  durch 
andere  Eiweisskorper  gelost  werden  kann.  In  dieser  Bindung  hat  das  Ferment,  unter 
der  (nicht  vollig  exakten)  Annahme  einer  linearen  Zcitfunktion,  eine  minima le  Rest- 
wirkung  von  13%. 

Ein  ahnlicher  Rest  wurde  bei  a-Glycerinphosphat,  bei  j3-Glycerinphosphat  und  bei 


230 


p.  OHLMEYER 


VOL.  4  (1950) 


Adenylsaure  als  Substrat  gefunden.  Er  zeigt  sich  ebenfalls  bei  den  Phosphatase!!  aus 
Muskel  und  aus  weiblichem  Harn.  Er  bleibt  ferner  erhalten,  wenn  das  Prostataferment 
durch  grossere  Mengen  Hefenukleinsaure  oder  durch  Tannin  gehemmt  wird.  Pikrinsaure 
und  Nikotin  haben  unter  analogen  Bedingungen  keine  Wirkung.  Der  Rest  ist  unab- 
hangig  von  der  Substratkonzentration  und  vom  p^  in  den  Grenzen  2.5  und  4.0. 

Werden  3.7  y  Ferment  bei  pn  =  3.7  ohne  Zusatz  und  mit  2  y  Nukleinsaure  20  min 
auf  der  Ultrazentrifuge  bei  115  000  g  zentrifugiert,  so  bleibt  ein  geringer  Anteil  des 
Ferments  in  Losung,  der  hemmbar  ist  wie  das  gesamte  Ferment  (Tab.  II).  Hieraus  und 
aus  der  Restwirkung  der  Verbindung  des  Ferments  mit  Zellkernen  geht  hervor,  dass 
nicht  13%  des  Ferments  ungebunden  bleiben,  sondern  dass  das  gebundene  Ferment 
einen  Wirkungsrest  von  13%  behalt. 

TABELLE  II 

DIE    VERBINDUNG    FERMENT-NUKLEINSAURE    IN    DER    ULTRAZENTRIFUGE 


Uberstand 

Nicht 

Nukleinsaure  (y) 

zentri- 

— 

2 

2 

60 

fugiert 

PH  im  Ansatz 

4.8 

4.8 

3-7 

4.8 

100 

102 

II 

2 

0 

Zu  Ansatzen  von  8  y  Ferment  mit  dei  maximal  hemmenden  Nukleinsauremenge 
(0.8  y)  wurde  eine  Anzahl  von  Eiweisstoffen  in  Verdiinnungsreihen  zugegeben  und  so 
dieMenge  ermittelt,  welche  die  Hemmung  auf  das  halbe  Maximum  emiedrigt.  In  Tab.  Ill 
ist  diese  Menge  in  Mikrogramm  angegeben. 


TABELLE  III 

ENTHEMMUNG    DURCH    EIWEISSKORPER 


Substanz 

y 

Substanz 

y 

Gliadin 

700 

Serumglobulin 

5 

Pepsin 

?  0  400) 

Hamoglobin 

2.8 

Tabakmosaikvirus 

36 

Salmin 

2.4 

Eicralbumin 

25 

Protamin  aus  Heringssperma 

0.7 

Inaktiviertes  Ferment 

24 

Protein  E 

0-3 

VOL.  4  (1950)  BINDUNG   VON    PROTEINEN    AN    NUKLEINSAUREN  23I 

Das  Tabakmosaikvirus  wurde  geprlift,  well  die  Frage  war,  ob  sein  Nukleotidanteil 
hemmen  oder  sein  Proteinanteil  enthemmen  wiirde.  Dass  (sauer)  inaktiviertes  Ferment 
enthemmen  wiirde,  war  zu  postulieren;  seine  Wiikung  wird  durch  Pepsinverdauung 
zerstort.  Die  Verbindung  Nukleinsaure-Serumglobulin  wurde  in  grosseren  Ansatzen 
gravimetrisch  bestimmt  und  zeigte  das  konstante  Gewichtsverhaltnis  1:3. 

ZUSAMMENFASSUNG 

Phosphatase  wird  durch  Bindung  an  Zellkerne  oder  an  Nukleinsaure  stark  gehemmt.  Die  Ver- 
bindung ist  nur  bei  pn  <  5  bestandig.  Durch  Eiweisstoffe  kann  das  Ferment  verdrangt  und  wieder 
mit  der  urspriingHchen  Aktivitat  erhalten  werden. 

SUMMARY 

Phosphatase  is  strongly  inhibited  by  combination  with  cell  nuclei  or  with  nucleic  acids.  The 
compound  is  only  stable  at  pn-values  less  than  5.  The  enzyme  can  be  displaced  by  proteins  and 
recovered  with  the  original  activity. 

RfiSUMfi 

La  phosphatase  est  fortement  inhibee  par  combinaison  avec  les  noyaux  cellulaires  au  avec  les 
acides  nucleiques.  Cette  combinaison  n'est  stable  qu'a  un  pH  inferieur  a  5.  Au  moyen  de  prot^ines 
le  ferment  peut  etre  deplace  de  cette  combinaison  et  reg^nere  avec  son  activity  primitive. 

Eingegangen  den  4.  April  1949 


232  BIOCHIMICA   ET   BIOPHYSICA   ACTA  VOL.  4  (1950) 


THE  BIOLOGICAL  INCORPORATION  OF 

PURINES  AND  PYRIMIDINES  INTO  NUCLEOSIDES 

AND  NUCLEIC  ACID 

by 

HERMAN  M.  KALCKAR 
Enzyme  Research  Division,  University  of  Copenhagen  (Denmark) 


INTRODUCTION 

The  fundamental  studies  by  Meyerhof  and  his  associates  on  the  metaboHsm  of 
phosphoric  esters  in  muscle  extracts  marked  the  beginning  of  a  very  fruitful  era  in 
which  the  pathway  of  breakdown  and  synthesis  of  carbohydrates  gradually  became 
known.  Meyerhof  showed  that  Harden  and  Young's  discovery  of  phosphate  uptake 
in  cell-free  yeast  fermentation  mixtures  could  be  extended  to  animal  tissues,  especially 
muscle.  Later  Meyerhof  and  his  associates  and  Needham  and  Pillai  in  Cambridge 
showed  that  esterification  of  phosphate  in  muscle  was  coupled  to  the  oxidation-reduction 
between  phosphotriose  and  cozymase.  This  development  led  to  the  discovery  of  the 
acylphosphates  (Warburg  and  coworkers,  Lipmann).  It  was  known,  however,  from 
Lundsgaard's  studies  that  muscle,  performing  alactacid  contractions  in  an  oxygen-free 
atmosphere  accumulates  large  amounts  of  hexosephosphoric  esters.  This  is  further 
accentuated  if  dinitrophenol  which  'uncouples'  oxidati^e-reductive  phosphorylation  is 
added  together  with  iodoacetate.  These  observations  which  were  made  by  Cori  and 
CoRi  in  1936  indicated  that  phosphate  can  also  be  incorporated  into  ester  linkage  by 
another  process  which  has  nothing  to  do  with  oxidation-reduction.  The  phenomenon 
of  phosphate  uptake  independent  of  oxidation-reduction  was  very  soon  encountered  in 
in  vitro  experiments  too.  Within  the  same  year  Parnas  and  Ostern  reported  that  the 
glycogen  present  in  aged  and  dialysed  muscle  extracts  canreact  with  inorganic  phosphate. 
A  few  months  later  Carl  and  Gerty  Cori  isolated  a-glucose-1-phosphate  from  muscle 
extracts  and  three  years  later  CoRi,  Cori,  and  Schmidt  demonstrated  the  synthesis  of 
a  polysaccharide  from  a-glucose-1-phosphate  by  means  of  a  muscle  enzyme.  Kiessling, 
a  student  of  Meyerhof,  performed  independently  in  1939  an  analogous  in  vitro  syn- 
thesis of  polysaccharide  using  a  yeast  enzyme.  During  the  subsequent  years  Cori  and 
his  associates  turned  their  attention  towards  the  kinetics  of  starch  and  glycogen  synthe- 
sis in  vitro.  A  number  of  important  studies  on  starch,  dextran  and  sucrose  formation  in 
enzyme  systems  from  plants  and  microorganisms  appeared  during  the  next  three  or 
four  years.  The  studies  on  the  enzymatic  synthesis  of  ribo-  and  desoxyribonucleosides 
can  also  be  considered  an  outgrowth  of  Cori's  fundamental  observ^ations  on  phospho- 
rolysis  of  glucosidic  linkages. 
References  p.  22y. 


VOL.  4  (1950)  PURINES    AND    PYRIMIDINES   IN    RIBOSIDIC   LINKAGE  233 

ENZYMATIC    SYNTHESIS    OF    PURINE    RIBO-NUCLEOSIDES 

The  presence  in  animal  tissues  of  an  enzyme,  called  nucleosidase  which  splits  of 
purines  from  purine  nucleosides  of  the  ribose  series  has  been  known  for  many  years. 
Klein^  who  made  a  detailed  study  of  this  enzyme  found  that  phosphate  and  arsenate 
enhance  the  enzymatic  splitting  of  purine  nucleosides.  When  I  spent  some  time  in  1943- 
1944  isolating  nucleosidases  from  liver  it  was  done  only  with  the  purpose  of  using  these 
enzymes  as  analytical  tools  in  an  optical  micromethod  which  I  was  trying  to  develop 
at  that  time.  I  had  no  knowledge  about  Klein's  work  at  the  time  when  I  came  across 
the  observation  that  nucleosidase  subjected  to  prolonged  dialysis  loses  its  activity.  In 
view  of  observations  by  Meyerhof  and  Cori  it  was  not  too  far-fetched  to  try  to  add 
inorganic  ortho-phosphate  to  the  system  and  it  turned  out  that  this  addition  completely 
restored  the  catalytic  activity  of  the  system.  Pursuing  the  analogy  to  Cori's  work  on 
the  polysaccharide  phosphorylase^  I  attempted  to  demonstrate  the  formation  of  ribose- 
1-phosphate  as  a  suspected  intermediate.  These  attempts  failed  quite  a  few  times.  For- 
tunately LowRY  who  was  my  colleague  at  that  time  at  The  Public  Health  Research 
Institute  had  worked  out  a  new  method  for  phosphate  determination  which  operates 
at  Ph  4-  This  method,  the  well-known  Lowry-Lopez  method^,  permits  an  estimation 
of  highly  labile  phosphoric  esters  such  as  phosphocreatine  and  acylphosphates  in  the 
presence  of  inorganic  phosphate.  With  the  Lowry-Lopez  procedure  it  became  possible 
to  show  a  clearcut  proportionality  between  liberation  of  purine  and  uptake  of  inorganic 
phosphate*.  It  was  fairly  obvious  therefore  that  a  new  and  highly  acid-labile  phosphoric 
ester  was  formed  as  a  product  of  the  enzymatic  phosphorolysis  of  nucleosides.  The  ester 
was  later  obtained  as  the  barium  salt.  It  contained  i  mole  pentose  for  each  mole  of 
labile  phosphate  and  for  each  equivalent  of  aldose  liberated  upon  mild  acid  hydrolysis. 
Lowry  has  investigated  the  lability  of  ribose-1-phosphate  in  dilute  hydrochloric  acid 
at  room  temperature  and  found  that  50%  of  the  ester  was  split  after  2.5  minutes  incuba- 
tion in  N  hydrochloric  acid.  In  view  of  these  properties  and  the  resynthesis  experiments 
described  below  the  new  ester  was  named  ribose-1-phosphate. 

The  next  step  was  an  attempt  to  resynthesize  purine  nucleosides  with  ribose-1- 
phosphate.  This  was  performed  by  incubating  hypoxanthine,  ribose-1-phosphate  and 
a  fractionated  sample  of  liver  nucleosidase  about  20  minutes  at  25°  and  subsequently 
analysing  free  and  incorporated  hypoxanthine^.  It  was  then  found  that  a  large  propor- 
tion of  the  hypoxanthine  was  incorporated  in  ribosidic  linkage  and  an  equimolar  amount 
of  labile  phosphate  was  liberated.  This  enzymatic  synthesis  of  inosine  (ribose-l-hypo- 
xanthine)  proceeded  very  far;  thus,  if  equimolar  amounts  of  hypoxanthine  and  ribose- 
1-phosphate  were  incubated  with  the  enzyme  about  80%  of  the  phosphoriboside  was 
converted  into  purine-riboside.  If  the  mixture  contained  twice  as  much  phosphoriboside 
as  hypoxanthine  more  than  95%  of  the  latter  was  incorporated  in  ribosidic  hnkage. 
The  equilibrium  can  be  formulated  as  follows :  ribose-1-phosphate  ~  h^'poxanthine  ^ 
ribose-1-hypoxanthine  -j-  phosphate.  The  enzyme  catalysing  this  equilibrium  was  named 
nucleoside  phosphorylase.  Nucleoside  phosphorylase  possesses  a  certain  specificity  with 
regard  to  the  nitrogenous  bases  added  as  well  as  to  the  pentoses  present.  Inosine  and 
guanine  riboside  are  the  only  ribosides  which  undergo  phosphorolysis  in  the  presence 
of  the  enzyme  used.  Adenosine  and  xanthosine  are  inert  in  this  system  as  are  pyrimidine 
ribosides.  Likewise  hypoxanthine  and  guanine  are  the  only  nitrogenous  bases  which  are 
incorporated,  i.e.,  which  in  the  presence  of  the  enzyme  undergo  an  exchange  with  the 
References  p.  2J7. 


234  ^-  ^^-  KALCKAR  VOL.  4  (1950) 

i-phospho  group  in  ribose-1-phosphate.  This  selective  trait  with  regard  to  purines  will 
be  discussed  a  little  later.  With  regard  to  the  sugar  component  the  furanoid  structure 
of  the  sugar  seems  to  be  imperative  for  the  reaction.  Thus,  pyranose-ribose-1-phosphate 
(synthesized  by  chemical  means  by  Todd  and  Lythgoe)  was  practically  inactive  in  the 
enzyme  test  as  was  a-glucose-1-phosphate.  Although  the  furanoid  structure  of  the  pentose 
seems  to  be  essential,  other  changes  in  the  sugar  molecule  seem  to  affect  the  enzymatic 
exchange  much  less.  Klein  had  already  observed  that  liver  and  spleen  nucleosidase 
catalyse  the  splitting  of  purine  desoxyribosides  just  as  well  as  purine  ribosides.  We  have 
found  too  that  nucleoside  phosphorylases  fractionated  by  various  means  catalyse  the 
phosphorolysis  of  purine  desoxyribosides  as  well  as  the  purine  riboside^' '.  If  we  assume 
that  the  enzymatic  catalysis  of  the  two  types  of  nucleosides  is  due  to  the  same  enzyme 
and  there  is  good  evidence  for  such  an  assumption,  the  substitution  of  an  OH  group  by 
a  H  at  carbon  no.  2  seems  to  be  unessential  for  the  activity  of  the  liver  nucleoside 
phosphorylase. 

ENZYMATIC   SYNTHESIS   OF   DESOXYRIBO-NUCLEOSIDES 

It  was  tempting  to  analyse  a  little  more  closely  the  phosphorolysis  of  desoxyri- 
bosides, and  if  possible  perform  an  enzymatic  synthesis  of  nucleosides  belonging  to  the 
desoxyribose  series.  Friedkin  who  joined  our  group  here  in  Copenhagen  as  a  research 
visitor  participated  in  this  project  and  undertook  a  closer  analysis  of  some  of  the  com- 
ponents of  the  system.  Guanine  desoxyriboside  was  isolated  and  subjected  to  an  enzym- 
atic phosphorolysis  analogous  to  that  used  for  ribosides.  After  removal  of  the  inorganic 
phosphate  the  Lowry-Lopez  phosphate  analysis  was  performed  in  order  to  disclose  the 
the  presence  of  a  highly  acid-labile  ester.  The  outcome  was  entirely  negative.  The  failure 
to  detect  any  ester  formation  by  this  method  could  be  due  to  the  fact  that  the  1-ester 
formed  in  this  case  was  more  stable  than  ribose-1-phosphate.  The  other  alternative 
was  that  the  1-ester  was  even  more  acid-labile  than  ribose-1-phosphate.  We  were  inclined 
towards  the  latter  possibility.  This  turned  out  to  be  correct.  If  free  phosphate  and  ester 
phosphate  are  estimated  separately,  using  precipitation  of  the  true  inorganic  phosphate 
by  means  of  ammoniacal  ammonium-magnesium  sulphate  it  is  possible  to  detect  the 
formation  of  a  desoxyribose  phosphoric  ester.  This  new  ester  was  found  to  undergo 
rapid  hydrolysis  in  an  acetate  buffer  of  pn  4  at  room  temperature.  Friedkin  found  that 
50%  of  the  desoxyribose  phosphate  ester  was  spHt  in  11  minutes  at  25°  at  pn  4-  This 
is  presumably  the  most  acid-labile  phosphoric  ester  yet  described.  It  has  been  possible 
to  show  that  this  ester  can  act  as  a  precursor  for  desoxynucleoside  synthesis  in  vitro. 
The  quantitative  assay  of  the  desoxyribose  ester  is  under  preparation  and  it  can  there- 
fore only  be  stated  that  if  hypoxanthine  is  incubated  with  liver  nucleoside  phosphorylase 
in  the  presence  of  a  moderate  excess  of  the  desoxyribose  ester  (but  no  inorganic  phos- 
phate) more  than  50%  of  the  hypoxanthine  is  incorporated  with  the  desoxysugar.  The 
enzymatic  formation  of  a  desoxynucleoside  was  further  substantiated  by  Hoff- 
Jorgensen  using  the  microbiological  technique^'  ^.  A  proper  estimation  of  the  amount 
of  aldose  present  before  and  after  mild  hydrolysis  of  the  new  desoxyribose  ester  is  under 
preparation.  It  is  felt  most  likely  that  the  new  ester  is  an  analogue  of  ribose-1-phosphate, 
i.e.,  a  desoxyribose-1-phosphate. 

Recently  Manson  and  Lampen^  in  Cori's  department  have  prepared  an  enzyme 
from  thymus  gland  which  brings  about  a  splitting  of  hypoxanthine  desoxyriboside 

References  p.  23^. 


VOL.  4  (1950)  PURINES   AND    PYRIMIDINES   IN    RIBOSIDIC   LINKAGE  235 

provided  that  either  phosphate  or  arsenate  is  present.  The  ester  formed  was  isolated 
and  identified  as  desoxyribose-5-phosphate.  The  authors  have  evidence  for  the  presence 
of  an  enzyme  which  catalyses  the  conversion  of  a  primarily  formed  1-ester  into  the 
5-ester.  The  same  two  authors  have  also  made  recent  contributions  towards  our  under- 
standing of  the  enzymatic  splitting  of  pyrimidine  desoxynucleosides,  especially  thymi- 
dine^". They  have  isolated  an  enzyme  from  bone  marrow  and  kidney  which  catalyses 
a  splitting  of  thymine  from  thymidine,  again  provided  that  either  phosphate  or  arsenate 
is  present.  The  enzyme  preparations  contain  both  purine  nucleoside  phosphorylase  and 
pyrimidine  nucleoside  phosphorylase.  Manson  and  Lampen's  observations  point  also 
towards  a  formation  of  a  desoxyribose-1-ester  from  pyrimidine  desoxynucleoside.  Thus, 
addition  of  hypoxanthine  enhances  the  liberation  of  thymine  from  thymidine  in  the 
presence  of  mixed  phosphorylases.  This  effect  indicates  at  least  that  an  enzymatic 
exchange  between  hypoxanthine  and  thymine  takes  place.  However,  since  the  incor- 
poration into  ribosidic  linkage  of  hypoxanthine  and  that  of  thymine  is  catalysed  by 
two  different  enzymes  the  assumption  of  a  formation  of  1-phospho-desoxyribose  as  a 
common  substrate  for  both  enzymes  can  explain  the  above  mentioned  effect. 

THE   BIOLOGICAL   PATHWAY   OF   PURINE    AND   PYRIMIDINE    INCORPORATION 

INTO    NUCLEIC   ACIDS 

The  pathway  of  purine  and  pyrimidine  incorporation  into  nucleic  acids  is  a  problem 
of  major  biological  importance.  The  isotope  technique  has  made  it  possible  to  make  an 
account  of  the  most  significant  steps  of  such  a  synthesis  in  the  intact  organism.  In  1941 
ScHOENHEiMER  and  his  colleagues  initiated  some  studies  on  purine  incorporation  in  the 
intact  adult  organism.  I  shall  not  go  into  a  discussion  of  the  interesting  feeding  experi- 
ments using  N^^  labelled  ammonia  and  C^^  or  C^*  labelled  carbon  dioxide  which  have 
shed  so  much  light  on  the  synthesis  of  the  purine  bases.  This  discussion  is  dealing  with 
results  of  feeding  experiments  with  labelled  purines.  These  studies  were  initiated  by 
Plentl  and  Schoenheimer^^  and  brought  into  a  very  successful  and  fruitful  develop- 
ment by  the  studies  performed  at  the  Sloan-Kettering  Institute  by  Brown  and 
coworkers.  It  will  be  recalled  that  Plentl  and  Schoenheimer  found  that  adult  rats 
fed  N^^  labelled  guanine  excreted  the  entire  amount  of  this  substance  as  uric  acid  and 
allantoin  and  correspondingly  the  guanine  of  the  nucleic  acids  was  found  to  be  devoid 
of  any  excess  N^^.  This  finding  was  substantiated  6  to  7  years  later  by  Brown  and  co- 
workers. Brown  and  his  colleagues  synthesized  N^-^  adenine  and  guanine  according  to 
recent  methods  developed  by  Todd  and  Lythgoe.  The  most  remarkable  result  of  their 
studies,  was  the  fact  that  N^^  labelled  adenine  was  readily  incorporated  into  the  ribo- 
nucleic acids  both  as  adenine  and  guanine^^.  If  a  moderate  amount  of  N^^  adenine  was 
administered  to  adult  rats  about  50%  was  incorporated  as  nucleic  acid  adenine  and 
guanine  and  the  other  50%  appeared  as  allantoin.  Bendich  and  Brown^^  have  recently 
made  the  interesting  observation  that  2-6  diamino  purine  labelled  with  N^^  appears 
in  large  amounts  in  the  nucleic  acid  guanine  but  not  in  the  adenine.  H5rpoxanthine 
seems  to  be  converted  exclusively  into  uric  acid  and  allantoin^*. 

How  are  the  present  results  of  the  studies  on  liver  nucleoside  phosphorylase  to  be 
interpreted  in  the  light  of  recent  findings  gained  from  isotope  experiments  performed  on 
intact  organisms?  It  will  be  recalled  that  the  liver  nucleoside  phosphorylase  catalyses 
the  incorporation  of  only  two  purine  bases,  hypoxanthine  and  guanine  —  exactly  the 
References  p.  257. 


236  H.  M.  KALCKAR  VOL.  4  (1950) 

two  purines  bases  which  according  to  the  studies  on  the  intact  organism  are  not  incor- 
porated into  the  nucleic  acids.  We  are  forced  to  conclude  therefore  that  the  type  of 
incorporation  of  purines  which  can  be  demonstrated  in  incubates  with  liver  enzymes  does 
not  represent  the  final  way  by  which  the  intact  organism  incorporates  purines  for  the 
maintenance  of  its  protoplasmic  nucleic  acids.  It  is  even  justified  to  question  whether 
the  nucleoside  phosphorylase  has  anything  whatever  to  do  with  the  incorporation  of 
purines  into  nucleic  acids.  The  nucleoside  phosphorylase  might  for  instance  play  a  role 
in  processes  other  than  the  incorporation  of  purines  into  nucleic  acids.  This  brings  us  to 
recall  the  situation  with  respect  to  the  amino  acid  oxidases  around  1936.  At  that  time 
Krebs  described  a  water  soluble  oxidase  which  catalysed  the  oxidation  of  the  d-amino 
acids  and  which  Warburg  and  Christian  purified  and  identified  as  a  flavine  enzyme. 
Six  to  seven  years  later  Green,  Ratner,  and  Nocito  isolated  the  oxidase  which  catal- 
ysed the  oxidation  of  1-amino  acids  and  this  also  proved  to  be  flavoprotein.  When  we 
talk  about  protein  metabolism  especially  combustion  of  proteins  in  the  animal  organism 
we  realize  that  the  oxidation  of  the  amino  acids  from  proteins  must  be  catalysed  by 
the  1-amino  acid  oxidase  and  not  by  the  d-amino  acid  oxidase.  The  physiological  function 
of  the  latter  enzyme  still  remains  obscure.  We  may  apply  the  same  point  of  view  to- 
wards the  nucleoside  phosphorylase.  It  appears  unlikely  that  the  enzyme  should  simply 
serve  in  the  breakdown  of  purine  compounds  since,  as  mentioned  earlier,  in  an  enzymatic 
mixture  of  free  purine,  phosphate,  nucleoside  and  phospho-riboside  the  equilibrium  is 
definitely  favourable  towards  nucleoside  formation.  The  possibility  should  not  be  over- 
looked that  formation  of  inosine  from  ribose-1-phosphate  catalysed  by  liver  nucleoside 
phosphorylase  might  represent  a  primary  step  in  the  synthesis  of  purine  ribosides 
prior  to  the  incorporation  of  adenine.  Adenine  might  then  be  exchanged  directly  with 
the  hypoxanthine  present  in  inosine  by  an  enzyme  which  does  not  occur  in  our  usual 
enzyme  preparations.  The  catalytical  action  of  inosine  on  the  deamination  of  adenine 
by  a  bacterial  enzyme^^  might  be  explained  on  this  assumption;  in  vitro  studies  with 
labelled  carbon  or  nitrogen  in  the  adenine  ring  should  be  able  to  clarify  this  problem. 
As  regard  to  the  incorporation  of  pyrimidine  into  nucleic  acid  little  is  known.  The  recent 
team  work  between  Bergstrom  and  Hammarsten  and  his  group^^  has  shed  interesting 
light  on  this  problem.  It  was  found  that  N^^  labelled  orotic  acid  can  be  used  as  a  pre- 
cursor of  the  ribonucleic  acid  pyrimidines  of  the  adult  rat.  The  question  regarding  in- 
corporation of  purines  and  pyrimidines  into  desoxyribonucleic  acids  brings  up  important 
new  problems  regarding  the  rejuvenation  of  nuclear  components.  It  is  known  from  the 
studies  by  Brues,  Tracy,  and  Cohn  and  as  well  as  by  Hammarsten  and  Hevesy 
that  the  phosphorus  in  the  desoxyribonucleic  acids  is  renewed  at  a  much  slower  rate 
than  that  incorporated  in  ribonucleic  acids.  In  regenerating  or  growing  tissues  the 
renewal  of  desoxynucleic  acid  phosphorus  is  increased  markedly.  Likewise  Brown  and 
coworkers^'  found  that  the  rate  of  incorporation  of  N^-^  adenine  into  desoxyribonucleic 
acid  in  the  adult  rat  is  negligible  as  compared  with  the  corresponding  processes  taking 
place  in  the  ribonucleic  acid.  These  observations  indicating  a  very  slow  turnover  of 
desoxyribonucleic  acid  components  in  the  adult  organism  coupled  with  the  knowledge 
of  the  existence  of  a  highly  active  desoxynucleoside  phosphorylase  poses  several  new 
questions.  For  example  the  enzymatic  system  catalysing  degradation  and  synthesis  of 
desoxynucleosides  in  liver  should  be  taken  into  account  in  considering  the  regulatory 
mechanisms  which  control  transitions  between  resting  and  growing  states. 

As  concluding  remarks  I  should  like  to  add  that  the  two  types  of  approaches,  the 
References  p.  237 . 


VOL.  4  (1950)  PURINES    AND    PYRIMIDINES   IN    RIBOSIDIC   LINKAGE  237 

study  of  enzymatic  step  reactions  in  vitro  and  the  study  with  isotope  labelled  precursors  in 
vivo  are  equally  indispensable  and  exert  a  mutual  and  valuable  influence  on  each  other. 
An  example  is  the  importance  of  the  Embden-Meyerhof  glycolysis  scheme  for  the 
interpretation  of  the  distribution  of  labelled  carbon  in  glycogen  from  rats  fed  with 
labelled  carbon  dioxide.  The  ingenious  analysis  by  Wood  and  coworkers  in  this  field 
may  well  serve  as  an  encouragement  for  investigators  working  in  allied  fields. 

SU?kLMARY 

The  mechanism  of  incorporation  of  purines  and  pyrimidines  into  ribosidic  Unkage  has  been 
discussed  from  various  points  of  view.  Results  gained  from  enzymatic  studies  are  not  in  direct 
agreement  with  observations  made  in  intact  organism  using  isotopes.  Various  ways  of  interpretations 
are  discussed. 

r£su!^i£ 

Le  mecanisme  de  I'incorporation  de  purines  et  de  pyrimidines  dans  la  liaison  ribosidique  a 
ete  discute  de  differents  points  de  vue.  Les  resultats  obtenus  par  des  etudes  enzymatiques  ne  con- 
cordent  pas  entierement  avec  les  observations  faites  dans  I'organisme  intact  au  moyen  d'isotopes. 
Difierentes  possibilites  d'interpretation  ont  ete  envisagees. 

ZUSAMMENFASSUNG 

Der  Mechanismus  der  Einverleibung  von  Purinen  und  Pyrimidinen  in  die  Ribosid-Bindung 
ist  von  verschiedenen  Gesichtspunkten  aus  erortert  worden.  Die  aus  enzymatischen  Untersuchungen 
gewonnenen  Ergebnisse  stimmen  nicht  voUig  iiberein  mit  Beobachtungen  welche  im  unversehrten 
Organismus  mittels  Isotopen  gemacht  wurden.  Verschiedene  Erklarungsmoglichkeiten  werden 
besprochen. 

REFERENCES 

1  W.  Klein,  Z.  physiol.  Chem.,  231  (1935)  125. 

2  C.  F.  CoRi,  Federation  Proc,  4  (1945)  226. 

^  O.  H.  LowRY  AND  J.  A.  Lopez,  /.  Biol.  Chem.,  162  (1946)  421. 

*  H.  M.  Kalckar,  /.  Biol.  Chem.,  167  (1947)  477. 
^  H.  M.  Kalckar,  /.  Biol.  Chem.,  167  (1947)  429. 

^  M.  Friedkin  and  H.  M.  Kalckar,  and  E.  Hoff-Jorgensen,  /.  Biol.  Chem.,  178  (1949)  527. 
■^  M.  Friedkin  and  H.  M.  Kalckar,  unpubhshed  experiments. 

*  E.  HOFF-J0RGENSEN,  /.  Biol.  Chem.,  178  (1949)  525. 

*  L.  A.  Manson  and  J.  O.  Lampen,  Abstracts  of  Sept.  1948,  Meeting  of  Am.  Chem.  Sac. 

^°  L.  A.  Manson  and  J.  O.  Lampen,  Abstract  of  April  1949,  Meeting  of  Fed.  Am.  Soc.  Exptl  Biol. 

^^  A.  A.  Plentl  and  R.  Schoenheimer,  /.  Biol.  Chem.,  153  (1944)  203. 

^2  G.  B.  Brown,  P.  M.  Roll,  A.  A.  Plentl,  and  L.  F.  Cavalieri,  /.  Biol.  Chem.,  172  (1948)  469. 

^^  A.  Bendich  and  G.  B.  Brown,  /.  Biol.  Chem.,  176  (1948)  1471. 

"  H.  Getler,  p.  Roll,  J.  F.  Tinker,  and  G.  B.  Brown,  /.  Biol.  Chem.,  178  (1949)  259. 

^°  M.  Stephenson  and  A.  R.  Trim,  Biochem.  J.,  32  (1938)  1740. 

^®  S.   Bergstrom,   H.  Arvidsen,   E.   Hammarsten,   N.  A.  Eliasson,   P.   Reichardt,   and  H.   v. 

Ubisch,  /.  Biol.  Chem.,  i-jj  (1949)  495. 
^^  G.  B.  Brown,  Mary  L.  Petermann,  and  S.  Sidney  Furst,  J.  Biol.  Chem.,  174  (1948)  1043. 

Received  April  i6th,  1949 


238  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


L'fiNERGIE  DE  FORMATION  DES 

complexes  dissociables  enzyme-substrat  et 
antig£:ne-anticorps 

par 

RENfi  WURMSER  et  SABINE  FILITTI-WURMSER 

Institut  de  Biologie  physicochimique,  Paris  (France) 


I.  LES   COMPLEXES   ENZYME-SUBSTRAT 

La  connaissance  des  energies  et  entropies  de  formation  des  complexes  proteiques 
dissociables  permet trait  de  preciser  la  nature  des  liaisons  qui  y  sont  impliquees,  et  de 
comprendre  I'effet  specifique  qui  en  resulte. 

Ainsi  Taction  catalytique  des  enzj^mes  est  generalement  expliquee  par  une  attrac- 
tion entre  I'enzyme  et  le  substrat.  Plusieurs  mecanismes  de  detail  ont  ete  proposes^. 
Par  exemple,  I'attraction  de  deux  substrats  juxtaposes  sur  I'enzyme  les  presse  I'un 
contre  I'autre  et  favorise  leur  union.  Dans  une  representation  plus  elaboree  de  Tactiva- 
tion,  Stearn^  considere  I'hydrolyse  d'une  liaison  peptide.  La  formation  du  groupe 
d'atomes  active  CONH  serait  facilitee  par  I'approche  d'un  dipole  de  I'enzyme  qui 
attire  I'oxhydrile  vers  le  groupe  C-N.  L'attraction  du  substrat  par  I'enzyme  servirait 
a  vaincre  les  forces  de  repulsion  qui  s'opposent  a  ce  rapprochement. 

D'autre  part,  on  pent  admettre  que  I'abaissement  de  I'energie  d'activation  ne 
depend  pas  directement  de  la  combinaison  de  I'enzyme  avec  le  substrat,  pris  comme  un 
tout  et  dans  son  etat  normal.  L'energie  potentielle  de  I'etat  active  serait  abaissee  par 
resonance  d'un  groupe  reactif  du  substrat  dans  I'etat  active  avec  un  groupe  correspon- 
dant  de  I'enzyme.  Le  mecanisme  suggere  par  DelbrOck^  pour  expliquer  I'auto- 
reproduction  des  proteines  s'apparente  a  cette  maniere  de  voir.  La  connaissance  exacte 
des  energies  de  liaison  serait  utile  pour  entreprendre  une  discussion  serree  de  ces  deux 
conceptions. 

Or,  on  ne  possede  pas  de  donnees  certaines  sur  les  energies  d'association  des  enzymes 
avec  leur  substrats.  Celles  dont  on  dispose  jusqu'ici  ont  ete  obtenues  en  appliquant  la  loi 
de  Van  't  Hoff  a  la  variation  de  la  constante  de  Michaelis  en  fonction  de  la  temperature. 
Comme  I'ont  mis  en  evidence  Briggs  et  Haldane^,  cette  constante  Ki^j  n'est  pas  neces- 
sairement  egale  a  I'inverse  de  la  constante  d'affinite  K  de  I'enzyme  pour  son  substrat. 
La  variation  de  K^  avec  la  temperature  ne  pent  done  servir  sans  reserves  a  calculer  la 
chaleur  de  formation  a  pression  constante  ou  enthalpie  z)  H  du  compose.  La  condition 
est  que  la  vitesse  kj  de  la  dissociation  du  compose  ES  en  E  et  S,  soit  grande  par  rapport 
a  la  vitesse  kg  de  decomposition  du  complexe  en  produit  final  de  la  reaction,  ou  que  la 
decomposition  du  complexe  ait  la  meme  energie  d'activation  que  sa  dissociation. 

La  constante  d'affinite  K  de  I'enz^^me  pour  son  substrat  a  bien  ete  determinee  directe- 
ment, dans  une  circonstance,  par  Chance^.  Elle  est  100  fois  plus  grande  que  i/K^.  II 
Bibliographic  p.  243. 


VOL.  4  (1950)  COMPLEXES   ENZYME-SUBSTRAT,  ANTIGENE-ANTICORPS  239" 

s'agit  de  la  peroxydase  et  du  peroxyde  d'hydrogene  dont  I'union  donne  un  compose 
caracterise  par  son  spectre  d'absorption.  Malheureusement  la  variation  de  la  constante 
avec  la  temperature  n'a  pas  ete  determinee,  si  bien  que  meme  dans  ce  cas  on  n'a 
pas  encore  I'enthalpie.  La  technique  employee  par  Chance  est  d'ailleurs  restreinte 
aux  associations  enzyme-substrat  qui  ont  un  spectre  d'absorption  caracteristique. 

Une  autre  technique,  applicable  specialement  aux  associations  des  enzymes  avec  de 
grosses  molecules,  pent  etre  fondee  sur  une  autre  propriete.  On  salt  mesurer,  en  principe,. 
les  poids  moleculaires  a  partir  de  I'intensite  de  la  lumiere  diffusee  et  tirer  des  indications 
sur  les  dimensions  des  molecules  a  partir  de  la  distribution  angulaire  de  cette  intensitc. 
Cette  technique,  actuellement  mise  en  oeuvre  dans  notre  laboratoire,  pourra  etre  appli- 
quee  aux  complexes  formes  entre  les  enz5mies  proteolytiques  et  leur  substrat. 


II.  L'UNION    DE    l'aGGLUTININE    AUX   HEMATIES 

I.  Equilihre  de  V agglutination 

Pour  un  autre  type  de  complexes  proteiques  dissociables,  celui  forme  par  un  anti- 
gene  avec  un  anticorps,  une  mesure  directe  de  la  chaleur  degagee  a  ete  effectuee  par 
Boyd  et  ses  collaborateurs^.  Ces  auteurs  ont  trouve  que  la  combinaison  de  I'hemocyanine 
avec  son  anticorps  chez  le  cheval,  degage  40000  calories  par  molecule  d'antihemocyanine. 

On  a  depuis  Arrhenius  cherche  a  obtenir  la  chaleur  de  reaction  a  partir  de  I'effet 
de  la  temperature  sur  I'equilibre  qui  s'etablit  entre  antigenes  et  anticorps.  La  difhculte 
est  d'expliciter  la  relation  qui  unit  les  constantes  d'equilibre  a  la  composition  du  com- 
plexe  forme.  En  particulier,  les  resultats  dependent  de  I'idee  que  Ton  se  fait  de  la  struc- 
ture de  ce  complexe,  de  la  valence  des  constituants,  et  des  interactions  entre  les  groupes 
reactifs  d'une  meme  molecule. 

Nous  avons  pense  que  le  procede  statistique  le  plus  simple  pouvait  etre  applique 
k  I'isohemagglutination.  Celle-ci  etant  une  reaction  de  surface,  on  devait  etre  a  meme  de 
calculer,  avec  un  minimum  d'hypotheses,  la  relation  existant  entre  la  grandeur  observee 
et  une  constante  d'equilibre.  Ce  phenomene  presentait  en  outre  I'avantage  que  sa 
reversibilite  avait  ete  tres  surement  prouvee. 

Soit  T  le  taux  d'a  ;glutination,  c'est-a-dire,  en  appelant  Nj  le  nombre  d'hematies 
libres,  le  rapport  entre  le  nombre  des  hematies  agglutinees  (N^ — Nj)  et  le  nombre  total 
d'hematies  N^.  Filitti-Wurmser  et  Jacquot-Armand^  ont  etabli  que,  par  numeration 
dans  un  hematimetre,  I'erreur  standard  sur  le  taux  d'agglutination  varie  entre  0.3% 
pour  T  =  0.99  et  7%  pour  r  =  0.45.  La  technique  est  done  utihsable  pour  une  etude 
quantitative.  Elle  a  servi  a  demontrer  la  reversibilite  de  I'agglutination  par  les  faits 
suivants. 

a)  Dissociation  de  I'agglutinat.  On  obtient  le  meme  etat  d'equilibre  quand  on  agglu- 
tine  des  hematies  ou  quand  on  dissocie  un  agglutinat. 

Pour  le  prouver  on  melange  dans  une  premiere  operation  un  serum  avec  un  nombre 
donne  d'hematies  et  une  solution  tampon  de  maniere  a  avoir  un  volume  V.  On  obtient 
un  certain  taux  d'agglutination.  Dans  une  deuxieme  operation  on  melange  le  meme 
serum  avec  le  meme  nombre  d'hematies  et  une  quantite  de  solution  tampon  telle  que 
le  volume  v  est  plus  petit  que  V.  II  se  forme  un  agglutinat  plus  abondant.  Lorsque  celui-ci 
a  atteint  son  equilibre,  on  dilue  jusqu'au  volume  V.  Le  nouveau  taux  d'agglutination 
qui  s'etablit  est  egal  a  celui  obtenu  dans  la  premiere  operation. 
Bibliographie  p.  243. 


240 


R.  WURMSER,  S.  FILITTI-WURMSER 


VOL.  4  (1950) 


h)  Deplacement  de  I'equilibre  par  la  temperature.  Lorsque,  a  un  serum  donne,  on  ajoute 
des  quantites  croissantes  d'hematies,  on  obtient,  suivant  la  temperature  a  laquelle  on 

opere,  les  resultats  representes  par  la 
Fig.  I.  Sur  ce  diagramme  on  a  porte  en 
abscisses  log  N^  et  en  ordonnees  log 
(Nj, — -Ni).  On  voit  que  le  nombre  maxi- 
mum d'hematies  agglutinees  augmente 
quand  la  temperature  s'abaisse. 

En  outre,  le  taux  d'agglutination 
est  d'abord  voisin  de  I'unite  (portion 
lineaire  des  courbes)  a  toutes  les  tem- 
peratures, mais  au-dela  d'une  certaine 
valeur  de  N^,  le  taux  d'agglutination  est 
nettement  plus  eleve  quand  la  tempera- 
ture est  plus  basse.  On  pent  mettre  en 
evidence  le  deplacement  reversible  de 
I'equilibre  par  le  fait  qu'un  meme  taux 
d'agglutination  est  atteint  soit  directe- 


«■ 

•  • 

•  •   • 

• 

5 

i 
0 

X 

«- 

■< 

0 
0       ^ 

° 

° 

0 

45 

.  ' 

< 

s 

• 

0 

0 

4.5 


53 


Log  No 


g.  I.  log  (hematies  agglutinees)  en  fonction  de  log 
(hematics  totales)  •  a  15°  C,  x  a  25°  C,  o  a  37°  C 


ment  a  37°,  soit  apres  une  mise  en  equilibre  a  5°  suivie  d'une  dissociation  partielle  de 
I'agglutinat  a  ^y°. 

II  fallait,  pour  I'application  de  la  statistique  que  nous  voulions  faire,  s'assurer  que 
I'effet  de  la  temperature  n'est  pas  du  a  I'existence  de  groupes  actifs  differents.  Plusieurs 
preuves  en  ont  ete  donnees:  en  particulier,  de  I'agglutinine  extraite  par  elution  d'un 
agglutinat  forme  a  37°  presente  le  meme  effet  de  temperature  que  le  serum  lui-meme. 

2.  Determination  de  I'energie  de  formation  du  complexe  agglutininc  — -  groupe  agglutinogene 

Nous  avons  done  admis'  que  I'agglutination  resulte  de  la  fixation  de  molecules 
d'agglutinine  A  sur  des  groupes  G  tous  pareils  situes  a  la  surface  des  hematies,  et  assez 
eloignes  les  uns  des  autres  pour  etre  sans  interactions.  Les  hematies  qui  s'agglutinent 
sont  celles  qui  ont  fixe  en  moyenne  un  nombre  minimum  /  de  molecules  d'agglutinine. 
II  suffit  alors  pour  obtenir  le  taux  d'agglutination  en  fonction  de  la  concentration  d'ag- 
glutinine (A)  d'appliquer  un  raisonnement  classique. 

S'il  existe  a  la  surface  de  chaque  hematic  m  groupes  capables  de  reagir  reversible- 
ment  avec  I'agglutinine,  il  y  aura  une  distribution  des  hematies  HA  portant  un  nombre 
n  de  molecules  d'agglutinine,  n  variant  de  0  (hematies  nues)  a  ;w  (hematies  saturees). 

Soit  K  la  constante  "intrinseque"  correspondant  a  I'equilibre: 

G  +  A  ^  G  A 

entre  I'agglutinine  et  les  groupes  agglutinogenes  supposes  reagir  comme  s'ils  etaient  des 
molecules  separees;  K(A)/i  +  K(A)  est  la  probabilite  pour  qu'un  groupe  individuel  fixe 
une  molecule  d'agglutinine.  En  portant  cette  valeur  dans  la  relation  de  Bernoulli,  on 
trouve  que  le  taux  d'agglutination  est : 

m! 


r  =  [I  +  K(A)]-    2 


'i  n!(m  —  n) ! 


[K(A)]" 


La  variation  du  taux  d'agglutination  en  fonction  de  la  concentration  d'agglutinine 
a  une  temperature  donnee,  pent  etre  obtenue  experimentalement.  On  sait  titrer  I'agglu- 
Bibliographie  p.  243. 


VOL.  4  (1950) 


COMPLEXES   ENZYME-SUBSTRAT,  ANTIGENE-ANTICORPS 


241 


/ 

^ 

/' 

i 

/ 

/D 

i 

/ 

I 

0 

tinine  en  valeurs  relatives  a  (A)  d'apres  le  nombre  maximum  d'hematies  agglutinees  a 
4°  C.  On  obtient  la  courbe  r  =/[a(A)]  de  la  maniere  suivante:  les  valeurs  de  t  sont 
determinees  directement  dans  une  premiere  agglu- 
tination en  comptant  les  hematies  restees  libres 
dans  les  melanges  constitues  par  une  quantite  fixe 
de  serum  et  des  quantites  croissantes  d'hematies  * 
dans  un  volume  constant.  Les  valeurs  correspon- 
dantes  de  a  (A)  proviennent  des  titrages  effectues  g 
par  une  serie  d'agglutinations  pratiquees  cette  fois 
sur  le  liquide  obtenu  en  centrifugeant  chacun  des 
melanges  ayant  servi  a  la  mesure  de  t,  apres  que 
I'equilibre  d'agglutination  a  ete  atteint. 

Les  courbes  de  la  Fig.  2  representent  les  resul-    < 
tats  obtenus  pour  des  agglutinations  d'un  meme 
serum  du  groupe  A,  a  25°  C  et  a  37°  C. 

On  determine  a  partir  de  ces  courbes  le  rapport 
des  valeurs  de  K  a  25°  C  et  a  37°  C,  en  faisant 

comme  seule  hypothese  que  le  nombre  I  ne  varie  pas  ou  varie  tres  pen  avec  la 
temperature.  Ce  rapport  K25/K37  est  egal  au  rapport  (A)37/(A)25  des  concentrations 
relatives  d'agglutinines  pour  un  meme  taux  d'agglutination.  La  valeur  trouvee  est 
3.5  i:  0.2,  ce  qui  correspond  a  une  enthalpie  J  H  de  — 19000  calories. 

Une  determination  de  J  H  qui  n'implique  pas  d'hypothese  sur  le  mecanisme 
de  I'agglutination  proprement  dite,  consiste  a  porter  en  abscisses  des  grandeurs 
proportionnelles  a  i/K(A)  et  en  ordonnees  des  grandeurs  proportionnelles  a  i/Af,  en 
appelant  Af  I'agglutinine  fixee  divisee  par  la  totalite  des  hematies.  Cette  quantite  est 

mesuree  par  difference  entre  I'agglutinine  initiale 
et  I'agglutinine  restante.  On  doit  obtenir  une 
droite,  si  les  groupes  sont  sans  interaction : 


1  2  3  (A) 

Fig.  2.  Taux  d'agglutination  en  fonc- 
tion  de  la  concentration  d'agglutinine 
non     fixee     (en     valeurs 

X  a  25°  C,  o  a  37°  C 


relatives) 


'At 

*k 

f 

J 

■/ 

1 

I  0 
1  ° 

Wo 
/• 

/ 

/ 

I 

a; 


I 


[mK(A) 


(I) 


7 


Le  rapport  des  pentes  a  2  temperatures  37°  C 
et  25°  C  est  egal  au  rapport:  K25/K37. 

La  Fig.  3  montre  les  points  experimentaux  et 
les  droites  calculees^  d'apres  la  methode  des  moin- 
dres  Carres,  pour  un  meme  serum  (2519)  a  deux 
temperatures  37°  C  et  25°  C,  et  pour  un  autre 
serum  (1028)  a  37°  C.  II  s'agit  de  2  serums  de 
titre  eleve  (NjQax4°est  egal  a  1254000  par /^l  pour 
le  serum  2519  et  a  1925000  pour  le  serum  1028). 

Les  pentes  correspondantes  pour  le  serum 
2519  sont:  a  37°  C,  1.851  avec  une  erreur  standard 
a  =  0.147  6t  a  25°  C,  0.573  3-vec  une  erreur  stan- 
dard a  =  0.017.  Le  rapport  K25/K37  est  done  3.23  et  I'enthalpie  A  H  — 18000  calories. 


V(^ 


Fig.  3.  Inverse  de  la  quantite  d'agglu- 
tinine  fix^e  (en  valeurs  relatives)  en 
fonction  de  I'inverse  de  la  concentra- 
tion d'agglutinine  non  fixee  (en  valeurs 
relatives).  Serum  No.  2519,  x  a  25°  C, 
•  a  37°  C;  Serum  No.  1028,  o  a  37°  C 


La  concordance  avec  le  resultat  precedent  — 19000  calories  est  satisfaisante. 


References  p.  243. 
16 


242  R.  WURMSER,  S.  FILITTI-WURMSER  VOL.  4  (1950) 

III.  DISCUSSION 

En  ce  qui  concerne  la  nature  des  liaisons,  on  notera  que  20  000  calories  correspondent 
a  la  formation  d'environ  4  liaisons  hydrogene  ou  a  une  vingtaine  d'attractions  de  Van 
der  Waals  (Pauling^).  Ces  valeurs  sont  raisonnables  si  Ton  admet,  par  exemple,  qu'un 
groupe  agglutinogene  renferme  un  polysaccharide. 

On  pent  avoir  une  idee  de  la  grandeur  de  la  constante  d'cquilibre  K.  Cette  constante 
"intrinseque"  caracterise  I'equilibre  entre  I'agglutinine  et  les  groupes  agglutinogenes 
supposes  independants.  Elle  est  egale  a  une  constante  d'equilibre  classique  entre  I'agglu- 
tinine et  les  hematics  portant  un  nombre de   groupes   combines,    c'est-a-dire 

les  hematics  demi-saturees,  parce  que  pour  ces  hematics  I'effet  statistique  sur  I'energie 
libre  est  elimine. 

Nous  avons  utilise  les  donnees  de  Kabat^"  sur  la  concentration  de  I'isoagglutinine 
dans  les  serums  pour  calculer  la  valeur  de  m  a  partir  du  rapport  (A)o/(A)  des  concen- 
trations d'agglutinine  avant  et  apres  agglutination  en  presence  d'un  petit  nombre 
d'hematies  (environ  4-10^  par  /d).  On  trouve  ainsi  que  m  est  de  I'ordre  de  10^,  qui 
correspond  d'ailleurs  sensiblement  au  maximum  de  place  disponible  pour  I'agglutinine 
a  la  surface  d'une  hematic.  Cette  valeur  de  m  portee  dans  la  relation  (i)  donne  alors 
pour  K  la  valeur  2  •  10^  a  4°  C,  soit  i  •  10''  a  37°  C. 

A  cette  derniere  temperature  la  variation  d'energie  libre  par  molecule-gramme 
d'agglutinine  est  J  F  =  — -10000  calories  et  la  variation  d'entropie  zl  S  =  — 30,  environ 
8  unites  par  liaison.  Toutes  ces  valeurs  apparaisscnt  vraisemblables. 

L'energie  libre  ainsi  trouvee  est  a  comparer  avec  la  valeur  calculee  selon  les  procedes 
ordinaires  de  la  theorie  statistique,  par  Morales,  Botts  et  Hill^^  pour  l'energie  libre 
de  combinaison  d'une  molecule  d'antihemocyanine  avec  une  molecule  d'hemocyanine. 
Ces  auteurs  partent  de  la  donnee  calorimetrique  de  Boyd  et  collaborateurs.  lis  tiennent 
seulement  compte,  pour  obtenir  la  fonction  de  partition,  des  effets  de  translation  et  de 
rotation  et  supposent  que  les  deux  molecules  ont  meme  masse  et  meme  rayon,  et  que 
le  moment  d'inertie  du  complexe  est  celui  d'une  sphere  equivalente.  Leur  resultat 
-II 000  calories  par  groupe  fixe  est  tout  a  fait  voisin  de  celui  que  nous  obtenons  pour  la 
combinaison  de  I'agglutinine  avec  un  groups  agglutinogene  d'une  hematic.  Mais  dans 
le  cas  de  I'hemocyanine,  l'energie  totale  etant  de  40000  calories,  8  liaisons  de  5000 
calories,  au  lieu  de  4,  sont  impliquees  dans  la  formation  du  complexe;  l'energie  libre 
par  liaison  est  done  moitie  de  celle  trouvee  pour  I'agglutinine. 

La  coherence  des  resultats  obtenus  dans  le  cas  de  I'isohemagglutination  presente 
un  autre  interet  que  celui  de  donner  une  base  aux  hypotheses  possibles  sur  la  nature  des 
liaisons  en  jeu.  II  sera  utile  d'introduire  la  mesure  de  ces  grandeurs  energetiques  dans 
la  comparaison  de  serums  d'origines  diverses.  Apres  un  examen  plus  approfondi  des 
facteurs  accessoires  (force  ionique,  presence  d'inhibiteurs),  susceptibles  de  les  faire  varier 
pour  une  meme  agglutinine,  il  n'est  pas  exclu  qu'il  se  degage,  d'une  telle  comparaison, 
des  caracteres  de  groupes  interessants,  meme  a  un  point  de  vue  strictement  biologique. 

r£sum£ 

On  ne  connait  pas  de  donnees  rigoureuses  sur  l'energie  de  liaison  des  enzymes  a  leur  substrats. 
En  ce  qui  concerne  I'union  des  antigenes  aux  anticorps,  il  n'existait  qu'une  determination  calorime- 
trique de  I'union  de  I'hemocyanine  a  I'antihdmocyanine.  L'^tude  de  I'isohemagglutination  a  permis 
de  calculer  l'energie  de  la  liaison  agglutinine-groupe  agglutinogene  et  d'6valuer  la  constante  d'cqui- 
libre correspondante,  soit  i  •  10'  a  37°  C. 

Bibliographie  p.  24.3.     ' 


VOL.  4  (1950)  COMPLEXES   ENZYME-SUBSTRAT,  ANTIGENE-ANTICORPS  243 

SUMMARY 

No  exact  data  are  known  about  the  energy  of  the  bonds  between  enzymes  and  their  substrates. 

As  to  the  attachment  of  antigens  to  antibodies  only  a  calorimetric  determination  of  the  bond 
haemocyanin-antihaemocyanin  was  known.  The  study  of  isohaemagglutination  has  permitted  the 
calculation  of  the  bond-energy  of  the  complex  agglutinin-agglutinogenic  group  and  the  estimation 
of  the  corresponding  equihbrium  constant,  being  i  •  10'  at  37"^  C. 

ZUSAMMENFASSUNG 

Man  kennt  keine  genauen  Angaben  iiber  die  Bindungsenergie  der  Enzyme  an  ihre  Substrate. 

Was  den  Komplex  Antigen-Antikorper  anbelangt,  so  ist  nur  eins  kalorimatrische  B^stimmung 
der  Bindung  von  Haemocyanin  an  Antihaemocyanin  bekannt. 

Die  Untersuchung  der  Isohaemagglutination  erlaubt  die  Energie  der  Verbindung  Agglutinin- 
agglutinogene  Gruppe  zu  berechnen  und  die  entsprechende  Gleichgewichtskonstante,  i-io'  bei 
37°  C,  anzugeben. 

BIBLIOGRAPHIE 

1  J.  B.  S.  Haldane,  Enzymes,  Longmans,  Green,  and  Co,  London  (1930)  182. 

2  A.  E.  Stearn,  Ergeb.  Enzyinforsch.,  VII  (1938)  i. 

^  M.  Delbruck,  Cold  Spring  Harbor  Symposia  Quant.  Biol.,  IX  (1941)  122. 

*  B.  Chance,  /.  Biol.  Chem.,  151  (1943)  553- 

^  W.  C.  Boyd,  J.  B.  Conn,  D.  C.  Gregg  et  G.  B.  Kistiakowsky,  /.  Biol.  Chem.,  139  (1941)  787. 

®  S.  Filitti-Wurmser  et  Y.  Jacquot-Armand,  Arch.  sci.  physioL,  i  (1947)  151. 

'  S.  Filitti-Wurmser,  Y.  Jacquot-Armand  et  R.  Wurmser,  Compt.  rend.  acad.  sci.,  226  (1948)  844. 

^  S.  Filitti-Wurmser  et  Y.  Jacquot-Armand,  travail  non  encore  public. 

"  L.  Pauling,  The  Specificity  of  Serological  Reactions ;  Landsteiner,  Harvard  University  Press,  1945. 
1°  E.  A.  Kabat  et  a.  E.  Bezer,  /.  Exptl.  Med.,  82  (1945)  207. 
^^  M.  F.  Morales,  J.  Botts  et  T.  L.  Hill,  /.  Am.  Chem.  Soc,  70  (1948)  2339. 

Re9u  le  21  mars  1949 


244  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


n£cessit£  d'un  coenzyme  pour  le  fonctionnement 

DE  LA  DfiSULFINICASE 

par 

BERNADETTE  BERGERET,  FERNANDE  CHATAGNER 
ET  CLAUDE  FROMAGEOT 

Laboratoire  de  Chimie  biologique  de  la  Faculte  des  Sciences,  Paris  [France) 


L' action  de  divers  extraits  de  foie  sur  I'acide  cysteinesulfinique^  est  susceptible  de 
presenter  des  irregularites  notables,  quoique  ces  extraits  aient  ete  obtenus  dans  des 
conditions  apparemment  identiques.  Recherchant  la  cause  de  ces  irregularites,  nous 
avons  constate  qu'elles  sont  dues,  au  moins  en  partie,  a  une  perte  plus  ou  moins  impor- 
tante  en  un  facteur  indispensable  au  fonctionnement  de  la  desulfinicase,  perte  qui  a  lieu 
au  cours  de  la  preparation  de  I'enzyme.  Ce  facteur  est  un  coenzyme  dont  nous  ignorons 
encore  la  nature ;  nous  savons  seulement  qu'il  est  constitue  par  une  molecule  organique 
et  que,  en  dehors  du  foie,  il  existe  egalement  dans  la  levure.  Dans  le  present  travail, 
nous  donnons  quelques  resultats  experimentaux  qui  mettent  en  evidence  I'importance 
de  ce  coenzyme  dans  la  desulfination  enzymatique  de  Tacide  cysteinesulfinique. 

PARTIE    EXPERIMENTALE 

La  solution  de  desulfinicase  est  obtenue  en  traitant  pendant  30  minutes  k  0°,  n  g  de  poudre 
acdtonique  de  foie  de  lapin-  par  n.  10  ml  d'eau  distillee.  On  centrifuge,  lave  le  culot  de  centrifugation 
avec  un  peu  d'eau  qu'on  ajoute  a  la  solution  enzymatique,  et  on  complete  le  volume  a  n.  10  ml  avec 
de  I'eau.  Le  poids  sec  d'un  tel  extrait  est  de  I'ordre  de  30  mg  par  ml. 

Le?  solutions  de  coenzyme  debarrassees  d'apoenzyme  sont  obtenues  en  traitant  au  bain-marie 
bouillant  pendant  4  minutes  la  solution  enzymatique  precedente.  On  ^limine  par  centrifugation  les 
proteines  coagulees  par  la  chaleur,  et  on  concentre  sous  vide,  de  telle  sorte  que  10  ml  d'une  telle 
preparation  corresponde  a  un  poids  donne  de  la  poudre  acetonique  extraite  initialement.  Le  poids 
sec  d'une  solution  de  coenzyme  contenant  I'extrait  de  i  g  de  poudre  acetonique  de  foie  dans  10  ml 
est  de  I'ordre  de  10  a  15  mg  par  ml. 

Les  tubes  utilises  dans  les  experiences  et  la  mesure  de  I'activite  des  systemes  enzymatiques  ont 
6te  decrits  anterieurement^.  Les  experiences  sont  faites  ici  en  solution  de  bicarbonate  de  sodium  a 
0.16%  et  sous  atmosphere  d'azote  contenant  10%  d'anhydride  carbonique;  le  pH  du  milieu  est  ainsi 
de  7.3.  La  temperature  est  de  35°,  et  la  duree  d'action  est  de  2  heures.  Les  resultats  sont  exprim6s 
en  micromoiecules  d'anhydride  sulfureux  degage. 

I.  Separation  du  coenzyme  par  dialyse  et  reactivation  de  V apoenzyme  par  addition  de 
coenzyme 

Dans  5  tubes  contenant  chacun  130  micromolecules  de  cysteinesulfinate  de  sodium 
dans  10  ml  de  solution  de  bicarbonate  de  sodium  a  0.16%,  on  introduit: 

Tube  1 :  10  ml  de  solution  d'enzyme  additionnee  de  bicarbonate  de  sodium  a  0.16%, 
■et  correspondant  a  i  g  de  poudre  acetonique ;  cette  solution  est  preparee  extemporane- 
ment.  Plus  5  ml  de  solution  de  bicarbonate. 
Bibliographie  p.  248. 


VOL.  4  (1950) 


COENZYME  POUR  LA  DESULFINICASE 


245 


Tube  II:  10  ml  de  solution  d' enzyme  analogue  a  la  precedente;  mais  cette  solution 
a  ete  prealablement  maintenue  pendant  7  heures  a  0°.  Plus  5  ml  de  solution  de  bicar- 
bonate. 

Tube  III :  10  ml  de  solution  d'enzyme  analogue  aux  precedentes;  mais  cette  solution 
a  ete  prealablement  dialysee  pendant  7  heures  a  0°  contre  une  solution  de  bicarbonate 
de  sodium  a  0.16%.  Plus  5  ml  de  solution  de  bicarbonate. 

Tube  IV:  10  ml  de  la  solution  d'enzyme  dialysee  comme  dans  le  tube  precedent, 
plus  5  ml  d'une  solution  de  coenzyme  correspondant  a  i  g  de  poudre  acetonique,  addi- 
tionnee  de  bicarbonate  de  sodium  a  0.16%. 

Tube  V:  5  ml  de  la  solution  de  coenzyme  utilisee  dans  le  tube  precedent,  plus  10  ml 
de  solution  de  bicarbonate. 

Les  resultats  obtenus  sont  donnes  dans  le  Tableau  I. 

TABLEAU  I 

INACTIVATION   ET   REACTIVATION   DE   LA   DESULFINICASE 
PAR   i:LIMINATION,  PUIS    ADDITION    DE    COENZYME 


Tube 

SO2  d^gage 

Tube 

SOj  degage 

I 

II 

III 

40 

29 

5 

IV 
V 

30 
6 

Les  chiffres  du  Tableau  I  montrent  que:  i.  la  desulfinicase  perd  son  activite  par 
dialyse;  2.  son  activite  reapparait  apres  addition  d'une  solution  de  coenzyme;  3.  la 
solution  de  coenzyme  ne  presente  elle-meme  qu'une  tres  faible  activite;  4.  le  maintien 
de  I'enzyme  pendant  7  heures  a  0°  provoque  une  certaine  inactivation. 


2.  Activation  par  le  coenzyme  d'un  extrait  non  dialyse 

Les  solutions  de  desulfinicase  obtenues  par  la  methode  utilisee  ici  donnent  normale- 
ment,  sans  addition  supplementaire  de  coenzyme,  un  degagement  d'anhydride  sulfureux 
de  50  a  55  /<mol  apres  2  heures,  pour  10  ml  de  solution  enzymatique  agissant  sur  130  ^^mol 
de  cysteinesulfinate  de  sodium  dans  les  conditions  decrites;  exceptionnellement,  on 
obtient  des  preparations  plus  actives ;  mais  on  rencontre  assez  souvent  des  preparations 
fermentaires  qui  presentent  une  activite  plus  faible.  Ces  diverses  preparations  peuvent 
etre  generalement  activees  par  addition  de  coenzyme.  En  voici  un  exemple: 

Dans  5  tubes  contenant  chacun  130  micromolecules  de  cysteinesulfinate  de  sodium 
dans  10  ml  de  solution  de  bicarbonate  de  sodium  a  0.32%  et  10  ml  de  solution  de 
desulfinicase,  on  introduit  5  ml  de  solution  de  coenzyme  en  concentrations  croissantes, 
additionnees  de  bicarbonate  a  0.16%.  Dans  un  sixieme  tube,  les  10  ml  de  solution  de 
desulfinicase  sont  remplaces  par  10  ml  de  solution  de  bicarbonate. 

Le  Tableau  II  presente  les  resultats  obtenus. 

Ces  resultats  montrent  que  I'addition  de  coenzyme  donne  a  la  preparation  enzy- 
matique une  activite  maximum  qui  ne  pent  etre  ensuite  depassee,  quelle  que  soit  la 
quantite  de  coenzyme  ajoutee  en  exces. 
Bibliographic  p.  248. 


246 


CL.  FROMAGEOT   et  al. 


VOL.  4  (1950) 


TABLEAU  II 

ACTIVATION    PAR    LE    COENZYME    D'UNE    SOLUTION    DE    D^SULFINICASE 

La  concentration  de  la  solution  de  coenzyme  representee  par  i  est  telle  que  10  ml  de  solution 

correspondent  a  i  g  de  poudre  acetoniquc. 


Solution  de 

Concentration 

SO2  degage 

Tube 

desulfinicase 
(ml) 

de  la  solution 
de  coenzyme 

absolu 

corrige  * 

I 

10 

0 

31 

31 

II 

10 

I 

51 

45 

III 

10 

2 

65 

54 

IV 

10 

4 

75 

53 

V 

10 

6 

88 

55 

VI 

0 

2 

II 

*  Corrections  tenant  compte  de  I'activite  residuelle  des  solutions  de  coenzyme;  les  chifTres 
corriges  representent  I'activite  propre  de  la  solution  de  desulfmicase  reactivee. 

3.  Stahilite  du  coenzyme  a  la  chaleur 

Les  experiences  precedentes  indiquent  que  les  solutions  de  coenzyme  obtenues 
apres  un  chauffage  de  4  minutes  presentent  encore  par  elles-memes  une  legere  action 
sur  I'acide  cysteinesulfinique.  Nous  avons  constate  qu'il  est  possible  de  supprimer  prati- 
quement  cette  action  en  traitant  la  solution  enzymatique  au  bain-marie  bouillant 
pendant  15  minutes  au  lieu  de  4.  Mais  on  obtient  alors  des  solutions  de  coenzyme  sen- 
siblement  moins  actives.  L'experience  presentee  ici  est  faite  dans  les  conditions  suivantes : 

Dans  5  tubes  contenant  chacun  65  micromolecules  de  cystcinesulfmate  de  sodium 
dans  10  ml  de  solution  de  bicarbonate  de  sodium  a  0.32%,  on  introduit  soit  5  ml  de 
solution  de  desulfinicase  et  5  ml  d'eau  (S),  soit  5  ml  de  solution  de  desulfinicase  et  5  ml 
de  solution  de  coenzyme,  cette  derniere  correspondant  a  I'extraction  de  2  g  de  poudre 
acetonique  (SC),  soit  5  ml  de  solution  de  coenzyme  et  5  ml  d'eau  (C).  Les  resultats 
obtenus  sont  fournis  par  le  Tableau  II L 

TABLEAU  III 

INFLUENCE  DU  TEMPS  DE  CHAUFFAGE  SUR  LE  COENZYME 


Contenu 

Temps  au 

SO2  d6gag6 

des  tubes 

bain-marie 

absolu 

corrige  * 

S 

SC 
SC 
C 
C 

4 
15 

4 
15 

15 

38 

27 

6 

2 

15 
32 
25 

Voir  note  du  Tableau  II. 


4.  Mise  en  evidence  de  la  nature  organique  du  coenzyme 

Les  cendres  de  la  solution  de  coenzyme  sont  incapables  d'activer  I'apoenzyme  de  la 
desulfinicase.  L'experience  est  faite  ici  avec  une  solution  de  desulfinicase  non  prealable- 
ment  dialysee,  mais  susceptible  toutefois  d'avoir  son  activite  notablement  accrue  par 
addition  de  coenzyme.  Les  cendres  sont  obtenues  par  calcination  dans  une  capsule  de 
Bibliographic  p.  248. 


VOL.  4  (1950) 


COENZYME    POUR   LA   DESULFINICASE 


247 


platine  de  I'extrait  sec  de  10  ml  de  solution  de  coenzyme  correspondant  a  4  g  de  poudre 
acetonique.  Le  produit  de  cette  calcination  est  dissous  dans  I'eau  acidulee  et  la  solution, 
ajustee  a  p^  7.0  est  ramenee  a  10  ml.  Chaque  tube  contient  65  micromolecules  de  cys- 
teinesulfinate  de  sodium  dans  10  ml  de  solution  de  bicarbonate  de  sodium  a  0.32%.  Les 
tubes  sont  additionnes  en  outre  de  soit  5  ml  de  solution  de  desulfinicase  et  5  ml  d'eau  (S), 
soit  5  ml  de  solution  de  desulfinicase  et  5  ml  de  solution  de  coenzyme  (SC),  soit  5  ml 
de  solution  de  desulfinicase  et  5  ml  de  solution  de  cendres  (SM).  Les  poids  sees,  en  mg 
par  ml,des  diverses  solutions, sont  les  suivants:  desulfinicase  30, coenzyme  38,cendres6.5. 
Le  Tableau  IV  indique  les  resultats  obtenus. 

TABLEAU  IV  <.  \3^  4 

ACTIONS  COMPAREES  DU  COENZYME  ET   DE  SES  CENDRES 


Contenu  des  tubes 

SO2  degage 

S 
SC 

SM 

-4 

58 

2 

II  apparait  ainsi  que,  non  seulement  les  cendres  n'ont  aucun  pouvoir  activant 
vis-a-vis  de  la  desulfinicase,  mais  que,  au  contraire,  elles  exercent  une  action  inhibitrice 
nette.  Le  mecanisme  de  cette  action  est  actuellement  a  I'etude. 


5.  Action  de  divers  coenzymes  sur  la  desulfinicase 

La  nature  organique  d'une  partie  au  moins  du  coenzyme  de  la  desulfinicase  ayant 
ete  etablie,  il  etait  interessant  de  rechercher  si  des  coenzymes  connus  etaient  capables 
d'activer  I'apoenzyme  de  la  desulfinicase.  Parmi  ces  coenzymes,  deux  sont  particuliere- 
ment  interessants  par  suite  de  I'analogie  des  reactions  auxquelles  ils  participent, 
reactions  de  decarboxylation,  avec  la  desulfination  de  I'acide  cysteinesulfinique,  ce  sont 
la  cocarboxylase  et  le  phosphate  de  pyridoxal.  Sans  qu'il  soit  utile  de  donner  ici  de 
chiffres,  disons  que  a  la  dose  de  500  //g  par  tube  (20  ml),  et  en  presence  ou  en  absence 
de  I  mg  de  chlorure  de  magnesium,  aucune  activation  de  la  desulfinicase  n'a  pu  etre 
mise  en  evidence  avec  les  substances  suivantes:  cocarboxylase,  phosphate  de  pyridoxal, 
pantothenate  de  calcium,  lactoflavine.  II  apparait  a  priori  peu  probable  que  le  phosphate 
de  lactoflavine  et  les  codehydrogenases,  que  nous  n'avons  pas  encore  essayes,  aient  ici 
une  action.  II  semble  done  que  la  codesulfinicase  differe  des  coenzymes  actuellement 
connus. 

6.  Presence  de  la  codesulfinicase  dans  la  levure 

On  traite  de  la  levure  de  boulangerie  en  la  chauffant  avec  son  poids  d'eau  a  100° 
pendant  15  minutes;  le  liquide  obtenu  apres  centrifugation  contient  le  coenz5mie, 
comme  le  montre  I'experience  suivante: 

Dans  3  tubes  contenant  chacun  65  micromolecules  de  cystcinesulfinate  de  sodium 
dans  10  ml  de  solution  tampon  de  phosphates  a  p^  7.35,  on  introduit: 

Tube  1 :  5  ml  de  solution  de  desulfinicase  moyennement  active,  plus  5  ml  d'eau. 

Tube  II:  5  ml  de  la  solution  precedente  de  desulfinicase,  plus  5  ml  du  liquide  d'ex- 
traction  de  la  levure. 

Bibliographic  p.  248. 


248  CL.  FROMAGEOT   et  al.  VOL.  4  (1950) 

Tube  III:  5  ml  du  liquide  d' extraction  de  la  levure,  plus  5  ml  d'eau. 

Les  quantites  d'anhydride  sulfureux  degage  apres  2  heures  a  35°  en  atmosphere 
d'azote,  sont  donnees  dans  le  Tableau  V. 

L'activation  par  I'extrait  de  levure,  qui  n'exerce  lui-meme  aucune  action  sur 
I'acide  cysteinesulfinique,  est  tres  nette. 

Nous  sommes  heureux  de  remercier  ici  MM.  Gunsalus  et  Westenbrink  qui  nous 
ont  aimablement  envoye  les  echantillons  de  phosphate  de  pyridoxal  et  de  cocarboxylase 
utilises  ici. 

TABLEAU  V 

ACTIVATION    DE    LA    DESULFINICASE    PAR    UN    EXTRAIT    DE    LEVURE 


Tube 

SO2  degage 

I 

16 

II 

47 

III 

0 

r£sum£ 

La  desulfinicase,  inactiv6e  par  dialyse,  recupere  son  activit6  apres  addition  d'un  extrait  de 
foie  incapable  par  lui-meme  d'agir  sur  I'acide  cysteinesulfinique.  L'activite  des  solutions  de  desul- 
finicase, meme  non  dialys^es,  est  generalement  accrue  par  addition  d'extrait  de  foie  ou  d'extrait  de 
levure.  II  apparait  ainsi  que  la  desulfinicase  necessite  pour  son  fonctionnement  la  presence  d'un 
coenzyme,  facUement  dissociable  de  I'apoenzyme.  Ce  coenzyme  est  de  nature  organique  et  differe 
de  la  cocarboxylase  et  du  phosphate  de  pyridoxal. 

SUMMARY 

Desulphinicase,  inactivated  by  dialysis,  regains  its  activity  after  addition  of  a  liver  extract 
which  itself  is  incapable  of  acting  on  cysteine-sulphinic  acid.  The  activity  of  desulphinicase  solutions, 
also  undialysed  ones,  is  generally  increased  by  addition  of  liver  extract  or  yeast  extract.  It  thus 
appears  that  desulphinicase  necessitates  for  its  functioning  the  presence  of  a  coenzyme,  readily 
dissociable  from  the  apoenzyme.  This  coenzyme  is  organic  in  nature  and  differs  from  cocarboxylase 
and  pyridoxal  phosphate. 

ZUSAMMENFASSUNG 

Durch  Dialyse  inaktivierte  Desulfinicase  erlangt  ihre  Wirksamkeit  wieder  nach  Beifiigung  eines 
Leberextraktes  der  fiir  sich  selbst  unfahig  ist,  auf  Cysteinsulfinsaure  einzuwirken.  Die  Wirksamkeit 
von  Desulfinicase-Losungen,  sogar  undialysierten,  wird  im  Allgemeinen  durch  Zusatz  von  Leber-  oder 
Hefeextrakten  verstarkt.  Es  scheint  also,  dass  die  Desulfinicase  zu  ihrer  Wirkung  ein  Coenzym 
braucht,  welches  leicht  vom  Apoenzym  dissozierbar  ist.  Dieses  Coenzym  ist  organischer  Natur,  jedoch 
verschieden  von  Cocarboxylase  und  von  Pyridoxalphosphat. 

BIBLIOGRAPHIE 

1  C.  Fromageot,  F.  Chatagner  et  B.  Bergeret,  Biochim.  Biophys.  Acta,  2  (1948)  294. 

2  C.  Fromageot  et  F.  Chatagner,  Compt.  rend.,  224  (1947)  367. 

Re9u  le  17  mai  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  249 


BODY  SIZE  AND  TISSUE  RESPIRATION 

by 

H.  A.  KREBS 

Medical  Research  Council  Unit  for  Research  in  Cell  Metabolism,  Department  of  Biochemistry, 

University  of  Sheffield  (England) 


It  has  long  been  known  that  in  homoiothermic  animals  the  basal  metabolic  rate, 
per  unit  of  body  weight,  decreases  with  the  size  of  the  animal,  and  the  question  has 
often  been  discussed  whether  the  respiration  of  individual  tissues  of  animals  of  different 
size  shows  the  same  differences  as  the  intact  organisms.  Terroine  and  Roches  and 
Grafe,  Reinwein,  and  Singer^  measured  the  respiration  of  various  tissues  in  vitro  and 
came  to  the  conclusion  that  homologous  tissues  of  different  animals  respire  in  vitro  at 
about  the  same  rate,  irrespective  of  the  size  of  the  animal.  They  ascribed  the  differences 
found  in  the  intact  animal  to  the  regulatory  influences  of  the  nervous  system  and  of 
hormones.  Kleiber^'  *  on  the  other  hand,  reported  that  the  rate  of  respiration  of  liver 
slices  of  rats,  rabbits,  sheep,  horses  and  cows,  per  unit  of  weight,  decreased  with 
increasing  size  of  the  animal.  The  decrease  observed  was  of  the  same  order  as  the 
decrease  of  the  basal  metabolism  of  the  living  animal. 

This  lack  of  agreement  is  not  due  to  discrepancies  in  experimental  observations  but 
arises  from  difficulties  of  procedure  and  interpretation.  Whilst  the  measurement  of  the 
basal  metabolic  rate  is  a  standardized  technique,  no  accepted  standards  exist  for  the 
measurement  of  the  oxygen  uptake  of  isolated  tissues  in  vitro.  It  has  often  been  demon- 
strated that  the  oxygen  uptake  of  tissues  in  vitro  is  not  a  constant  value.  Specimens  of 
the  same  tissue  can  show  wide  and  reproducible  variations,  depending  on  the  conditions 
under  which  the  measurements  are  made.  Among  the  factors  responsible  for  these 
variations  two  are  of  special  importance:  the  composition  of  the  medium  in  which  the 
tissue  is  suspended  and  the  physical  treatment  of  the  material.  As  the  part  played  by 
these  factors  was  not  fully  appreciated  in  previous  investigations  it  was  thought  that 
new  measurements  of  the  rate  of  respiration  of  isolated  tissues  under  standard  conditions 
are  needed.  As  a  preliminary  it  was  necessary  to  define  standard  conditions  which 
would  resemble  as  closely  as  possible  the  state  of  the  tissues  in  the  intact,  possibly 
resting,  animal,  and  which  would  yield  a  "standard  rate"  of  tissue  respiration. 

A.    GENERAL   CONSIDERATIONS   CONCERNING   THE    MEASUREMENT   OF   THE 
"standard   rate"    OF  TISSUE   RESPIRATION 

I.  Treatment  of  tissue 

In  order  to  measure  the  rates  of  metabolic  processes  in  isolated  tissues  it  is,  as  a 
rule,  unavoidable  to  subject  the  tissues  to  procedures  like  shcing,  mincing  or  homo- 
genizing, so  that  the  cells  can  be  satisfactorily  supplied  with  oxygen  and  substrates. 

References  p.  267— 26g. 


250  H.  A.  KREBS  VOL.  4  (1950) 

These  procedures  affect  different  tissues  in  different  ways.  In  the  case  of  kidney  cortex, 
Hver,  striated,  and  cardiac  muscle,  mince  and  homogenates  show  higher  initial  rates  of 
respiration  than  sliced  material  when  phosphate  saline  without  a  combustible  substrate 
is  used  as  the  medium.  If  the  medium  contains  substrates  which  stimulate  respiration 
of  slices,  such  as  lactate,  pyruvate,  fumarate,  and  glutamate,  homogenates,  mince  and 
slices  give  approximately  the  same  rates  of  respiration^'  ^' '.  In  these  tissues  minced  or 
homogenized  materials  give  the  maximum  rate  of  respiration.  In  other  tissues,  e.g., 
spleen^,  and  lung^,  minced  and  homogenized  material  gives  consistently  lower  rates 
of  respiration  than  sliced  material.  The  low  values  have  been  attributed  to  the  hydrolysis 
of  coenzymes  by  nucleosidases  released  on  the  destruction  of  the  tissue^. 

It  is  reasonable  to  assume  that  slicing  leaves  the  tissue  nearer  to  the  'natural'  state 
than  mincing  or  homogenizing,  because  the  number  of  physically  damaged  cells  is 
bound  to  be  much  smaller  in  slices  than  in  mince  or  homogenates.  Slicing  is  therefore 
suggested  as  the  procedure  of  choice  for  the  measurement  of  the  standard  rate  of 
metabolism. 

2.  Choice  of  medium 

In  this  section  5  different  media  are  considered  for  the  measurement  of  a  standard 
rate  of  respiration.  They  are: 

Serum 

Supplemented  serum 

Saline  serum  substitute  (later  referred  to  as  'medium  I') 

Phosphate  saline  without  Ca,  low  in  bicarbonate  and  CO2  (later  referred  to  as 
"medium  IF) 

Saline  low  in  phosphate,  bicarbonate,  and  CO,  (later  referred  to  as  'medium  IIP). 

Serum.  Plasma  or  serum,  being  the  natural  environment  of  animal  tissues,  suggest 
themselves  as  the  most  physiological  standard  media.  Plasma  requires  the  addition  of  an 
anticoagulant  and  several  of  these,  e.g.,  sodium  fluoride  and  sodium  oxalate,  are  unsuit- 
able as  they  inhibit  metabolic  processes.  Among  the  remaining  substances  heparine  is 
least  likely  to  affect  tissue  metabolism,  but  relatively  large  amounts  are  required  to 
prevent  coagulation  in  the  presence  of  tissues.  In  general  serum  is  preferable  to  plasma 
because  the  absence  of  fibrinogen  from  the  medium  is  less  likely  to  affect  the  activities 
of  the  tissue  than  the  addition  of  an  anticoagulant. 

Supplemented  serum.  Although  serum  resembles  the  physiological  environment 
more  closely  than  any  other  medium  it  is  by  no  means  a  perfect  medium  for  in  vitro 
experiments.  A  tissue  suspended  in  plasma  or  serum  may,  by  its  metabolism,  soon  cause 
major  changes  in  the  concentration  of  important  constituents,  such  as  glucose,  pyruvate, 
lactate,  and  the  acids  of  the  tricarboxylic  cycle,  and  also  of  bicarbonate.  In  the  intact 
body  the  balance  of  activities  of  all  organs  maintains  a  relative  constancy  of  the  con- 
centration of  serum  constituents ;  thus,  glucose  used  up  by  some  tissues,  is  replenished 
from  liver  stores  and  by  the  absorption  from  the  gut.  But  in  vitro  the  metaboHc  activity 
of  a  single  tissue  can  rapidly  convert  serum  into  an  'unphysiological'  medium  by 
exhausting  the  available  substrates. 

Another  factor  to  be  taken  into  consideration  is  the  circumstance  that  in  the  intact 
organ  the  path  of  diffusion  is  much  shorter  than  in  vitro,  the  average  distance  between 
capillary  wall  and  tissue  cell  being  much  shorter  than  the  average  distance  between  the 
References  p.  267-269. 


VOL.  4  (1950) 


BODY    SIZE    AND   TISSUE    RESPIRATION 


251 


surface  and  the  centre  of  the  sUce.  Hence  a  concentration  gradient  and  a  rate  of  diffusion 
which  might  be  sufficient  to  saturate  the  cells  in  vivo  may  become  a  limiting  factor 
in  vitro. 

Both  difficulties — rapid  exhaustion  and  slow  diffusion — can  be  overcome  by 
increasing  the  concentration  of  the  'relevant'  metabolites  in  the  medium.  This  con- 
sideration raises  the  question  of  what  are  'relevant'  substrates.  Among  the  very  large 
number  of  organic  substances  known  to  occur  in  plasma  and  serum  (listed  in  Table  I) 
only  a  few  have  been  found  to  influence  the  oxygen  uptake  in  vitro.  They  are  glucose, 
lactate,  pyruvate,  the  acids  of  the  tricarboxylic  cycle,  and  glutamate  (or  glutamine),and 
some  closely  related  substances  such  as  phosphorylated  intermediates  of  glycolysis 
which  need  not  be  considered  separately.  A  few  special  amino  acids  {e.g.,  tyrosine, 
phenylalanine,  proline)  can  increase  the  respiration  of  liver,  kidney,  and  sperma- 
tozoa^^' ®^'  ®^'  ^*,  but  although  these  effects  may  be  of  importance  in  relation  to  the 


TABLE  I 

COMPOSITION    OF    HUMAN    BLOOD    PLASMA 


Substance 


mg/ioo  ml 


Average  or 

representative 

value 


Range  or 
standard 
deviation 


References 


Nitrogenous  substances 

Protein  (total) 
Albumin 
ai-Globulin 
Qg -Globulin 
^-Globulin 
y-GlobuIin 
Fibrinogen 
Non-protein  nitrogen  (total) 

Amino-N  (as  N,  ninhydrin  method) 
Amino-N  (as  N,  nitrous  acid  method) 

Alanine 

Arginine 

Citrulline 

Glutamic  acid 

Glutamine 

Glycine 

Histidine 

Iso-leucine 

Leucine 

Lysine 

Methionine 

Phenylalanine 

Threonine 

Tryptophane 

Tyrosine 

Valine 
Ammonia  (as  N,  whole  blood) 
Creatine 
Creatinine 
Glycocyamine 
Urea  (as  N) 
Uric  acid 
Allantoin 
Allantoin  (dog) 

References  p.  26y-26g. 


below 


6720 
4040 
310 
480 
810 
740 
340 
25 
4.1 
4.4 
3-97 
2-34 
0.50 

3-41 
5-78 
1.77 
1.42 
1.60 
1.91 

2.95 
0.85 

1.38 
2.02 
1.08 
1.48 
2.83 
0.05 
0.9 
0.4 
0.26 
12 
4 


S.D. 

S.D. 

S.D. 

S.D. 

S.D. 

S.D. 
18- 
3-4- 
3-7- 

S.D. 

S.D. 

0.38- 

S.D. 

S.D. 

S.D. 

S.D. 

S.D. 

S.D. 

S.D. 

0.46- 

S.D. 

S.D. 

S.D. 

S.D. 

S.D. 


270 
51 
83 

126 

151 
59 
30 
5-5 
5-9 
0.70 
0.62 
0.59 
1-39 
1-55 
0.26 
o.i8 
0.31 

0.34 
0.42 
1.48 
0.32 

6.45 
0.21 

0.37 
0.34 


0.62-1.02 

0.28-0.62 

0.24-0.28 

10-17 

2-6 

0.3-0.6 

1. 1-3.0 


10 

10 

10 

10 

10 

10 

10 

n 

12, 

13 

12 

14. 

15 

16. 

17.  I 

19 

20 

14 

16, 

18 

16. 

18 

16, 

18 

16, 

18 

21, 

18 

16 

16, 

18 

16, 

22 

16 

16, 

18 

23. 

24 

25 

25 

17 

II, 

26 

27 

19 

19 

252 


H.  A.  KREBS 
TABLE  I   (continued) 


VOL.  4  (1950) 


mg/ioo  ml 

Substance 

Average  or 

Range  or 

References 

representative 

standard 

value 

deviation 

Carbohydrate  and  related  substances 

Glucose,  fasting,  venous  blood 

83 

S.D.  4 

28 

Glucose,  fasting,  capillary  blood 

93 

S.D.  3 

28 

Total  reducing  substances  (as  glucose) 

90-120 

29 

Lactic  acid  (resting) 

8-17 

30 

Pyruvic  acid 

I.O 

0.77-1.23 

31.  32 

Citric  acid 

2.5 

1.9-2.8 

33.  34 

a-Ketoglutaric  acid 

0.8 

35.  36 

Succinic  acid 

0-5 

36,  37 

Fat  and  related  substances 

Fat  (total) 

570 

360-820 

II 

Fatty  acids  (total),  as  stearic  acid 

340 

200-800 

II 

Phospho-Lipids  (total) 

215 

123-293 

38 

Lecithin 

50-200 

39,  compare 
38  and  40 

Cephalin 

50-130 

39,  compare 
38  and  40 

Sphingomyelin 

15-35 

39,  compare 
38  and  40 

Lipid-P 

9.2 

6.1-14.5 

II 

Cholesterol,  total 

194 

107-320 

II,  41 

Cholesterol,  free 

69 

26-106 

II,  41 

Ketone  bodies  (as  j5-hydroxybutyric  acid) 

0.33-0.87 

42 

BUe  acids  (as  cholic  acid) 

0.2-3.0 

43 

Vitamins 

Vitamin  A 

0.019-0.036 

0.025 

44 

Carotene  (total  carotenoids) 

0.06-0.18 

0.09 

44 

Ascorbic  acid 

0.1-0.70 

45 

Inositol 

0.42-0.76 

46 

Folic  acid 

1.75- 10-3 

1.62-1.95-10-3 

22 

Biotin 

1.27- 10-^ 

0.95-1.66-10-3 

22 

Pantothenic  acid 

12- 10-3 

6-22- TO-3 

22 

Mineral  constituents 

Na 

309 

300-330 

47.  48.  49 

K 

18 

12-29 

47.  48 

Ca 

10 

8.2-II.6 

47 

Mg 

2.0 

1.6-2.7 

47 

Fe,  men 

0.0945 

S.D.  0.0295 

50 

Fe,  women 

0.0895 

S.D.  0.0269 

50 

Cu 

0.09 

0.07-0.12 

51 

Mn  (whole  blood) 

0.005-0.020 

52 

Zn 

0.30 

S.D.  0.16 

53.  54 

CI 

366 

350-375 

47,  see  also 
49 

I  (total) 

0.006-0.008 

55 

I  (protein  bound) 

0.007 

0.006-0.008 

56 

F  (whole  blood) 

• 

0.04-0.15 

57 

HCO3-  (as  vol.  %  CO2) 

61 

55-75 

29,  47.  49 

Phosphate,  inorganic  (as  P) 

3-7 

2.9-4-3 

47 

Phosphate,  lipid  (as  P) 

9.2 

6.I-I4-5 

II 

SO4  (as  S) 

1-57 

1. 00-1.85 

58,  59.  60 

References  p.  267-269. 


VOL.  4  (1950)  BODY   SIZE    AND   TISSUE    RESPIRATION  253 

Specific  dynamic  action,  they  are  insignificant  for  the  conditions  of  basal  metabolism 
because  the  concentration  of  these  substances  in  plasma  is  too  low  except  during  the 
period  of  absorption  from  the  intestine. 

The  above  list  of  'relevant'  substances  can  be  simplified  because  lactate  and 
pyruvate  have  very  similar  effects  which  are  not  additive,  and  only  one  of  the  two 
therefore  needs  to  be  added.  Of  the  two,  pyruvate  has  the  advantage  over  L-lactate  of 
being  more  readily  available.  Furthermore,  all  the  acids  of  the  tricarboxylic  acid  cycle 
have  very  similar  effects^^,  as  may  be  expected  from  their  interconvertibility.  Thus  the 
addition  of  one  of  the  acids  should  be  sufficient.  As  for  the  choice,  only  three  of  the  eight 
main  acids  of  the  cycle  are  readily  available:  citrate,  succinate,  and  fumarate.  Citrate 
has  the  disadvantage  that  it  forms  complexes  with  calcium  and  magnesium  ions  and 
thereby  upsets  the  ionic  balance  of  the  medium.  Succinate  occupies  a  rather  special 
position  in  that  the  first  stage  of  its  oxidation,  the  formation  of  fumarate,  may  proceed 
much  more  rapidly  than  the  other  stages  of  the  cycle^^;  it  may  cause  a  brief  period  of 
rapid  oxygen  consumption  followed  by  a  steady  rate  at  a  lower  level.  There  remains 
fumarate  as  the  most  suitable  representative  of  the  cycle. 

From  the  point  of  view  of  tissue  respiration  the  list  of  relevant  metabolites  can  thus 
be  reduced  to  four:  glucose,  pyruvate  or  lactate,  fumarate,  glutamate.  x^s  regards  the 
concentrations  to  be  used,  experiments  on  kidney  and  brain  cortex  show  that  increasing 
the  concentrations  of  pyruvate,  lactate,  fumarate  or  glutamate  above  0.005  ^  makes 
no  difference  to  the  rate  of  respiration,  except  in  very  prolonged  experiments.  Glucose 
is  usually  not  a  limiting  factor  when  its  concentration  is  above  0.2%. 

It  is  therefore  suggested  that  serum  be  supplemented  by  adding  isotonic  substrate 
solutions  in  the  following  proportions : 


100  parts  of  serum 

3  parts  of  0.16  M  Na-pyruvate  (or  Na-L-lactate) 
6  parts  of  o.i  M  Na- fumarate 
3  parts  of  0.16  M  Na-L-glutamate 
5  parts  of  0.3  M  glucose 


The  mixture  must  be  in  equilibrium  with  a  gas  mixture  containing  about  5%  COo. 
The  additions  cause  a  dilution  of  the  serum  of  about  15%.  It  is  not  possible  when  making 
additions  to  maintain  both  isotonicity  and  concentrations,  and  preference  is  given  to 
the  former. 

The  blood  from  which  the  serum  is  prepared  should  be  cooled  immediately  after 
collection,  otherwise  the  glycolytic  activity  of  the  blood  cells  will  reduce  the  concen- 
trations of  glucose  and  bicarbonate  and  increase  that  of  lactate.  The  bicarbonate  content 
of  the  serum  should  be  determined  and  if  below  0.025  M  it  should  be  adjusted  to  that 
level  by  the  addition  of  1.3%  NaHCOg  solution.  It  is  advisable  to  sterilize  the  medium 
by  passing  it  through  a  Seitz-filter. 

Saline  serum  substitute  [Medium  I).  Serum  contains  unknown  and  variable,  and 
thus  uncontrolled,  constituents.  It  is  furthermore  difficult  to  obtain  in  sufficient  quan- 
tities in  the  case  of  small  animals,  and  heterologous  serum  may  contain  inhibitory 
antibodies.  There  is  therefore  a  case  for  a  serum  substitute  which  can  be  easily  prepared 
and  whose  composition  is  exactly  known. 

As  a  rule  serum  does  not  preserve  the  metabolic  activities  of  isolated  tissues  more 
effectively  than  do  saline  media  supplemented  with  substrates.  The  rates  of  the  metabo- 
Re/erences  p.  2()'j-26g. 


254  ^-  ^-  KREBS  VOL.  4  (iQSO) 

lie  processes  in  isolated  material  which  have  so  far  been  studied  have  usually  been  found 
to  be  of  the  same  order  in  serum  and  in  suitable  saline  media,  at  least  for  the  usual 
experimental  periods  of  under  two  hours.  But  some  tissues,  in  particular  brain,  retina, 
choroid  plexus,  and  foetal  membranes,  assume  an  opaque  appearance  on  incubation  in 
saline  and  tend  to  break  up  into  fragments  whilst  appearance  and  texture  remain 
unchanged  in  serum.  The  use  of  serum  may  therefore  be  advantageous  in  some  investi- 
gations. 

The  earlier  serum  substitutes,  such  as  Ringer's  solution,  were  designed  on  an 
empirical  basis.  Ringer"  tested  the  effect  of  various  saline  media  on  the  beat  of  the 
isolated  frog  heart,  and  found  that  solutions  containing  certain  quantities  of  Ca  and 
K  ions,  in  addition  to  NaCl  maintained  the  beat  for  longer  periods  than  NaCl  solutions. 
Later,  when  precise  data  on  the  chemical  composition  of  blood  serum  became  available, 
saline  media  were  modelled  on  these  data^^'  ^^'  ^"'  ''^.  It  has  been  found  repeatedly  that 
the  closer  the  medium  resembles  serum  the  better  does  it  maintain  tissue  activities 
in  vitro.  The  previous  attempts  to  copy  the  composition  of  serum,  however,  considered 
only  the  inorganic  constituents  and  glucose. 

The  saline  medium  of  Krebs  and  Henseleit'^^  closely  reproduces  the  inorganic 
constituents  of  mammalian  serum  except  that  the  concentration  of  CI  is  about  20% 
higher.  A  discrepancy  of  this  kind  is  unavoidable  in  a  purely  inorganic  medium  because 
in  serum  a  fraction  of  the  anions,  amounting  to  about  22  milliequivalents,  consists  of 
organic  substances.  Replacement  of  part  of  the  NaCl  by  the  Na  salts  of  pyruvic  (or 
L-lactic),  fumaric  and  glutamic  acids  and  addition  of  glucose  eliminates  the  discrepancy 
in  the  chloride  concentration  and  introduces  the  'relevant'  metabolites.  The  following 
composition  is  suggested  for  the  saline  serum  substitute.  Mix 

1.  80  parts  of  0.9%  NaCi  (0.154  M) 

2.  4  parts  of  1.15%  KCl  (0.154  M) 

3.  3  parts  of  o.ii  M  CaCl., 

4.  I  part  of  2.11%  KH2P64  (0.154  M) 

5.  I  part  of  3.82%  MgS04.7H20 

6.  21  parts  of  1.3%  NaHCOg  (0.154  M) ;  treated  with  CO2  until  pn  is  7.4 

7.  4  parts  of  0.16  M  Na-pyruvate  (or  L-lactate)    1    Prepared  by 

8.  7  parts  of  o.i  M  Na-fumarate 
g.    4  parts  of  0.16  M  Na-L-glutamate 

10.    5  parts  of  0.3  M  (5.4%)  glucose 


neutralizing  a 

solution  of  the  acids  with  M 

NaHCOg  solution 


The  mixture  must  be  saturated  with  a  gas  mixture  containing  about  5%  COg.  The 
stock  solutions  are  approximately  isotonic. 

Solutions  7  to  10,  unless  sterilized,  cannot  be  kept  at  room  temperature.  In  the 
refrigerator  they  keep  for  about  a  week  if  gross  bacterial  infections  are  avoided. 

Solutions  I  to  6  are  mixed  in  the  same  proportion  as  the  medium  of  Krebs  and 
Henseleit'^1,  except  that  80  parts  NaCl  solution  instead  of  100  parts  are  used.  The 
difference  of  20  ml  is  made  up  by  the  solutions  7  to  10.  The  concentrations  of  the  con- 
stituents of  this  medium  are  shown  in  Table  II.  For  comparison,  data  for  human  and 
rat  sera  are  also  given  and  it  will  be  seen  that  the  concentration  of  the  electrolytes  in 
the  sera  and  the  'serum  substitute'  are  very  similar. 

Sera  of  different  mam.Tialian  species  show  relatively  small  variations  except  in  the 
case  of  inorganic  sulphate.  Normal  human  serum  is  reported  to  contain  i  to  1.5  mg  SO4 
References  p.  26y—26g. 


VOL.  4  (1950) 


BODY    SIZE    AND   TISSUE    RESPIRATION 


255 


TABLE  II 

COMPARISON    OF    THE    COMPOSITION    OF    SERUM    AND    SERUM    SUBSTITUTE 


Concentration  in  serum 

Substance 

Concentration  in  medium 
(milliequivalent/litre) 

(milliequivalent/litre) 

Human'5 

Rat's, " 

Na 

141. 0 

142 

134 

K 

5-93 

5 

5-1 

Ca 

5.0S 

5 

6.05 

Mg 

2.36 

3                              2.57 

CI 

104. 8 

103 

102 

Phosphate*  (inorganic) 

2.22 

2 

4-3 

Sulphate  (inorganic) 

2.36 

I 

HCO3 

24.9 

27 

22 

CO2  (at  40°) 

i.o 

Pyruvate 

4-9 

Glutamate 

4-9 

Fumarate 

5-4 

Total  organic  anions 

20.7 

22 

Glucose 

9.2 

In  accordance  with  common  usage  one  P  is  taken  as  1.8  equivalent. 


(expressed  as  S)  per  100  ml  =  0.7  to  i.o  milliequivalent  per  litre^^'  ^";  somewhat  higher 
figures  are  given  by  Guillaumin'^.  For  dog,  ox,  goat,  and  horse  figures  between  3  and 
4  mg  S  per  100  ml  are  reported'^^'  ''*.  The  serum  substitute,  being  primarily  intended  for 
use  with  animal  tissues,  copies  the  sulphate  concentration  of  animal  serum.  If  a  substi- 
tute for  human  serum  is  required  half  of  the  MgS04  should  be  replaced  by  an  equivalent 
amount  of  MgClg  solution. 

Owing  to  the  danger  of  bacterial  infection  the  solutions  containing  organic  sub- 
stances should  be  freshly  prepared  before  use.  A  composite  stock  solution  containing 
solutions  1-5  in  the  proportion  stated  and  3  parts  of  solution  6  is  stable;  the  use  of  this 
mixture  shortens  the  procedure  for  preparing  the  full  medium. 

Phosphate  saline  without  Ca,  and  loio  in  bicarbonate  and  CO^  {Medium  II).  Serum 
and  the  saline  serum  substitute  may  be  inconvenient  in  the  manometric  measurement 
of  respiration  because  they  must  be  kept  in  equilibrium  with  gas  mixtures  containing 
about  5%  CO2.  The  measurement  of  the  oxygen  uptake  is  simpler  and  more  accurate  if 
the  CO2  pressure  of  the  gas  phase  can  be  kept  near  zero  by  absorbing  the  gas  with  alkali. 
A  reduction  of  the  CO2  pressure  necessitates  an  equivalent  reduction  in  the  bicarbonate 
concentration  if  p^  is  to  remain  within  the  physiological  range.  The  following  two  types 
of  media  with  low  bicarbonate  and  CO,  concentrations  have  been  in  use : 

Type  A.  The  greater  part  of  the  bicarbonate-COg  buffer  system  is  replaced  by  a 
phosphate  buffer  of  the  same  pn  and  approximately  equivalent  concentration.  As  a  high 
concentration  of  phosphate  is  incompatible  with  the  physiological  concentration  of 
calcium  ions  the  latter  are  usually  omitted  from  such  media.  Ca-free  phosphate  salines 
are  especially  valuble  as  a  medium  for  minced  tissues  and  homogenates,  as  they  give 
higher  and  steadier  rates  than  calcium  containing  media"^'  ^^'  ^>  ^^>  ^^'  ^^. 

Type  B.  The  bicarbonate  content  is  reduced  to  about  one-tenth  of  the  physiological 
value,  with  no  change  in  the  other  constituents^^'  ^^.  Such  a  medium  has  the  advantage 
of  having  a  physiological  concentration  of  calcium,  but  its  buffering  capacity  is  much 
below  that  of  the  media  of  Type  A.  The  pn  is  not  precisely  defined  but  indicator  tests 
References  p.  26/—26g. 


256  H.  A.  KREBS  VOL.  4  (1950) 

show  that  if  the  medium  is  shaken  with  respiring  tissues  which  produce  CO2  continuously 
pjj  remains  about  7.3.  When  the  medium  is  allowed  to  stand  for  long  periods  or  shaken 
without  tissues  pn  rises. 

Comparative  measurements  have  shown  in  many  cases^^'  ^®  that  tissues  kept  in 
these  types  of  media  respire  at  about  the  same  rate  as  serum  or  saline  serum  substitutes 
containing  Ca  and  bicarbonate  in  physiological  concentrations. 

A  medium  of  the  type  A  is  prepared  by  omitting  CaClj  from  medium  I  and  replacing 
18  parts  of  the  NaHCOg  solution  by  an  isotonic  phosphate  buffer.  Mix 

83  parts  of  0.9%  NaCl 
4  parts  of  1.15%  KCl 
I  part    of  2.11%  KH2PO4 
I  part    of  3.82%  MgS04.7H20 

3  parts  of  1.3%  NaHCOg 

18  parts  of  Na-phosphate  buffer  (100  parts  of  o.i  M  Na2HP04  (1.78%  Na2HP04.2  H2O) 
and  25  parts  of  o.i  M  NaH2P04  (1.38%  NaH2P04.H20)) 

4  parts  of  0.16  M  Na-pyruvate  (or  L-lactate) 
7  parts  of  O.I  M  Na-fumarate 

4  parts  of  0.16  M  Na-L-glutamate 

5  parts  of  0.3  M  (5.4%)  glucose 

In  this  calcium-free  medium  the  concentrations  of  Na,  K,  Mg,  CI  and  SO4  approxi- 
mate to  those  of  serum;  the  concentration  of  phosphate  is  about  20  times  higher,  and 
that  of  HCO3  about  10  times  lower,  than  the  physiological  values. 

Saline  low  in  phosphate,  bicarbonate,  and  CO^  {Medium  III).  Many  previous  obser- 
vations indicate  that  calcium  ions  can  influence  the  rate  of  respiration^'^'  ^>  ^^'  ^''.  It  is 
therefore  useful  to  have  a  medium  which,  like  the  synthetic  serum  substance,  contains 
Ca  in  physiological  concentrations  but  can,  at  the  same  time,  be  used  in  manometric 
experiments  where  CO2  is  being  absorbed  by  alkali.  The  medium  suggested  differs  from 
medium  II,  apart  from  the  inclusion  of  Ca,  by  a  lower  phosphate  concentration  and 
therefore  lowering  buffering  capacity.  These  differences  are  necessitated  by  the  limited 
solubility  of  Ca-phosphates.  Mix 

95  parts  of  0.9%  NaCl 
4  parts  of  1.15%  KCl 
3  parts  of  0.1 1  M  CaCla 
I  part    of  2.11%  KH2PO4 
I  part    of  3.82%  MgS04.7H20 
3  parts  of  1.3%  NaHCOg 

3  parts  of  Na-phosphate  buffer  (as  described  for  medium  II) 

4  parts  of  0.16  M  Na-pyruvate 
7  parts  of  0.1  M  Na-fumarate 

4  parts  of  0.16  M  Na-L-glutamate 

5  parts  of  0.3  M  (5.4%)  glucose 

O2  pressure 

In  order  to  safeguard  saturation  of  tissue  slices  with  O,  it  is  generally  necessary  to 
have  an  O2  pressure  of  one  atmosphere  in  the  cup.  It  is  known®^'  ^^'  ^^  that  O2  of  this 
pressure  has  a  poisoning  effect  on  some  of  the  oxidative  enzymes.  As  these  effects  are 
References  p.  26y-26g. 


VOL.  4  (1950)  BODY    SIZE    AND   TISSUE    RESPIRATION  257 

small  when  the  medium  contains  Mg  ions  and  the  period  of  observation  is  below  2  hours^^ 
they  may  be  neglected  in  many  cases. 

B.    MEASUREMENT   OF   Qq^   OF   FIVE    MAMMALIAN   TISSUES 

1.  Procedure 

At  the  start  of  this  investigation  it  was  decided  to  use  medium  II  for  the  main 
measurements  in  preference  to  medium  I  because  the  absorption  of  COg,  permissible  in 
the  case  of  medium  II,  simplifies  the  manometric  technique.  It  was  expected,  on  the 
basis  of  the  results  of  previous  investigators  on  similar  media^^'  ^^,  that  the  three  media 
would  all  give  approximately  the  same  Qq^  values,  but  later  comparative  measurements 
of  Qq2  in  the  three  different  salines  gave  consistent  differences  in  the  case  of  some 
tissues,  especially  brain. 

The  measurements  of  the  O2  uptake  were  carried  out  on  sliced  material  in  conical 
Warburg  flasks  of  20  to  26  ml  capacity,  provided  with  a  centre  well.  The  main  compart- 
ment contained  4  ml  medium,  the  centre  well  0.3  ml  2  N  NaOH,  the  gas  space  Og.  The 
temperature  was  40°.  All  measurements  were  done  in  duplicate. 

Five  tissues,  brain  cortex,  kidney  cortex,  liver,  spleen,  and  lung,  were  examined. 
They  were  removed  from  the  fasting  animal  as  soon  as  possible  after  death  and  placed 
in  ice-cold  saline  (medium  III,  in  which  the  organic  substrate  solutions  were  replaced 
by  an  equal  volume  0.9%  NaCl).  Slices  were  made  free-hand  or  by  the  method  of 
Deutsch^*.  During  the  slicing  operation  the  tissue  and  razor  blades  were  bathed  in  the 
modified  medium  III.  Readings  began  after  an  equilibration  period  of  15  min  and  were 
continued  at  5  or  10  min  intervals  for  45  min,  so  that  the  total  period  of  incubation  was 
60  min.  Q02  was  calculated  from  the  pressure  change  observed  during  the  45  m.in  period 
of  recording. 

Abattoir  material  was  collected  in  Dewar  vessels  containing  250  ml  water,  250  g  ice, 
3.5  g  NaCl,  15  ml  1.15%  KCl  and  12  ml  o.ii  M  CaCU.  On  addition  of  the  tissue  most 
of  the  ice  melted  and  the  resulting  solution  contained  Na,  K,  Ca  and  CI  in  approximately 
physiological  concentrations.  The  material  usually  reached  the  laboratory  within  about 
one  hour  after  killing.  To  test  to  what  extent  this  treatment  affected  the  rate  of  respi- 
ration samples  of  guinea  pig  and  rat  tissue  were  sliced  immediately  after  death  and 
another  portion  of  the  organ  was  subjected  to  storage  in  iced  saline  in  the  same  way  in 
the  abattoir  material,  except  that  the  period  of  storage  was  4  hours.  The  results  are 
shown  in  Table  III.  It  will  be  seen  that  small  losses  of  activity  exceeding  the  limits  of 
error  occurred  in  storing  guinea  pig  liver  and  guinea  pig  lung.  As  the  delay  in  the 
examination  of  abattoir  material  was  usually  only  one  quarter  of  the  time  allowed  for 
storing  in  this  experiment  it  may  be  assumed  that  the  losses  in  activity  due  to  storage 
were  negligible.  If  losses  actually  occurred  the  value  given  for  abattoir  material  would 
be  too  low.  Prolonged  storage  in  iced  saline  caused  considerable  losses  of  activit}'.  In  an 
experiment  in  which  guinea  pig  tissue  was  examined  after  a  storage  period  of  24  hours 
Q02  of  brain  cortex  fell  Z7%>  of  kidney  cortex  11%,  of  liver  77 °o,  of  spleen  43%,  and 
of  lung  29%. 

2.  Qq^  in  phosphate  saline  without  calcium  [medium  II) 

Data  obtained  on  9  different  mammalian  species  are  given  in  Table  IV.  Of  each 
tissue  6  specimens  were  examined  in  the  case  of  the  rat,  guinea  pig,  rabbit,  sheep,  cattle 

References  p.  26^-26^. 

17 


258 


H.  A.  KREBS 


VOL.  4  (1950) 


and  horse,  7  in  the  case  of  the  mouse,  5  in  the  case  of  the  dog  and  2  in  the  case  of  the  cat. 
The  mean  Q02  values  for  each  tissue  are  given  in  Table  V,  together  with  mean  values  of 
heat  production,  for  animals  of  the  same  average  weight.  The  heat  values  are  taken 
from  Benedict®^. 


TABLE  III 

EFFECT    OF    STORAGE    OF    TISSUES    ON    Q02 


Q02  (average  of  duplicate) 

Change  in  Q02 

Tissue 

Sliced                      Stored  4  hours 
immediately                 in  iced  saline 

due  to  storing 
(%) 

Brain  cortex,  guinea  pig 
Kidney  cortex,  guinea  pig 
Liver,  guinea  pig 
Lung,  guinea  pig 
Liver,  rat 

—  25-1 

—  32.9 

—  13-7 

—  9.1 

—  19.7 

—  23-9 

—  34-8 

—  II. 7 

—  8.0 

—  19.2 

—  4-7 
+     5-7 

—  145 

—  12. 1 

—  2.5 

TABLE  IV 

QO2    OF    5    TISSUES    OF    9    MAMMALIAN    SPECIES 

Slices  suspended  in  medium  II  (phosphate  buffered,  no  calcium) ;  the  data  are  the  averages  of  dupli- 
cate determinations) 


Species 

Breed 

Sex 

Age 

Weight 
(kg) 

'        Q02 

No 

years 

months 

Brain 
cortex 

Kidney 
cortex 

Liver 

Spleen 

Lung 

1 

Mouse 

Albino 

m 

0-035 

—  32.2 

—  50.3 

—  22.2 

—  20.3 

—  13-5 

2 

,, 

m 

0.034 

—  30.2 

—  35-5 

—  20.2 

—  15-3 

—  10.6 

3 

,, 

m 

0.012 

—  30.0 

—  53-4 

—  21.9 

—  17.1 

—  10.4 

4 

,, 

m 

0.028 

—  35-6 

—  48.1 

—  23-7 

—  14.8 

—  II. 6 

5 

,, 

m 

0.015 

—  30.2 

—  41-5 

—  20.9 

-16.3 

—     9-2 

6 

,, 

m 

0.009 

—  331 

—  37-0 

—  25.6 

—  17.1 

—  15-4 

7 

•• 

m 

0.031 

—  39-6 

—  56.8 

—  27.5 

— 19.0 

—  13-6 

1 

Rat 

Albino 

m    i 

0.22 

—  24.8 

—  33-8 

—  15-7 

—  11.9 

—  9.0 

2 

,, 

m 

0.22 

—  25.6 

—  26.8 

—  13-9 

—  12.3 

—  9.6 

3 

,, 

m 

0.20 

—  21.5 

—  41.6 

—  19.7 

—  12.2 

—  90 

4 

,, 

f 

0.19 

-35-8 

—  43-1 

—  16.5 

— 12.0 

—  8.0 

5 

,, 

f 

0.24 

—  20.2 

-38.5 

—  14.7 

—  12.8 

—  7.0 

6 

" 

m 

0.18 

—  30.0 

—  45-3 

—  22.6 

—  14.7 

—  9.1 

I 

Guinea  pig 

m 

0.74 

-28.7 

—  33-5 

—  13-6 

—  9.9 

-8.5 

2 

,, 

f 

0.54 

-27.8 

—  31-3 

—  137 

—  13-5 

—  7.6 

3 

,, 

m 

0.42 

—  27-3 

—  27-5 

—  12.6 

—  12.2 

—  9.2 

4 

,, 

m 

0.40 

—  22.5 

—  31.0 

—  II. 0 

— 11.7 

—  7-4 

5 

,, 

m 

0.49 

—  25-1 

—  32.9 

—  13-5 

—  10.9 

—  9.1 

6 

" 

m 

0.41 

—  32.1 

—  34-3 

-13-6 

—  11.6 

—  9-4 

I 

Rabbit 

Chinchilla 

f 

1.03 

—  26.6 

—  31-4 

—  15-5 

—  7-5 

2 

,, 

m 

1-34 

—  30.6 

—  330 

—  12.7 

— •  16.4 

—  7-9 

3 

,, 

m 

I-3I 

—  33-2 

—  36-7 

—  9.6 

—  19.1 

—  g.i 

4 

,, 

f 

1.23 

—  27.0 

—  40.0 

—  10.9 

—  12.5 

—  8.6 

5 

,, 

f 

1.36 

—  28.2 

—  31.0 

—  I3-I 

—  12.6 

—  8.2 

6 

•• 

f 

I. II 

—  23-3 

—  34-8 

—  II. 8 

—  9-3 

-6.5 

I 

Cat 

m 

3-ri 

—  29.4 

—  21.0 

—  13-3 

—  7-3 

—  3-1 

2 

m 

2.39 

—  24-3 

—  24.4 

—  13.0 

—  9-5 

—  4-7 

References  p.  267—269. 


VOL.  4  (1950) 


BODY    SIZE   AND   TISSUE    RESPIRATION 
TABLE  IV  (continued) 


259 


Age 

Weight 
(kg) 

Q02 

No 

Species 

Breed 

Sex 

years 

months 

Brain 
cortex 

Kidney 
cortex 

Liver 

Spleen 

Lung 

I 

Dog 

Mongrel 

m 

12. 1 

—  20.7 

—  24.7 

—  12.0 

—  7.2 

—  4-5 

2 

,, 

^, 

m 

12.5 

—  19.9 

—  24.5 

—  12.2 

—  6.2 

—  4.9 

?, 

,, 

,, 

f 

18.2 

-18.3 

—  25-3 

—  12.7 

—  6.2 

—  3-9 

4 

,, 

,. 

m 

22.5 

—  22.4 

—  32.0 

—  10.5 

-6.5 

—  4.6 

5 

- 

" 

f 

14.1 

—  24.5 

—  28.7 

—  II. I 

—  7-1 

—  6.4 

I 

Sheep 

f 

2 

6 

72 

—  19-3 

—  26.9 

—  9-3 

—  7.2 

—  7.0 

0 

^^ 

m 

6 

36 

—  18.6 

—  31-3 

-8.3 

—  10.5 

—  51 

^ 

,, 

Cheviot 

f 

4 

0 

63 

—  19.6 

—  27.1 

-7.8 

—  6.8 

—  4-7 

4 

^^ 

Scotch 

m 

8 

36 

—  22.4 

—  26.1 

—  9.2 

—  5-5 

—  5-2 

5 

^^ 

Massam 

f 

7 

41 

—  20.2 

—  29.9 

—  9.6 

—  6.5 

—  5.8 

6 

■• 

Cheviot 

f 

I 

6 

45 

—  18.3 

—  23.6 

—  6.6 

—  4.8 

—  4-7 

I 

Cattle 

Cross 

f 

3 

6 

320 

—  17.9 

—  22.8 

—  8.2 

—  4.2 

—  4.9 

2 

,, 

Short  horn 

f 

4 

6 

380 

—  20.1 

—  22.0 

—  7-9 

—  4.2 

—  3-9 

3 

,, 

,, 

f 

4 

6 

510 

—  16.5 

—  23.6 

—  8.0 

—  4.2 

—  3-9 

4 

„ 

,, 

m 

4 

0 

440 

— 18.1 

—  21.9 

—  7-3 

—  4.9 

—  3-3 

5 

,, 

Aberdeen  angus 

m 

2 

0 

570 

—  13-4 

—  30-3 

—  9.6 

—  4.4 

-4.8 

6 

•• 

Short  horn 

f 

3 

0 

320 

—  17-3 

—  19.2 

—  8.1 

—  4-7 

—  4.2 

I 

Horse 

Shire 

f 

25 

0 

610 

—  16.4 

—  18.2 

—  6.1 

—  4.4 

—  5-3 

2 

,, 

Cross 

f 

15 

0 

610 

—  17.6 

—  21.0 

—  5-7 

-3-8 

—  4.6 

3 

,, 

Shire 

m 

10 

0 

790 

—  17.4 

—  23-5 

—  6.1 

—  4.9 

—  4.1 

4 

,, 

,, 

m 

6 

0 

790 

—  12.0 

—  22.6 

—  4.0 

—  4.4 

—  4.0 

5 

,, 

,, 

f 

7 

0 

760 

-16.5 

—  19.1 

—  5-9 

—  4.1 

—  4.6 

6 

•• 

m 

18 

0 

790 

—  14.1 

—  24-5 

—  4-5 

-3-8 

—  4.1 

TABLE  V  ' 

AVERAGE   Q02  OF  5   TISSUES   OF  9  MAMMALIAN   SPECIES  COMPARED  WITH   AVERAGE  BASAL  HEAT 
PRODUCTION    (Q02    MEASURED    IN    MEDIUM    II) 

(The  average  Q02  values  are  computed  from  Table  IV.  The  data  on  average  basal  heat  production 
per  kg  bodyweight  are  taken  from  Benedict's  graphs*^.  The  heat  data  refer  to  animals  of  the  average 
body  weight  given  in  the  third  vertical  column). 


Bodyweight  (kg) 

Q02 

Basal  heat 

production/kg 

Species 

Range 

Mean 

Brain 
cortex 

Kidney 
cortex 

Liver 

Spleen 

Lung 

bodyweight  in 
24  hours  (Cal) 

Mouse 

0.012-0.035 

0.021 

—  32.9 

—  46.1 

—  23.1 

—  16.9 

— 12.0 

158 

Rat 

0.18-0.22 

0.21 

—  26.3 

-38.2 

—  17.2 

—  12.7 

—  8.6 

100 

Guinea  pig 

0.42-0.74 

0.51 

—  27-3 

-31.8 

—  13.0 

—  II. 6 

-8.5 

82 

Rabbit 

1. 03-1. 36 

1.05 

—  28.2 

—  34-5 

—  11.6 

—  14.2 

—  8.0 

60 

Cat 

2.39-3. 1 1 

2-75 

—  26.9 

• —  22.7 

—  13.2 

-8.4 

—  3-9 

50 

Dog 

12. 1-22.5 

15-9 

—  21.2 

—  27.0 

—  II. 7 

—  6.6 

—  4-9 

34 

Sheep 

36-72 

49 

—  19.7 

—  27-5 

-8.5 

—  6.9 

—  5-4 

25 

Cattle 

320-570 

420 

—  17.2 

—  23-5 

—  8.2 

—  4.4 

—  4-3 

20 

Horse 

610-790 

725 

—  15-7 

—  21.5 

—  5-4 

—  4.2 

—  4.4 

17 

As  already  stated  the  values  for  Qq^  were  calculated  from  the  pressure  changes 
recorded  between  15  and  60  min  after  the  start  of  the  incubation  period.  The  rate  of 
oxygen  uptake  often  showed  a  progressive  fall  during  the  45  min  of  observation,  and 
References  p.  26y—26g. 


26o 


H.  A.  KREBS 


VOL.  4  (1950) 


Q02  values  calculated  for  the  period  of  incubation  between  20  to  40  min  were  therefore 
as  a  rule  somewhat  higher  than  those  given  in  the  Table.  In  the  case  of  brain  and  kidney 
the  difference  was  no  greater  than  5%.  In  the  case  of  the  other  three  tissues  it  was  of 
the  order  of  10%. 

3.  Qq^  in  saline  containing  calcium  and  low  in  phosphate  and  bicarbonate  {medium  III) 

On  each  of  the  9  species  i  or  2  experiments  were  carried  out  in  which  Q02  was 
measured  at  the  same  time  in  media  II  and  III.  These  experiments  showed  that  in 
general  the  Q02  values  calculated  from  the  early  readings  (20  to  40  min)  tended  to  be 
somewhat  lower  in  medium  III,  but  the  progressive  fall  with  time  was  less  in  this 
medium,  and  the  Q02  values  calculated  for  the  15  to  60  min  period  were  within  10% 
the  same  in  the  case  of  kidney  cortex,  lung  and  spleen  in  all  9  species.  On  the  other  hand 
Q02  for  brain,  and  some  species  of  liver,  was  considerably  lower  in  medium  III,  and  of 
these  2  tissues  further  specimens  were  examined.  The  results  are  given  in  Tables  VI 
and  VII. 

TABLE  VI 

QO2    OF    BRAIN    CORTEX    AND    LIVER    OF    Q    MAMMALIAN    SPECIES 

(Slices  suspended  in  medium  III  (low  in  bicarbonate;  containing  calcium) ;  the  data  are  the  averages 


of  duplicate  determinations). 

Age 

Weight 
(kg) 

Q02 

No 

Species 

Breed 

Sex 

years 

months 

Brain 
cortex 

Liver 

I 

Mouse 

Albino 

m 

0.045 

—  19.9 

—  15-6 

2 

,, 

,, 

m 

0.044 

—  22.9 

3 

,, 

,, 

m 

0.031 

—  24.4 

4 

,, 

,, 

m 

0.040 

—  23.2 

—  20.2 

5 

,, 

,, 

f 

0.009 

—  24-3 

—  22.3 

6 

,, 

Black 

m 

0.020 

-18.3 

7 

.' 

■' 

m 

0.013 

—  20.3 

I 

Rat 

Albino 

m 

0.27 

—  20.6 

—  12.3 

2 

,, 

,, 

m 

0.18 

—  20.8 

—  17.4 

3 

,, 

,, 

m 

0.37 

—  17.9 

—  13-9 

4 

,, 

,, 

m 

0.24 

-18.3 

— 14.0 

5 

'■ 

" 

m 

0.25 

-18.5 

—  15-5 

I 

Guinea  pig 

m 

0.58 

-18.5 

—  6.07 

1 

,, 

m 

0.58 

—  20.0 

—  6.60 

3 

,, 

m 

0.41 

—  17.4 

—  9-50 

4 

,, 

m 

0.28 

—  16.4 

— 11.6 

5 

" 

m 

0.50 

-15-8 

—  9-95 

I 

Rabbit 

Chinchilla 

I. II 

—  15-3 

-7.6 

2 

,, 

,, 

0.93 

—  15.0 

—  8.1 

3 

,, 

,, 

1. 12 

-15-6 

—  7-5 

4 

,, 

1-53 

—  15.6 

-7.8 

5 

■• 

" 

1-35 

—  14.2 

—  6.9 

I 

Cat 

m 

311 

—  14.9 

—  9.4 

2 

" 

m 

2.39 

— 16.1 

—  II. 0 

I 

Dog 

Mongrel 

f 

18.2 

—  16.0 

—  12.9 

2 

,, 

,, 

m 

22.5 

—  13.8 

—  9-5 

3 

" 

" 

f 

14.1 

—  14-5 

—  9.9 

References  p.  26y-26g. 


VOL.  4  (1950) 


BODY    SIZE   AND   TISSUE    RESPIRATION 


261 


TABLE  VI  (continued) 


Age 

Weight 
(kg) 

Q02 

No. 

Species 

Breed 

Sex 

years 

months 

Brain 
cortex 

Liver 

I 

Sheep 

Scotch 

m 

0 

9 

27 

—  12.4 

—  7.2 

2 

„ 

f 

2 

6 

36 

—  10. 0 

—  8.6 

3 

Cheviot 

f 

I 

6 

45 

—  10. 0 

—  6.7 

4 

Sussex 

f 

0 

8 

41 

.^  10. 1 

—  3-5 

5 

'■ 

Lincolnshire  crossbred 

m 

0 

8 

27 

—  14.1 

—  5-^ 

I 

Cattle 

Shorthorn 

m 

4 

6 

280 

—  2.6 

•7 

Shorthorn  crossbred 

m 

3 

0 

290 

—  1^-3 

—  4-3 

3 

Shorthorn 

f 

3 

0 

320 

—  15-4 

-3-8 

4 

m 

2 

6 

380 

—  10.8 

—  4.0 

5 

,, 

f 

4 

6 

320 

—  10.6 

— ^2.2 

6 

" 

f 

3 

6 

290 

—  11.4 

—  3-5 

I 

Horse 

Shire 

m 

18 

0 

790 

—  10.0 

—  1.8 

2 

,, 

,, 

f 

7 

0 

760 

—  8.64 

—  2.4 

3 

Belgian 

m 

15 

0 

710 

—  13-7 

—  2.5 

4 

,, 

Shire 

f 

10 

0 

760 

—  "•5 

—  3-2 

5 

" 

" 

m 

13 

0 

760 

—    8.78 

—  2.9 

TABLE  VII 

AVERAGE    Q02    OF   BRAIN    CORTEX    AND    LIVER    OF    9    MAMMALIAN  SPECIES    COMPARED    WITH    AVERAGE 
BASAL    HEAT    PRODUCTION     (Qq^    MEASURED    IN    MEDIUM    III) 

(The  average  Q02  values  are  computed  from  Table  VI.  The  average  basal  heat  production  is  taken 
from  Benedict's*^  graphs 


Body  weight 

Oo- 

Basal  heat  pro- 

Species 

(kg) 

duction/kg  body- 

Range 

Mean 

Brain  cortex 

Liver 

weight  in  24  hours 

Mouse 

0.009-0.045 

0.038 

—  22.9 

145 

0.009-0.045 

0.026 

—  19-3 

152 

Rat 

0.176-0.365 

0.26 

—  19.2 

— 14.6 

92 

Guinea  pig 

0.279-0.58 

0.44 

—  17.4 

—  9.5 

85 

Rabbit 

0.93-I-53 

1. 21 

—  15. 1 

-7.6 

57 

Cat 

2.39-3. II 

2-75 

—  15-5 

—  10.2 

50 

Dog 

14. 1-22.5 

18.3 

—  14.8 

—  10.8 

31 

Sheep 

27-45 

35 

—  II-3 

—  6.2 

27 

Cattle 

280-380 

320 

— 12. 1 

—  3-6 

21 

Horse 

710-790 

760 

—  10.5 

—  2.6 

17 

4.  Qq^  in  saline  serum  substitute  {medium  I) 

In  order  to  decide  whether  the  difference  between  the  Q02  values  obtained  for  brain 
and  liver  in  media  II  and  III  were  due  to  the  differences  in  the  calcium  content,  or  in 
the  bicarbonate  and  phosphate  content,  comparative  measurements  were  made  on  the 
same  tissue  sample  in  media  I,  II  and  III.  The  'indirect'  method  of  Warburg^^  was  used 
for  the  measurements  in  medium  I,  in  preference  to  those  of  Dickens  and  Simer"  or 
Dixon  and  Keilin^^,  because  with  this  method  it  is  possible  to  follow  the  time  course 
of  the  oxygen  uptake.  Duplicate  sets  of  vessels  were  used  in  each  measurement.  Q02  was 
again  calculated  for  the  15  to  60  min  period  of  incubation.  The  results  of  the  comparative 
measurements  are  given  in  Table  VIII. 
References  p.  26y~26g. 


262 


H.  A.   KREBS 


VOL.  4  (1950) 


TABLE  VIII 

COMPARATIVE    MEASUREMENTS    OF    Q02    IN    3    DIFFERENT    SALINE    MEDIA 


Species 

Q02 

Tissue 

Medium  I 
(Containing  physio- 

Medium II 
(Phosphate 

Medium  III 
(Low  in  bicarbonate; 
containing  calcium) 

logical  concentrations 
of  HCO3'  and  CO2) 

buffered, 
no  calcium) 

Brain  cortex 

Guinea  pig 

—  18.6 

—  34-2 

—  16.4 

»          It 

Rabbit 

—  17-5 

—  23.9 

—  15-6 

ft          It 

Sheep 

—  13-5 

-17.6 

—  12.4 

I. 

Cattle 

—    9-9 

—  15-9 

—  10.8 

,. 

Horse 

—  137 

—  16.5 

—  13-7 

Liver 

Mouse 

—  19.6 

—  18.6 

—  20.2 

J, 

Guinea  pig 

—  10.8 

— ■  12.2 

—  11.6 

jj 

Rabbit 

—  10.3 

—  9.9 

—  8.1 

jj 

Sheep 

—  5-2 

—  6.0 

—  5-1 

,, 

Cattle 

-3-6 

—  4-7 

—  3-5 

" 

Horse 

—  2.7 

—  3-2 

—  2.9 

C.    DISCUSSION    OF    RESULTS 

I.  Comparison  of  the  Qq^  values  obtained  in  the  3  media 

Kidney  cortex,  spleen  and  liver  gave  about  the  same  Q02  in  all  three  media,  but 
differences  exceeding  10%  were  found  in  brain  cortex  and  in  liver.  A  comparison  of  the 
data  from  Tables  V  and  VII  (see  Table  IX)  shows  that  the  average  Q02  values  for  brain 
cortex  in  medium  II  were  between  37  and  87%  higher  than  those  obtained  in  medium  III. 
In  the  case  of  the  liver  the  differences  were  smaller;  they  are  of  doubtful  significance  in 
the  small  animals  (mouse,  rat)  and  increase  approximately  (though  not  strictly)  parallel 
with  the  body  weight  of  the  species,  being  greatest  in  cattle  and  horse. 


TABLE  IX 

DIFFERENCES  IN  THE  AVERAGE  QO2  VALUES  IN  MEDIA  II   (CONTAINING  NO  Ca)   AND  III  (CONTAINING  Ca) 

(The  figures  show  the  level  of  gMedium  II  expressed  as  per  cent  of  gMedium  III_  calculated  from  the 
data  in  Tables  V  and  VII). 


/-^Medium  II 

pvMedium  III 

V^Oa 

Species 

Brain  cortex 

Liver 

Mouse 

144 

120 

Rat 

137 

118 

Guinea  pig 

157 

137 

Rabbit 

187 

153 

Cat 

174 

129 

Dog 

143 

108 

Sheep 

174 

137 

Cattle 

142 

227 

Horse 

150 

208 

References  p.  26y-26g. 


VOL.  4  (1950)  BODY    SIZE   AND   TISSUE    RESPIRATION  263 

According  to  Table  VIII,  media  I  and  III  give  approximately  the  same  Q02  values. 
The  considerable  differences  in  the  concentration  of  bicarbonate,  CO2  and  phosphate  in 
these  two  media  have  thus  no  major  effect  on  the  Qq^  under  the  conditions  tested.  Since 
medium  I  resembles  the  physiological  environment  more  closely  than  the  other  media, 
Qop,  values  obtained  with  this  medium  might  be  regarded  as  approximating  more  closely 
to  the  physiological  value  than  higher  values  found  for  brain,  and  the  liver  of  the  larger 
animals  in  medium  II.  The  latter  are  not  likely  to  be  standard  Q02  values  but  no  definite 
statement  can  be  made  on  this  point  because  reliable  data  on  the  O2  consumption  of 
tissue  in  vivo  are  too  scanty.  In  experiments  of  Noell  and  Schneider^^  the  Og  con- 
sumption of  dog  brain  cortex  in  vivo  was  4.5  ml  per  minute  per  100  g  fresh  weight,  and 
on  the  assumption  that  the  dry  weight  of  dog  brain  cortex  is  21%  of  the  fresh  weight^°° 
Q02  i^  vivo  was  -12.9.  This  value  is  in  good  agreement  with  the  figure  of  -14.8  found 
in  medium  III  (Table  VII)  and  favours  the  view  that  the  values  found  for  brain  in  the 
Ca-free  medium  II  are  abnormally  high. 

Effects  of  calcium  in  the  Q02  of  slices  and  homogenates  have  been  described  before 
and  have  recently  been  reviewed  by  Cutting  and  McCance^".  Elliott  and  Libet^ 
found  that  Ca  depresses  the  initial  rate  of  respiration  of  brain  homogenates,  but  delays 
the  falling  off  at  the  later  stages  of  incubation,  thus  steadying  the  rate  of  respiration. 
It  does  not  seem  to  have  been  noted  before  that  the  effect  of  Ca  on  tissue  slices  is  greater 
in  brain  than  in  other  tissues. 

Whilst  there  is  some  uncertainty  as  to  which  of  the  values  obtained  in  the  different 
media  constitute  the  'basal'  Qq^,  it  should  be  stated  that  the  conclusions  drawn  in  the 
following  sections  are  not  affected  by  this  uncertainty. 

2.  Absolute  level  of  Qq^ 

The  Q02  values  in  all  3  media  tend  to  be  considerably  higher  than  the  values  re- 
ported in  the  literature  for  saline  media^"^,  especially  in  the  case  of  brain,  liver  and 
kidney.  However,  no  strict  comparison  is  possible  because  different  substrates  were  used 
in  previous  measurements.  The  combination  of  substrates  added  in  the  present  experi- 
ments give,  in  general,  higher  values  than  the  substrates  added  in  most  previous  work 
(glucose  or  lactate).  The  Q02  values  observed  in  the  new  media  are  of  the  same  order 
as  the  highest  values  recorded  for  serum.  Thus  the  intention  to  include  in  the  new  media 
the  substances  in  serum  which  stimulate  respiration^^  seems  to  have  been  accomplished. 

3.  Qq^  and  body  size 

General  survey.  The  data  given  in  Tables  IV,  V  and  VII  show  that  the  Q02  values 
of  the  tissues  of  the  larger  species  are,  in  general,  somewhat  lower  than  the  homologous 
values  of  the  smaller  species.  But  there  are  many  exceptions  to  this  general  rule.  No 
strict  parallelism  exists  between  the  Q02  values  of  the  homologous  tissues  and  the  basal 
heat  p-odaction  per  unit  body  weight  of  the  intact  animal.  The  Q02  values  for  brain, 
kidney,  spleen,  and  lung  change  much  less,  and  those  for  liver  slightly  less,  with  the 
body  weight  than  the  rate  of  basal  heat  production.  Neither  is  there  a  simple  correlation 
between  body  size  and  Q02  within  the  same  species.  The  body  weights  of  the  7  mice 
listed  in  Table  IV  varied  between  9  and  35  g  and  that  of  the  5  mice  listed  in  Table  VI 
between  9  and  45  g.  There  were  variations  between  36  and  72  kg  in  the  body  weight  of 
the  6  sheep  of  the  first  series.  These  differences  of  the  body  weight  within  one  species 
are  not  reflected  by  differences  in  the  Q02  values,  with  the  doubtful  exception  of  the 
References  p.  267-269. 


264  H.  A.  KREBS  VOL.  4  (1950) 

values  for  brain  in  Table  VI,  where  the  brains  of  the  2  smaller  sheep  show  higher  values 
than  the  3  brains  from  the  larger  animals. 

Brain  cortex.  In  the  largest  species  (horse)  the  average  Q02  of  brain  cortex  was  about 
half  the  average  Q02  value  of  the  smallest  species  (mouse)  nam.ely  48  %  for  the  measure- 
ments in  medium  II,  and  46%  for  the  measurements  in  medium  III.  In  contrast,  the 
basal  heat  production  per  kg  bodyweight  of  the  horse  is  only  11%  and  12%  respectively 
of  that  of  the  mouse. 

Kidney  cortex.  The  changes  of  the  Q02  values  from  species  to  species  in  this  tissue 
were  similar  to  those  of  brain  cortex.  The  average  Q02  value  of  horse  kidney  cortex  was 
47%  of  that  of  mouse  kidney.  The  average  Q02  value  for  sheep  kidney  was  only  14% 
below  that  for  guinea  pig  kidney,  whilst  the  basal  heat  production  per  kg.  bodyweight 
of  the  sheep  is  only  37%  of  the  guinea  pig.  Thus  the  decrease  of  the  Q02  values  with  body 
size  was  again  much  smaller  than  the  decrease  in  the  rate  of  the  basal  heat  production. 

Spleen,  Mug.  For  the  horse  the  Q02  value  of  spleen  tissue  was  about  a  quarter,  and 
for  lung  about  one  third,  of  the  corresponding  values  for  the  mouse.  In  these  two  tissues 
the  discrepancies  between  the  changes  in  Q02  and  the  changes  in  basal  heat  production 
in  relation  to  body  size  are  thus  not  as  great  as  in  brain  and  kidney,  but  they  are  still 
considerable. 

Liver.  Liver  shows  greater  Q02  changes  with  body  weight  than  any  other  tissue 
tested,  especially  in  medium  III  (Tables  V  and  VII).  In  medium  II  Q02  of  horse  liver 
was  23%,  and  in  medium  III  it  was  13.5%  of  that  of  mouse  liver.  Thus,  when  comparing 
the  Q02  values  obtained  in  medium  II  for  these  two  species,  about  the  same  percentage 
change  is  found  as  for  the  basal  rate  of  heat  production.  But  the  parallelism  over  the 
whole  series  of  species  is  very  imperfect.  For  example,  the  Q02  values  for  guinea  pig,  cat 
and  dog  are  about  the  same  (-9.5;  -10.2;  -10.8),  whilst  the  basal  rate  of  heat  production 
shows  a  progressive  fall  with  body  weight  (85;  50;  31). 

The  changes  of  Q02  of  liver  with  body  weight  reported  in  this  paper  are  similar  to 
those  found  by  Kleiber^,  but  owing  to  the  differences  in  the  media  used  the  present 
Q02  values  are  all  higher  than  those  reported  by  Kleiber. 

4.  Rdle  of  muscle  tissue  in  chemical  temperature  control 

As  the  rate  of  respiration  of  a  number  of  homologous  tissues  of  animals  of  different 
sizes  fails  to  show  a  strict  parallelism  with  the  basal  rate  of  heat  production  of  the  intact 
body,  it  remains  to  be  explained  how  the  characteristic  differences  in  the  basal  rates  of 
heat  production  of  animals  of  different  sizes  arise.  One  kind  of  explanation  is  contained 
in  various  publications  by  Kestner^''^'  '^^^  and  Blank^"'*,  who  stated  that  the  proportion 
of  highly  active  organs  is  somewhat  greater  in  the  body  of  small  animals  than  in  that 
of  large  animals.  He  expressed  the  view  that  the  "relative  size  of  the  brain  and  the  large 
glands  can  give  a  complete  explanation  of  the  different  heights  of  metabolism  in  different 
animals^"^".  This  view  is  not  substantiated  by  quantitative  measurements  and  such 
data  as  are  available  cannot  be  reconciled  with  Kestner's  hypothesis  (see  Kleiber*). 

An  alternative  explanation  is  offered  by  the  conception  that  the  relation  between 
Q02  and  body  weight  found  for  the  5  tissues  tested  does  not  hold  for  every  tissue ;  that 
there  is  at  least  one  major  tissue  whose  "basal"  Q02  changes  with  the  body  weight 
approximately  parallel  with  the  basal  heat  production;  that  this  organ  is  the  striated 
musculature. 

The  substance  of  this  conception  is,  of  course,  not  new  in  that  it  is  commonly 
References  p.  262-269. 


VOL.  4  (1950)  BODY    SIZE    AND   TISSUE    RESPIRATION  265 

accepted  that  the  muscles  play  a  leading  part  in  the  regulation  of  heat  production. 
Evidence  in  support  of  this  conception  is  the  increased  muscular  activity  on  exposure 
to  cold,  manifesting  itself  by  increased  tension  and  shivering,  and  the  failure  of  the 
curarized  animal  to  maintain  the  physiological  temperature  level  on  exposure  to  cold. 
It  has  not  been  directly  demonstrated  that  the  basal  respiration  of  the  musculature 
varies  with  body  size  in  the  postulated  fashion,  and  no  satisfactory  experimental  proce- 
dure has  as  yet  been  devised  to  carry  out  the  necessary  measurements.  Data  obtained 
on  isolated  muscles  bear  no  simple  relation  to  the  basal  respiratory  rate  of  the  muscle 
in  situ  because  the  Qq^  of  muscle  depends  more  than  that  of  any  other  tissue  on  the  state 
of  activity  of  the  tissue.  Activity  may  cause  a  thirty-fold  rise  of  the  resting  rate  of 
respiration  (Barcroft^"^,  Meyerhof^"'^).  As  the  state  of  activity  is  controlled  by  the 
higher  nervous  centres  detachment  from  the  nervous  system  is  bound  to  affect  the  rate 
of  respiration. 

5.  Factors  determining  the  level  of  tissue  respiration 

If  body  size  is  not  a  major  factor  determining  the  O02  of  the  5  tissues  tested  it 
remains  to  be  examined  which  other  factors  control  the  level  of  respiration  of  these 
tissues.  As  the  respiration  of  living  tissues  primarily  serves  to  supply  energy,  the  level 
of  tissue  respiration  is  expected  to  be  determined  by  the  energy  requirements.  A  variety 
of  factors  contribute  to  the  requirements.  They  may  be  classed  in  three  groups : 

1.  Energy  is  required  when  tissues  perform  mechanical,  osmotic,  chemical,  or  other 
kinds  of  external  work. 

2.  Energy  is  required  to  maintain  structures  which  are  thermodynamically 
unstable.  An  example  is  the  maintenance  of  concentration  gradients  between  tissue  and 
blood  plasma  of  readily  diffusable  substances,  such  as  inorganic  ions,  amino  acids, 
coenzymes. 

3.  Energy  is  required  to  maintain  the  body  temperature. 

Energy  generated  for  the  first  two  purposes  always  yields  heat  as  a  by-product  and 
in  homeotherms  this  heat  is  partly,  or  wholly,  utilised  to  maintain  the  body  temperature. 
In  an  organism  performing  some  physical  exercise,  and  living  at  a  temperature  not  far 
removed  from  that  of  the  body  temperature,  the  heat  arising  as  a  by-product  may  be 
enough  for  the  upkeep  of  the  body  temperature.  In  a  cold  environment  the  heat  arising 
as  a  by-product  in  a  resting  organism  ma}^  no  longer  be  sufficient  to  maintain  the  body 
temperature,  and  extra  heat  has  to  be  formed.  It  is  reasonable  to  assume  that  the  highly 
differentiated  cells  whose  task  it  is  to  carry  out  specialised  functions,  as  do  those  of 
brain  or  the  glands,  are  designed  to  deal  solely  with  these  specialized  functions  rather 
than  to  act  as  heat  generators  in  the  case  of  exceptional  loss  of  heat.  The  extra  source 
of  heat  might  be  expected  to  be  the  muscle  tissue  which,  for  other  reasons,  has  the 
capacity  of  varying  the  rate  of  heat  production.  If  this  assumption  is  correct,  in  other 
words,  if  the  level  of  respiration  of  highly  specialized  tissues  is  determined  by  the  energy 
requirement  falling  under  categories  (i)  and  (2),  it  is  to  be  expected  that  the  rate  of 
energy  production  of  the  highly  differentiated  cells  is  not  dependent  on  the  size  of  the 
animal,  because  the  energy  needed  for  the  performance  of  a  given  piece  of  work  is  inde- 
pendent of  the  size  of  the  body. 

However,  somewhat  different  from  the  question  of  energy  requirements  of  the 
highly  differentiated  cells  is  the  problem  of  the  energy  requirements  of  organs  as  a  whole. 
Homologous  organs  of  different  species  have  by  no  means  identical  structures.  For 
References  p.  26y-26g. 


266  H.  A.  KREBS  VOL.  4  (1950) 

example,  in  a  larger  species,  tissue  structures  accessory  to  the  main  functional  cells  are 
bound  to  constitute  a  relatively  larger  portion  of  the  organ  than  in  the  homologous 
tissue  of  a  smaller  animal.  Such  accessory  structures  are,  among  others,  blood  vessels, 
glandular  ducts,  connective  tissues. 

Thus  some  changes  of  the  Qq^  values  with  body  size  may  be  expected  in  homologous 
tissues  even  if  the  Q02  of  homologous  cells  is  the  same.  In  general  the  change  will  be  a 
decrease  with  body  size  because  cells  with  lower  respiration,  like  those  of  connective 
tissue,  blood  vessels  and  ducts,  are  bound  to  became  more  preponderant  in  the  larger 
species.  The  changes  in  the  Q02  with  body  size,  seen  in  Tables  V  and  VII,  may  in  part 
be  due  to  this  factor. 

SUMMARY 

The  factors  affecting  the  rate  of  respiration  in  isolated  tissues  are  discussed  with  reference  to 
the  measurement  of  a  "standard  rate"  of  metaboUc  processes  in  vitro.  Media  for  the  suspension  of 
tissues  are  devised ;  their  composition  is  essentially  based  on  the  available  analytical  data  for  blood 
plasma. 

Q02  of  liver,  brain  cortex,  kidney  cortex,  spleen,  and  lung  was  measured  for  9  mammalian  species 
of  different  body  size  (mouse,  rat,  guinea-pig,  rabbit,  cat,  dog,  sheep,  cattle,  horse).  Three  different 
media  were  used  ("phosphate  saline  without  Ca",  "saline  low  in  phosphate,  bicarbonate  and  COj" 
and  "saline  serum  substitute"  containing  physiological  concentrations  of  inorganic  ions  in  addition 
to  organic  substrates).  Kidney  cortex,  spleen,  and  liver  gave  about  the  same  Q02  values  in  all  three 
media.  Q02  for  brain  cortex  was  for  all  species  higher  in  the  medium  containing  no  Ca,  the  average 
level  being  37-87%  higher.  Q02  for  liver  was  also  higher  in  the  absence  of  Ca,  especially  in  the  larger 
species. 

Q02  values  of  the  tissues  of  larger  animals  were  in  general  somewhat  lower  than  the  homologous 
values  of  the  smaller  species  but  no  strict  parallelism  between  Q02  values  and  basal  heat  production 
of  the  intact  animal  was  found.  The  Q02  values  for  most  tissues  changed  much  less  with  the  body 
weight  than  the  rate  of  basal  heat  production. 

The  absolute  level  of  Q02  in  the  new  media  (which  apart  from  glucose  contain  pyruvate,  fumarate 
and  L-glutamate)  was  higher  than  the  values  reported  in  the  literature  for  saline  media.  They  are 
of  the  same  order  as  the  highest  values  recorded  for  serum. 

The  characteristic  differences  in  the  basal  rate  of  heat  production  in  animals  of  different  size 
are  to  be  attributed  mainly  to  variation  in  the  Q02  of  the  musculature.  It  is  suggested  that  the  Q02 
of  tissues  other  than  muscle  is  in  the  first  place  governed  by  the  specific  energy  requirements  of  the 
tissues,  and  not  by  the  heat  requirements  of  the  whole  body. 

RfiSUMfi 

Les  facteurs  qui  influencent  la  vitesse  de  la  respiration  dans  les  tissus  isol6s  sont  discut6s  par 
rapport  aux  mesures  d'une  "vitesse  standard"  des  processus  metaboliques  in  vitro.  L'auteur  d^crit 
des  milieux  de  sus{tnsion  de  tissus;  leur  composition  se  base  essentiellement  sur  les  donn^es  analyti- 
ques  connues  pour  le  plasma  sanguin. 

Le  Q02  a  ^te  determine  pour  le  foie,  le  cortex  du  cerveau  et  du  rhein,  la  rate  et  le  poumon  de 
9  especes  de  mammiferes  de  taille  diff6rente  (souris,  rat,  cobaye,  lapin,  chat,  chien,  mouton,  b^tail, 
cheval).  Trois  milieux  differents  ont  6te  employes,  le  "phosphate  salin  sans  Ca",  "la  solution  saline 
faible  en  phosphate,  bicarbonate  et  COg  et  "la  solution  saline,  rempla9ant  le  serum"  qui  contient  des 
concentration  physiologiques  d'ions  inorganiques  en  plus  du  substrat  organique.  Dans  les  trois 
milieux  le  cortex  rhenal,  la  rate  et  le  foie  donnerent  environ  les  memes  valeurs  de  Qo2-  Pour  le  cortex 
cervical  ce  facteur  ^lait  plus  ^lev^  pour  toutes  les  especes  animales  examinees  dans  les  milieux 
exempts  de  Ca.  En  m^yenne  les  valeurs  ^taient  de  37  a  87%  superieures.  Pour  le  foie  le  Q02  ^tait 
aussi  sup^rieur  en  absence  de  Ca,  surtout  dans  les  especes  plus  grandes. 

En  g^n^ral  les  valeurs  de  Q02  ^taient  plus  basses  pour  les  tissus  des  animaux  plus  grands  que 
les  valeurs  homologues  pour  les  animaux  plus  petits.  Cependant  nous  n'avons  pas  trouve  un  parallfe- 
lisme  stricte  entre  les  valeurs  de  Q02  et  la  production  de  chaleur  des  animaux  intacts. 

Dans  les  nouveaux  milieux  (qui,  a  part  le  glucose,  contiennent  du  pyruvate,  du  fumarate  et 
du  L-glutamate)  le  niveau  absolu  du  Q02  ^tait  plus  eleve  que  les  valeurs  rapportees  dans  la  litt^rature 
pour  une  solution  saline.  Elles  sont  du  meme  ordre  que  les  valeurs  les  plus  61evees  rapportees  dans  la 
litterature  pour  le  serum. 

References  p.  26^—26^. 


VOL.  4  (1950)  BODY    SIZE    AND   TISSUE    RESPIRATION  267 

Les  differences  caract^ristiques  dans  la  vitesse  de  base  de  la  production  de  chaleur  des  animaux 
de  differente  taille  doivent  etre  attributees  surtout  a  la  variation  du  Qo^  de  la  musculature.  L'auteur 
suggere  I'idee  que  le  Q02  des  tissus  autres  que  le  muscle  serait  gouverne  en  premier  lieu  par  les  besoins 
specifiques  d'energie  des  tissus  et  non  par  les  besoins  de  chaleur  du  corps  entier. 


ZUSAMMENFASSUNG 

Die  Faktoren,  welche  die  Geschwindigkeit  der  Atmung  in  isolierten  Geweben  beeinflussen, 
werden  diskutiert  und  zwar  mit  Riicksicht  auf  die  Messungen  einer  "Standardgeschwindigkeit" 
metabolischer  Prozesse  in  vitro.  Medien  fiir  Gewebesuspensionen  werden  vorgeschlagen,  deren 
Zusammensetzung  sich  hauptsachlich  auf  die  fiir  Blutplasma  bekannten  analytischen  Werte  grijndet. 

Der  Faktor  Q02  von  Leber,  Gehirnrinde,  Nierenrinde,  Milz,  und  Lunge  wurde  fiir  9  Saugetier- 
arten  verschiedener  Korpergrosse  (Maus,  Ratte,  Meerschweinchen,  Kaninchen,  Katze,  Hund,  Schaf, 
Vieh,  Pferd)  bestimmt.  Drei  verschiedene  Medien  wurden  verwendet,  namlich  "Phosphat-Salz- 
Losung  ohne  Ca",  "Salzlosung  mit  geringem  Gehalt  an  Phosphat,  Bicarbonat  und  CO2"  und  "Salz- 
losung-Serumersatz",  welche  ausser  anorganischen  lonen  in  physiologischen  Konzentrationen  orga- 
nische  Substrate  enthalt.  Nierenrinde,  Milz  und  Leber  gaben  ungefahr  dieselben  Q02- Werte  in  alien 
drei  Medien.  Der  Q02  der  Gehirnrinde  war  fiir  alle  Arten  in  dem  Ca-freien  Medium  hoher  und  zwar 
betrug  der  Unterschied  durchschnittlich  37  bis  87%.  Auch  fiir  die  Leber  lagen  die  Werte  hoher  in 
Abwesenheit  von  Ca  und  zwar  insbesondere  in  den  grosseren  Arten. 

Im  Allgemsinen  lagen  die  Q02- Werte  der  Gewebe  grosserer  Tiere  etwas  niedriger  als  die  homo- 
logen  Wtrte  kleinerer  Arten;  es  konnte  aber  kein  strenger  Parallelismus  zwischen  den  Qoj-Werten 
und  der  Warmebildung  unverletzter  Tiere  gefunden  werden.  Die  Q02" Werte  der  meisten  Gewebe 
varieren  viel  weniger  mit  dem  Korpergewicht  als  die  Geschwindigkeit  der  Warmebildung. 

Die  absolute  Lage  der  O02- Werte  war  in  den  neuen  Medien,  die  ausser  Glucose  nocn  Pyruvat, 
Fumarat  und  L-Glutamat  enthalten,  hoher  als  die  in  der  Literatur  fiir  Salzlosungen  beschriebenen 
Werte.  Sie  sind  von  der  gleichen  Grossenordnung  wie  die  hochsten  in  der  Literatur  fiir  Serum  ange- 
fiihrten  Werte. 

Die  charakteristischen  Unterschiede  in  der  Geschwindigkeit  der  Warmebildung  von  Tieren 
verschiedener  Korpergrosse  miissen  hauptsachlich  auf  die  Anderungen  des  Q02  in  der  Muskulatur 
zuriickgefiihrt  werden.  Die  Ansicht  wird  ausgesprochen,  dass  der  Q02  von  anderen  Geweben  als 
Muskeln  an  erster  Stelle  durch  die  Energiebediirfnisse  der  Gewebe  und  nicht  durch  den  Warmebedarf 
des  ganzen  Korpers  bedingt  wird. 


REFERENCES 

^  F.  Terroine  and  J.  Roche,  Conipt.  rend.  Acad.  Sci.  Paris,  180  (1925)  225. 
-  E.  Grafe,  H.  Reinwein,  and  V.  Singer,  Biochem.  Z.,  165  (1935)  102. 
^  M.  Kleiber,  Proc.  Soc.  exptl  Biol.  Med.,  48  (1941)  419. 

*  M.  Kleiber,  Physiol.  Rev.,  27  (1947)  511. 

5  K.  A.  C.  Elliott  and  F.  H.  Elliott,  /.  Biol.  Chem.,  127  (1939)  457. 

*  K.  A.  C.  Elliott  and  B.  Libet,  /.  Biol.  Chem.,  143  (1942)  227. 
''   H.  A.  Krebs,  Biochem.  J .,  29  (1935)  1620. 

*  A.  E.  Bender  {unpublished). 

^  E.  S.  G.  Barron,  Z.  B.  Miller,  and  G.  R.  Bartlett,  /.  Biol.  Chem.,  171  (1947)  791. 
^°  V.  P.  Dole,  /.  din.  Invest.,  23  (1944)  708. 

^^  J.  P.  Peters  and  D.  D.  van  Slyke,  Quantitative  Clinical  Chemistry  I  (1946). 
^^  P.  B.  Hamilton  and  D.  D.  van  Slyke,  /.  Biol.  Chem.,  150  (1943)  231. 
^^  F.  B.  Cramer  and  T.  Winnick,  /.  Biol.  Chem.,  150  (1943)  259. 
"  H.  N.  Christensen,  P.  F.  Cooper,  Jr.,  R.  D.  Johnson,  and  E.  L.  Lynch,  /.  Biol.  Chem.,  168 

(1947)  191- 
^*  G.  E.  GuTMAN  AND  B.  ALEXANDER,  /.  Biol.  Chem.,  168  (1947)  527. 
^^  S.  W.  HiER  AND  O.  Bergeim,  /.  Biol.  Chem.,  163  (1946)  129. 
'^  H.  D.  Hoberman,  /.  Biol.  Chem.,  idj  (1947)  721. 

^^  L.  Sheffner,  J.  B.  Kirsner,  and  W.  L.  Palmer,  /.  Biol.  Chem.,  175  (1948)  107. 
"  R.  M.  Archibald,  /.  Biol.  Chem.,  156  (1944)  '^^^■ 

2"  H.  A.  Krebs,  L.  V.  Eggleston,  and  R.  Hems,  Biochem.  J.,  44  (1949)  156. 
-^  H.  A.  Harper,  L.  W.  Kinsell,  and  H.  C.  Barton,  Science,  106  (1947)  319. 
2^  C.  W.  Denko,  W.  E.  Grundy,  and  J.  W.  Porter,  Arch.  Biochem.,  13  (1947)  481. 
2'  J.  K.  Parnas  and  J.  Heller,  Biochem.  Z.,  152  (1924)  i. 
2^  E.  J.  Conway  and  R.  Cooke,  Biochem.  J.,  33  (1939)  457. 


268  H.  A.  KREBS  VOL.  4  (1950) 

25  M.  J.  C.  Allinson,  /.  Biol.  Chem.,  157  (1945)  169. 

26  E.  M.  Mackay  and  L.  L.  Mackay,  /.  Clin  Invest.,  4  (1927)  295. 
2'  H.  A.  Bulger  and  H.  E.  Johns,  /.  Biol.  Chem.,  140  (1941)  427. 

28  B.  F.  Miller  and  D.  D.  van  Slyke,  /.  Biol.  Chem.,  114  (1936)  583. 

28  P.  B.  Hawk,  B.  L.  Oser,  and  W.  H.  Summerson,  Practical  Physiological  Chemistry,  J.  and   A. 

Churchill  Ltd.,  12th  Ed.  1947. 
•'"  L.  C.  Cook  and  R.  H.  Hurst,  /.  Physiol.,  79  (1933)  443. 

31  E.  Bueding,  H.  Wortis,  and  M.  Stern,  /.  Clin.  Invest.,  21  (1942)  85. 

32  A.  Vinet  and  Y.  Raoul,  Bull.  soc.  chim.  biol.,  24  (1942)  357. 

33  L.  Hagelstam,  Acta  chirurg.  Scand.,  go  (1944)  37. 

34  J   Thunberg,  Kgl.  Fysiograf.  Sallskap.  Lund  Forh.,  3  (1933)  No.  17. 

35  H.  A.  Krebs,  Biochem.  J.,  32  (1938)  108. 

36  H.  A.  Krebs,  unpublished. 

3'  T.  Thunberg,  Acta.  Med.  Scand.  Suppl.,  90  (1938)  122. 

38  A.  Taurog,  C.  Entenman,  and  I.  L.  Chaikoff,  /.  Biol.  Chem.,  156  (1944)  385. 

38  S.  J.  Thannhauser,  J.  Benotti,  and  H.  Reinstein,  /.  Biol.  Chem.,  129  (1939)  709. 

*°  R.  G.  Sinclair,  /.  Biol.  Chem.,  174  (1948)  343. 

"  J.  Schmidt-Thome,  G.  Schettler,  .\nd  H.  Goebel,  Hoppe-Seyler's  Z.  physiol.  Chem.,  283  (1948)63. 

*2  T.  E.  Weichselbaum  and  M.  Somogyi,  /.  Biol.  Chem.,  140  (1941)  5. 

■*3  S.  Sherlock  and  V.  Walshe,  Clin.  Sci.,  6  (1948)  223. 

**  E.  M.  M.  Hume  and  H.  A.  Krebs,  Medical  Research  Council  Special  Report  Series,  No.  264  (1948). 

•**    Vitamin  C  Subcommittee,  Medical  Research  Council,  unpublished  (1946). 

^^  S.  Sonne  and  H.  Sobotka,  Arch.  Biochem.,  14  (1947)  93. 

"  P.  M.  Hald,  /.  Biol.  Chem.,  103  (1933)  471. 

*^  P.  M.  Hald,  /.  Biol.  Chem  163  (1946)  429. 

^^  P.  M.  Hald,  A.  J.  Heinsen,  and  J.  P.  Peters,  /.  Clin.  Invest.,  26  (1947)  983 

5"  K.  Hoyer,  Act.  Med.  Scand.,  119  (1944)  562. 

51  H.  a.  Krebs,  Klin.  Wochschr.,  7  (1928)  584. 

52  C.  K.  Reiman  and  a.  S.  Minot,  /.  Biol.  Chem.,  42  (1920)  329. 

53  B.  L.  Vallee  and  J.  G.  Gibson,  2nd,  /.  Biol.  Chem.,  176  (1948)  445. 
5*  A.  I.  Burnstein,  Biochem.  Z.,  216  (1929)  449. 

55  A.  Taurog  and  I.  L.  Chaikoff,  /.  Biol.  Chem.,  163  (1946)  313. 

56  N.  B.  Talbot,  A.  M.  Butler,  A.  H.  Saltzman,  and  P.  M.  Rodigues,/.  Biol.  Chem.,  153  (1944)  479. 
5'  H.  WuLLE,  Z.  physiol.  Chem.,  260  (1939)  169. 

58  A.  Sturm  and  A.  Pothmann,  Z.  klin.  Med.,  137  (1940)  467. 

58  M.  H.  Power  and  E.  G.  Wakefield,  /.  Biol.  Chem.,  123  (1938)  665. 

6"  T.  V.  Letonoff  and  J.  G.  Reinhold,  /.  Biol.  Chem.,  114  (1936)  147. 

61  H.  A.  Krebs,  Hoppe-Seyler's  Z.  physiol.  Chem.,  217  (1933)  191. 

62  F.  Bernheim  and  M.  a.  L.  Bernheim,  /.  Biol.  Chem.,  106  (1934)  79;  107  (1934)  275. 

63  H.  Weil-Malherbe  and  H.  a.  Krebs,  Biochem.  J .,  29  (1935)  2077. 
6*  J.  Tosic,  Nature,  159  (1947)  544- 

65  H.  a.  Krebs,  Advances  in  Enzymol.,  HI  (1943)  191. 

66  O.  Rosenthal,  Biochem.  J.,  31  (1937)  1710. 

67  S.  Ringer,  /.  Physiol.,  3  (1882)  380;  4  (1883)  29  and  222;  7  (1886)  291. 

68  F.  S.  Locke,  Zentr.  Physiol.,  14  (1900)  670. 

68  G.  Barkan,  p.  Broemser,  and  a.  Hahn,  Z.  Biol.,  74  (1921)  i. 

"^^  O.  Warburg,  Biochem.  Z.,  152  (1924)  51. 

'1  H.  A.  Krebs  and  K.  Henseleit,  Hoppe-Seyler's  Z.  physiol.  Chem.,  210  (1932)  33. 

72  C.  O.  Guillaumin,  Bull.  soc.  chim.  biol.,  22  (1940)  571. 

73  L.  Reed  and  W.  Denis,  /.  Biol.  Chem.,  73  (1927)  623. 

''*  M.  H.  Power  and  E.  G.  Wakefield,  /.  Biol.  Chem.,  123  (1938)  665. 

75  J.  L.  Gamble,  Extracellular  Fluid,  Cambridge,  Mass.,  Harvard  University  Press,  1947. 

76  P.  K.  Smith  and  A.  H.  Smith,  /.  Biol.  Chem.,  107  (1934)  673. 

77  P.  J.  Boyle  and  E.  J.  Conway,  /.  Physiol.,  100  (1941)  i. 

78  F.  J.  Stare  and  C.  A.  Baumann,  Proc.  Roy.  Soc.  B.,  121  (1936)  33S. 
78   G.  D.  Greville,  Biochem.  J.,  31  (1937)  2274. 

8"  S.  R.  Elsden,  Biochem.  J.,  33  (1939)  1890. 

81  H.  A.  Krebs  and  L.  V.  Eggleston,  Biochem.  J.,  34  (1940)  442. 

82  A.  Kleinzeller,  Biochem.  J.,  34  (1940)  1241. 

83  M.  V.  Tyrode,  Arch,  intern,  pharmacodynamic,  20  (1910)  205. 
8*  O.  Warburg,  Biochem.  Z.,  142  (1923)  317. 

85  C.  O.  Warren,  /.  Biol.  Chem.,  156  (1944)  559. 

86  F.  Dickens  and  F.  Simer,  Biochem.  J.,  25  (1931)  985. 

87  A.  Lasnitzki  and  O.  Rosenthal,  Biochem.  Z.,  207  (1929)  120. 


VOL.  4  (1950)  BODY   SIZE    AND   TISSUE    RESPIRATION  269 

^^    J.  Leibowitz,  Biochem.  Z.,  226  {1930)  338. 

**^    B.  KiscH,  Biochem.  Z.,  273  (1934)  338. 

*"  M.  Cutting  and  R.  A.  McCance,  /.  Physiol.,  106  (1947)  405. 

*^  P.  J.  G.  Mann  and  J.  H.  Quastel,  Biochem.  J.,  40  (1946)  139. 

^^  F.  Dickens,  Biochem.  J.,  40  (1946)  145. 

®*  W.  C.  Stadie,  B.  C.  Riggs,  and  N.  Haugaard,  Am.  J.  Med.  Sci.,  207  (1944)  84. 

**  W.  Deutsch,  /.  Physiol.,  87  (1936)  56P. 

"5  F.  G.  Benedict,   Vital  Energetics,  Washington,  Carnegie  Institution,  1938. 

^^  O.  Warburg,  Biochem.  Z.,  152  (1924]  51. 

^'   F.  Dickens  and  F.  Simer,  Biochem.  J.,  25  (1931)  973. 

®^   M.  Dixon  and  D.  Keilin,  Biochem.  J.,  27  (1933)  86. 

®*   W.  NoELL  and  M.  Schneider,  quoted  in  FIAT  Review  of  German  Science,  Physiology .  Part  II 

(1948)  38;  Plliiger's  Arch.  ges.  Physiol,  250  (1948)  35. 
"^"^  E.  Abderhalden  and  A.  Weil,  Hoppe-Seyler's  Z.  physiol.  Chem.,  83. 
1°^  H.  A.  Krebs  and  W.  a.  Johnson,  Tabulae  Biolog.  Period.,  21. 
^"2  O.  Kestner,  Pfliiger's  Arch.  ges.  physiol.,  234  (1934)  290. 
^"^  O.  Kestner,  /.  Physiol.,  87  (1936)  39P. 
1"*  H.  Blank,  Pfliiger's  Arch.  ges.  Physiol.,  234  (1934)  310. 
1°^  J.  Barcroft,  Ergeb.  Physiol.,  i  (1907)  699. 
^o*  O.  Meyerhof,  Die  Chemischen  Vorgdnge  im  Muskel,  Berlin  1930. 

Received  April  29th,  1949 


270  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


SYNTHASE  ET  UTILISATION 

DE  L'AMIDON  CHEZ  UN  FLAGELLfi  SANS  CHLOROPHYLLE 

INCAPABLE  D'UTILISER  LES  SUCRES 

par 

ANDRfi  LWOFF,  h£l£:NE  IONESCO  et  ANTOINETTE  GUTMANN 
Service  de  Physiologie  microbienne,  Institut  Pasteur,  Paris  {France) 


On  connait  un  certain  nombre  de  microorganismes  incapables  d'utiliser  les  sucres 
comme  aliment  carbone.  Ce  sont  des  bacteries  comme  les  Moraxella  ou  des  flagellc3 
appartenant  a  des  groupes  divers  et  possedant  ou  non  de  la  chlorophylle :  Euglena  et 
Astasia  (Eugleniens),  Polytoma,  Chlorogonium  et  Hyalogonium  (Chlamydomonadines), 
Les  flagelles  synthetisent  tons  des  reserves  glucidiques  figurees:  paramylon  chez  les 
Eugleniens,  amidon  chez  les  autres,  dont  ils  sont  tres  abondamment  pourvus  a  certains 
stades  de  revolution  des  cultures  et  qui  diminuent  ou  meme  disparaissent  a  d'autres. 

Le  probleme  de  la  S5m these  et  de  I'utilisation  des  polysaccharides  par  un  micro- 
organisme  incapable  d'utiliser  les  sucres  se  trouvait  pose.  Nous  avons  etudie  de  ce  point 
de  vue  le  flagelle  Polytomella  coeca.  Nos  resultats  ont  ete  exposes  dans  une  note  prelimi- 
naire^.  Polytomella  est  un  flagelle  sans  chlorophylle.  II  se  developpe  en  culture  bacterio- 
logiquement  pure  dans  des  milieux  synthetiques  avec  un  sel  d'ammonium  comme  aliment 
azote,  de  I'acide  acetique  ou  de  I'ethanol  comme  aliment  carbone  energetique,  les  sels 
mineraux  habituels  et  deux  facteurs  de  croissance,  le  methyl-4  /3-hydroxyethyl-5 
thiazole  et  la  methyl-2  amino-4  aminomethyl-5  pyrimidine^.  II  possede  la  propriete  de 
se  multiplier  bien  entre  p^  3-0  et  8.0^,  propriete  precieuse  qui  a  deja  ete  mise  a  profit 
pour  I'examen  d'un  certain  nombre  de  problemes*. 

Aucun  Sucre  ne  pent  remplacer  I'acide  acetique  ou  I'ethanol  pour  la  nutrition  car- 
bonee.  En  particulier,  ni  le  glucose,  ni  le  maltose,  ni  le  saccharose,  ni  le  trehalose  ne  sont 
utilisables.  Des  essais  pour  obtenir  des  mutants  utilisant  les  sucres  ont  cchoue.  Dans  des 
milieux  pauvres  en  aliment  carbone,  par  exemple,  ethanol  a  i  p.  5000  ou  a  i  p.  10 000, 
c'est  la  teneur  en  ethanol  qui  limite  la  croissance.  Dans  ces  milieux  pauvres,  les  flagelles 
restent  vivauts  pendant  plus  de  3  mois.  Si  un  mutant  capable  d'utiliser  un  glucide 
apparaissait  dans  un  milieu  pauvre  additionne  de  glucose,  il  y  aurait  multiplication 
abondante.  Nous  n'avons  jamais  observe  ce  phenomene.  II  n'est  naturellement  pas 
possible  d'exclure  son  existence,  mais  Ton  pent  dire  que  la  probabilite  de  I'apparition 
d'un  mutant  utilisant  les  glucides  est  faible.  Enfin,  du  glucose  ajoute  a  des  cultures  en 
voie  de  developpement  en  presence  d'ethanol  ne  disparait  pas. 

L'incapacite  d'utihser  les  glucides  pour  la  nutrition  est  done  absolue.  Comment  les 
flagelles  synthctisent-ils  I'amidon,  et  comment  I'utilisent-ils  s'ils  sont  incapables  de 
metaboliser  les  glucides.  Tel  est  le  probleme  qui  va  etre  examine. 
Bibliographic  p.  2741275. 


VOL.  4  (1950) 


SYNTHESE    D'AMIDON    CHEZ   UN    FLAGELLE 


271 


TECHNIQUE 

Le  milieu  suivant  a  ete  utilis6:  sulfate  d'ammonium  i  g,  SO^Mg  +  7  HgO  o.i  g,  PO^HjK 
0.5  g,  ac6tate  de  sodium  i  g,  ^thanol  3  ml,  thiamine  0.0 r  mg,  eau  bidistiH6e  i  1,  soude  pour  p^  7.0. 
Apres  sterilisation  on  ajoute  du  fer  sous  forme  de  citrate  ferrique,  sterilise  a  part,  pour  obtenir  una 
concentration  finale  de  10  mg/1. 

On  utilise  pour  I'alimentation  carbon6e  un  melange  d'ac6tate  de  sodium  et  d'6thanol  afin  que 
le  Ph  ne  s'eloigne  pas  trop  de  la  neutralite.  Le  pn  augmente  en  efiet  notablement  lorsque  I'aliment 
carbone  est  represente  par  de  I'acdtate  de  sodium  —  liberation  d'ions  Na~  —  et  diminue  lorsque 
I'aliment  carbone  est  represente  par  de  I'^thanol  par  suite  de  la  liberation  non  compensee  des  ions 
SO4 —  de  I'aliment  azote.  Avec  le  melange  utilise,  il  n'y  a  pas  de  variation  impDrtante  du  pn.  et  11 
n'est  pas  necessaire  de  tamponner  le  milieu.  Seuls  des  milieux  acides  pauvent  d'ailleurs  etre  tamponnes 
efficacement  sans  inconvenient.  Les  flagell^s  ne  supportent  pas  une  concentration  de  phosphate 
M/25  a  ph  7-0  alors  qu'ils  se  developpent  a  pn  4-6  dans  des  milieux  renfermant  des  phosphates  a 
concentration  M/3.5. 

TABLEAU  I 

VARIATIONS    DE    LA    RESISTANCE    AU    PHOSPHATE    EN    RELATION    AVEC    LE    pjj 


M/ 

Apres  2  jours 

Apres  6  jours 

Apres  4  jours 

7-4 

7.2 

7.0 

6.6 

4.6 

7-4 

7.2 

7.0 

6.6 

4.6 

45 

+  + 

+  + 

+  + 

+  + 

+  + 

25 

+  + 

+  + 

20 

0 

0 

± 

+  + 

+  + 

+  + 

15 

0 

0 

0 

± 

+  + 

0 

0 

+ 

10 

0 

0 

+  + 

0 

+ 

4-5 

± 

3-5 

± 

+  + 

-\-  +  Culture  abondante,  plus  de  1 000  fiagelles///! 
+  200  a  1 000  flagelles//il 
i  I  a  20  fiageiles//il 
o  moins  de  i  flagelle/al 

Les  phosphates  utilises  sont  tous  des  phosphates  R.A  .L.  ou  Merck,  quality  Sorensen  ou  "pour  analyse". 

L'ensemencement  est  effectue  avec  20  ml  d'une  culture  jeune  dans  des  ballons  renfermant  4 
litres  de  milieu  aeres  par  barbotage  d'air  et  maintenus  a  24°.  Le  barbotage  doit  etre  menage  au  debut 
afin  de  ne  pas  diminuer  trop  la  tension  de  COg.  Dans  ces  conditions,  on  obtient  en  3  jours  des  cultures 
tres  abondantes.  Celles-ci  sont  centrifugees  dans  une  centrifugeuse  Sharpless.  A  grande  vitesse,  les 
flagelles  eclatent.  La  pate  blanchatre  est  broyee  avec  du  sable  fin  lave.  Le  broyat  est  mis,  suivant 
les  cas,  en  suspension  dans  un  tampon  de  phosphate  M/ioo  ou  de  citrate  M/50  de  pH  7-0.  Une  premiere 
centrifugation  a  faible  vitesse  ^limine  avec  le  culot  les  grains  d'amidon  et  des  debris  cellulaires.  Le 
liquide  trouble  qui  surnage  est  centrifuge  a  12000  tours  dans  une  centrifugeuse  angulaire  et  le  culot 
remis  en  suspension  dans  un  tampon. 


MISE    EN    EVIDENCE    D'UNE    PHOSPHORYLASE 


Les  preparations  enzymatiques  sont  additionnees  d'un  tampon  de  phosphate  et 
d'amidon  soluble.  La  concentration  en  phosphate  est  donnee  dans  le  Tableau  IL  La 
concentration  finale  en  amidon  est  de  2  a  5  mg/ml.  On  constitue  un  temoin  sans  amidon. 
Les  tubes  additionnes  de  toluene  sont  places  au  bain-marie  a  24°.  Apres  traitement  par 
I'acide  trichloracetique,  le  phosphate  mineral  est  dose  par  la  methode  de  Fiske  et 
SubbaRow^.  Le  Tableau  II  montre  les  resultats  de  quelques  experiences.  On  voit  qu'il 
y  a  disparition  du  phosphate  mineral  en  presence  d'amidon. 
Bibliographic  p.  2y4i2y5. 


2/2 


A.  LWOFF   Ct   al. 


VOL.  4  (1950) 


TABLEAU  II 


disparition  du  phosphore  mineral  en 
d'amidon 

PRESENCE 

A 

B 

c 

Enzyme  temoin* 
Enzyme  +  amidon 

47-5 
37-5 

58-5 
52.5 

49-5 
39 

P  mineral  en  yumol/ml. 

*  La  teneur  en  P  mineral  dans  les  preparations  temoins  est  identique  au  depart  et  a  la  fin  de 
1 'experience. 


RECHERCHE    DU    GLUCOSE-I-PHOSPHATE 


Nous  avons  done  cherche  a  mettre  en  evidence  le  glucose-i-phosphate.  II  n'est  pas 
possible  d'eliminer  des  preparations  brutes  le  phosphate  mineral  par  le  reactif  ammo- 
niaco-magnesien  car  le  glucose-i-phosphate  est  coprccipite. 

Nous  avons  done  utilise  la  technique  de  Le  Page  et  Umbreit,  variante  comportant 
la  precipitation  par  I'ethanol,  au  premier  stade,  des  sels  de  Ba.  Si  les  operations  sont 
repetees,  la  plus  grande  partie  du  P  mineral  est  eliminee.  Le  surnageant,  apres  la  derniere 
centrifugation  destinee  a  eliminer  le  sulfate  de  Baryum  est  amene  a  pn  8.2  avec  de  la 
potasse  et  le  sel  de  K  de  I'acide  glucose-i-phosphorique  precipite  par  I'alcool  est  seche 
sous  vide. 

L'hydrolyse  par  CIH  M  a  100°  (et  non  M/io  ainsi  qu'il  a  ete  imprime  par  erreur  dans 
notre  note  preliminaire)  libere  du  phosphate  mineral.  L'hydrolyse  est  complete  en  7  mi- 
nutes. Elle  libere  aussi  un  sucre  reducteur  qui  a  ete  identifie  au  glucose  par  la  forme 
cristalline  de  son  osazone  et  aussi  par  Taction  specifique  de  la  glucose-oxydase  (notatine). 
Le  dosage  a  ete  effectue  par  la  methode  de  Keilin  et  Hartree*'  a  la  notatine  et  par  la 
methode  de  Somogyi.  Le  tableau  suivant  montre  que  les  deux  techniques  donnent  des 
resultats  identiques  et  que  le  glucide  et  le  phosphate  sont  en  quantites  sensiblement 
equi-moleculaires. 

TABLEAU  III 

DOSAGE  DU  GLUCOSE  ET  DU  PHOSPHORE  DANS  DEUX  HYDROLYSATS 

(/imol/ml) 


Glucose:  methode  de  Somogyi 

Glucose:  methode  de  Keilin  et  Hartree 

P  mineral* 


*  Apres  soustraction  du  P  mineral  trouve  avant  hydrolyse:  A  0.15;  B  1.7  /^mol. 

Le  compose  isole  a  partir  des  preparations  presente  done  les  proprietes  suivantes: 
sels  de  Ba  soluble  a  p^  8.2  et  precipite  par  4  vol.  d'alcool.  Pas  de  precipitation  par  le 
reactif  ammoniaco-magnesien  (apres  purification).  Hydrolyse  complete  en  7  minutes 
a  100°  par  CIH  M.  Presence  de  glucose  et  de  phosphate  en  quantites  equimoleculaires. 
II  s'agit  done  bien  de  glucose-i-phosphate. 

En  presence  de  glucose- 1 -phosphate  (prepare  avec  la  phosphorylase  de  la  pomme  de 
terre  suivant  la  technique  de  Hanes)  et  de  dextrine  comme  amorce,  les  preparations 
Bibliographie  p.  27412^5. 


VOL.  4  (1950)  SYNTHESE    D'AMIDON    CHEZ   UN    FLAGELLE  273 

enzymatiques  liberent  du  phosphate  mineral.  Les  preparations  n'ayant  pu  etre  debarras- 
sees  des  traces  d'amylase,  la  synthase  d'amidon  n'a  pu  etre  mise  en  evidence.  Notons 
aussi  qu'il  n'apparait  pas  de  sucres  reducteurs  au  cours  de  la  liberation  du  P  mineral. 
II  nous  parait  fort  vraisemblable  que  Faction  de  la  phosphorylase  de  Polytontella  comme 
celle  des  phosphorylases  classiques  etudiees  par  W.  Kiessling',  C.  S.  Hanes^  et  par 
C.  F.  CoRi,  G.  Schmidt  et  G.  T.  Cori^  est  reversible. 

essai  de  purification  de  l'enzyme 

a)  La  preparation  est  traitee  par  le  sulfate  d'ammonium  au  tiers  de  saturation.  Le 
surnageant  reste  actif.  Par  contre,  I'activite  passe  dans  le  culot  apres  precipitation  par 
le  sulfate  d'ammonium  a  demi-saturation. 

b)  Si  la  preparation  est  centrifugee  dans  une  centrifugeuse  angulaire  de  Servall 
a  12000  tours  par  minute  pendant  10  minutes,  la  fraction  active  est  dans  le  culot. 
L'enzyme  est  lie  a  des  granules  qui  sont  visibles  au  microscope,  mais  que  nous  n'avons 
pas  identifies. 

Cette  centrifugation  elimine  la  plus  grande  partie  d'une  amylase  que  nous  n'avons 
cependant  pas  pu,  meme  apres  centrifugations  repetees,  eliminer  des  granules  contenant 
la  phosphorylase.  D'autres  essais  de  purification  n'ont  pas  ete  tentes. 

remarques  sur  l' amylase 

Les  preparations  d'amylase  dans  un  tampon  citrate  M/20  ne  donnent  pas  de  sucre 
reducteur  en  2  h  aux  depens  de  I'amidon,  mais  uniquement  des  dextrines.  Par  contre, 
les  preparations  maintenues  24  h  sous  toluene  en  1' absence  de  phosphate  mineral 
montrent  une  legere  activite  reductrice.  Le  sucre,  qui  est  vraisemblablement  du  maltose, 
n'a  pas  ete  identifie.  On  sait  que  le  maltose  n'est  pas  utilise  par  Polytontella  coeca.  Si  done 
la,  ou  les  amylases  intervenaient  seules  dans  I'utilisation  de  I'amidon,  leur  action  abouti- 
rait  a  un  glucide  qui  serait  perdu  pour  les  fiagelles  en  culture  bacteriologiquement  pure. 
II  est  fort  probable  que  I'amylase,  ou  des  amylases,  interviennent  dans  les  premiers 
stades  de  I'utilisation  des  grains  d'amidon,  et  que  les  dextrines  produites  au  debut  de 
I'attaque  sont  phosphorylees  et  donnent  du  glucose- i-phosphate  avant  que  le  stade 
glucide  reducteur  ne  soit  atteint. 

discussion 

On  connait  jusqu'ici  deux  voies  de  biosynthese  de  I'amidon:  par  la  phosphorylase 
classique  (C.Hanes,C.etG.Cori)  et  par  I'amylomaltase  (J.Monodet  A.M.Torriani^"). 
Ces  deux  enzymes  representent  d'ailleurs  conformement  aux  idees  exprimees  par 
DouDOROFF,  Barker  et  Hassid^^  et  par  A.  M.  Torriani  et  J.  Monod^^  jgg  trans- 
glucosidases. 

Le  defaut  de  I'utilisation  du  maltose  et  des  autres  disaccharides  permet  d'exclure 
I'hypothese  d'une  synthese  de  I'amidon  chez  Polytomella  par  une  amylomaltase  ou  par 
un  enzyme  du  meme  type.  L'existence  d'une  phosphorylase  suffit  a  rendre  compte  de  la 
synthese  du  polysaccharide. 

Admettons  que  cette  phosphorylase  soit  responsable  de  la  synthese  de  I'amidon 
chez  le  flagelle.  Deux  questions  restent  posees. 
Bibliographic  -b.  2']4\2'j$. 
18 


274  A.  LWOFF  et  al.  vol.  4  (1950) 

1.  Pourquoi  les  flagelles  sont-ils  incapahles  d'utiliser  les  glucides  et,  en  particulier ,  le 
glucose?  Si  les  flagelles  possedaient  une  hexokinase  et  une  phosphoglucomutase,  ils 
seraient  bien  entendu  capables  de  synthetiser  le  glucose- 1 -phosphate.  L'absence  de  ces 
deux  enzymes,  ou  d'un  seul  d'entre  eux,  suffit  a  expliquer  le  defaut  d'utilisation  du 
glucose.  Nous  avons  en  tous  cas  constate  que  I'hexose-diphosphate  mis  en  presence  de 
preparations  enzymatiques  du  flagelle  n'est  pas  attaque. 

2.  Comment  les  flagelles  synthetisent-ils  le  glucose- i-phosphate?  Cette  question  est 
actuellement  a  I'etude.  L'hypo.these  la  plus  simple  est  celle  d'une  synthese  par  conden- 
sation aldolique  sous  I'influence  d'un  enzyme,  d'acide  dioxyacetone-phosphorique  et  de 
D-aldehyde-glycerique.  L'existence  de  cette  reaction  chez  les  levures  a  ete  demontree 
par  les  recherches  d'OxTO  Meyerhof^^. 

Quoi  qu'il  en  soit,  le  flagelle  Polytomella  coeca,  comme  beaucoup  de  flagelles  avec 
ou  sans  chlorophylle,  synthetise  de  I'amidon  et  est  incapable  d'utiliser  les  glucides.  Le 
glucose  n'apparait  done  pas  comme  un  metabolite  intermediaire  oblige  entre  les  aliments 
carbones  mineiaux  ou  organiques  et  les  polysaccharides.  Des  organismes  peuvent 
synthetiser  I'amidon  et  I'utiliser  sans  que  le  glucose  apparaisse  dans  ce  cycle  autrement 
que  sous  forme  phosphorylee. 

r£sum£ 

1.  Le  flagelle  Polytomella  coeca  synthetise  et  utilise  Tamidon.  Ce  flagell6  est  incapable  d'utiliser 
les  glucides  pour  son  alimentation  carbon6e. 

2.  Le  flagelle  possede  une  phosphorylase :  du  glucose- 1 -phosphate  a  6te  isol6  a  partir  de  prepa- 
rations enzymatiques  additionn^es  d'amidon  soluble  et  de  phosphate  mineral. 

3.  Le  probleme  de  la  synthese  du  glucose- 1 -phosphate  n'a  pas  6t6  r6solu. 

4.  Des  organismes  peuvent  synthetiser  I'amidon  et  I'utiliser  sans  que  le  glucose  apparaisse  dans 
ce  cycle  autrement  que  sous  forme  phosphorylee. 

SUMMARY 

1.  The  flagellate  Polytomella  coeca  synthesizes  and  utilizes  starch.  This  flagellate  is  unable 
to  utilize  the  sugars  for  its  carbon-nutrition. 

2.  The  flagellate  contains  a  phosphorylase.  Glucose- 1 -phosphate  has  been  isolated  from  enzyme 
preparations  to  which  soluble  starch  and  mineral  phosphate  were  added. 

3.  The  problem  of  the  synthesis  of  glucose- 1 -phosphate  has  not  been  solved. 

4.  Organisms  exist,  which  can  synthesize  and  utilise  starch,  glucose  appearing  in  the  cycle  only 
in  phosphorylated  form. 

ZUSAMMENFASSUNG 

1.  Der  Flagellat  Polytomella  coeca  baut  Starke  auf  und  verwendet  sie.  Dieser  Flagellat  ist 
unfahig,  die  Zucker  fiir  seine  Kohlenstoff-Nahrung  zu  verwenden. 

2.  Der  Flagellat  enthalt  eine  Phosphorylase:  Glucose- i-phosphat  wurde  aus  Enzym-Prapa- 
raten  isoliert,  zu  denen  losliche  Starke  und  mineralisches  Phosphat  zugegeben  worden  waren. 

3.  Das  Problem  der  Synthese  des  Glucose- i-phosphats  ist  noch  ungelost. 

4.  Es  gibt  Organismen,  welche  Starke  aufbauen  und  abbauen  konnen,  ohne  dass  Glucose  in 
diesem  Zyklus  erscheint,  ausser  in  phosphorylierter  Form. 

BIBLIOGRAPHIE 

1  A.  LwoFF,  H.  loNEsco  ET  A.  GuTMANN,  Compt.  tend.,  228  (1949)  342-344. 

^  A.  LwoFF  ET  H.  Dusi,  Compt.  rend.,  205  (1937)  630;  Compt.  rend.  soc.  biol.,   127   (1938)   1408. 

'  A.  LwoFF,  Ann.  inst.  Pasteur,  66  (1941)  407. 


VOL.  4  (1950)  SYNTHESE    D'AMIDON   CHEZ    UN    FLAGELL^  275 

*  A.  LwoFF,  F.  NiTTi,  Mme  J.  Trefou£l  et  V.  Hamon,  Ann.  inst.  Pasteur,  67  (1941)  9. 
5  C.  H.  FiSKE  ET  Y.  SuBBAROW,  /.  Biol.  Chem.,  81  (1929)  629. 
^  D.  Kellin  et  E.  F.  Hartree,  Biochem.  J.,  42  (1948)  230. 
'  W.  KiESSLiNG,  Natiirwissenschaften,  27  (1939)  129. 
8  C.  S.  Hanes,  Proc.  Roy.  Soc,  B,  12S  {1939-1940)  421-450. 
®  C.  F.  CoRi,  G.  Schmidt  et  G.  T.  Cori,  Science,  89  (1939)  464. 
^°  J.  MoNOD  ET  A.  M.  ToRRiANi,  Compt.  rend.  acad.  sci.,  227  (1948)  240-242. 

11  M.  DouDOROFF,  H.  A.  Barker  et  W.  Z.  Hassid,  /.  Biol.  Chem.,  168  (1947)  725-746. 

12  A.  M.  ToRRiANi  ET  J.  MoNOD,  Compt.  rend.  acd.  sci.,  228  (1949). 
1*  O.  Meyerhof,  Bull.  soc.  chim.  biol.,  20  {1938)  1033-1042. 

Re^u  le  9  mars  1949 


276  BIOCHIMICA   ET   BIOPHYSICA   ACTA  VOL.  4  (1950) 


INHIBITION  OF  THE  METABOLISM  OF  NUCLEATED 

RED  CELLS  BY  INTRACELLUAR  IONS  AND  ITS  RELATION  TO 

INTRACELLULAR  STRUCTURAL  FACTORS 

by 

GILBERT  ASHWELL  and  ZACHARIAS  DISCHE 

Department  of  Biochemistry,  College  of  Physicians  and  Surgeons,  Columbia  University* , 

New  York.  N.Y.  {U.S.A.) 


INTRODUCTION 

The  fundamental  investigations  of  Meyerhof,  Embden,  Warburg,  Cori  and 
others  on  the  anaerobic  metabolism  of  the  skeletal  muscle,  yeast  and  blood  cells,  and 
the  discovery  of  the  role  of  dicarboxylic  and  tricarboxylic  acids  in  the  oxidative  meta- 
boHsm  of  animal  cells  by  Szent-Gyorgi  and  Krebs  and  of  the  mechanism  of  the  hy- 
drogen transfer  to  oxygen  by  Keilin  and  Warburg  laid  the  foundations  of  our 
knowledge  of  the  nature  of  chemical  reactions  providing  the  energy  for  cell  activities. 
Meyerhof's  work  elucidated  the  correlation  between  certain  oxidative  and  anaerobic 
enzyme  reactions  and  certain  phases  of  muscle  activity.  In  general,  however,  our  knowl- 
edge of  the  integration  of  enzyme  reactions  involved  in  aerobic  metabolism  into  the 
organisation  of  the  cell  and  its  mechanism  is  rather  inadequate. 

The  cell  metabolism  is  not  a  static  phenomenon.  Any  increase  in  cell  activity 
following  stimulation  is  accompanied  by  a  very  considerable  increase  of  the  oxidative 
cell  metabolism.  The  latter  goes  on  mainly  at  the  expense  of  glucose  taken  up  from  the 
environment  or  glycogen  present  in  the  cell.  There  is  some  evidence  scattered  in  the 
literatme  that  the  mechanism  of  this  part  of  the  oxidative  metabolism  of  sugar,  which 
appears  after  stimulation  may  not  be  completely  identical  that  with  of  the  oxidative 
metabolism  of  the  resting  cell.  This  evidence  was  obtained  from  the  study  of  the  meta- 
bolism of  cells  stimulated  in  vitro.  In  1936  Deutsch  and  Raper^  made  the  important 
observation  that  slices  of  glandular  tissue  (salivary  gland,  pancreas,  liver)  increase 
their  O2  uptake  several  times,  when  treated  with  certain  hormones  like  acetyl  choline, 
adrenaline  and  secretin,  which  in  vivo  stimulate  the  specific  activities  of  those  glands. 
Specific  pharmacological  stimulants  of  glands  like  pilocarpine  showed  the  same  effect. 
The  increase  is  temporary,  lasting  about  30-60  minutes.  It  can,  however,  repeatedly 
be  fully  reproduced  by  a  new  dose  of  a  stimulant  some  times  after  the  preceding  stimula- 
tion. Adrenaline  provokes  the  increase  in  respiration  only  with  salivary  glands  which 
can  be  physiologically  stimulated  by  the  sympathetic  and  adrenaline. 

This  fact,  the  reproducibility  of  the  metabolic  response  to  stimulants  after  a  period 
of  recovery  and  its  temporary  character,  strongly  suggest  that  this  metabolic  process 

*  This  work  was  supported  by  a  grant  of  the  Donner  Foundation  Inc.,  Cancer  Research  Division. 
References  p.  292. 


VOL.  4  (1950)  METABOLISM    OF    NUCLEATED    RED   CELLS  277 

in  vitro  is  essentially  with  the  metabolic  response  to  stimulation  in  vivo.  This  is  further 
borne  out  by  observations  of  Brock,  Druckerey  and  Herken^  who  confirmed  the 
findings  of  Deutsch  and  Raper.  They  calculated  the  metabolic  turnover  of  the  whole 
salivary  gland  from  the  values  obtained  in  vitro  on  slices  and  found  after  stimulation 
values  which  agreed  well  with  values  obtained  by  Barcroft  and  Peper^  on  the  salivary 
gland  stimulated  in  vivo  by  chorda  tympani.  They  found,  furthermore,  that  the  "stimu- 
lation metabolism",  as  they  call  the  metabolic  response  of  tissue  slices  to  stimulants, 
depends  on  the  ionic  equilibrium  in  the  Ringer  solution  in  which  the  slices  are  suspended. 
Complete  removal  of  the  Ca  from  the  Ringer  suppresses  completely  the  stimulation 
response,  which  can  be  restored  by  the  subsequent  addition  of  Ca.  The  removal  of  K  ions 
does  not  suppress  the  first  response  but  prevents  the  recovery.  The  ionic  equilibrium 
in  the  medium  is  essential  for  the  structural  integrity  of  the  cell  or  at  least  its  surface 
membrane.  It  is  therefore  clear  that  the  stimulation  response  requires  the  integrity 
of  the  cell  structure  and  cannot  be  a  consequence  of  injury  and  structural  disintegration. 

The  stimulation  metabolism  shows  two  significant  features  as  compared  with  the 
basic  or  rest  metabolism:  i.  the  latter  has  a  R.Q.  below  i  while  the  excess  respiration 
after  stimulation  has  a  R.Q.  of  i,  indicating  a  pure  carbohydrate  metabolism;  2.  the 
increase  in  Og  uptake  is  always  paradoxically  accompanied  by  a  production  of  free 
acids,  of  which  at  least  half  was  shown  by  Deutsch  and  Raper  to  be  lactic  acid^. 
Brock,  Druckerey,  and  Herken*  have  shown  that  this  production  of  acid  does  not 
occur  when  K  ions  are  removed  from  the  surrounding  medium,  although  the  increase 
in  respiration  appears  unchanged  in  size  after  the  first  stimulus. 

The  characteristic  metabolism  response  to  hormonal  or  pharmacological  stimuli  is 
by  no  means  a  peculiarity  of  glandular  tissues.  The  increased  respiration  of  the  sea 
urchin  egg  after  fertilization  shows  all  the  characteristic  properties  of  the  stimulation 
metabolism  of  glands^.  The  production  of  free  acid  in  this  case  was  found  by  Runnstrom, 
although  the  nature  of  the  acid  was  not  definitely  established.  As  in  glands  there  is  also 
a  marked  difference  in  the  sensitivity  towards  HCN  between  the  respiration  of  the 
unfertilized  and  that  of  the  fertilized  egg.  And  according  to  Brock  ei  al.  a  hormonal 
extract  of  the  anterior  pituitary  which  influences  the  division  of  the  egg  provokes  the 
same  characteristic  metabolic  response  in  it  as  fertilization.  This  cannot  be  obtained 
with  extracts  which  do  not  influence  the  cleavage  of  the  eg^. 

Finally  a  similar  metabolic  response  was  observed  in  1937  by  Gottdenker  and 
Marchi^  on  mammalian  heart  lung  preparations.  They  found  that  adrenaline,  which  is 
a  heart  stimulant,  increased  the  O2  uptake  of  these  preparations  and  at  the  same  time 
provoked  an  intensive  lactic  acid  production. 

The  fact  that  the  increased  respiration  in  stimulated  tissue  slices  goes  on  at  the 
expense  of  carbohydrates  and  is  accompanied  by  formation  of  lactic  acid  only  under 
physiological  conditions  of  the  medium  suggests  a  certain  interpretation  of  the  mecha- 
nism of  this  metabolic  phenomenon.  The  anaerobic  glycolysis  of  the  glands  is  completely 
suppressed  by  the  basic  respiration  due  to  the  Pasteur  effect.  Any  factor  leading  to 
a  deteriorization  of  the  structural  integrity  of  the  cell  tends  to  provoke  an  aerobic 
glycolysis.  This  is  the  case  for  instance  with  liver  or  brain  slices  when  K  is  removed 
from  the  medium.  The  aerobic  glycolysis  accompanying  the  stimulation  response  differs 
in  this  respect  fundamentally  in  being  dependent  on  the  presence  of  K  ions  in  the  me- 
dium and  is  suppressed  completely  after  their  elimination.  This  indicates  clearly  that 
aerobic  glycolysis  of  stimulation  is  not  due  to  structural  damage  or  increase  of  per- 
References  p.  2g2. 


278  G.  ASHWELL,  Z.  DISCHE  VOL.  4  (1950) 

meability,  but  to  a  specific  coupling  between  the  oxidative  breakdown  of  sugar  and 
glycolysis.  Now  it  is  reasonable  to  assume  that  phosphorylation  of  glucose  to  hexo- 
sediphosphate  constitutes  the  first  steps  in  glycolysis.  Any  coupling  between  glycolysis 
and  respiration  therefore  will  consist  primarily  in  a  coupling  between  certain  oxidative 
processes  and  phosphorylation  of  glucose.  It  is  well  known  that  the  oxidation  of  pyruvic 
acid  in  the  Krebs  cycle  is  coupled  with  an  intensive  phosphorylation  of  glucose  and' 
adenylic  acid  (to  ATP).  Certain  individual  enzyme  reactions  in  the  Krebs  cycle,  like 
oxidation  of  the  succinic  and  a-ketoglutaric  acid,  have  been  shown  to  be  coupled  with 
phosphorylation  of  glucose  and  adenylic  acid'.  Quite  recently  the  same  was  shown  for 
the  electron  transfer  from  dihydrocozymase  to  the  cytochrome  system^.  Ochoa^  has 
shown  for  heart  muscle  extracts  that  complete  oxidation  of  one  molecule  of  pyruvate 
can  be  coupled  with  the  phosphorylation  of  9  molecules  of  glucose  to  hexosediphosphate. 
The  oxidation  of  i  molecule  of  glucose  over  the  Krebs  cycle  therefore  can  phosphorylate 
18  molecules  of  glucose.  That  this  excess  phosphorylation  does  not  appear  in  resting 
cells  must  be  ascribed  to  the  coupling  of  the  phosphorylation  of  glucose  with  oxidative 
processes  in  such  a  way  that  the  speed  of  these  processes  does  not  exceed  the  maximal 
speed  of  oxidation  of  pyruvate.  If  the  Krebs  cycle  is  operating  and  these  controls  are 
eliminated,  aerobic  glycolysis  or  accumulation  of  hexosephosphate  must  result.  All 
these  considerations  suggest  that  the  metabolic  response  to  stimulation  in  organs  may 
be  due  to  a  release  or  increase  of  the  activity  of  the  tricarboxylic  acid  system  and 
accompanying  phosphorylation.  In  the  metabolism  of  resting  cells  this  system  may  play 
only  a  minor  role  or  be  lacking  altogether.  This  view  appears  supported  by  the  fact 
that  cells  like  embryonic  and  tumor  cells,  et  al.,  which  according  to  Brock  do  not  show 
any  stimulation  response  in  vitro,  show  only  very  weak  activity  of  enzymes  belonging 
to  the  tricarboxylic  acid  system. 

Turning  to  the  consideration  of  possible  mechanisms  involved  in  the  release  of 
the  metabolic  response  to  stimulation  we  must  keep  in  mind  that  every  cell  responds 
to  stimulation  by  the  electric  current  essentially  in  the  same  way  as  to  that  by  nervous 
impulses  or  hormonal  and  pharmacological  stimuli.  The  primary  effect  of  the  electric 
stimulus  consists  in  shifts  of  intracellular  ions.  It  is  generally  assumed  that  such  shifts, 
with  consecutive  accumulation  of  certain  ions  on  intracellular  membranes,  are  respon- 
sible for  the  functional  response  to  stimulation.  It  may  reasonably  be  assumed  that  such 
shifts  of  intracellular  ions  are  also  instrumental  in  provoking  the  metabolic  response. 
As  the  latter  can  be  more  protracted  than  the  functional  response  the  effects  of  ionic 
shifts  must  be  more  complex  in  this  case  and  consist  in  a  chain  of  reactions  released 
by  the  primary  shift.  The  ions  could  exert  their  influence  either  directly  on  enzymes 
involved  in  the  stimulation  metabolism  or  indirectly  by  changing  the  permeability  of 
intracellular  membranes  and  thus  facilitating  the  access  of  substrates  to  certain  enzymes. 

It  was  observed  recently^^  that  hemolysates  of  nucleated  red  cells  of  pigeon  glyco- 
lyse  only  in  presence  of  oxygen.  This  aerobic  glycolysis  disappears  in  presence  of  M/500 
NaCN.  It  was  further  found  that  all  intracellular  polyvalent  ions  like  Mg,  Ca,  ortho- 
phosphate,  ribonucleate  inhibit  the  aerobic  glycolysis  in  physiological  concentration. 
Colo  WICK,  Kalckar  and  Cori^^  found  in  1941  a  similar  obligatorily  aerobic  glycolysis 
in  kidney  extracts  and  showed  that  it  is  dependent  upon  the  oxidation  of  succinic  acid. 
As  it  was  known  that  nucleated  red  cells  are  able  to  oxidise  pyruvic  acid  to  CO2  and  that 
their  respiration  is  coupled  with  the  synthesis  of  ATP  it  seemed  reasonable  to  assume 
that  the  aerobic  glycolysis  in  hemolysates  of  these  cells  is  the  result  of  the  coupling 

References  p.  292. 


VOL.  4  (1950)  METABOLISM   OF    NUCLEATED    RED    CELLS  279 

of  phosphorylation  of  glucose  with  the  oxidative  processes  of  the  Krebs  cycle.  The 
inhibitory  effects  of  ions  on  the  aerobic  glycolysis  suggested  that  we  are  here  in  presence 
of  an  enzymatic  system  displaying  this  sensitivity  towards  ions  which  underlies  the 
mechanism  of  the  metabohc  response  to  cell  stimulation. 

The  possible  general  physiological  significance  of  this  phenomenon  invites  closer 
investigation  of  its  mechanism.  The  present  report  deals  with  experiments  in  this 
direction. 

EXPERIMENTAL 

A .  Preparation  of  the  material 

Red  blood  cells  of  pigeons  were  used  for  the  experiments.  The  animals  were  kept  fasting  for 
at  least  12  hours  preceding  the  bleeding,  which  was  carried  out  by  cutting  the  throat  on  one  side 
after  removal  of  feathers.  The  blood  was  caught  in  a  dish  containing  0.3  ml  of  3.6%  sodium  citrate. 
It  was  centrifuged  and  the  upper  stratum  of  the  sediment,  containing  the  white  cells,  was  removed 
as  far  as  possible  by  pipetting.  The  remaining  red  cells  were  first  washed  twice  with  a  fivefold  volume 
of  a  mixture  of  i  part  3.6%  sodium  citrate  and  9  parts  of  0.9%  NaCl  and  then  3  times  with  the  NaCl 
solution.  The  washed  cells  were  hemolyzed  by  adding  1.5  parts  of  distilled  water  to  i  part  of  cells. 
The  pH  of  these  hemolysates  was  found  to  vary  between  7.25  and  7.15.  As  it  was  intended  to  investi- 
gated the  effect  of  salts  on  the  metabolism  of  the  hemolysate  it  was  not  possible  to  use  buffers  in 
our  experiments  and  we  had  to  rely  for  the  stabilization  of  pn  during  the  experimental  period  on  the 
considerable  buffering  capacity  of  hemoglobin.  Orienting  experiments,  however,  showed  that  the 
shift  of  PH  due  to  acid  formation  during  4  hours  at  25°  did  not  exceed  0.2.  The  optimal  pH  for  the 
aerobic  metabolism  was  found  to  be  about  6.8.  In  most  of  our  experiments  the  pn  at  time  o  was 
therefore  that  of  the  original  hemolysate  or  slightly  lower,  i.e.,  6.9-7.0.  The  latter  was  obtained  by 
adding  an  appropriate  amount  of  diluted  HCl  to  the  water  used  for  hemolysis. 

B.  Analytical  methods 

In  a  certain  number  of  experiments  a  complete  balance  of  O2  uptake,  COj  production,  and  glu- 
cose consumption  was  carried  out.  In  these  and  most  of  the  other  experiments  the  total  volume 
of  either  water  or  of  respective  solutions  added  to  the  hemolysate  was  0.2  ml  per  i  ml  of  the  original 
hemolysate.  The  final  dilution  of  the  original  cell  suspension  was  therefore  threefold.  All  experiments 
were  done  at  25°  and  lasted  as  a  rule  four  hours.  The  Oj  uptake  was  measured  on  2  ml  of  the  hemo- 
lysate in  standard  B.\rcroft-W.\rburg  manometers  with  absorption  of  COj  and  NH3.  This  shifted 
the  PH  of  the  hemolysate  no  more  than  o.i  to  the  alkaline  side.  COj  production  was  determined  by 
the  direct  method.  To  account  for  the  retention  of  COj  by  the  hemolysate  the  manometer  in  which 
CO2  was  not  absorbed  contained  in  a  second  sidearm  0.4  ml  of  diluted  H2SO4.  At  the  end  of  the  ex- 
periment the  acid  was  tipped  in  from  the  sidearm  into  the  hemolysate.  The  pH  of  the  latter  was  then 
shifted  below  4.  The  hemolysate  became  very  viscous  at  this  pn  but  came  into  the  equilibrium  with 
the  gas  phase  after  about  30  minutes.  As  the  hemolysate  contained  from  the  beginning  a  certain 
amount  of  bound  COg  the  same  procedure  was  carried  out  on  a  sample  of  the  hemolysate  at  time  o. 
The  difference  of  the  increase  in  gas  volume  after  addition  of  acid  in  the  two  samples  gave  the  amount 
of  CO2  produced  by  oxidation  and  retained  by  the  hemolysate.  .\t  the  end  of  the  experiment  i  ml 
of  the  hemolysate  was  pipetted  out  of  the  manometer  vessels,  deproteinezed  with  4  ml  of  7.5% 
trichloracetic  acid.  The  centrifugate  served  for  the  determination  of  lactic  acid  and  glucose  and  phos- 
phate fractions.  The  lactic  acid  determination  was  carried  out  by  the  procedure  of  B.\rker  and 
SuMMERSON^^,  glucose  by  the  new  spectrophotometric  micromethod  of  Dische,  Shettles  and 
OsNOS^^  based  on  a  specific  reaction  of  hexoses  with  cysteine  in  H2SO4.  In  this  reaction  fructose 
gives  only  12%  more  absorption  than  the  equivalent  of  glucose,  so  that  the  phosphorylation  of  a 
small  amount  of  the  latter  to  Harden-Young  ester  will  not  influence  significantly  the  accuracy 
of  the  determination.  In  some  experiments  we  tested  for  this  ester  and  triosephosphate  by  a  new 
highly  sensitive  reaction  with  carbazole,  which  allows  the  determination  of  fructose  and  triosephos- 
phate in  the  same  sample.  Inorganic  and  the  labile  phosphate  were  determined  by  the  Fiske- 
SubbaRow  method  in  the  modification  of  King,  ribose  and  adenosine-5-phosphate  by  the  orcinol 
reaction. 

C.  Results 

In  a  first  series  of  experiments  the  aerobic  metabolism  of  the  hemolysate  was 
examined  to  obtain  information  about  the  nature  of  enzyme  reactions  involved  in  this 
References  p.  2g2. 


280  G.  ASHWELL,  Z.  DISCHE  VOL.  4  (1950) 

metabolism.  In  a  second  series  the  influence  of  various  cations  and  anions  on  those 
reactions  was  investigated. 

1.  The  aerobic  metabolism  in  the  hemolysate 

a)  O2  uptake,  lactic  acid  formation  in  the  hemolysate  in  absence  of  glucose.  The  hemo- 
lysate to  which  0.2  ml  of  liquid  per  ml  was  added  shows  a  marked  respiration  which 
varied  in  our  experiments  between  19  and  92  cmm  per  i  ml  and  4  hours.  The  respiration 
is  in  general  much  higher  during  the  first  hour  and  drops  afterwards  to  a  lower  but 
constant  level.  The  R.Q.  varies  considerably  between  0.82  and  i  (Table  I).  The  erythro- 
cytes contain  very  little  hexoses  soluble  in  trichloracetic  acid.  Less  than  i  y/ml  of 
hexose  (calculated  as  glucose)  was  found  in  the  hemolysate.  This  amount  does  not 
change  during  the  4  hours  of  the  experiment.  On  the  other  hand  there  is  a  considerable 
decrease  in  the  amount  of  adenosine-5-phosphate.  In  experiment  VI  (Table  I)  84  y/ml 
of  this  compound,  corresponding  to  35  y/ml  pentose,  disappeared  in  4  hours.  If  all 
of  this  pentose  had  been  oxidized  to  CO2  half  of  the  total  O2  uptake  in  this  experiment 
would  be  accounted  for.  The  breakdown  of  adenosine-5-phosphate  can  be  explained 
by  the  fact  that  it  is  formed  in  the  hemolysate  by  the  ATPase  and  dephosphorylated 
to  adenosine  which,  as  was  shown  for  human  erythrocytes,  can  be  split,  with  phosphory- 
lation, to  form  triosephosphate  and  hexosediphosphate.  One  part  of  the  respiration  of 
the  hemolysate  in  absence  of  glucose  must  be  due  to  the  oxidation  of  either  fat  or  pro- 
tein'. The  hemolysate  contains  from  the  beginning  very  small  amounts  of  lactic  acid 
(about  5  y/ml).  In  some  cases  small  amounts  of  this  acid  are  formed  during  incubation, 
but  not  more  than  about  5  y/ml. 

b)  The  tricarboxylic  acid  cycle  in  the  hemolysate.  The  presence  of  this  enzyme  system 
in  the  hemolysate  can  be  demonstrated  after  addition  of  citrate  or  one  of  the  dicarboxylic 
acids  metabolised  by  the  system.  When  M/1200  of  succinic,  fumaric,  malic,  oxaloacetic, 
citric  and  a-ketoglutaric  acid  is  added  the  O2  uptake  increases  considerably  (Table  I). 
In  presence  of  ketoglutaric  and  citric  acid  much  more  than  in  that  of  other  acids  this 
additional  Og  uptake  increases  with  the  concentration  of  the  acid.  It  is  about  twice 
as  great  in  presence  of  M/600  succinate  than  of  M/i  200.  At  the  same  time  lactic  acid 
is  formed  in  significant  amounts.  This  increases  with  the  concentration  of  succinate 
or  malate.  The  amount  of  lactic  acid  varies  with  the  nature  of  the  acid  in  the  following 
sense :  malate,  fumarate  >  succinate  >  a-ketoglutarate  >  citrate.  This  can  be  explained 
by  the  assumption  that  oxaloacetic  is  formed  from  malic  acid,  with  reduction  of  co- 
enzyme I  to  dihydrocoenzyme  I.  One  part  of  the  oxaloacetic  acid  is  decarboxylated  to 
pyruvate  and  COg.  As  the  cytochrome  system  is  not  able  to  oxidize  dihydrocozymase 
rapidly  enough,  one  part  of  it  reduces  pyruvate  to  lactate.  The  same  sequence  of  reactions 
was  observed  by  E.  A.  Evans^*  in  liver  extracts.  As  the  increase  in  succinate  increases 
the  O2  uptake  as  well  as  lactic  acid  formation  the  cytochrome  system  apparently  com- 
petes with  the  pyruvate  for  dihydrocozymase.  Thus  the  fact  that  lactic  is  formed  from 
citrate  indicates  that  the  whole  series  of  reactions  from  citrate  to  oxaloacetates  goes 
on  in  the  hemolysate.  Pyruvic  acid  also  increases  the  respiration  and  lactic  acid  for- 
mation, though  less  than  any  one  of  the  polycarboxylic  acids,  and  the  increase  is  ob- 
served only  during  the  last  3  hours  of  the  4  hour  period. 

2.  Aerobic  metabolism  in  presence  of  glucose 

When  50  mg  %  of  glucose  is  added  to  the  hemolysate  it  is  broken  down  at  a  rate 
References  p.  292. 


VOL.  4  (1950) 


METABOLISM    OF    NUCLEATED    RED    CELLS 


281 


TABLE  I 

INFLUENCE  OF  MgClj  M/25O  ,OF  PYRUVIC,  CITRIC  AND  DICARBOXYLIC  ACIDS  OF  THE  KrEBS  CYCLE 
ON  THE  O2  CONSUMPTION  OF  THE  HEMOLYSATE  IN  PRESENCE  AND  ABSENCE  OF  GLUCOSE  AND  ON 
AEROBIC    GLYCOLYSIS.    TIME    OF    EXP.:    4    h    THE    BRACKETED    VALUES    REPRESENT    THE    Oj  UPTAKE   IN 

THE    LAST    3    h 


Substance 
added 

Og  uptake  in  i  /ml  of  hemolysate  in  /xl 

Aerobic  glycolysis 

Exp. 
No. 

by 
hemolysate 

change 
0/ 

glucose 
in  the 

change 

0/ 

y  lactic  acid 
y/ml  of 

change 
0/ 

itself 

/o 

hemolysate 

/o 

hemolysate 

/o 

I 

0 

43-5  (31) 

8.2     (3.0) 

168 

a.  MgCl2  M/250 

54      (32-4) 

+  24  (+  5) 

15      (12.5) 

+  84  (+  320) 

245 

+  46 

b.  Na  succinate 

M/1540 

52      (35-6) 

+  20  (+  16) 

218 

+  30 

a  +  b 

75      (47-5) 

+  70  (+  53) 

259 

+  54 

II 

0 

34.2  (23.8) 

8.3  (2.4) 

220 

MgClj  M/250 

45      (28.5) 

+  31.6  (+19-9) 

13-9  (11) 

+  67  (+  360) 

265 

+  21 

Na  succinate 

M/770 

60.2  (34) 

+  76  (+  43) 

III 

0 

29      (18.7) 

6.7  (8) 

139 

MgCl2  M/250 

34-3  (25-5) 

+  18  (+  36) 

12.6  (lO.l) 

+  88  (+  26) 

185 

+  33 

Na  Pyruvate 

21.7  (21.7) 

-25(+  16) 

14      (5) 

+  10.9  (—37) 

156 

+  12 

M/1200 

IV 

0 
Na  succinate 

M/1200 
Na  citrate 
Na  /1200 
Na  a-keto  glu- 
tarate  M/1200 

44-3  (27) 
54      (37) 
60      (41) 

67      (42-5) 

+  22  (+  37) 
+  35(+  51) 
+  52  (+  57) 

V 

0 
Na  succinate 

M/i  200 
Na  citrate 

M/i  200 
Na  a-keto  glu- 
tarate  M/i  200 

32.4  (21) 
49      (34-3) 
55-4  (41) 
64      (43) 

+  51  (63) 
+  71  (+  95) 
+  100  (4- 102) 

VI 

0 

45-6  (30.1) 

6        (10.5) 

262 

Na  pyruvate 

48      (34.6) 

+  5-3  (+  15) 

0        (0) 

— 100  ( — 100) 

258 

—  1-5 

M/i  200 

VII 

0 
Na  succinate 

35      (i9-i) 

17-5  (12.3) 

164 

M/1200 

55      (36.1) 

+  57  (+  89) 

13-3  (12.4) 

-  24  (+  I) 

218 

+  33 

Na  pyruvate 

M/i  200 

46.5  (29.9) 

+  33  (+  55) 

6.7  (6.1) 

—  62  (-  50) 

169 

+  3 

VIII 

0 
Na  pyruvate 

75-6  (38.6) 

8.6  (20.7) 

276 

M/i  200 

78.7  (47) 

+  4-1  (+  22) 

14-3  (8.9) 

+  66  (-  58) 

246 

—  II 

IX 

0 

27.8  (18.7) 

13-3  (IO-3) 

159 

NaCN  M/500 

2-5  (2-9) 

-91  (—87) 

3-3  (1-2) 

-  75  (-  88) 

NaCN  (M/250 

0      (0) 

— 100  ( — 100)       0      (0) 

— 100  ( — 100) 

131 

—  92 

References  p.  2g2. 


282  G.  ASHWELL,  Z.  DISCHE  VOL.  4  (1950) 

of  75-150  y/ml  per  hour.  The  O2  consumption  increases  at  the  same  time  considerably 
by  13-50%  in  4  hours.  At  the  same  time  an  intensive  aerobic  glycolysis  and  sometimes 
esterification  of  inorganic  P  to  difficultly  hydrolyzable  esters  is  observed.  Up  to  260 
y/ml  of  lactic  is  produced  in  4  hours.  The  rate  of  O2  consumption  during  the  first  hour 
is  different  from  the  rate  in  the  following  3  hours  during  which  it  remains  almost  con- 
stant. The  rate  of  glycolysis  is  in  general  smaller  during  the  first  hour  than  later.  If 
we  assume  that  the  additional  0^  consumption  in  presence  of  glucose  is  due  to  the  total 
oxidation  of  the  latter  and  calculate  the  total  breakdown  of  glucose  by  oxidation  and 
glycolysis  the  latter  turns  out  to  be  considerably  smaller  than  the  amount  of  glucose 
which  really  disappeared.  The  R.Q.  of  the  additional  respiration  due  to  glucose  is  only 
about  0.7  (Table  VI).  The  discrepancy  between  the  observed  values  and  those  calculated 
for  glucose  which  disappears  indicates  that  only  one  part  of  it  is  completely  oxidized 
while  another  part  is  oxidized  either  to  phosphogluconic  or  pyruvic  acid. 

3.  The  coupling  between  aerobic  glycolysis  and  respiration 

The  glycolysis  of  the  hemolysate  is  obligatorily  aerobic  and  disappears  almost 
completely  when  the  oxidation  processes  in  the  hemolysate  are  suppressed  either  by 
inhibitors  or  by  elimination  of  O^.  Thus  NaCN  at  M/250  almost  completely  suppresses 
the  glycolysis  and  90%  of  the  total  O2  consumption.  (Table  I)  Further  increase  of  the 
concentration  does  not  have  any  significant  effect.  The  small  residual  glycolysis  amounts 
to  only  a  few  per  cent  of  the  total  and  is  probably  due  to  the  leucocytes  which  were 
not  removed.  The  leucocytes  which  are  siphoned  off  in  the  beginning  of  the  blood  wash- 
ing display  in  fact  a  powerful  anaerobic  glycolysis  which  is  partly  suppressed  in  aero- 
biosis.  That  the  effect  of  cyanide  on  glycolysis  is  due  to  the  blocking  of  respiration  could 
be  shown  in  experiments  in  which  O^  was  removed  from  the  hemolysate.  These  were 
carried  out  in  the  following  way.  4  ml  of  the  hemolysate  +  0.8  ml  of  0.3%  glucose 
solution  were  pipetted  into  a  500  ml  flask  which  was  closed  by  a  ground  stopper  with 
stopcock.  The  flask  was  weighed  and  then  evacuated  first  with  a  water  pump.  When 
the  foaming  of  the  fluid  became  too  intense  the  evacuation  was  interrupted  until  the 
foam  broke  down  and  the  evacuation  then  resumed  until  no  more  gas  escaped.  The 
evacuation  was  continued  with  the  oil  pump  until  a  vacuum  of  about  i  mm  Hg  was 
obtained.  The  flask  was  then  weighed  again  to  determine  the  loss  in  water.  The  hemo- 
lysate was  kept  in  vacuo  for  4  hours  at  room  temperature  and  then  the  flask  opened, 
the  evaporated  water  replaced  and  the  hemolysate  deproteinized  simultaneously  with 
a  control,  which  stayed  during  the  same  period  in  presence  of  oxygen  and  one 
to  which  NaCN  M/500  was  added.  The  determination  of  lactic  acid  showed  that  the 
glycolysis  was  suppressed  in  the  sample  in  vacuo,  though  not  quite  as  far  as  in  the 
sample  with  NaCN. 

While  suppression  of  the  Og  consumption  inhibits  the  glycolysis  in  our  hemolysate 
any  increase  of  Og  consumption  after  addition  of  pyruvate,  citrate  and  dicarboxylic 
acids  of  the  Krebs  cycle  is  accompanied  by  a  strong  increase  of  glycolysis  (Table  II). 
If  the  final  dilution  of  the  hemolysate  is  no  more  than  the  threefold  of  the  original 
volume  of  the  suspension,  a-ketoglutarate  is  most  effective,  with  succinate  and  fumarate 
following,  and  pyruvate  the  least  effective.  It  was  found  for  the  succinate  that  the 
promoting  effect  on  glycolysis  increases  with  the  concentration,  as  also  does  the  Og 
consumption. 
References  p.  2g2. 


VOL.  4  (1950) 


METABOLISM   OF   NUCLEATED    RED    CELLS 


283 


TABLE  II 

INFLUENCE    OF    PYRUVATE,    CITRATE    AND    DICARBOXYLIC    ACIDS     OF    THE    KrEBS    CYCLE    ON    AEROBIC 
GLYCOLYSIS    IN    THE    HEMOLYSATE.  TIME    2    HOURS,  T    25° 


Experiment 
No. 

Substance  added 

mg  lactic  acid/ml 
of  hemolysate 

Change 
% 

PH 

I 

0 

46 

7.0 

M/600  MgClg 

73. 

+    58 

M/i  200  succinate 

96 

+  109 

M/600  MgClj  +  M/i  200  succinate 

131 

+  185 

II 

0 

58 

7.0 

M/600  MgClj 

81 

+    40 

M/1200  succinate 

100 

+    72 

M/600  +  M/1200  succinate 

120 

+  108 

III 

0 

42 

7.0 

M/i  200  succinate 

54 

+    29 

M/300  MgClj 

80 

+    90 

M/1200  Na  pyruvate 

39-5 

—     6 

M/i  200  Na  pyruvate  +  M/i  200  succinate 

53 

+    29 

IV 

0 

103 

6.8 

M/i  200  succinate 

151 

+    46 

M/800  succinate 

158 

+    53 

V 

0 

35 

7-1 

M/400  succinate 

47 

+    34 

M/400  a-ketoglutarate 

44 

+    26 

VI 

0 

7-5 

7.0 

M/400  succinate 

29.5 

+  300 

M/iooo  pyruvate 

15 

+  100 

VII 

0 

48 

7.2 

M/1200  succinate 

123 

+  156 

M/800  succinate 

132 

+  175 

VIII 

M/800  a-ketoglutarate 

168 

+  250 

M/i  200 

100 

+  108 

IX 

0 

42 

7.2 

M/450  succinate 

86 

+  105 

M/450  malate 

64 

+    57 

X 

0 

24 

7.2 

M/450  succinate 

52 

+  116 

M/900  succinate 

38 

+    58 

M/450  malate 

41 

+    70 

M/900  malate 

28 

+    17 

4.  Influence  of  ions  on  the  aerobic  metabolism  in  presence  and  absence  of  glucose 

Two  different  effects  of  ions  on  the  aerobic  metabolism  in  the  hemolysate  can  be 
observed.  The  first  is  specific  for  magnesium  ions  and  the  second  is  common  to  all 
multivalent  ions.  In  this  second  group,  the  nature  and  the  charge  of  the  ion  is  important 
for  the  intensity  of  the  effect. 

a)  Magnesium.  In  concentrations  up  to  M/200  MgClg  increases  the  basic  0.^  consump- 

References  p.  2g2. 


284  G.  ASHWELL,  Z.  DISCHE  VOL.  4  (1950) 

tion  in  the  hemolysate  as  well  as  the  additional  uptake  in  presence  of  glucose  and  the 
dicarboxylic  acids.  The  increase  ranges  from  18  to  24%  for  the  basic  respiration  and 
from  67  to  88%  for  the  additional  respiration  due  to  glucose  (Table  I).  At  the  same 
time  there  is  an  increase  of  the  aerobic-glycolysis  amounting  to  21-46%  of  the  original 
value  (Table  I,  Exp.  I-III).  This  effect  of  Mg  reaches  its  maximum  at  M/200  to  M/150. 
The  additional  O2  uptake  as  well  as  the  accompanying  aerobic  glycolysis  are  inhibited 
by  M/500  NaCN  to  the  same  extent  as  is  the  case  without  addition  of  Mg. 

b)  Univalent  cations.  When  so  much  KCl  is  added  to  the  hemolysate  that  the  con- 
centration of  the  added  salt  in  the  hemolysate  becomes  i/ii  M  and  the  hemolysate 
therefore  isotonic  no  inhibition  of  the  basic  O2  uptake  with  and  without  glucose  can  be 
observed.  The  aerobic  glycolysis  is  in  general  somewhat  decreased.  In  some  cases,  how- 
ever, a  decrease  of  60%  was  observed.  NaCl  at  the  same  concentration  decreases 
the  O2  uptake  moderately  and  inhibits  the  aerobic  glycolysis  33-50%.  It  must  be 
noted  that  this  concentration  of  Na  ions  cannot  be  considered  any  more  as  physiological. 
If  the  concentration  of  the  added  NaCl  was  only  M/25  no  significant  inhibition  of  the  O2 
uptake  or  aerobic  glycolysis  could  be  observed.  These  observations  indicate  that  CI  ions 
in  physiological  concentrations  do  not  have  any  significant  effect  on  the  aerobic  meta- 
bolism of  the  hemolysate. 

c)  Calcium  and  other  multivalent  cations.  When  the  concentration  of  Mg  exceeds 
M/150  the  aerobic  glycolysis  in  the  hemolysate  begins  te  decline.  At  M/80  an  inhibition 
of  about  15-25%  appears.  This  inhibitory  effect  is  a  property  of  all  multivalent  cations. 
(Table  IV).  Of  all  the  cations  investigated  Ca  shows  the  strongest  inhibitory  effect. 
M/i  ooo-M/i  500  shows  almost  complete  inhibition  of  the  aerobic  glycolysis.  Sr  is  almost 
as  strong  but  Ba++,  Ce+++  and  La+++  are  ten  times  weaker  inhibitors.  However,  our 
figures  merely  correlate  the  strength  of  the  inhibition  with  the  overall  concentration 
of  the  salt.  The  latter  is  almost  identical  with  the  concentration  of  the  bivalent  ions  for 
the  earth  alkalis  and  rare  earth  but  not  for  the  other  metals,  the  hydroxides  of  which 
possess  low  second  dissociation  constants.  The  ion  Mn++  and  Cd++  as  such  are,  therefore, 
probably  stronger  inhibitors  than  Ca++.  This  however  does  not  seem  of  any  physiological 
significance.  The  inhibitory  effect  of  Ca  on  the  glycolysis  is  still  perceptible  at  M/8000. 
After  having  ascertained  that  the  inhibitory  effect  of  Mg  and  Ca  on  glycolysis  is  related 
to  their  multivalence  the  effects  on  the  O2  consumption  of  those  two  as  representatives 
of  multivalent  cations  were  studied.  The  basic  O2  consumption  was  inhibited  28-52% 
by  Ca  M/iooo.  The  oxidation  due  to  glucose,  however,  may  completely  disappear  at 
this  concentration  while  that  of  succinate  and  a-ketoglutarate  is  reduced  to  about  the 
same  extent  as  the  basic  respiration  (Table  III). 

d)  Anions.  All  multivalent  anions  inhibit  strongly  the  aerobic  glycolysis  (Table  III). 
The  importance  of  valency  is  more  marked  with  anions  than  cations.  The  bivalent 
HPO4 —  and  SO4 —  show  a  significant  inhibition  only  at  M/ioo  and  M/50  respectively, 

while  the  tetravalent  Fe(CN) at  M/250,  ribonucleate,  diphosphoglycerate  and  ino- 

sitolhexaphosphate  strongly  at  M/1500,  M/700  and  M/iooo  respectively.  The  nature 
of  the  ion  plays,  however,  also  a  considerable  role.  The  bivalent  oxalate  for  example 
shows  at  M/iooo  a  stronger  inhibition  than  malonate  at  M/200.  The  physiological 
polycarboxylic  acids  like  succinate  and  citrate,  which  up  to  M/500  increase  the  aerobic 
glycolysis,  inhibit  at  higher  concentrations.  At  M/50  the  inhibition  is  considerable  with 
citrate.  That  multivalency  is  only  one  of  the  factors  promoting  the  inhibitory  effect 
on  the  metabolism  is  shown  by  the  behaviour  of  the  CNS~  ion.  While  KCl  at  M/ii  and 
References  p.  2g2. 


VOL.  4  (1950) 


METABOLISM   OF   NUCLEATED    RED   CELLS 


285 


TABLE  III 

EFFECT    OF    KCl,  NaCl,  MgClj  and    of   multivalent   ions    on    the  O2  CONSUMPTION   BY  THE   HEMOL- 
YSATE  ITSELF  AND  BY  GLUCOSE,   SUCCINATE,  a-KETOGLUTARATE  IN  THE  HEMOLYSATE.   TIME  4  HOURS 


Experi- 

Substance  added 

By  hemol- 
ysate  itself 

Bygh 

icose 

By  succinate 
M/1200 

Bya-keto  glu- 
tarate  M/1200 

No. 

O2 

used 

Inhibi- 
tion % 

0, 

used 

Inhibi- 
tion % 

O2 
used 

Inhibi- 
tion % 

0, 

used 

Inhibi- 
tion % 

I 

Mg  M/250 

Mg  M/250  +  Ca  M/2000 

54 

48-5 

10 

15 

8.2 

45 

II 

Mg  M/250 

Mg  M/250  +  Ca  M/i  000 

45 

21-5 

52 

136 

2 

93 

III 

0 
Ca  M/iooo 

92 
50.6 

45 

21.8 
143 

34 

40.2 
25-4 

37 

IV 

0 

Ca  M/iooo 

Mg  M/250 

Mg  M/250  +  Ca  M/iooo 

45-6 
24.7 
45-6 
23.6 

46 
48 

5-9 

0.3 

21.3 

II-5 

95 
46 

V 

0 

Ca  M/ 1 000 

35 
18.9 

46 

17-5 
4-3 

76 

20 
5-9 

70 

VI 

0 

Ca  M/iooo 

75-6 
34 

55 

8.7 
4.1 

53 

VII 

0 
Ca  M/ 1 000 

32.1 
23 

28 

10.5 
0.0 

100 

VIII 

0 

KCl  M/12 

27.8 
29.4 

— 

13-4 
14 

— 

IX 

0 
PO4  M/500 
Oxalate  M/500 

48 

40-3 
41.6 

16 
14 

II 
I 

6.2 

90 
38 

29.4 
16.3 

45 

X 

0 
Oxalate  M/500 

44-3 
29.5 

34 

10. 1 
19 

— 

233 
32.5 

— 

XI 

0 
Oxalate  M/250 

32.4 
17.8 

46 

10 
0.7 

93 

16.8 
II 

34 

31-4 
32.6 

— 

XII 

0 
Na2S04  M/24 

60.2 
47-4 

21 

21 
4.1 

80 

19.2 
16 

17 

29-5 
26.6 

10 

XIII 

0 
NaCl  M/12 
NaCl  M/25 
KCl  M/12 

563 
51.6 
68.4 
56.3 

8 

XIV 

0 
Ribosenucleic  acid  M/1500 

56 
431 

23 

14.2 
3 

80 

Ph  7-2  does  not  inhibit  at  all  or  only  little,  KCNS  at  the  same  concentration  completely 
inhibits  glycolysis  (Table  III). 

The  Oo  consumption  is  suppressed  by  anions  to  about  the  same  extent  as  glycolysis. 
References  p.  2g2. 


286 


G.  ASHWELL,  Z.  DISCHE 


VOL.  4  (1950) 


TABLE  IV 

INHIBITION  OF  AEROBIC  GLYCOLYSIS  BY  VARIOUS  CATIONS  AND  ANIONS.  TIME :  EXPERIMENT  I-X  4  HOURS, 

EXPERIMENT    XI    2    HOURS 


Experiment 
No. 

Substance  added 

y  Lactic  acid  formed  in 
I  ml  of  hemolysate 

Inhibition 
% 

PH 

I 

0 

62 

7-25 

HCN  M/500 

II. 2 

82 

CaCljM/iooo 

II 

82 

BaClj  M/333 

20.7 

66 

SO4  M/333 

18.6 

70 

MnClj.  M/333 

II 

82 

CaClj  M/333 

II 

82 

II 

0 

71.8 

7-1 

SrClj  M/800 

5 

93 

FeSO^  M/333 

II. 9 

83 

CdSO,  M/333 

5-8 

92 

HCN  M/500 

0.07 

0.64 

99 

0 

III 

2,3  diphosphoglycerate  M/500 

39 

39 

7.2 

Inositol  hexaphosphate  M/700 

22.1 

65.5 

IV 

0 

37-2 

CaCU  M/4000 

19.4 

48 

Phosphate  M/50 

28.3 

23 

Na^SO,  M/50 

18.4 

50 

Na  Citrate  M/50 

5-2 

86 

V 

0 

46.5 

Ribosenucleic  acid  M/1600 

37-2 

20 

7-25 

Yeast  adenyUc  acid  M/400 

48 

—  3 

VI 

0 

164 

CaCljM/iooo 

30 

82 

7 

Na  Succinate  M/i  200 

218 

CaCljMioo  -f-  Na  Succinate  M/i  200 

27 

83 

VII 

0 
NaCl  i/ii 

64 
65 

7.2 

KCl  1/9 

33 

48 

VIII 

0 

36 

KCl  i/ii 

35 

3 

7.2 

MgClj  M/250 

45 

MgCl2M/25o  +  KCl  i/ii 

37 

17 

IX 

0 

227 

6.9 

NaCl  M/ 1 1 

128 

43 

KCl  M/ 1 1 

85 

63 

X 

0 

290 

6.9 

KCl  M/I  I 

158 

45 

NaCl  M/ii 

163 

43 

Ribonucleate  M/1540 

169 

42 

XI 

0 

103 

M/300  MgClg 

177 

7.0 

M/1200  Na  Succinate 

151 

M/iooo  CaClj 

34 

97 

M/I  000  CaClj  +  MgClj  M/300 

13-6 

87 

M/iooo  CaClj  +  Ml  200  Succinate 

16 

85 

References  p.  2g2. 


VOL.  4  (1950) 


METABOLISM    OF    NUCLEATED    RED    CELLS 


287 


Different  oxidation  processes,  however,  are  influenced  to  a  very  different  degree.  The 
oxidation  of  glucose  suffers  much  more  than  the  basic  oxidation.  M/480  sodium  oxalate 
suppresses  the  additional  respiration  by  glucose  80-100%,  the  basic  only  0-15%. 
Essentially  the  same  relation  is  vahd  for  M/25  Na2S04,  M/50  Na  phosphate  and  M/1700 
Na  ribonucleate.  The  oxidation  of  succinate  is  less  suppressed  than  that  of  glucose  but 
more  so  than  that  of  a-ketoglutarate  and  citrate. 

e)  Synergy  between  Mg  and  Ca  and  anions  in  their  inhibitory  effects.  Effects  of  ions 
on  colloidal  particles  are  in  general  counteracted  by  ions  of  opposite  charge  if  the 
effects  are  due  to  neutralization  of  electric  charges.  It  is,  therefore,  surprising  that 
inhibitory  effects  of  anions  on  the  metabolism  of  red  cells  are  not  eliminated  or  de- 
creased, but  on  the  contrary  strongly  enhanced  by  Mg  and  Ca  (other  multivalent  cations 


TABLE  V 

SYNERGY  BETWEEN   Mg''"''  AND   Ca*"''  AND   MULTIVALENT  ANIONS  IN  THEIR  EFFECTS   ON  THE   AEROBIC 
GLYCOLYSIS   OF  THE   HEMOLYSATE   IN   PRESENCE   OF  GLUCOSE.   TEMP.       25°.   TIME!    EXP.   I-V   4   HOURS, 

EXP.  II    2    HOURS 


Experiment 

Inhibitor 

Lactic  acid  formed  in 

Inhibition 

PH 

No. 

mg/ml  of  hemolysate 

% 

I 

a.        0 

36 

7.2 

b.  Na  phosphate  M/50 

25 

30 

c.  CaClg  M/4000 

34 

6 

b.  +  c. 

4-3 

88 

d.  MgS04  M/150 

38.5 

e.  KCIM  i/io 

35 

3 

d.  +  e. 

33 

8 

II 

a.        0 

71 

7-15 

b.  CaClj  M/4000 

50 

30 

c.  Na  phosphate  M/50 

55-4 

22 

b.  +  c. 

28.3 

60 

III 

a.        0 

46.5 

7.2 

b.  CaClj  M/4000 

34 

27 

c.  Inositol  hexaphosphate  M/iooo 

31 

33 

b.  +  c. 

6.6 

86 

d.  NajSO^  M/ioo 

41.4 

II 

b.  +  d. 

9.2 

80 

e.  MgClsj  M/250 

62 

e.  +  d. 

38.1 

38 

IV 

a.        0 

33 

7.2 

b.  phosphate  M/50 

41 

—  24 

c.  MgClj  M/250 

66 

b.  +  c. 

40 

40 

V 

a.         0 

202 

725 

b.  K,Fe(CN)8M/iooo 

17-5 

13 

c.  KCXS  M/90 

20.4 

—  I 

d.  MgClj  M/250 

56.3 

b.  +  d. 

33-8 

40 

c.  +  d. 

33-8 

40 

VI 

a.        0 

112 

6.8 

b.  CaClj  M/4000 

99 

12 

c.  phosphate  M/go 

90 

20 

b.  -f  c. 

41 

635 

References  p.  2g2. 


288 


G.  ASHWELL,  Z.  DISCHE 


VOL.  4  (1950) 


were  not  investigated).  M/ioo  NagSO^  and  Na  phosphate,  M/iooo  K4Fe(CN)6  and  M/90 
KCNS  which  by  themselves  show  little  or  no  inhibition  of  aerobic  glycolysis,  strongly 
inhibit  in  presence  of  M/250  MgClg,  which  by  itself  increases  the  glycolysis.  The  inhibi- 
tion by  M/4000  Ca  in  presence  of  M/ioo  Na2S04  or  Na  phosphate  is  much  stronger  than 
corresponds  to  the  sum  of  inhibitions  of  the  two  kinds  of  ions  (Table  IV).  This  synergy 
manifests  itself  also  towards  the  oxidation  of  glucose  as  well  as  towards  the  original  O2 
consumption  of  the  hemolysate.  On  the  other  hand  no  synergy  was  found  between  K 
and  Na2S04  or  Na  phosphate. 

/)  Reversibility  of  the  inhibitory  effect  of  Ca  against  the  aerobic  glycolysis.  That  the 
inhibition  of  the  metabolism  in  the  hemolysate  by  ions  is  not  due  to  an  irreversible 
destruction  of  enzymes  is  clearly  indicated  by  the  fact  that  the  degree  of  the  inhibition 
does  not  increase  with  the  time  even  when  the  inhibition  was  not  complete.  The  rever- 
sibility of  the  inhibition  was,  furthermore,  demonstrated  directly  for  Ca  in  the  following 
way.  Two  samples  of  washed  red  cells  were  taken.  One  sample,  hemolysate  I,  was 
hemolyzed  with  1.5  volumes  of  water  containing  enough  Ca  to  yield  a  final  concentration 
of  2  mg  %  in  the  hemolysate.  The  other  sample,  hemolysate  II,  was  hemolyzed  with 
1.5  volumes  of  water.  4  samples  of  i  ml  each  were  pipetted  from  every  hemolysate. 
0.03  ml  of  a  2%  glucose  solution  were  added  to  samples  of  hemolysate  I  and  i  sample  of 
hemolysate  II  (glucose  samples)  while  to  the  remaining  five  samples  0.03  ml  of  water 
was  added  (water  samples).  All  samples  were  left  for  2  hours  at  25°  and  then  the  glucose 
sample  and  one  water  sample  of  II  and  one  of  the  glucose  samples  of  I  and  one  water 
sample  were  deproteinized  (2  hours  samples).  0.6  ml  of  water  was  now  added  to  the 
glucose  and  water  samples  of  I  and  to  the  one  of  the  water  samples  of  II  while  the  other 
water  sample  of  II  received  0.6  ml  of  a  glucose  solution  of  0.1%.  The  Ca  concentration 
in  I  was  thus  reduced  from  2  to  1.2  mg  %.  If  the  inhibition  of  the  aerobic  glycolysis 
by  Ca  was  reversible  then  the  reduction  of  the  Ca  concentration  in  I  should  result  in 
a  decrease  of  the  inhibition  in  the  following  2  hours.  This  was  in  fact  the  case. 

It  must  be  noted  that  the  4  hour  glucose  sample  of  I  contained  more  lactic  acid 
in  the  second  2  hour  period  than  the  corresponding  sample  of  II.  This  tended  to  make 
the  inhibition  by  Ca  rather  stronger  than  weaker. 

TABLE  VI 

BALANCE  OF  GLUCOSE  CONSUMPTION,   Oj  UPTAKE  AND  LACTIC  ACID  FORMATION  IN  THE  HEMOLYSATE. 

4    HOUR    EXPERIMENTS    AT    25°    PH    7-0 


O2  uptake  in  y/ml 
of  hemolysate 

Increase 
of  CO2 
produc- 
tion by 
glucose 

Glucose 

con- 
sumed 
in  y/ml 

Lactic 

acid 

formed 

C 

Og uptake 

due  to 

glucose 

in  /tmol 

D 

mol 
glucose 
oxydized 

Ratio 

Exp. 
No. 

A 

by  hemo- 
lysate 
itself 

B 

hemoly- 
sate + 
glucose 

B-A 

C 

d" 

33 
35 
40 

50 
46 
86 

75 
61 

"5 

25 
15 
29 

21-5 

255 
250 

525 

163 

207 
320 

0.78 
0.47 
0.9 

0.51 
0.24 
1. 14 

1-53 
1.96 
0.79 

References  p.  2g2. 


VOL.  4  (1950)  METABOLISM   OF   NUCLEATED    RED    CELLS  289 

DISCUSSION 

5.  Mechanism  of  the  aerobic  glycolysis  in  the  hemolysate 

On  the  basis  of  our  expeiiments  we  can  draw  the  conclusion  that  the  aerobic 
metabohsm  in  nucleated  erythrocytes  consists  of  several  distinct  enzymatic  systems. 
If  no  glucose  is  added  to  the  hemolysate  no  significant  amounts  of  preformed  hexoses 
are  available  for  oxidation,  but  adenosine-5-phosphate,  derived  from  ATP,  breaks 
down  and  its  ribose  disappears.  This  process  and  oxidation  of  fat  and  protein  should 
be  responsible  for  the  observed  respiration  of  the  hemolysate  in  absence  of  glucose. 
The  increase  after  addition  of  glucose  can  be  traced  again  to  at  least  two  different 
reactions,  namely,  complete  oxidation  to  CO2  and  oxidation  of  glucose  to  a  phosphoric 
ester,  whereby  one  atom  of  oxygen  combines  with  one  mol  of  glucose.  It  is  probable 
that  the  latter  reaction  consists  in  the  oxidation  of  glucose  to  phosphogluconic  acid. 

The  powerful  aerobic  glycolysis  in  the  hemolysate  in  presence  of  glucose  can  be 
due  to  the  fact  that  the  oxidation  of  one  molecule  of  glucose  is  coupled  with  the  phos- 
phorylation of  many  molecules  of  this  sugar  and  the  triosephosphate  dehydrogenase 
is  much  more  efficient  in  the  hemolysate  than  the  system  oxidizing  pyruvate.  The 
excess  of  the  latter  is  therefore  reduced  to  lactic  acid.  As  the  hemolysate  contains  the 
enz5nne  system  of  the  tricarboxylic  acid  cycle  it  is  reasonable  to  assume  that  the  oxida- 
tion of  glucose  to  CO2  goes  over  this  cycle.  It  is  known  from  experiments  on  other  tissue 
extracts  that  the  oxidation  of  i  mol  of  glucose  in  this  way  can  be  coupled  with  the 
phosphorylation  of  18  molecules  of  glucose  to  hexose  diphosphate.  This  would  explain 
the  fact  that  the  addition  of  all  those  acids  which  increase  the  turnover  of  the  Krebs 
cycle,  and  of  Mg  which  is  an  activator  of  the  oxidation  of  pyruvic  acid,  considerably 
increases  the  aerobic  glycolysis. 

The  inability  of  the  hemolysate  to  glycolyse  anaerobically  can  be  explained  easily. 
The  hemolysis  of  nucleated  erythrocytes  is  accompanied  by  an  explosive  increase  in 
the  activity  of  ATPase.  At  room  temperature  practically  all  of  ATP  originally  present 
in  the  cells  is  dephosphorylated  in  a  few  minutes;  the  glycolysis  of  one  molecule  of 
glucose  can  maximally  resynthesize  2  molecules  of  ATP.  As  long,  therefore,  as  the 
speed  of  the  simple  dephosphorylation  of  ATP  exceeds  the  speed  of  transphosphoryla- 
tion  with  glucose  the  latter  process  must  stop  in  anaerobiosis  due  to  the  total  disappear- 
ance of  ATP.  The  efficiency  of  the  oxidative  breakdown  of  glucose  as  far  as  synthesis 
of  ATP  is  concerned  makes  it  possible  to  keep  up  under  aerobic  conditions  a  certain 
minimum  concentration  of  ATP  necessary  for  the  phosphorylation  of  glucose.  This 
amount,  however,  is  very  small,  even  under  aerobic  conditions,  and  not  detectable 
by  the  usual  colorimetric  procedures  of  determination. 

Point  of  attack  of  ions 

The  realization  of  this  multitude  of  enzymatic  processes  involved  in  the  aerobic 
metabolism  is  important  for  the  consideration  of  the  possible  mechanism  of  the  inhibi- 
tory effects  of  ions  on  this  metabolism.  It  appears  significant  that  all  ions,  cations  as  well 
as  anions,  are  able  to  suppress  not  one  but  many  of  the  enzyme  reactions  constituting 
the  oxidative  metabolism.  On  the  other  hand,  the  degree  of  inhibition  is  different  for 
different  enzyme  reactions  or  systems  of  reactions.  The  aerobic  glycolysis  is  in  general 
more  strongly  inhibited  than  the  oxidation  of  glucose,  which  in  turn  suffers  more  than 
the  O2  consumption  without  glucose.  The  oxidation  of  succinate  and  a-ketoglutarate 
References  p.  zgz. 
19 


290  G.  ASHWELL,  Z.  DISCHE  VOL.  4  (1950) 

are  least  affected.  It  is  very  significant  that  this  sequence  in  the  susceptibility  to  inhi- 
bitory effects  is  the  same  for  all  kinds  of  ions  and  the  reactions  affected  are  of  very 
different  tjrpes.  The  oxidation  of  glucose,  for  example,  is  as  was  shown  due  to  two 
completely  different  reactions.  It  appears  most  improbable  that  so  many  and  so  different 
reactions  should  be  influenced  in  the  same  way  by  all  the  ions.  We  have  rather  to  assume 
that  the  ions  exert  their  influence  on  a  substrate  the  activity  of  which  is  again  correlated 
is  some  way  with  the  activities  of  all  the  enzymes  of  the  oxidative  system.  Such  a 
substrate  for  example  is  the  cytochrome  system,  which  serves  as  Hg  carrier  to  the  oxida- 
tion of  the  preformed  substrates  of  the  hemolysate  as  well  as  that  of  added  glucose. 
It  seems  impossible,  however,  to  consider  the  cytochrome  system  as  the  point  of  attack 
in  the  ionic  inhibition,  because  of  the  great  differences  between  various  enzymes  in  their 
sensitivity  towards  the  ions.  M/iooo  of  Ca  almost  completely  inhibits  the  oxidation 
of  glucose,  but  the  inhibition  of  the  basic  respiration  of  the  hemolysate  is  not  complete 
even  at  M/200.  M/500  HCN,  on  the  other  side,  inhibits  both  to  the  same  extent.  All 
this  suggests  that  the  inhibitory  action  of  ions  is  directed  against  one  single  substrate 
which  in  changing  its  physicochemical  properties  influences  in  its  turn  all  the  enzymes 
of  the  oxidative  system.  The  enzymes  are  in  fact  not  in  solution  inside  the  cell,  but  are 
attached  to  insoluble  particles,  the  mitochondria.  These  contain,  apart  from  proteins, 
considerable  amounts  of  lipids  and  ribosenucleic  acids.  In  these  subcellular  structural 
and  functional  units  the  enzyme  proteins  are  probably  attached  to  a  stroma  consisting 
of  lipo-  and  nucleoproteins  and  may  be  surrounded  by  a  surface  membrane.  One  way 
to  explain  the  effects  of  ions  on  the  oxidative  processes  would  therefore  be  to  assume  a 
decrease  in  the  permeability  of  such  a  surface  membrane  under  their  influence.  The  fact 
that  the  aerobic  glycolysis  coupled  with  the  oxidations  is  quite  generally  more  stiongly 
inhibited  than  the  oxidative  processes  themselves  is  in  agreement  with  this  concept. 
This  glycolysis  depends  on  the  coupled  phosphorylation  of  a  phosphate  carrier  which 
transfers  the  phosphate  to  glucose.  Any  decrease  in  the  permeability  of  the  surface 
membrane  will  decrease  the  speed  of  the  penetration  not  only  of  the  substrate  but  also 
of  the  phosphate  carrier  and  the  speed  with  which  it  leaves  the  particle  after  being 
phosphorylated.  The  amounts  of  the  phosphate  carrier  available  for  the  reaction  with 
glucose  must  decrease  to  a  much  higher  degree  than  the  corresponding  oxidative 
process.  It  could  also  be  that  the  ions  change  not  the  permeability  but  the  physical 
properties  of  the  hypothetical  stroma  to  which  the  enzyme  proteins  are  attached.  Any 
change  in  the  water  binding  capacity  or  shape  of  the  protein  molecules  of  the  stroma 
would  have  a  considerable  influence  on  the  shape  and  arrangement  of  the  respective 
enzyme  proteins  and  tend  to  change  their  activity. 

If  we  assume  that  in  one  way  or  the  other  the  proteins  of  mitochondria  are  the 
point  of  attack  of  inhibiting  ions  the  most  probable  mechanism  of  this  inhibitions  appears 
to  be  elimination  of  local  electric  fields  on  the  surface  of  this  protein,  due  to  the  ad- 
sorption of  the  ions.  Thus  CNS""  which  forms  stable  complexes  with  proteins,  inhibits 
the  aerobic  glycolysis  at  low  concentrations,  whereas  CI""  is  ineffective.  This  can  also 
explain  the  characteristic  sjmergy  between  cations  and  anions  in  their  effects.  Even 
at  the  isoelectric  point  of  a  protein  the  charged  groups  on  its  surface  will  exert  con- 


*  More  recent  experiments  on  the  mechanism  of  the  inhibition  of  the  oxidative  enzymes  by  Ca"*""*" 
suggest,  that  the  specific  protein  in  the  mitochondria,  affected  by  ions,  influences  the  energy  transfer 
during  the  enzymereactions  of  the  Krebs  cycle  rather,  than  the  enzymes  themselves  or  the  access 
of  substrates.  The  results  of  these  new  experiments  will  be  reported  in  a  subsequent  paper. 

References  />.  2g2. 


VOL.  4  (1950)  METABOLISM  OF  NUCLEATED  RED  CELLS    •  29I 

siderable  forces  of  repulsion  on  ions  of  the  same  charge  and  thus  counteract  their 
adsorption.  This  repulsion  will  obviously  be  decreased  by  the  simultaneous  presence 
in  solution  of  ions  of  opposite  charge  of  great  surface  activity.  The  adsorption  of  cations 
will  therefore  be  facilitated  by  the  presence  of  easily  adsorbable  polyvalent  anions,  and 
vice  versa,  and  thus  a  higher  degree  of  elimination  of  polarized  groups  on  the  protein 
surface  may  be  achieved.  This  again  will  affect  the  water  binding  capacity  and  the  shape 
of  the  respective  protein  molecule. 

This  view  appears  supported  by  the  rather  striking  analogy  between  the  inhibition 
of  the  aerobic  metabolism  by  ions  and  the  effect  of  certain  ions  on  proteins  like  myosin, 
actin,  actomyosin  and  the  so  called  structural  proteins  of  kidney  and  brain  investigated 
by  Szent-Gyorgyi  and  his  associates'^.  These  proteins  adsorb  physiological  cations 
(Na,  K,  Ca,  Mg)  from  solutions  of  physiological  concentrations.  Ca  is  more  strongly 
adsorbed  than  Mg  and  this  again  more  strongly  than  the  monovalent  cations.  This 
adsorption  neutralizes  charges  of  polar  groups  on  the  protein  surface  and  changes  the 
affinity  to  water  and  in  the  case  of  actin  the  ability  to  polymerize.  A  striking  analogy 
to  the  synergy  between  ions  in  our  case  can  be  seen  in  the  influence  of  the  cations 
(K,  Ca)  on  the  adsorption  of  the  polyvalent  ATP  ion  by  myosin.  In  this  case  the  anion 
of  ATP  does  not  counteract  the  effect  of  K  on  myosin  but  enhances  it. 

The  affinity  of  structural  proteins  to  ions  depends  upon  a  certain  specific  state  of 
the  protein  surface  and  is  easily  suppressed  by  procedures  tending  to  denature  the 
protein.  The  adsorption  of  cations  by  myosin  for  example  decreases  strongly  during 
24  hour  storage  at  0°'^.  This  may  be  the  reason  why  such  general  inhibitory  effects 
of  ions  on  oxidative  enzymes  have  not  yet  been  observed  in  tissue  homogenates.  In  this 
case  the  subcellular  structural  units  may  suffer  considerable  injury  by  the  mechanical 
crushing  of  the  tissue.  Hemolysis  on  the  other  hand  appears  as  a  much  milder  procedure 
for  getting  access  to  a  little  altered  inner  parts  of  the  cell. 

SUMMARY 

1.  The  hemolyzed  nucleated  erythrocytes  of  the  pigeon  show  considerable  Oj  consumption, 
which  is  considerably  increased  by  MgClj  M/250,  glucose  and  constituents  of  the  tricarboxylic  acid 
cycle  and  completely  inhibited  by  NaCN  M/250. 

2.  This  oxidative  metabolism  is  coupled  with  a  strong  aerobic  glycolysis. 

3.  All  multivalent  cations  and  anions  inhibit  the  Oj  consumption  as  well  as  the  aerobic  glycolysis. 

4.  CaClg,  orthophosphate  and  ribonucleate  inhibit  strongly  at  physiological  concentrations. 

5.  Different  oxidative  reactions  in  the  hemolysate  are  inhibited  by  ions  to  a  different  degree. 

6.  These  inhibitory  effects  of  ions  ma}^  be  due  to  disturbances  of  the  local  electric  fields  of  proteins 
which  are  constituents  either  of  membrane  or  stroma  of  subcellular  structural  units  which  are  carriers 
of  enzymes  of  the  oxidative  system  of  the  cell. 

RfiSUMfi 

1.  Les  nucleo-erythrocytes  hemolyses  du  pigeon  montrent  une  consommation  d'oxygfene  con- 
siderable, qui  est  encore  fortement  accrue  par  MgClj  M/250,  le  glucose  et  les  constituants  du  cycle 
des  acides  tricarboxyliques,  mais  completement  inhibee  par  NaCN  I\I/25o. 

2.  Ce  metabolisme  d'oxydation  est  coupl6  avec  une  forte  glycolyse  aerobique. 

3.  Tous  les  cations  et  anions  plurivalents  inhibent  la  consommation  d'oxygene  aussi  bien  que 
la  glycolyse  aerobique. 

4.  Le  CaClj,  I'ion  orthophosphorique  et  I'ion  ribonucleique  sont  de  forts  inhibiteurs  aux  con- 
centrations physiologiques. 

5.  Differentes  reactions  d'oxydation,  dont  I'h^molysat  est  le  siege,  sont  inhibees  par  les  ions  a 
des  degrds  diff^rents. 

6.  Ces  effects  inhibitoires  d'ions  sont  peut-etre  dus  a  des  perturbations  des  champs  electriques 
locaux  des  proteines  qui  sont  des  constituants  soit  de  la  membrane,  soit  du  tissus  conjonctif  d'unites 
structurales  subcellulaires,  supports  d'enzymes  du  systeme  d'oxydation  de  la  cellule. 

References  p.  2g2. 


292  G.  ASHWELL,  Z.  DISCHE  VOL.  4  (1950) 

ZUSAMMENFASSUNG 

1.  Die  hamolysierten,  kernhaltigen  Erythrocyten  der  Taube  zeigen  einen  bedeutenden  Og- 
Verbrauch,  welcher  durch  MgClj  M/250,  Glucose  und  Bestandteile  des  Tricarboxylsaure-Zyklus 
betrachtlich  erhoht,  durch  NaCN  M/250  dagegen  voUig  unterbunden  wird. 

2.  Dieser  oxydative  Metabolismus  ist  mit  starker  aerober  Glykolyse  gekuppelt. 

3.  Alle  mehrwertigen  Kationen  und  Anionen  hemmen  den  Og-Verbrauch  sowohl  als  die  aerobe 
Glykolyse. 

4.  CaClj,  Orthophosphat  und  Ribonukleinat  hemmen  bei  physiologischer  Konzentration  stark. 

5.  Verschiedene  oxydative  Vorgange  im  Hamolysat  werden  durch  lonen  verschieden  stark 
gehemmt. 

6.  Diese  hemmenden  Wirkungen  der  lonen  beruhen  vielleicht  auf  Storungen  lokaler  elektrischer 
Felder  von  Proteinen,  welche  Bestandteile  sind  von  Membran  oder  Bindegewebe  von  subcellularen 
Struktureinheiten,  die  Trager  von  Enzymen  des  oxydativen  Systems  in  der  Zelle  sind. 

REFERENCES 

1  W.  Deutsch  and  Raper,  /.  Physiol..  87  (1936)  275;  92  (1938)  i39- 

2  N.  Brock,  H.  Druckerey,  and  H.  Herken,  Biochem.  Z.,  300  (1939)  i;  Arch,  exptl.  Path.  Phar- 
makol.,  198  (1941)  601. 

3  J.  Barcroft  and  H.  Piper,  /.  Physiol.,  44  (1913)  359- 

*  N.  Brock,  H.  Druckerey,  and  H.  Herken,  Biochem.  Z.,  302  (1939)  393- 
5  D.  Runnstrom,  Protoplasma,  20  (1934)  i. 

8  Gottdenker  and  De  Marchi,  Klin.  Wochschr.,  16  (i937)  1282. 
'  V.  A.  Belitzer  and  E.  T.  Tsibakowa,  Biokhimiya,  4  (i939)  516. 
8  A.  L.  Lehninger  and  M.  E.  Friedkin,  Proc.  Fed.  Biol.  Soc,  8  (1949)  218. 
»  S.  OCHOA,  /.  Biol.  Chem..  151  (i943)  493- 
1°  Z.  Dische,  /.  Biol.  Chem.,  163  (1946)  575- 

11  S.  CoLOwiCK,  H.  Kalckar,  and  C.  F.  Cori,  /.  Biol.  Chem.,  137  (194O  343- 

12  S.  B.    Barker  and  W.  H.  Summerson,  /.  Biol.  Chem.,  138  (1941)  535- 

13  Z.  Dische,  L.  Shettles,  and  M.  Osnos,  Arch.  Biochem.,  22  (i949)- 

1*  E.  a.  Evans,  B.  Vennesland,  and  L.  Slotin,  /.  Biol.  Chem.,  147  (1943)  77i- 

15  A.  Szent-Gyorgyi,  Chemistry  of  Muscular  Contraction,  3-38  (i947)- 

16  V.  S.  Hermann,  Hung.  Acta  Physiol.,  I  (1946)  25. 

Received  June  loth,  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  293 


THE  BIOCHEMISTRY  OF  ABNORMALITIES  IN  CELL  DIVISION 

by 

E.  BOYLAND 

Chester  Beatty  Research  Institute,  Royal  Cancer  Hospital,  London  {England) 


Carbohydrates  have  been  considered  for  a  long  time  to  be  the  fuel  of  the  tissues 
of  the  body,  but  it  is  only  during  the  last  few  years  that  some  of  the  mechanisms 
whereby  the  energy  from  carbohydrate  catabolism  is  utilized  have  been  revealed. 
Meyerhof  has  done  more  than  any  other  biochemist  to  show  how  carbohydrate  meta- 
boHsm  involves  phosphorylation  and  how  the  phosphorylated  products  can  yield  energy 
for  other  biological  processes.  A  remarkable  property  of  living  machinery  is  that  it 
can  make,  repair  and  maintain  its  own  working  parts.  Cancer  tissue  has  a  high  carbo- 
hydrate metabolism  and  a  high  rate  of  cell  division.  The  carbohydrate  metabolism, 
partly  aerobic  and  partly  anaerobic,  yields  the  energy  necessary  for  cell  division  and  the 
maintenance  of  the  nuclei  which  seem  to  control  the  processes  of  cell  division.  The  main 
constituents  of  cell  nuclei  of  both  normal  and  cancer  cells  appear  to  be  proteins  and  nu- 
cleic acids,  and  the  carbohydrate  metabohsm  is  possibly  merely  concerned  with  pro- 
duction of  high  energy  phosphate  bonds  which  will  yield  energy  in  a  form  available  for 
synthesis  of  nucleic  acids  and  possibly  of  proteins.  Inhibition  of  these  processes  will 
stop  cell  division  and  so  inhibit  growth.  If  the  inhibition  is  such  that  cell  division  is 
impeded  but  not  stopped  then  the  incidence  of  abnormalities  such  as  damaged  chromo- 
somes, mutations  or  cancer  might  be  increased. 

Normal  cells  are  not  capable  of  continuous  growth.  If  they  continue  to  receive 
surplus  nourishment  after  attaining  a  certain  limiting  size  they  divide.  If  the  process 
of  cell  division  is  inhibited,  then  growth  is  also  inhibited.  In  the  cell  division  or  mitosis 
in  which  nuclei  and  plasmagenes  play  a  dominant  role  there  is  exact  partitioning  of.  the 
chromosome  material  between  the  daughter  cells.  The  occurrence  of  spontaneous  chro- 
mosome abnormahties  and  mutations  shows  that  chromosomes  are  not  absolutely 
stable.  The  induction  of  changes  or  mutations  by  physical  and  chemical  agents  indicates 
that  the  nuclear  material  is  sensitive  and  vulnerable  to  conditions  of  the  environment. 

Perhaps  the  most  sensitive  indication  of  abnormalities  of  cell  division  is  the  occur- 
rence of  mutations,  as  these  are  functional  manifestations  of  such  abnormalities.  If  the 
change  of  normal  cells  to  cancer  cells  is  a  somatic  mutation  then  the  fact  that  an  agent 
is  carcinogenic  is  an  indication  that  it  is  mutagenic.  Actually  most  of  the  mutagenic 
agents  which  lend  themselves  to  testing  have  been  found  to  be  carcinogenic  and  many 
carcinogenic  agents  have  been  shown  to  induce  mutations. 

Many  of  the  means  which  will  induce  cancer  and  increase  the  mutation  rate  of 
animals  will  inhibit  the  growth  of  animals  or  of  tumours  growing  in  animals.  Such 
inhibition  of  growth  by  carcinogenic  hydrocarbons  was  described  by  Haddow^.  Inhibi- 
tion of  growth  in  this  way  may  form  the  basis  for  therapy  of  cancer. 

The  more  complete  correlation  between  the  actions  we  are  considering  was  first 

References  p.  300. 


294 


E.  BOYLAND 


VOL.  4  (1950) 


shown  with  X-rays.  Radiotherapy  of  cancer  started  (Grubbe^)  soon  after  Rontgen's 
discovery  of  X-rays.  Seven  years  later  Frieben^  reported  that  a  skin  cancer  had  devel- 
oped in  a  man  who  had  been  exposed  to  X-rays.  Muller*  showed  that  X-rays  increased 
the  incidence  of  mutations  in  Drosophila  and  Painter  and  Muller^  and  Roller^ 
found  that  X-radiation  caused  visible  abnormalities  in  chromosomes. 

All  these  effects  can  be  produced  by  certain  chemical  agents,  such  as  the  nitrogen 
mustards  and  urethane,  which  for  this  reason  have  been  called  radiomimetic.  The  carci- 
nogenic hydrocarbons  such  as  1:2:5: 6-dibenzanthracene  are  also  radiomimetic  agents. 
Table  I  shows  the  grouping  of  the  different  effects. 

TABLE  I 

REFERENCES    TO    EFFECTS    PRODUCED    BY    X-RAYS    AND    BY    CHEMICAL 
COMPOUNDS    WITH    RADIOMIMETIC    ACTIONS 


Treatment  of 
Cancer  or 

Induction  of 

Induction 

of 
Mutations 

Chromosome 

Inhibition  of 
growth 

Cancer 

Damage 

X-rays 

Grubbe^ 

Frieben^ 

MULLER* 

Painter  and 
Muller^ 

Nitrogen  Mustard 

Rhoads" 

BOYLAND  AND 

Auerbach, 

Boyland,   Clegg 

Horning* 

Robson  and 
Carr' 

Koller,  Rhoden 
AND  Warwick^" 

Urethane 

Paterson, 

Nettleship  and 

Oehlkers^^ 

Boyland  and 

ApThomas, 

Henshaw^^ 

Koller" 

Haddow,  and 

Watkinson^^ 

1 :2:5:6-Dibenz- 

Haddow,  Scott, 

KennawayI® 

Carr" 

Koller^* 

anthracene 

and  Scott^s 

Methylcholanthrene 

Haddow,  Scott, 
and  ScottI* 

Cook  and 
Haslewood^* 

Strong^" 

— 

N:N-di  (2-chloro- 

Haddow,  Kon, 

Haddow, 



KollerI* 

ethyl)-2-naph- 

AND  Ross^^ 

Horning,  and 

thylamine 

Koller22 

4-Dimethylamino- 

Haddow,  Harris, 

Haddow,  Harris, 



KOLLER^^ 

stilbene 

Kon,  AND  RoE^s 

Kon,  and  Roe^^ 

Another  effect  which  many  of  these  agents  produce  is  the  bleaching  or  greying  of 
hair.  This  was  described  in  mice  exposed  to  X-rays  by  Hance  and  Murphy^*.  A  similar 
effect  occurs  with  nitrogen  mustard  derivatives  either  aliphatic  (Boyland  et  al^^)  or 
aromatic  (Haddow  et  al.^^).  This  greying  of  hair  is  a  permanent  effect,  remaining  with 
the  mouse  for  the  remainder  of  its  life.  It  may  be  perhaps  considered  as  a  somatic 
mutation  and  in  this  respect  is  analogous  to  an  induced  tumour.  The  change  from  col- 
oured to  white  hair  which  is  induced  is  unlikely  to  be  due  to  selective  survival  of  more 
resistant  white  hair  follicles  as  the  skin  of  the  black  (C57)  or  agouti  (CBA)  mice  used  do 
not  appear  to  contain  white  hairs.  This  change  of  colour  in  a  part  of  the  body  is  a  dis- 
continuous variation  in  properties  like  the  change  of  normal  into  cancer  cells.  Both 
changes  are  brought  about  by  the  same  agents  which  also  induce  germinal  mutations. 
References  p.  300  . 


VOL.  4  (1950)  ABNORMALITIES   IN    CELL    DIVISION  295 

These  agents  also  cause  visible  damage  to  chromosomes  and  it  is  probable  that  the 
inherited  variations  are  due  to  change  of  plasmagenes  or  to  chromosome  damage  which 
might  not  have  been  visible  if  the  affected  cell  had  been  examined.  The  dose  of  mutagenic 
agent  which  is  required  to  produce  visible  abnormalities  will  cause  death  in  many  of 
the  treated  cells  and  the  new  forms  arise  in  the  cells  which  have  received  a  sublethal  dose. 

The  tumours  which  arise  as  the  result  of  treatment  of  cells  with  a  mutagenic  agent 
are  possibly  derived  from  a  normal  host  cell  which  has  produced  daughter  cells  differing 
from  the  parent  cell  because  of  some  accidental  error  or  abnormality  of  cell  division. 
When  the  total  number  of  cell  divisions  in  the  whole  mammalian  body  is  taken  into 
account  these  abnormalities  are  very  infrequent.  The  chance  of  their  occurrence  seems 
to  be  made  much  more  probable  by  the  presence  of  a  carcinogenic  or  mutagenic  agent. 

If  the  changes  brought  about  by  carcinogenic  agents  are  random  variations  of  the 
original  cells  as  suggested  it  is  perhaps  surprising  that  different  tumours  are  so  similar 
in  their  morphology  and  biochemistry.  Each  tumour  has  its  own  specific  characters 
but  the  differences  between  tumours  induced  by  carcinogenic  agents  are  relatively 
small.  Different  tumours  resemble  each  other  more  closely  than  they  resemble  the  tissue 
of  their  origin.  Thus  tumours  have  less  of  the  specific  functions  of  the  cell  from  which 
the  tumour  arose  and  tumours  have  the  property  of  producing  lactic  acid  aerobically. 
Of  the  mutations  which  occur  in  somatic  cells  probably  many  are  unable  to  survive ;  many 
will  die  normally  and  others  will  be  unable  to  withstand  the  attacks  of  defence  processes 
of  the  host.  Of  the  numerous  mutations  which  occur  only  those  which  produce  cells  able 
to  survive,  grow,  and  induce  the  host  to  provide  a  blood  supply,  will  become  detectable 
cancers,  and  for  these  biological  processes  specific  characters  of  function  and  morphology 
may  be  required.  As  the  changes  are  induced  by  substances  which  damage  the  chromo- 
some material  (either  directly  or  indirectly)  and  probably  the  genes,  the  chang  esare 
probably  the  result  of  loss  or  inactivation  of  genes,  as  it  seems  unlikely  that  a  toxic 
agent  should  add  something  to  the  nuclear  material.  Such  changes  would  be  analogous 
to  the  mutations  induced  in  Neurospora  which  result  in  the  loss  of  ability  to  carry  out 
some  specific  chemical  process. 

The  biochemical  mechanism  which  operates  when  radiations,  nitrogen  mustards 
or  carcinogenic  hydrocarbons  induce  mutations  or  cancer,  is  still  obscure.  The  nitrogen 
mustards  or  chloroethylamines  are  chemically  reactive  and  combine  with  many  tissue 
constituents  and  inactivate  many  enzymes,  but  particularly  the  phosphokinases  and  the 
pyruvic  oxidase  enzyme  system.  In  order  to  produce  the  chromosome  damage  and 
inhibition  of  the  growth  of  tumours  in  animals  the  aliphatic  chlorethylamines  must  have 
two  chloroethyl  groups  (Boyland  et  al}'^)  and  the  necessity  of  two  reactive  or  polar 
groups  for  chemotherapeutic  action  against  cancer  was  suggested  earlier  (Boyland^^). 
GoLDACRE,  Loveless,  and  Ross^^  have  suggested  that  the  two  active  groups  join 
chromosome  parts  by  cross  linkage  of  protein  or  other  constituents.  As  a  result  of  these 
additional  cross  linkages  the  division  of  chromosomes  is  hindered  and  breakages  and 
damage  to  the  chromosomes  occurs.  This  theory  would  not  account  for  the  action  of 
urethane  (which  seems  to  have  no  chemically  reactive  groups)  and  it  is  difficult  to  see  how 
arsenicals  such  as  sodium  arsenite  could  act  in  this  way.  Sulphydryl  compounds  are  the 
only  known  tissue  constituents  with  which  arsenite  is  known  to  react.  As  there  is  very 
little  cysteine  or  other  sulphydryl  compound  in  chromosomes  (D.widson  and  Lavvrie") 
combination  of  chromosome  chains  by  union  of  sulphydryl  groups  through  an  arsenic 
atom  is  unlikely  to  occur.  It  also  seems  improbable  that  X-rays  would  cause  stable  cross 
References  p.  300. 


296  E.  BOYLAND  VOL.  4  {1950) 

linkages  between  chromosome  parts  to  be  formed.  The  current  theory  of  the  action  of 
radiations  on  cells  is  that  they  oxidize  sulphydryl  groups  through  the  production  of 
peroxide  or  other  oxidizing  agent  within  the  cells.  They  could  therefore  unite  peptide 
chains  by  conversion  of  sulphydryl  groups  to  the  disulphide  forms.  The  low  concen- 
tration of  cysteine  in  the  chromosomes  which  was  suggested  as  a  difficulty  in  the  theory 
as  applied  to  the  action  of  arsenicals  would  also  apply  to  X-rays.  A  linkage  through 
arsenic  might,  however,  be  more  stable  than  a  disulphide  link  which  would  probably 
be  reduced  in  processes  of  cell  metabolism.  This  hypothesis  of  cross  linkage  within 
chromosomes  being  the  cause  of  abnormalities  may  be  of  value  in  investigating  the 
action  of  drugs  on  tumour  cells,  but  it  is  possibly  of  no  more  value  than  the  knowledge 
that  in  the  chloroethylamine  series  and  other  compounds  two  active  groups  are  required 
for  the  biological  actions  considered. 

The  hypothesis  which  the  author  put  forward  (Boyland^^)  postulates  that  the 
effects  of  these  substances  are  due  to  inhibition  of  enzymes,  particularly  the  phospho- 
kinases  or  enzymes  involving  oxidative  phosphorylations  necessary  for  production  and 
metabolism  of  the  nucleic  acid  required  for  the  maintenance  of  normal  chromosomes 
and  genes.  Since  then  Barron,  Dickman,  and  Singer^^  have  shown  that  phospho- 
glyceraldehyde  dehydrogenase  is  particularly  sensitive  to  the  action  of  X-rays,  and 
Meyerhof  and  Wilson^"  have  described  the  inhibition  of  hexokinase  and  phospho- 
hexokinase  with  phenyl  urethane. 

Investigations  carried  out  during  the  war  showed  that  two  enzyme  systems  were 
particularly  sensitive  to  the  poisoning  action  of  vesicants.  Of  the  phosphokinases, 
hexokinase  was  first  shown  by  Dixon  and  Needham^^  to  be  inhibited  by  low  concentra- 
tions of  mustard  gas  and  nitrogen  mustard.  Later  Cori  and  his  co-workers^^  found 
that  phosphokinases  in  general  are  inhibited  by  vesicants.  Peters,  Sinclair,  and 
Thompson^^  found  that  the  arsenical  vesicant,  lewisite  and  other  vesicants  inhibit  the 
pyruvic  oxidase  system.  The  known  phosphate  transferring  enzymes  are  concerned  with 
the  building  up  of  energy  rich  phosphate  bonds  in  phosphoric  anhydrides  and  acylphos- 
phates.  Enzymes  of  this  type  must  be  concerned  in  the  biosynthesis  of  the  nucleotides 
and  nucleic  acids.  Although  we  know  very  little  of  the  specific  phosphokinases  involved 
in  nucleic  acid  synthesis,  the  fact  that  all  known  phosphokinases  are  easily  inhibited 
by  sulphur  mustard  and  nitrogen  mustards  would  suggest  that  nucleic  acid  synthesis 
should  be  inhibited  by  these  substances.  The  synthesis  of  proteins  may  also  involve 
phosphorylation  of  the  terminal  carboxyl  group  of  a  peptide  chain  and  reaction  of  the 
resulting  acyl  phosphate  with  a  fresh  amino  acid  molecule  to  give  a  new  peptide  link 
and  liberate  phosphate.  A  model  for  this  reaction  is  the  formation  of  glutamine  from 
phosphoryl  glutamic  acid  and  ammonia  (Speck^*,  Elliot^^).  The  enzymes  concerned 
with  nucleic  acid  and  protein  synthesis  need  investigation  and  for  this  the  mitotic  poi- 
sons may  be  useful  tools. 

The  substances  which  induce  mitotic  abnormalities  differ  greatly  in  their  apparent 
chemical  reactivity.  The  aliphatic  nitrogen  mustards  are  very  reactive  substances,  the 
aromatic  chloroethylamines  react  slowly,  but  the  aromatic  carcinogenic  hydrocarbons 
are  rather  inert.  The  French  theoretical  chemists  Daudel,  Pullman  and  then  associates 
(Daudel^^)  have  shown  that  the  carcinogenic  hydrocarbons  have  regions,  known  as  the 
K  regions,  in  which  there  is  high  electron  density,  which  in  the  majority  of  the  carcino- 
genic hydrocarbons  includes  an  activated  phenanthrene  double  bond.  The  activation 
is  enhanced  by  substituents  such  as  benzene  rings  or  methyl  groups  (which  repel  elec- 
Rejerences  p.  300. 


VOL.  4  (1950) 


ABNORMALITIES   IN    CELL    DIVISION 


297 


trons)  in  such  positions  that  they  increase  the  electron  density  of  the  phenanthrene 
double  bond.  This  double  bond  in  the  more  potent  carcinogenic  hydrocarbons  such  as 
9 :  lo-dimethylbenzanthracene  has  a  chemical  reactivity  for  some  addition  reactions 
approaching  that  of  an  aliphatic  ethylenic  bond  and  even  greater  than  that  of  the  ethyl- 
ene bond  of  some  stilbenes.  This  theory  which  is  now  substantiated  by  experimental 
evidence,  suggests  that  the  more  active  carcinogens  in  any  particular  series  of  aromatic 
compounds  are  those  which  are  on  the  whole  the  more  chemically  reactive. 

Phenanthrene  itself  reacts  readily  with  osmic  acid  (Criegee,  Marchand,  and 
Wannowius")  and  the  carcinogenic  hydrocarbons  react  even  more  rapidly  (Badger^^). 
Osmic  acid  adds  on  to  the  double  bond  of  the  K  region  to  form  an  adduct,  which  can  be 
easily  hydrolysed  to  give  cjs-dihydroxydihydro-derivatives.    . 

Perbenzoic  acid  is  another  reagent  which  appears  to  react  with  carcinogenic  hydro- 
carbons at  rates  varying  with  the  carcinogenic  activity.  This  reagent  was  shown  to 
react  with  20-methylcholanthrene  and  3.4-benzpyrene  more  rapidly  than  with  anthra- 
cene and  phenanthrene  (Eckhardt^^)  before  the  theory  of  the  K  region  of  carcinogens 
had  been  developed.  In  looking  for  a  means  of  measuring  the  relative  reactivity  of  the 
K  region,  the  reaction  of  perbenzoic  acid  with  a  series  of  carcinogens  has  been  deter- 
mined. Some  of  the  data  obtained  are  shown  in  Table  IL  The  figures  show  that  the 
carcinogenic  hydrocarbons  react  at  about  the  same  rate  as  the  carcinogenic  aminostil- 
benes.  This  suggests  that  the  bond  of  the  K  region  of  the  hydrocarbons  is  as  reactive  as 
the  ethylenic  bond  of  the  stilbene  molecule  and  as  the  azo  group  of  the  carcinogenic 
dimethylaminoazobenzene. 

The  fact  that  dimethylaminoazobenzene  dosed  to  animals  in  which  it  induces 
hepatoma  is  found  in  a  combined  form  in  the  protein  of  the  liver  (Miller  and  Miller^"), 


TABLE  II 

REACTION    OF    CARCINOGENS    AND    RELATED    SUBSTANCES    WITH    PERBENZOIC    ACID 

M/50  solutions  of  substances  dissolved  in  carbon  tetrachloride  with  M/50  perbenzoic  acid  at  25°  C. 

The  remaining  perbenzoic  acid  was  estimated  iodometrically  and  the  results  are  expressed  as  millimols 

of  perbenzoic  acid  used  per  mol  substrate. 


Compound 

Time  in  hours 

Carcinogenic 

3 

24 

48 

72 

activity 

9:  lo-Anthraquinone 

5 

0 

0 

5 



Naphthalene 

0 

0 

20 

10 

— 

Phenanthrene 

5 

15 

20 

25 

— 

9:  lo-Phenanthraquinone 

0 

0 

25 

35 

— 

Anthracene 

5 

35 

60 

80 

— 

1 : 2-Benzanthracene 

0 

25 

70 

95 

— 

1:2:5: 6-Dibenzanthracene 

0 

15 

35 

95 

+ 

5-Methyl-i :  2-benzanthracene 

0 

40 

40 

no 

+ 

4-Aminostilbene 

32 

85 

120 

140 

+ 

2-Acetylaminofiuorene 

0 

40 

100 

160 

+ 

3:4-Benzpyrene 

5 

90 

130 

202 

+ 

3 : 4-Benzphenanthrene 

45 

90 

145 

220 

+ 

Stilbene 

5 

20 

no 

295 

? 

3:4:5: 6-Dibenzcarbazole 

95 

215 

265 

322 

+ 

20-  Methylcholanthrene 

105 

275 

340 

405 

+ 

2'-Methyl-4-dimethyl-aminostilbene 

215 

405 

465 

535 

+ 

2'-Chlor-4-dimethyl-aminostilbene 

175 

390 

500 

590 

+ 

Dimethylaminoazobenzene 

455 

590 

615 

+ 

References  p.  300. 


298  E.  BOYLAND  VOL.  4  {1950) 

shows  that  a  carcinogen  can  react  with  tissue  protein.  As  the  hydrocarbons  react  with 
perbenzoic  acid  almost  as  rapidly  as  dimethylaminobenzene  and  the  azo  group  of  the 
latter  compound  is  expected  on  theoretical  grounds  to  have  an  electron  density  of  the 
same  order  as  the  carcinogenic  hydrocarbons,  the  carcinogenic  hydrocarbons  might  also 
be  expected  to  combine  with  some  tissue  protein  in  a  similar  way. 

Although  the  French  theoretical  chemists  have  concentrated  on  the  K  region  of  a 
particular  carcinogenic  hydrocarbon  it  is  perhaps  worth  noticing  that  these  substances 
have  two  active  regions.  Many  carcinogens  such  as  1:2:  5 : 6-dibenzanthracene  and  3:4- 
benzphenanthrene  contain  two  active  phenanthrene  double  bonds  or  K  regions.  In  those 
carcinogenic  hydrocarbons  with  only  a  single  K  region  the  groups  which  activate  that 
region  may  also  increase  the  activity  of  a  second  part  of  the  molecule.  Thus,  in  the  potent 
carcinogen  9:  io-dimethyl-i:2-benzanthracene,  the  two  methyl  groups  not  only  make 
the  3 : 4  bond  more  active  than  in  the  unsubstituted  i :  2-benzanthracene  but  also 
increase  the  chemical  reactivity  of  the  9:10  or  meso  positions.  Such  meso  substituted 
anthracene  derivatives  are  extremely  susceptible  to  many  chemical  reactions,  such  as 
photo-oxidation.  The  metabolism  of  carcinogens  also  shows  that  another  region  of  the 
molecule  (the  benzene  ring  adjoining  the  K  region)  is  liable  to  attack  in  vivo.  Although 
it  is  quite  clear  that  carcinogenic  hydrocarbons  must  have  one  centre  of  high  chemical 
reactivity,  they  also  have  a  second  active  centre,  either  a  second  phenanthrene  double 
bond,  active  meso  positions,  or  an  amino  group  as  in  the  aminostilbenes  or  the  amino- 
azobenzene  derivatives. 

The  reactivity  of  hydrocarbons  is  also  shown  by  metabolism  experiments  with  non- 
carcinogenic  hydrocarbons  such  as  naphthalene  (Booth  and  Boyland^^);  (Young*^) 
and  anthracene  (Boyland  and  Levi*^)  as  well  as  with  the  carcinogenic  hydrocarbon 
3 : 4-benzpyrene  (Weigert  and  Mottram^*).  These  hydrocarbons  undergo  the  reaction 
of  perhydroxylation  involving  the  addition  of  the  elements  of  hydrogen  peroxide  with 
formation  of  dihydroxydihydro  derivatives  or  diols.  In  the  case  of  the  non-carcinogenic 
hydrocarbons  the  addition  of  the  hydroxyl  groups  occurs  at  the  centres  with  highest 
electron  density.  But  in  the  carcinogenic  hydrocarbons  which  have  been  examined  the 
oxidation  occurs  in  positions  in  a  ring  adjacent  to  the  K  region  —  not  in  the  reactive 
K  region  itself.  This  may  be  because  the  more  reactive  carcinogens  combine  with  some 
tissue  constituent  through  the  double  bond  so  that  only  regions  of  secondary  activity 
are  available  for  the  oxidative  process.  The  investigation  of  3 : 4-benzpyrene  metabolism 
showed  that  the  dihydroxydihydro-benzpyrene  formed  by  metabolism  in  isolated  skin 
was  combined  to  some  tissue  constituent.  The  combination,  however,  could  be  destroyed 
by  treatment  with  wet  acetone.  Studies  with  i :  2 :  5 : 6-dibenzanthracene  containing 
radioactive  carbon  (Heidelberger  and  Jones^^)  have  shown  that  a  small  part  of  the 
carcinogen  remains  in  animals  for  many  months  after  injection.  Thus  there  are  several 
indications,  that  the  carcinogenic  hydrocarbons  can  react  with  some,  as  yet  unidentified, 
tissue  constituents. 

Although  these  hydrocarbons  have  some  of  the  biological  effects  of  nitrogen  mus- 
tards they  do  not  appear  to  inhibit  the  hexokinase  of  tumours;  the  anaerobic  glycolysis 
and  respiration  of  tumours  is  the  same  whether  they  are  growing  normally  or  are  in- 
hibited by  1 : 2 :  5 : 6-dibenzanthracene  (Boyland  and  Boyland*^).  On  the  other  hand 
inhibition  of  tumour  growth  by  nitrogen  mustard  is  accompanied  by  a  decrease  in  the 
anaerobic  glycolysis  of  the  tissue  (Boyland  et  al.''-^).  This  inhibition  of  tumour  growth 
by  carcinogens,  such  as  4-dimethylaminostilbene  or  1:2:5: 6-dibenzanthracene,  is  only 

References  p.  300. 


VOL.  4  (1950)  ABNORMALITIES   IN    CELL    DIVISION  299 

seen  if  the  treated  animals  are  maintained  on  a  low  protein  diet  (Elson  and  Haddow*^). 
This  finding  indicates  that  the  inhibition  of  growth  is  probably  due  to  interference  with 
protein  metabolism  which  can  be  overcome  if  the  protein  intake  of  the  host  is  sufficiently 
high.  As  1:2:5: 6-dibenzanthracene  causes  abnormalities  of  chromosomes  these  ex- 
periments suggest  that  chromosomes  require  an  adequate  supply  of  amino-acids  for 
their  proper  maintenance. 

The  rates  of  diffusion  and  reaction  are  probably  important  characteristics  of  the 
nuclear  poisons  which  have  been  discussed.  The  compounds  must,  presumably,  react  in 
or  near  the  nucleus  to  produce  their  effects.  For  this  they  must  diffuse  through  the  cell 
to  the  nucleus  more  rapidly  than  they  react  with  the  constituents  of  the  tissue  through 
which  they  are  passing,  unless  they  have  a  specific  affinity  for  the  particular  constituents 
concerned  with  nuclear  behaviour.  The  aliphatic  nitrogen  mustards  react  very  rapidly  in 
the  body,  having  a  life  of  only  a  few  minutes,  but  they  do  not  react  instantaneously 
with  any  reagent  and  diffuse  rapidly  so  that  some  unchanged  molecules  may  reach 
the  nucleus. 

The  evidence  put  forward  supports  the  theory  that  chemical  carcinogenic  and 
therapeutic  agents  for  cancer  combine  with  tissue  constituents  and  that  physical  agents 
cause  some  chemical  change  in  chromosome  constituents. -Goldacre,  Loveless,  and 
Ross^^  suggest  that  it  is  the  chromosomes  themselves  which  are  affected  while  the 
author  considers  that  the  effects  are  due  to  inhibition  of  enzymes  concerned  in  metabolic 
processes  involved  in  maintenance  and  functioning  of  the  chromosomes. 

This  investigation  has  been  supported  by  grants  to  the  Royal  Cancer  Hospital  from 
the  British  Empire  Cancer  Campaign,  the  Jane  Coffin  Childs  Memorial  Fund  for  Medical 
Research,  the  Anna  Fuller  Fund,  and  the  U.S.  Public  Health  Service. 

SUMMARY 

1.  The  association  of  the  effects  of  chromosome  damage,  induction  of  mutations  and  induction 
of  cancer  with  a  number  of  agents  is  discussed. 

2.  Examination  of  the  reaction  of  a  series  of  carcinogenic  compounds  with  perbenzoic  acid  shows 
that  carcinogenic  hydrocarbons  react  more  rapidly  than  simpler  non-carcinogenic  hydrocarbons  and 
at  about  the  same  rate  as  nitrogenous  aromatic  carcinogens. 

3.  The  suggestion  that  the  nitrogen  mustards  and  possibly  other  carcinogens  produce  their 
effects  by  inhibition  of  enzymes  necessary  for  normal  functioning  of  cell  nuclei  is  considered. 

RESUME 

1.  La  relation  entre  les  lesions  des  chromosomes,  I'induction  de  mutations  et  I'induction  du 
cancer  par  un  nombre  d'agents  est  discutee. 

2.  L'examen  de  la  reaction  d'une  serie  de  composes  cancerigenes  avec  I'acide  perbenzoique 
demontre  que  les  hydrocarbures  cancerigenes  reagissent  plus  rapidement  que  les  hydrocarbures  non- 
canc^rigenes  et  a  la  meme  vitesse  a  peu  pres  que  les  substances  cancerigenes  azotees  aromatiques. 

3.  La  suggestion  que  les  moutardes  azotees  et  peut-etre  d'autres  substances  cancerigenes  pro- 
duisent  leurs  effets  en  inhibant  les  enzymes  necessaires  pour  le  fonctionnement  normal  du  noyau 
cellulaire  est  consideree. 

ZUSAMMENFASSUNG 

1.  Das  Verhaltnis  zwischen  Chromosomenverletzung,  Hervorrufen  von  Mutationen  und  Krebs- 
bildung  durch  verschiedene  Agentien  wird  diskutiert. 

2.  Die  Untersuchung  der  Reaktionen  einer  Reihe  von  cancerogenen  Verbindungen  mit  Per- 

References  p.  300. 


300  E.  BOYLAND  VOL.  4  (1950) 

benzoesaure  zeigt,  dass  die  cancerogenen  Kohlenwasserstoffe  schneller  reagieren  als  einfachere,  nicht 
cancerogene  Kohlenwasserstoffe  und  ungefahr  ebenso  schnell  wie  stickstoffhaltige  aromatische 
Krebsstoffe. 

3.  Der  Verfasser  schlagt  vor,  dass  die  Chlorathylamine  und  moglicherweise  auch  andere  cancer- 
ogene Substanzen  ihre  Wirkung  durch  Hemmung  der  fiir  die  normale  Funktion  der  Zellkerne  not- 
wendigen  Enzyme  ausiiben  konnten. 

REFERENCES 

1  A.  Haddow,  Nature,  136  (1935)  868. 

2  E.  H.  Grubbe,  Trans.  Am.  Rontgen  Roy.  Soc.  (1903)  66. 

^  Frieben,  Fortschr.  Gebiete  Rontgenstrahlen,  6  (1902)  106.  ' 

*  H.  J.  MuLLER,  Proc.  Natl  Acad.  Sci.  U.S.,  14  (1928)  714. 

5  T.  S.  Painter  and  H.  J.  Muller,  /.  Heredity,  20  (1929)  287. 

8  P.  C.  KoLLER,  Genetica,  16  (1934)  447- 

'  C.  P.  Rhoads,  J.  Am.  Med.  Assoc,  131  (1946)  656. 

^  E.  BoYLAND  AND  E.  S.  HoRNiNG,  Brit.  J.  Cancer,  3  (1949)  118. 

^  C.  AuERBACH,  J.  M.  Robson,  AND  J.  G.  Carr,  Scieuce,  106  (1947)  243. 
1°  E.  BoYLAND,  J.  W.  Clegg,  P.  C.  KoLLER,  E.  Rhoden,  AND  O.  H.  WARWICK,  Brit.  J.  Cancer,  2 

(1948)  17. 
1^  E.  Paterson,  I.  ApThomas,  A.  Haddow,  and  J.  M.  Watkinson,  Lancet,  i  (1946)  677. 
12  A.  Nettleship  and  P.  S.  Henshaw,  /.  Natl  Cancer  Inst.,  4  (1943)  309. 
^^  F.  Oehlkers,  Z.  Induktive  Abstammungs-  und  Vererhungslehre,  81  (1943)  313. 
1^  E.  Boyland  and  p.  C.  Koll'er  (1949)  (In  preparation). 

^5  A.  Haddow,  C.  M.  Scott,  and  J.  D.  Scott,  Proc.  Roy.  Soc.  B.,  122  {1937)  477- 
^^  E.  L.  Kennaway,  Biochem.  J.,  24  (1930)  497. 
^'  J.  G.  Carr,  Brit.  J.  Cancer,  i  (1947)  152. 
^*  P.  C.  KoLLER  (1948)    Personal  communication. 
^^  J.  W.  Cook  and  G.  A.  D.  Haslewood,  /.  Chem.  Soc,  (1934)  4^8. 
2°  L.  C.  Strong,  Proc.  Natl  Acad.  Sci.,  31  (1945)  290. 

21  A.  Haddow,  G.  A.  R.  Kon,  and  W.  C.  J.  Ross,  Nature,  162  (1948)  824. 
"2  A.  Haddow,  E.  S.  Horning,  and  P.  C.  Koller  (1949)  (In  press). 

23  A.  Haddow,  R.  J.  C.  Harris,  G.  A.  R.  Kon,  and  E.  M.  F.  Roe.  Phil.  Trans,  A,  241  (1948)  i47- 
2*  R.  T.  Hance  and  J.  B.  Murphy,  /.  Exptl  Med.,  41  (1926)  339. 

25  E.  Boyland,  Biochem.  J.,  36  (1942)  7. 

26  R.  J.  Goldacre,  a.  Loveless,  and  W.  C.  J.  Ross,  Nature,  163  (1949)  667. 
2'  J.  N.  Davidson  and  R.  A.  Lawrie,  Biochem.  J.,  43  (1948)  XXIX. 

28  E.  Boyland,  Yale  J.  Biol,  and  Med.,  20  (1948)  321. 

28  E.  S.  G.  Barron,  S.  Dickmans,  and  T.  P.  Singer,  Federation  Proc,  6  236. 

30  O.  Meyerhof  and  J.  R.  Wilson,  Arch.  Biochem.,  17  (1948)  153. 

31  M.  Dixon  and  D.  M.  Needham,  Nature,  158  (1946)  432. 

32  C.  F.  CoRi,  S.  P.  Colowick,  L.  Berger,  and  M.  W.  Stein  (1942-44).  By  communication. 

33  R.  A.  Peters,  H.  M.  Sinclair,  and  R.  H.  S.  Thompson,  Biochem.  J.,  40  (1946)  516. 

34  J.  F.  Speck,  /.  Biol.  Chem.,  168  (1947)  403. 

35  W.  H.  Elliot,  Nature,  161  (1948)  128. 
38  R.  Daudel,  Rev.  Sci.,  84  (1946)  37. 

3^  R.  Criegee,  B.  Marchand,  and  H.  Wannowius,  Ann.,  550  (1942)  99. 

38  G.  M.  Badger,  Brit.  J.  Cancer,  i  (1949)  309. 

39  H.  J.  Eckhardt,  Ber.,  7313. 

40  E.  C.  Miller  and  J.  A.  Miller,  Cancer  Research,  7  (1947)  468. 

41  J.  Booth  and  E.  Boyland,  Biochem.  J .,  44  (1949)  (In  press). 

42  L.  Young,  Biochem.  J .,  41  (1947)  417. 

43  E.  Boyland  and  A.  A.  Levi,  Biochem.  J .,  29  (1935)  2679. 

44  F.  Weigert  and  J.  C.  Mottram,  Cancer  Research,  6  (1946)  109. 

45  C.  Heidelberger  and  H.  B.  Jones,  Cancer,  1  (1948)  252. 

46  E.  Boyland  and  M.  E.  Boyland,  Biochem.  J.,  33  (1939)  618. 
4'  L.  A.  Elson  and  a.  Haddow,  Brit.  J.  Cancer,  1  (1947)  97. 

Received  April  25th,  1949- 


VOL.  4  (1950)  BIOCHI  MICA   ET  BIOPHYSICA   ACTA  3OI 


LIPASE-  CATALYSED 
CONDENSATION  OF  FATTY  ACIDS  WITH  HYDROXYLAMINE 

by 

FRITZ  LIPMANN*  and  L.  CONSTANCE  TUTTLE** 

Biochemical  Research  Laboratory, 

Massachusetts  General  Hospital  and  Department  of  Biological  Chemistry, 

Harvard  Medical  School,  Boston,  Massachusetts  [U.S.A.) 


Some  time  ago  we  reported  preliminarily  on  two  different  types  of  enz5miatic 
reactions  leading  to  a  condensation  with  hydroxylamine^.  Acetate  when  incubated  with 
adenosine  triphosphate  and  hydroxylamine  was  found  to  yield  acet-hydroxamic  acid 
in  fresh  pigeon  liver  extracts.  This  reaction  is  specific  for  acetate,  depends  strictly  on 
ATP,  and  occurs  only  in  fresh  liver  extract  of  the  pigeon  but  not  of  rat,  rabbit  or  hog. 
The  reaction  is  lost  with  aging  but  is  regenerated  on  addition  of  coenzyme  A  and  thus 
belongs  in  a  class  with  the  coenzyme  A  dependent  acetyl  transfer  reaction.  The  charac- 
teristics of  this  type  of  hydroxamic  acid  acid  formation  will  be  reported  on  elsewhere  in 
more  detail. 

The  second  reaction  was  of  an  entirely  different  type.  It  occurred  only  with  higher 
concentrations  of  hydroxylamine  and  was  fully  independent  of  ATP.  In  the  meantime 
we  studied  this  reaction  extensively  and  are  reporting  here  the  results  obtained.  It  is 
found  to  occur  only  weakly  with  acetate  but  increasingly  with  the  lengthening  of  the 
fatty  acid  chain,  up  to  an  optimum  at  octanoate.  It  is  present  in  comparable  strength 
in  all  liver  extracts  studied  so  far.  It  does  not  diminish  appreciably  on  aging  or  dialysis. 
In  contrast  to  the  acetate  reaction  with  ATP,  it  was  strongly  inhibited  by  fluoride. 
This  and  other  observations  eventually  led  to  the  conclusion  that  we  were  dealing  here 
with  a  lipase-catalysed  condensation  of  the  fatty  acid  carboxyl  with  hydroxylamine. 


METHODS  AND  ENZYME  PREPARATIONS 

Hydroxamic  Acid  determination.  —  The  previously  described  method^  was  designed  for  a  deter- 
mination of  acyl  phosphate  formed  during  enzymatic  incubation.  Hydroxylamine  was  added  at  the 
end  of  incubation  to  react  non-enzymatically  with  pre-formed  acyl  phosphate  at  a  pH  of  slightly 
above  6.  Subsequently,  after  deproteinization  with  trichloracetic  acid,  the  color  was  developed  with 
acid  ferric  chloride.  In  contrast  to  this  earlier  set-up,  the  hydroxylamine  now  is  part  of  the  reaction 
system  and  is  present  during  incubation ;  the  method  is  modified  to  determine  the  enzymatically 
formed  hydroxamic  acid.  The  experiment  is  generally  terminated  by  addition  of  a  mixture  of  trich- 
loracetic acid,  hydrochloric  acid  and  additional  hydroxylamine.  Finally  ferric  chloride  is  added.  The 
addition  of  hydroxylamine  serves  only  to  stabilize  the  color  but  does  not  participate  in  primary 

*  I  am  happy  for  the  opportunity  to  express  with  this  contribution  my  gratitude  and  increas- 
ingly realized  indebtedness  to  Professor  Otto  Meyerhof  and  his  laboratory  for  what  I  imbibed 
there  during  my  apprenticeship  from  192 7- 1930. 

**  Present  address:  Department  of  Chemistry,  University  of  Nebraska. 

References  p.  jog. 


302 


F.  LIPMANN,  L.  C.  TUTTLE 


VOL.  4  (1950) 


condensation.  As  previously  described,  the  precipitate  is  eventually  removed  by  filtration  or  centri- 
fugation  and  the  color  determined  in  the  supernatant. 

Determination  in  50^^  alcoholic  solution. —  When  it  appeared  desirable  to  follow  the  hydroxamic 
acid  formation  with  fatty  acids  of  increasing  chain  length,  it  was  observed  that  these  hydroxamic 
acids  became  increasingly  insoluble  in  water  and  on  removing  the  protein  precipitate,  considerable 

amounts  were  lost.  It  was  found,  however, 

2^0  i  that  these  longer  chain  hydroxamic  acids 

are  easily  soluble  in  50%  ethyl  alcohol. 
Therefore  in  the  experiments  dealing  with 
higher  fatty  acids,  a  revised  procedure  was 
used  where,  after  incubation,  the  medium 
was  brought  to  a  concentration  of  appro- 
ximately 50%  in  ethyl  alcohol. 

Procedure  of  Hydroxamic  Acid  Deter- 
mination in  Alcoholic  Solution. —  To  0.5  ml 
of  enzyme-substrate-hydroxylamine  mix- 
ture, 3  ml  of  95%  ethanol  are  added  and 
well  mixed.  Then 

1.  1.5  ml  are  added  of  a  mixture  of 
equal  volumes  of  28%  hydroxylamine-HCl, 
3.5  normal  NaOH  and  a  hydrochloric  acid, 
obtained  by  dilution  of  concentrated  HCl 
with  2  volumes  of  water, 

2.  0.5  ml  of  24%  trichloracetic  acid 
and  finally, 

3.  0.5  ml  of  10%  ferric  chloride  in  0.2 
normal  HCl  are  added.  The  precipitate  is 
filtered  or  centrifuged  off  and  the  color 
measured  in  the  supernatant.  The  main 
change  of  procedure  is  in  the  use  of  more 
highly  concentrated  solutions  in  order  to 
keep  the  volume  down  and  give  space  for 
the  addition  of  ethanol. 

Since  the  appearance  of  our  original 
method,  an  interesting  application  of  the 
hydroxamic  acid-iron  colour  for  colori- 
metry  of  fatty  acid  esters  appeared^.  Esters  were  found  to  react  quantitatively  with  hydroxylamine 
in  strongly  alkaline  solution  and  this  reaction  is  used  by  Hill'  for  a  determination  of  fatty  acid 
esters.  An  extensive  and  very  instructive  discussion  of  the  reaction  between  hydroxylamine  and 
carboxyl  derivatives  may  be  found  in  the  spot  test  analysis  of  Fritz  Feigl*. 


0  0.5  1.0  15  2.0 

jjM  hydroxamic  acid  in  6cc 

Fig.  I.  Standard  curve  for  hydroxamic  acid  determi- 
nation  in   50%    ethanol.   Lithium  acetyl  phosphate 
was  used. 


ENZYME    PREPARATIONS 


Pigeon  and  rat  liver  homogenate  were  prepared  as  described  previously^  using  3  to  4  volumes 
of  1%  potassium  chloride  and  0.02  M  sodium  bicarbonate  solution. 

Hog  liver  fractionation.  —  In  this  fractionation  we  followed  roughly  the  procedure  elaborated 
for  the  purification  of  liver  lipase  by  King  and  his  collaborators®-^.  Fresh  hog  liver  was  obtained 
from  the  slaughterhouse  and  100  grams  were  homogenized  in  a  Waring  blender  with  200  ml  of  o.  i 
molar  disodium  hydrogen  phosphate.  The  homogenate  was  frozen  overnight  and  then  centrifuged 
for  half  an  hour  after  thawing. 

Fraction  L-i,  obtained  by  removal  of  inactive  protein  by  acidification.  —  75  ml  of  the  extract  were 
further  diluted  with  2  volumes  of  o.i  molar  secondary  phosphate  and  recentrifuged.  To  the  super- 
natant 75  ml  of  water  were  added  and  the  mixture  was  now  acidified  with  11. 5  ml  of  normal  acetic 
wherewith  the  pn  was  brought  to  4.8.  A  voluminous  precipitate  formed  and  was  centrifuged  off  and 
discarded.  127  ml  of  strongly  reddish,  almost  clear  supernatant  were  collected.  The  extract  was 
neutralized  with  5  ml  of  normal  ammonia  to  pn  6.8.  10  ml  were  taken  for  analysis. 

Fraction  L-2,  obtained  by  removal  of  inactive  protein  by  half  saturation  with  ammonium  sulphate. — • 
122  ml  of  fraction  L-i  were  mixed  with  an  equal  volume  of  saturated  ammonium  sulphate  solution. 
The  mixture  was  shortly  warmed  to  30°  and  filtered.  The  filtrate  was  dialysed  against  distilled  water. 

Fraction  L-3,  50^^  ammonium  sulphate  precipitate. —  The  precipitate  on  the  filter  was  squeezed 
between  filter  paper  layers  and  dried  as  far  as  possible.  The  precipitate  was  dissolved  in  about  10  ml 
of  water  and  dialysed  in  cellophane  against  4  liter  of  distilled  water  overnight  in  the  cold  room.  Next 
morning  the  globulin  precipitate  formed  on  dialysis  was  centrifuged  and  once  washed  with  water. 

References  p.  309. 


VOL.  4  (1950) 


ENZYMATIC   CONDENSATIONS   WITH    NH2OH 


303 


The  precipitate  was  dissolved  with  Krebs-Ringer  containing  o.oi  molar  ammoniuni  hydroxide  in 
a  10  ml  of  Krebs-Ringer  containing  o.oi  ammonium  hydroxide.  Most  of  it  went  into  solution  and  a 
little  undissolved  was  discarded.  This  fraction  L-3  was  practically  inactive. 

Fraction  L-4  obtained  by  full  saturation  with  ammonium  sulphate.  —  This  is  the  most  active 
fraction.  To  the  half  saturated  ammonium  sulphate  solution  (L-2)  37  grams  per  100  ml  of  solid 
ammonium  sulphate  were  added.  The  total  volume  of  250  ml  obtained.  This  was  warmed  to  30-35° 
and  filtered  overnight  in  the  cold  room.  The  almost  colourless  filtrate  was  discarded.  The  precipitate 
was  dissolved  in  15  ml  water;  it  dissolved  very  completely  to  a  dark  red  fluid.  It  was  dialysed  again.st 
distilled  water  with  agitation  at  room  temperature  for  T,y2  hours.  The  volume  increased  to  32  ml  and 
very  little  precipitate  was  formed,  which  we  centrifuged  off  and  discarded.  This  is  fraction  L-4. 

Pancreas  Lipase 

Pancreatine  Parke-D.wis  as  obtainable  on  the  market  was  used.  Some  fractionation  of  this 
product  is  described  later  on  in  the  text.. 


RESULTS 

In  the  first  two  tables,  the  Upase-catalysed  hydroxamic  acid  formation  is  compared 
with  the  acetate  +  ATP  reaction.  In  Table  I,  the  inactivity  of  ATP  with  octanoate  is 
contrasted  with  its  action  on  acet-hydroxamic  acid  formation.  It  appears  that  the  op- 
timum concentration  of  hydroxylamine  with  ATP  and  acetate  is  0.02  molar  and  that 
at  0.05  molar  already  an  inhibition  is  observed.  Table  II  shows  the  effect  of  increased 
concentrations  of  hydroxylamine  on  the  condensation  with  octanoate.  The  strong 
dependence  of  this  reaction  on  the  high  concentration  of  hydroxylamine  will  be  noted 
as  well  as  its  independence  on  the  presence  of  ATP.  In  the  further  study  generally 
an  0.4-0.6  molar  concentration  of  hydroxylamine  was  used. 

TABLE  I 

HYDROXAMIC  ACID  FORM.\TION  WITH  ACETATE  -|- ATP  AT  VARIOUS  CONCENTRATIONS  OF  HYDROXYLAMINE 

All  tubes  contained  0.5  ml  of  10%  fresh  acetone  pigeon   liver  extract  in  a  total  volume  of  i.i  ml, 
PH  7-3.  temperature  37°,  60  minutes  incubation. 


Octanoate 
M 

Acetate 
M 

ATP 
M 

Hydroxylamine 
M 

Hydroxamic  Acid  Formed 
^M 

O.OI 

O.OI 
O.OI 
O.OI 

O.OI 
O.OI 
O.OI 
O.OI 

0.05 
0.02 

O.OI 

0.02 

0.48 
1.08 
0.78 
0.02 

TABLE  II 

HYDROXAMIC    ACID    FORMATION    FROM    OCTANOATE   AT   HIGHER    CONCENTRATION    OF   HYDROXYLAMINE 

Each  tube  contained  0.5  ml  rat  liver  homogenate  (i  :3  in  1%  KCl,  frozen  for  4  days)  in  a  total  volume 
of  1.4  ml,  adjusted  to  pH  7-3,  37°,  60  minute  incubation  in  air. 


Octanoate 
M 

ATP 
M 

Hydroxylamine 
M 

Hydroxamic  Acid  Formed 
juM 

0.014 
0.014 
0.014 
0.014 

O.OOI 
O.OOI 

0-43 
0.43 
0.14 
0.14 

1.99 
1.90;  2.1* 

0.54 
0.48 

Parallel  experiment  in  a  Warburg  vessel  with  nitrogen  in  the  gas  phase. 

References  p.  jog. 


304 


F.  LIPMANN,  L.  C.  TUTTLE 


VOL.  4  (1950) 


Table  III  shows  the  pjj  optimum  of  the  Hpase  reaction  to  be  at  7.2.  The  measure- 
ments at  the  more  acid  range,  however,  do  not  give  a  true  impression  of  the  pn  depend- 
ence. A  decrease  of  activity  here  is  partly  caused  by  the  higher  concentrations  of  free 
fatty  acid  which  is  rather  strongly  inhibitory^. 

TABLE  III 

THE    Ph    OPTIMUM    OF    HYDROXAMIC    ACID    FORMATION    WITH    PORK    LIVER    EXTRACT 

Each  tube  contained  0.25  ml  liver  extract,  o.i  ml  of  o.i  M  octanoate,  and  0.15  ml  of  2  M  hydroxy  1- 
amine  hydrochloride-NaOH  buffer,  60  minute  incubated  at  37°.  The  buffer  was  prepared  by 
neutralizing  a  4  M  hydroxylamine  HCl  solution  with  increasing  amounts  of  NaOH  and  adjusting 
the  volume  with  water 


Hydroxylamine 

HCl 

Hydroxamic  Acid  Formed 

NaOH 

Ph 

/iM 

2:0.5 

5-9 

1-39 

2: 1 

6.4 

2.4 

2:1.5 

7.2 

2.99 

2:1.75 

7-5 

2.76 

2:1.95 

8 

1.42 

In  Table  IV,  the  activity  of  some  lipase  inhibitors  is  recorded.  Like  lipase  the 
hydroxamic  acid  reaction  is  strongly  inhibited  by  fluoride^"  and  hexyl  resorcinoF.  The 
action  of  benzoate  is  of  some  interest.  An  inhibitory  effect  of  benzoate  on  the  oxidation 
of  butyric  but  none  or  less  of  octanoic  acid  was  observed  by  Quastel  and  his  colla- 
borators^^. The  hydroxamic  acid  reaction  follows  the  same  pattern  of  decreased  inhibi- 
tion with  increasing  chain  length  of  the  fatty  acids.  The  inhibition  of  hydrolytic  lipase 
action  of  this  liver  extract  was  checked  manometrically  with  tributyrin  in  bicarbonate 
solution.  It  was  found  to  a  similar  extent  to  be  affected  by  fluoride  and  hexyl  resorcinol; 
but  benzoate  showed  only  a  small  inhibition  of  about  10%. 

TABLE  IV 

ACTION    OF    LIPASE    INHIBITORS    ON    HYDROXAMIC    ACID    FORMATION    WITH    HOG   LIVER    EXTRACTS 


Inhibitor 

Concentration 

Substrate 

%  Inhibition 

Sodium  fluoride 

0.05   M 
O.OI   M 
0.003 M 

octanoate 
octanoate 
octanoate 

71 
51 
35 

Sodium  benzoate 

O.OI   M 
O.OI    M 
O.OI   M 
O.OI   M 

propionate 
butyrate 
hexanoate 
octanoate 

87 
83  ' 
20 

15 

Hexylresorcinol  0.25% 

octanoate 

40 

In  Table  V,  the  lipase  action  and  hydroxamic  acid  formation  are  compared  with 
the  various  fractions,  obtained  as  described  above  from  hog  liver  extract.  The  parallel 
is  rather  striking.  It  may  be  noted  that  the  absolute  activity  expressed  in  /uM  turnover 

References  p.  jog. 


VOL.  4  (1950) 


ENZYMATIC    CONDENSATIONS   WITH    NHjOH 


305 


is  considerably  smaller  in  the  case  of  hydroxamic  acid  formation.  The  dependence  of 
lipatic  hydroxamic  acid  condensation  on  higher  concentrations  of  hydroxylamine  sug- 
gested a  near  equilibrium  situation.  Therefore,  the  influence  of  the  concentration  of 

TABLE  V 

COMPARISON  OF  HYDROXAMIC  ACID  FORMATION  AND  TRIBUTYRIN 
HYDROLYSIS    WITH    VARIOUS    HOG    LIVER    FRACTIONS 


Hog  Liver  Fraction 

Hydroxamic  Acid 
/iM/6o' 

Tributyrin  Split 

Li 

L2 

L3 
L4 

0.73 
0.75 

O.OI 

1.2 

1.67 
1.6 
0.03 
3-25 

For  the  hydroxamic  acid  determination  o.i  of  the  original  fraction  was  used  in  a  total  volume  of 
0.5  ml,  hydroxylamine  0.6  M,  and  octanoate  0.02  M,  and  incubated  for  60  minutes  at  37°. 
Tributyrin  hydroh'sis  was  measured  mano metrically  with  the  manometer  containing  the  fraction 
in  appropriate  dilutions,  Li:i/i2;  L2:i/i2;  L3:none;  L4:i/2o.  The  vessels  contained  o.i  ml  of 
the  diluted  fraction,  0.6  ml  of  0.1  M  Na  bicarbonate  and  0.05  tributjTin  was  dipped  in  from  the  side 
arm.  The  gas  room  contained  5%  COg  in  N^.  To  make  the  two  series  comparable  the  values  recorded 
in  the  table  for  the  manometric  experiment  were  obtained  by  multiplication  with  the  respective 
dilution  factors. 


the  other  reaction  partner,  the  carboxyl  ion, 
was  likewise  tested.  In  Fig.  2,  two  concen- 
tration levels,  0.02  and  0.2  molar  are  com- 
pared. The  expected  increase  with  carboxylate 
concentration  is  most  evident  at  intermediate 
chain  lengths.  With  longer  chain  lengths  the 
often  observed  inhibition  by  free  long-chain 
fatty  acid  overlaps.  This  also  explains  the 
change  of  the  chain  length  optimum  toward 
shorter  chains  at  higher  concentration,  due 
to  increasing  hydrolysis  of  the  salt  at  higher 
concentration  levels.  It  is  of  special  interest 
that  the  acetate  ion  starts  to  show  appreciable 
activity  at  the  0.2  molar  level. 

In  Fig.  3  the  time  curve  of  the  reaction 
is  traced.  It  appears  that,  with  the  reactants 
present  in  excess,  the  condensation  occurs 
practically  proportionally  with  time,  indi- 
cating, as  would  be  expected,  an  enzymatic 
reaction  of  the  zero  order. 

Although  in  the  experiment  with  carb- 
oxylate ion  an  intermediate  formation  of 
an  ester  was  seemingly  excluded,  it  appeared 
nevertheless  of  interest  to  explore  the  pos- 
sibility of  rapid  enzymatic  conversion  of  ester 
into  hydroxamate.  For  this  purpose,  the  en- 
zyme was  incubated  with  equivalent  amounts  of  tributyrin  and  butyrate.  As  shown 
References  p.  309. 


4      5 

Fig.  2.  Comparison  of  hydroxamic  acid 
formation  with  0.02  and  0.2  M  octanoate, 
0.6M  hydroxylamine,  and  0.1  ml  enzyme 
solution  in  0.5  ml  total  volume,  60  minutes 
incubabation  at  37°. 


3o6 


F.  LIPMANN,  L.  C.  TUTTLE 


VOL.  4  (1950) 


in  an  earlier  table,  the  ester  hydrolysis  is  much 
more  rapid  as  the  condensation  reaction  and 
very  soon  the  tributyrin  was  split  to  com- 
pletion. An  appreciable  exchange  should, 
however,  have  been  shown  by  a  considerable 
increase  of  hydroxamate  formation  with  the 
ester.  The  values  found  (Table  VI)  are  prac- 
tically identical,  due  to  the  presence  of  nearly 
equivalent  amounts  of  butyrate  during  the 
major  part  of  the  incubation  period.  In  the 
sample  with  tributyrin,  the  butyrate  obviously 
originated  from  hydrolysis. 

In  similar  experiments  with  equivalent 
amounts  of  ethyl  and  sodium  butyrate,  similar 
results  were  obtained.  A  slight  increase  of 
hydroxamate  formation  was  observed  in  the 
earlier  part  of  the  incubation  period,  which 
evened  out,  however,  with  the  progress  of 
time.  This  may  be  due  to  a  non-enzymatic 
reaction  of  the  ester  with  hydroxylamine, 
recently  observed  under  analogous  conditions 
by  Chantrenne^^  or  to  a  slow  enzymatic  exchange  reaction. 


15  30  45 

TIME,    MINUTES 

Fig.    3.    Time    curve    of    hydroxamic   acid 
formation.  Conditions  as  in  Fig.  2.  0.02  M 
octanoate. 


TABLE  VI 

COMPARISON  OF  EQUIVALENT  AMOUNTS  OF  TRIBUTYRIN  AND  BUTYRATE 


Added 

Hydroxamic  Acid  Formed 

Tributyrin 
Butyrate 

3-4 

1-7 

10. 0 

0.76 
0.33 
0.75 

0.1  ml  of  hog  liver  extract  in  0.5  ml  total  volume,  0.6  M  hydroxylamine.  The  tributyrin  was  diluted 
with  9  volumes  of  95%  ethanol  of  which  0.0 1  ml  was  added.  The  same  amount  of  ethanol  was  added 
to  the  butyrate  sample  to  equalize  conditions. 


EXPERIMENTS   WITH    PANCREAS    LIPASE    PREPARATIONS 


In  order  further  to  check  the  ability  of  lipase  to  condense  carboxyl  groups  with 
hydroxylamine  we  turned  to  an  exploration  of  the  action  of  pancreas  lipase  on  fatty  acid 
and  hydroxylamine.  As  source  of  the  enzyme,  the  marketed  pancreatine  of  Parke-Davis 
was  used.  The  condensation  with  hydroxylamine  was  easily  observed  likewise  with 
pancreas  enzyme,  although  somewhat  less  actively  than  with  the  liver  enzyme.  Sig- 
nificantly, the  chain  length  optimum  was  sh'fted  to  the  longer  chains  in  accordance 
with  the  more  truly  lipatic  nature  of  the  pancreas  enzyme. 

By  using  an  untreated  suspension  of  pancreatine  a  rather  la^ge  blank  value  was 
obtained.  Th's  could,  however,  be  reduced  considerably  by  wash'ng  with  sl'ghtly  ac'd 
fluid.  Generally,  not  too  much  activity  went  into  solution  in  this  manner.  The  residue 
References  p.  3og. 


VOL.  4  (1950) 


ENZYMATIC    CONDENSATIONS   WITH    NH.,OH 


307 


was  used  as  a  suspension.  In  Table  VII,  the  hydroxamic  acid  formation  with  dodecanoate 
is  described  using  various  fractions.  The  results  are  analogous  to  those  obtained  with 
the  liver  enzyme. 

TABLE  VII 

HYDROXAMIC    ACID    FORMED    WITH    PANCREATINE,  PaRKE-DaVIS 


No. 

Preparation 

Dodecanoate 
M 

Hydroxamic  Acid 

I 

Orginal  Suspension,  5% 

O.OI 

1-39 
2.32 

2 

Supernatant 

O.OI 

I-3I 
1.63 

3 

Residue  resuspended  to  volume 

O.OI 

0.16 
0.74 

4 

Residue  resuspended  in  ^/g  original  volumec 

O.OI 

0.40 
1.94 

0.5  g  of  pancreatine  was  suspended  in  10  ml  water,  an  aliquot  was  used  in  experiment  i.  20  drops  of 
0.02  molar  acetic  acid  were  added  and  the  suspension  shaken  up.  The  suspension  was  centrifuged 
for  half  an  hour  in  the  cold  room.  The  supernatant  was  neutralized  and  used  for  experiment  2.  The 
residue  was  resuspended  in  0.02  M  ammonia  buffer  with  final  pH  of  8,  and  used  for  experiments 
3  and  4. 

Each  tube  contained  0.14  ml  of  2  M  hydroxylamine  buffer  of  pH  6.6,  0.25  ml  enzyme  solution  and 
o.i  ml  of  o.i  M  dodecanoate  or  o.i  ml  water.  The  dodecanoate  solution  had  to  be  warmed  up  before 
addition.  Incubation  for  60  minutes  at  37°;  hydroxamic  acid  determination  in  alcoholic  solution. 


30 


20 


HOG  LIVER 
EXTRACT 


PANCREATINE 


6        7        8        S       10 
CHAIN      LENGTH 

Fig.  4.  Chain  length  optimum  for  liver  and  pancreas  lipase.  The  conditions  for  liver  extract  were  as 
described  in  Fig.  3,  60  minutes  incubation  time.  Pancreatine,  5%  suspension,  0.75  ml  in  0.05  M 
secondary  sodium  phosphate,  0.45  ml  2  M  hydroxylamine,  pjj  7,  0.3  ml  of  0.05  M  fatty  acid  salts, 

60  minutes  incubation. 

References  p.  jog. 


308  F.  LIPMANN,  L.  C.  TUTTLE  VOL.  4  (1950) 

A  survey  and  comparison  of  results  obtained  with  the  Hver  and  pancreas  enzyme 
appear  in  Fig.  4.  Particularly  the  difference  in  the  chain  length  optimum  may  be  noted, 
the  optimum  being  found  at  octanoate  for  liver  and  at  dodecanoate  for  pancreas  lipase. 
The  previously  mentioned  inhibitory  effect  of  free  long-chain  fatty  acids  surely  affects 
somewhat  the  situation  of  this  optimum.  In  the  experiments  with  solutions  of  the  salts 
of  higher  members  of  the  fatty  acid  series,  the  solution  was  prepared  by  warming  the 
acid  with  equivalent  amounts  of  sodium  hydroxide.  Such  solutions  jelled  on  cooling 
and  had  to  be  re  warmed  for  use  in  the  experiment. 

DISCUSSION 

There  are  primarily  two  points  that  seem  to  deserve  comment;  one,  the  low  energy 
requirement  of  the  hydroxamic  acid  condensation  and  tico,  the  apparent  non-specificity 
of  this  reaction  for  an  esterase.  Although  no  attempts  were  made  here  to  determine 
accurately  the  equilibrium  point,  it  is  quite  obvious  from  the  relatively  low  concentra- 
tion of  the  reactants  which  are  sufficient  to  support  condensation  on  the  catalyst  that 
the  change  of  free  energy  with  this  condensation  cannot  be  more  than  a  few  hundred 
calories.  It  nevertheless  is  well  known  that  spontaneous  reaction  between  the  free  car- 
boxyl  group  and  hydroxylamine  will  not  occur*  and  that  therefore  hydroxylamine  re- 
mains to  be  regarded  a  trapping  reagent  for  activated  carboxyl  groups.  It  is  true  that 
such  activation  need  not  mean  the  actual  input  of  considerable  energy  by  a  creation  of 
an  energy-rich  link.  However,  the  acetate^  or  glutamate^^  activation  by  primary  reaction 
with  ATP,  so  easily  measured  by  use  of  the  hydroxamic  acid  reaction,  bears  evidence  how 
valuable  a  tool  hydroxylamine  has  become  for  a  detection  of  this  type  of  reaction.  Never- 
theless as  rightly  emphasized  by  Chantrenne^^,  a  judicious  evaluation  of  the  particular 
experimental  conditions  is  required  and  the  use  of  lower  concentration  of  hydroxylamine 
may  be  recommended  in  cases  where  an  activation  of  carboxyl  by  primary  formation 
of  an  energy-rich  linkage  is  suspected. 

The  "non-specificity"  of  the  here  described  esterase  activity  appears  of  some 
significance.  The  link  formed  here  by  esterase  action  may  be  considered  rather  a  peptidic 
link.  It  is  thus  tempting  to  look  at  this  reaction  as  the  reverse  phenomenon  to  the 
esterase  activity  of  chymotrypsin,  uncovered  recently  by  Neurath  and  his  group^^. 


SUMMARY 

A  lipase-catalysed  condensation  of  fatty  acid  and  hydroxylamine  is  described.  Reaction  in  liver 
extracts  follows  the  inhibition  pattern  of  liver  lipase,  hexyl  resorcinol  and  fluoride  acting  as  powerful 
inhibitors.  On  fractionation  of  hog  liver  extract,  the  esterase  and  condensation  activities  remain 
associated.  An  analogous  reaction  is  found  with  pancreatine. 

The  condensation  with  hy  roxylamine  on  lipase  occurs  only  with  relatively  high  concentrations 
of  hydroxylamine  and  the  reaction  is  further  enhanced  by  increase  of  the  fatty  acid  concentration. 
To  obtain  considerable  hydroxamic  acid  formation,  the  concentration  of  0.4  to  0.6  molar  of  hydroxyl- 
amine is  required.  Witli  liver  esterase,  the  chain  length  optimum  is  found  with  octanoate,  while 
pancreas  lipase  reacts  little  with  compounds  containing  below  8  carbons,  and  shows  optimum  activity 
with  dodecanoate. 

The  observations  indicate  that  a  relatively  small  change  of  free  energy  occurs  with  condensation 
of  fatty  acids  with  hydroxylamine  to  form  hydroxamic  acid. 

For  the  determination  of  the  hydroxamic  acid  of  long-chain  fatty  acids,  a  50%  alcoholic  medium 
is  required  because  of  the  water  insolubility  of  this  compound.  The  hydroxamic  acid  determination 
was  modified  for  50%  ethanol-water. 

References  p.  jog. 


VOL.  4  (1950)  ENZYMATIC   CONDENSATIONS   WITH    NHgOH  3O9 

RfiSUMfi 

Les  auteurs  d^crivent  une  condensation  d'acide  gras  et  d'hydroxylamine  catalys^e  par  une 
lipase.  La  reaction  dans  les  extraits  de  foie  suit  le  schema  d'inhibition  de  la  lipase  de  foie,  I'hexyl- 
resorcine  et  le  fluorure  agissant  comme  inhibiteurs  puissants.  Lors  du  fractionnement  d'un  extrait 
de  foie  de  pore  les  activites  d'esterase  et  de  condensation  restent  associ6es.  L'on  trouve  une  reaction 
semblable  pour  la  pancreatine. 

La  condensation  avec  I'hydroxylamine  sous  Taction  de  la  lipase  se  produit  seulement  a  des 
concentrations  relativement  elevees  d'hydroxylamine  et  elle  est  acceleree  par  une  augmentation 
de  la  concentration  en  acide  gras.  Pour  obtenir  une  formation  d'acide  hydroxamique  considerable, 
l'on  doit  avoir  une  concentration  0.4  a  0.6  molaire  en  hydroxylamine.  Avec  la  lipase  de  foie  I'optimuni 
de  longueur  de  chaine  est  atteint  avec  I'octanoate,  tandis  que  la  lipase  de  pancreas  reagit  peu  avec 
les  composes  contenant  moins  de  8  atomes  de  carbone  et  montre  une  activite  optima  pour  le  dode- 
canoate. 

Les  observations  que  nous  avons  pu  faire  indiquent  qu'un  changement  relativement  faible 
d'energie  libre  se  produit  lors  de  la  condensation  des  acides  gras  avec  I'hydroxylamine  pour  former 
les  acides  hydroxamiques  correspondants. 

Pour  la  determination  des  acides  hydroxamiques  d'acides  gras  a  longue  chaine,  il  faut  employer 
nn  milieu  contenant  50%  d'alcool,  parceque  ces  produits  sont  insolubles  dans  I'eau.  La  determination 
d'acide  hydroxamique  a  ete  modifiee  pour  un  miUeu  ethanol/eau  a  50%. 

ZUSAMMENFASSUNG 

Eine  durch  Lipase  katalysierte  Kondensation  der  Fettsauren  mit  Hydroxjdamin  wird  be- 
schrieben.  Die  Reaktion  in  Leberextrakten  folgt  dem  Hemmungsschema  der  Leberlipase;  Hexyl- 
resorcin  und  Fluorid  wirken  als  starke  Hemmstoffe.  Bei  der  Fraktionierung  eines  Schweineleber- 
extraktes  bleiben  die  Esterase-  und  Kondensationsaktivitaten  vereinigt.  Eine  analoge  Reaktion 
wurde  fiir  Pankreatin  gefunden. 

Die  Kondensation  mit  Hydroxylamin  iiber  Lipase  findet  nur  bei  verhaltnismassig  hohen 
Hydroxylaminkonzentrationen  statt  und  wird  durch  Zunahme  der  Fettsaurekonzentration  weiter 
gesteigert.  Zur  Eriangung  einer  erheblichen  Hydroxamsaurebildung  ist  eine  0.4  bis  0.6  molare 
Hydroxylaminkonzentration  erforderlich.  Fiir  Leberlipase  ist  die  optimale  Kettenlange  mit  dem 
Oktanoat  erreicht,  wahrend  Pankreaslipase  nur  schwach  mit  Verbindungen  reagiert,  die  weniger 
als  8  Kohlenstoffatome  enthalten  und  fiir  das  Dodekanoat  eine  optimale  Aktivitat  zeigt. 

Unsere  Beobachtungen  weisen  darauf  hin,  dass  bei  der  Kondensation  von  Fettsauren  mit 
Hydroxylamin  unter  Bildung  von  Hydroxamsauren  verhaltnismassig  geringe  Anderungen  der  freien 
Energie  stattfinden. 

Zur  Bestimmung  der  Hydroxamsauren  von  Fettsauren  mit  langen  Ketten  muss,  wegen  der 
Unloslichkeit  dieser  Verbindungen  in  Wasser,  in  50%  igem  Alkohol  gearbeitet  warden.  Die  Hydroxam- 
saurebestimmung  wurde  fiir  50%  iges  Athanol/Wasser  angepasst. 

REFERENCES 

1  F.  LiPMANN  AND  L.  C.  TuTTLE,  /.  Biol.  Chcm.,  161  (1945)  415. 

2  F.  LiPMANN  AND  L.  C.  TuTTLE,  /.  Btol.  Chem.,  159  (1945)  21. 

3  U.  T.  Hill,  Ind.  Eng.  Chem.  Anal.  Ed.,  18  (1946)  317. 

*  F.  Feigl,  Quantitative  Analysis  by  Spot  Tests,  Elsevier  Publ.  Co.,  New  York  1946,  cf.  pages  352-353 
and  particularly  355-359- 

^  F.  LiPMANN,  /.  Biol.  Chem.,  160  (1945)  173. 

®  D.  Click  and  C.  G.  King,  /.  Biol.  Chem.,  94  (1931)  497. 

'  D.  Click  and  C.  C.  King,  /.  Biol.  Chem.,  95  (1932)  477. 

8  H.  H.  R.  Weber  and  C.  C.  King,  /.  Biol.  Chem.,  108  (1935)  131. 

*  Z.  Baker  and  C.  G.  King,  /.  Am.  Chem.  Soc,  57  (1935)  358. 
^°  F.  Li?MANN,  Biochem.  Z.,  206  (1929)  171. 

1^  M.  JowETT  AND  J.  H.  QuASTEL,  Biochem.  J.,  29  (1935)  2143. 

^2  H.  Chantrenne,  Compt.  rend.  trav.  lab.  Carlsber^,  26  (1948)  231. 

^^  J.  F.  Speck,  /.  Biol.  Chem.,  168  (1947)  403. 

"  W.  H.  Elliott,  Nature.  161  (1948)  128. 

^5  S.  Kaufman,  H.  Neurath,  and  G.  W.  Schwert,  /.  Biol.  Chem.,  177  (1949)  793. 

Received  May  gth,  1949 


310  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


ACYLATION  REACTIONS  MEDIATED  BY  PURIFIED  ACETYLCHOLINE 

ESTERASE  11* 

by 

SHLOMO  HESTRIN** 

Department  of  Neurology,  College  of  Physicians  and  Surgeons,  Columbia  University, 

New  York  (U.S.A.) 


The  probability  that  acetylchoUne  esterase  plays  a  role  in  the  generation  of  the 
action  potential^  lends  special  interest  to  the  study  of  the  nature  of  this  enzyme  and  of 
the  reactions  which  it  may  mediate.  In  an  earlier  communication^  the  ability  of  the 
electric  tissue  esterase  of  Electrophorus  electricus  to  mediate  acylations  of  choline  and 
hydroxylamine  was  noted.  In  the  present  report,  factors  which  govern  the  rate  and 
extent  of  these  reactions  are  considered. 

The  specificity  and  affinity  of  purified  electric  tissues  esterase  for  a  wide  range  of 
substrates  and  inhibitors  have  been  studied  by  Nachmansohn  et  al.^>  ^  and  more 
recently  by  Augustinsson^'^.  An  important  function  of  the  enzyme  —  the  hydrolysis 
of  esters  as  a  function  of  p^  —has  not  been  described  previously. The  manometric  method 
of  esterase  assay  is  conveniently  applicable  within  a  narrow  range  of  p^-  Characterization 
of  the  Ph  function  of  the  enzyme  by  the  potentiometric  technique  for  the  determination 
of  the  acid  reaction  product  would  be  feasible  but  laborious.  A  colorimetric  method'  for 
the  assay  of  ester  in  the  presence  of  excess  of  products  of  ester  hydrolysis  affords  a 
convenient  procedure  for  assay  of  esterase  activity  at  any  desired  p^.  The  method  is 
applicable  equally  to  measurement  of  both  hydrolysis  and  synthesis  of  the  ester  and 
with  its  aid  information  concerning  the  p^  function  of  an  esterase  is  easily  obtainable. 

METHODS 

Acetycholine  and  propionylcholine  were  determined  according  to  the  procedure  previously 
described'.  Aliquots  of  0.5  or  i.o  ml  of  the  test  solution  containing  0.3  to  4.0  jjM.  of  ester  were  used 
for  the  determinations. 

Acethyldroxamic  and  propionhydroxamic  acid  were  measured  in  aliquots  of  0.5  or  o.i  ml  con- 
taining 0.3  to  4.0  /iM.  The  samples  were  brought  to  pn  i. 0-1.4  with  hydrochloric  acid  and  then  esti- 
mated colorimetrically  with  i  °/^^  ferric  chloride  essentially  as  in  the  method  for  the  determination  of 
acetylcholine'. 

The  Klett  photoelectric  colorimeter  was  used  with  green  filter  54. 

Enzyme 

Acetylcholine  esterase  of  the  electric  tissue  of  Electrophorus  electricus  was  used.  The  enzyme  was 
purified  according  to  the  method  described  by  Rothenberg  and  Nachmansohn*.  The  enzyme  was 
dissolved  in  a  medium  of  sodium  pho.sphate  0.05  M,  magnesium  chloride  0.02  M,  and  sodium  chloride 
0.1  M  at  ph  7-0  and  stored  in  the  cold  at  4^^  C.  Stock  enzyme  solutions  were  diluted  into  2.8%  gelatin 
freshly  before  use.  In  the  hydrolysis  experiments  the  final  dilution  of  the  enzyme  solution  was  in  the 
order  of  magnitude  of  one  part  in  ten  thousand ;  in  the  experiments  on  acylation  a  much  higher  enzyme 
concentration  —  an  order  of  magnitude  of  one  part  in  ten  —  was  used. 

*  This  work  has  been  carried  out  under  grants  from  the  U.  S.  Public  Health  Service  and  the 
Office  rf  Naval  Research. 

Present  address:  The  Hebrew  University,  Jerusalem,  Israel. 

References  p.  321. 


VOL.  4  (1950) 


ACYLATIONS   BY   ACETYLCHOLINE    ESTERASE  II 


311 


A.    HYDROLYSIS   OF   ACETYLCHOLINE    AS   A   FUNCTION    OF   Pn 

An  enzyme  concentration  assay  curve  is  reproduced  in  Fig.  i.  The  hydrolysis-time 
curves  in  phosphate  solution  at  Ph  74  depart  from  a  stra'ght  line  to  a  measurable  extent 
only  alter  about  30%  of  the  substrate  at  an  initial  concentration  of  4  ^M  per  ml  has 
been  split.  The  plot  of  the  initial  reaction  velocity  against  enzyme  concentration  in  the 
range  studied  yields  a  straight  line. 


Fig.  I.  Acetylcholine  hj'drolysis  as  a  function 
of  enzyme  concentration.  Mixtures  contain  i  .0 
M  potassium  dihydrogen  phosphate  adjusted 
with  sodium  hydroxide  to  pn  7-4.  gelatin 
0.07%,  acetylcholine  4  //M/ml.  Temperature 
23°  C.  The  Ph  remained  constant  within  0.2  pn 
units  during  the  course  of  the  hydrolysis.  The 
non-enzymatic  hydrolysis  in  these  conditions 
was  barely  detectable.  Curves  1-5  show  fin- 
dings with  enzyme  dilutions  i :  4  000,  i :  8  000, 
1:12000,  1:20000  and  1 :  30000  respectively. 
In  the  inset,  relative  enzyme  concentration 
is  plotted  on  the  abscisca  and  the  corres- 
ponding relative  initial  reaction  velocity  on 
the  ordinate. 


,t?80 


36  i,2 

Minctes 


TABLE  I 

ACETYLCHOLINE    HYDROLYSIS    IN    PHOSPHATE    SOLUTION    AS    A    FUNCTION    OF    pH    IN    THE    ACID    RANGE 

The  solutions  contained  a  constant  amount  of  enzyme,  0.07%  gelatin,  o.i  M  potassium  phosphate, 
sodium  hydroxide  in  varying  amounts  and  acetylcholine  chloride  in  a  concentration  of  4  //M/ml. 
The  ph  remained  constant  during  the  course  of  the  hydrolysis  within  0.2  pn  units.  Temperature 
21°  C.  Non-enzj^matic  hydrolysis  proved  negligible  in  the  conditions  used.  Control  mi.xtures  to  which 
no  acetylcholine  was  added  failed  to  produce  colour  when  examined  with  the  reagent.  The  solutions 
remained   clear   and   removal   of  the   protein   present  in   the   reaction   mixture   was   unnecessary. 


Per  cent  hydrolysis  at  times  (min) 

Ph 

10' 

20' 

30' 

40' 

7.8 

7-4 
6.8 

6.3 
5-8 
5-5 

17 
16 

15 

13 

9 

32 
31 
28 

25 
17 
13 

45 
45 
39 
35 
23 

57 
55 
49 
44 
30 

Variation  of  esterase  activity  accompanied  shift  of  pn  on  the  acid  side  of  the  scale 
in  a  range  which  is  still  of  physiolog'cal  interest.  The  course  of  the  reaction  in  phosphate 
buffer  is  illustrated  by  the  experiment  recorded  in  Table  I.  It  is  evident  that  increase 
of  Ph  from  5.5  to  7.4  results  in  a  progressive  and  marked  rise  of  reaction  rate  in  phosphate 
buffer.  Between  pn  7.4  and  7.8  in  phosphate  and  between  p^  7.6  and  9.4  in  borate  the 
enzyme-mediated  hydrolysis  exhibited  a  constant  initial  reaction  rate.  At  pn  higher 
than  9.4  inactivation  of  enzyme  occurred  at  21°  C,  the  inactivation  was  retarded  con- 
siderably at  17°  C.  Non-enzymatic  hydrolysis  of  the  substrate  was  found  to  become 
relatively  appreciable  at  pn  9.2  and  rose  rapidly  with  further  increase  of  the  pn  (Table  II). 
A  summary  of  findings  is  presented  in  Fig.  2.  The  p^  range  in  which  the  acetylcholine 
Jie/erences  p.  321. 


312 


S.  HESTRIN 


VOL.  4  (1950) 


TABLE  II 

ACETYLCHOLINE    HYDROLYSIS    IN   BORATE    SOLUTION   AS    A    FUNCTION    OF   PH   IN    THE   ALKALINE    RANGE 

a)  Reaction  mixtures  contained  a  constant  amount  of  enzyme,  acetylcholine  chloride  4  ^M/ml., 
0.07%  gelatin  and  2  ml  of  Sorensen  borate  buffer  in  4  ml  of  final  mixture.  Temperature  21°  C. 
PH  remained  unchanged  within  0.2  pH  units  throughout  the  course  of  reaction.   Non-enzymatic 

hydrolysis  was  negligible. 


Percentage  hydrolysis  at  times  (min) 

PH 

5' 

10' 

20' 

30' 

35' 

7.6 

7-9 
8.1 

8 
8 
9 

17 

18 
18 

30 
31 
33 

43 

44 

51 
49 
50 

b)  As  in  a)  but  with  borate-potassium  chloride-sodium  carbonate  solutions  of  Atkins  and  Pantin^^ 
as  the  buffer.  Enzyme  was  added  to  the  reaction  mixtures  as  the  last  component.  By  use  of  a  high 
enzyme  concentration  and  a  rather  low  temperature  for  the  incubation  the  relative  role  of  the  non- 
enzymatic  hydrolysis  could  be  kept  to  a  minimum.  The  same  device  served  also  to  prevent  undue 
interference  at  highly  alkaline  pn  by  progressive  inactivation  of  the  enzyme.  The  temperature  was 
17°  C.  ph  remained  unchanged  within  0.2  pjj  units  throughout  the  observed  course  of  the  reaction. 


Percentage  hydrolysis  at  times  (min) 

PH 

.1                   0 

3' 

4 

6                    8' 

9 

10 

12 

Total  hydrolysis 

8.5 

17 

— 

30 

— 

42 

— 

53 

9-4 

15 

— 

28 

— 

41 

— 

50 

10. 0 

— ■ 

21 

31 

38 

■ — 

45 

— 

10.4 

— 

22 

31 

39 

— 

44 

— 

Non-enzymatic 

hydrolysis 

8.5 

— 

2 

— 

0 

— 

— 

0 

9-4 

— 

0 

— 

I 

— 

— 

2 

10. 0 

— 

5 

— 

9 

— - 

— 

13 

10.4 

— 

6 

— 

II 

— 

— 

16 

Enzymatic 

hydrolysis 

8.5 

15 

— 

30 

— 

42 

— 

53 

9-4 

15 

— 

28 

— 

40 

— 

48 

10. 0 

— 

16 

24 

29 

— 

34 

— 

10.4 

— 

16 

22 

28 

~ 

31 

hydrolysis  was  essentially  independent  of  pn  is  relatively  wide.  The  pn  function  of  the 
acetylcholine  esterase  from  electric  tissue  differs  in  this  respect  from  some  other  esterases 
which  have  been  studied  by  Glick^. 

The  effect  of  addition  of  choline  and  acetate  on  acetylcholine  hydrolysis  has  been 
studied  in  detail  by  Augustinsson^".  It  seemed  of  interest  to  ascertain  whether  p^ 
influences  the  role  played  by  the  hydrolysis  products.  In  an  experiment  reported  in 
Table  III  the  effect  of  choline  chloride  (12.5  /iM/ml)  on  the  hydrolysis  of  acetylcholine 
(4  fjMjmY)  at  three  selected  pn  values  is  shown.  Choline  proved  to  be  about  equally 
inhibiting  at  pjj  7-7  and  6.8;  the  choline  was  only  about  one-half  as  active  an  inhibitor 
at  Ph  5.9.  Acetate  even  in  high  concentrations  (o.i  M)  failed  to  inhibit  acetylcholine 
hydrolysis  by  electric  tissue  esterase  in  phosphate  solution  either  at  pn  5-5  or  7.7.  Since 
References  p.  321. 


VOL.  4  (1950) 


ACYLATIONS   BY   ACETYLCHOLINE   ESTERASE  II 


313 


moderation  of  the  action  of  esterase  inhibitors  by  way  of  regulation  of  p^  might  be 
a  matter  of  some  practical  as  well  as  theoretical  interest,  further  study  of  the  pn- 
dependence  of  esterase-inhibitor  inter- 
actions appears  desirable. 


Fig.  2.  Acetylcholine  hydrolysis  as  a  function  o, 
of  ph-  Curve  i :  Hydrolysis  of  acetylcholine  i 
in   the  presence  of  enzyme.  Relative  initial  ^ 
reaction  rates  corrected  for  enzymatic  hydro- 
lysis are  plotted  on  the  ordinate.  The  curve 
is  a  composite  of  data  given  in  Tables  I  and 
II.  Values  for  pn  7-8  in  phosphate,  and  pn 
8.1  and  8.5  in  borate  are  taken  equal  to  10. 
Curve  2 :  Hydrolysis  of  acetylcholine  in  ab- 
sence of  enzyme.  Acetylcholine  concentration 
4  //M/ml.  Ph  was  regulated  with  borate  buffer. 
Initial  reaction  rates  are  plotted  on  the  ordi- 
nate. The  value  for  pn  10.6  is  taken  equal 
to  10.  The  temperature  was  21°  C. 


TABLE  III 

INFLUENCE    OF    CHOLINE    ON    ACETYLCHOLINE    HYDROLYSIS    AT    DIFFERENT    pH    VALUES 

Reaction  mixtures  contained  a  constant  amount  of  enzyme,  acetylcholine  chloride  4  //M/ml,  choline 
chloride  (or  sodium  chloride)  12.5  /^M/ml,  potassium  phosphate  o.i  M,  sodium  chloride  0.05  M,  magne- 
sium chloride  0.02  M,  gelatin  0.07%  and  different  amounts  of  sodium  hydroxide.  Temperature  37°  C. 


Choline 

Percent  hydrolysis  at  times  (min) 

Ph 

10' 

20' 

30' 

40' 

50' 

60' 

70' 

7-7 



20 

40 

58 

71 







7-7 

+ 

— 

15 

22 

32 

39 

— 

— 

6.8 

— 

20 

38 

53 

65 

— 

— 

— 

6.8 

+ 

— 

15 

21 

— 

39 

— 

— 

5-9 

— 

— 

— 

22 

— 

38 

— 

49 

5-9 

+ 

— 

16 

— 

26 

38 

B.    SYNTHESIS    OF   ACETYL-  AND    PROPIONYLCHOLINE   BY   THE    ACTION    OF   PURIFIED 

ACETYLCHOLINE   ESTERASE 

The  equilibrium  constant  of  esterification  reactions  favours  strongly  the  reaction 
direction  of  hydrolysis^^.  Earlier  investigators^^  observed  that  the  pharmacological 
activity  of  choline  is  enhanced  by  incubation  with  acetate  in  the  presence  of  crude 
tissue  preparations  of  esterase.  Demonstration  of  this  synthesis  and  measurement  of 
the  equilibrium  was  greatly  facilitated  in  the  present  work  by  the  availability  of  the 
hydroxylamine  method  which  could  be  applied  to  the  determination  of  the  ester  in  the 
presence  of  a  large  excess  of  the  products  of  the  hydrolysis. 

Figs  3  and  4  analyse  the  effect  of  pn  on  the  equilibrium  position  of  the  hydrolysis 
of  acetylcholine  and  propionylcholine  respectively  by  the  purified  esterase.  The  approach 
to  equilibrium  at  three  selected  p^was  realized  in  each  case  from  both  reaction  directions. 
References  p.  321. 


314 


S.  HESTRIN 


VOL.  4  (1950) 


It  is  apparent  that  acid  shift  of  pn  within  the  range  studied  displaces  the  equihbrium 
in  the  direction  of  synthesis.  In  the  experiments  of  Figs  3  and  4  the  speed  of  the 
approach  to  the  equihbrium  was  found  to  be  dependent  upon  the  esterase  concen- 
tration. To  insure  a  close  approach  to  the  equilibrium  in  a  conveniently  short  time, 
a  much  greater  enzyme  concentration  than  is  conveniently  used  in  a  hydrolysis  assay 
was  taken. 


6« 
a> 

g   L 

0  s 

c; 

» 

>< 
s 

\\ 

\ 

It 

»\ 
\\ 

u 
l\ 
\\ 

l\ 
\\ 
\\ 
l\ 

-- 

y 

•1 

J- — 

f. 

r 

1 

n 

'■ '■  \ 

s- 

"■■-9 

^— - 

0  30  90  150  210 

Minutes 

Fig.  3.  Synthesis  of  acetylcholine  as  a  function  of  pn-  Solutions  were  made  with  1.15  g  each  of  choline 
chloride  and  sodium  acetate  trihydrate  at  pn  5-i  in  a  total  volume  of  6.0  ml,  and  with  1.2 1  g  each  of 
these  substrates  in  the  same  total  volume  at  pn  5-9  and  7.0.  p^  was  set  with  hydrochloric  acid  and 
measured  with  a  glass  electrode  in  samples  diluted  for  the  purpose  with  three  volumes  of  water.  In 
one  control  mixture  at  each  pn,  8  /tM  of  acetylcholine  per  ml  was  added  at  the  outset.  Enzyme  was 
added  in  an  amount  per  ml  sufficient  to  effect  hydrolysis  of  2  g  of  acetylcholine  chloride  per  hr  in 
optimum  conditions.  Temperature  23''  C.  Ester  was  determined  on  aliquots  of  0.5  ml.  A  standard 
curve  was  constructed  with  known  acetylcholine  amounts  in  the  same  medium.  Care  is  taken  in  the 
ester  determination  to  bring  the  pn  of  the  sample  to  i. 0-1.2  at  the  step  prior  to  ferric  chloride  addi- 
tion in  order  to  avoid  interfering  colour  by  reaction  between  fatty  acid  and  ferric  chloride.  In  several 
cases,  water  was  added  to  a  reaction  mixture  in  which  the  synthesis  had  come  to  a  rest.  A  rapid 
shift  of  the  equilibrium  in  the  reaction  direction  of  hydrolysis  could  then  be  observed.  In  the  absence 
of  either  acetate,  choline,  or  esterase,  no  ester  formation  was  observed. 


Concentration  at  equilibrium  (molarity) 

K 

(a-e) 

zdF 

PH 

water 
(a) 

choline 
(b) 

acetic  acid         acetic 
plus  acetate        acid 
(c)                  (d) 

acetyl- 
choline 
(e) 

=  -4.58Tlog^ 

(b-d) 

51 
5-9 

41 
39 

1-35 
1-45 

1.4 
1-5 

0.45 
o.i 

3.7-10-3         0.25 
I.o- 10—3          0.27 

—3160 
—3140 

References  p.  321. 


VOL.  4  (1950) 


ACYLATIONS   BY   ACETYLCHOLINE   ESTERASE  II 


315 


The  effect  of  pn  on  the  equilibrium  might  be  interpreted  as  follows.  On  general 
grounds,  it  seems  reasonable  to  suppose  that  the  immediate  product  of  ester  hydrolysis 
is  the  undissociated  acid  molecule  rather  than  its  ion : 


RCOOR'  +  H2O  ^  RCOOH  +  R'OH 
RCOOH  ^  RCOO-  +  H  + 


(I) 
(2) 


where  RCOOR'  represents  the  ester  and  RCOOH  and  R'OH  the  acid  and  alcohol  products 
of  hydrolysis.  Equilibrium  in  the  synthesis  will  then  be  defined  by  the  relationship: 

H2O]  [RCOOR'] 


K  = 


R'OH]  [RCOOH] 


where  K  is  the  Nernst  equilibrium  constant  calculated  from  concentrations  in  molarity. 
As  Pjj  is  decreased,  the  concentration  of  the  undissociated  acid  rises  and  an  accompanying 


120.  140 

Minutes 


Fig.  4.  Synthesis  of  propionylcholine  as  a  function  of  pjj-  Solutions  were  made  with  1.21  g  of  choline 

chloride  and  0.85  g  of  sodium  propionate  at  pn  7  and  5.8  in  a  total  volume  of  6.0  ml,  and  with  i.oi  g 

of  choline  chloride  and  0.71  g  of  sodium  propionate  at  pn  5-o  in  the  same  total  volume.  Temperature 

18°  C.  Procedure  otherwise  as  described  under  Fig.  3. 


Concentration  at  equilibrium  (molarity) 

K 

(a-e) 

^F 

PH 

water 
(a) 

choline 
(b) 

propionic  acid 

plus  propionate 

(c) 

propionic 
acid 

(d) 

propionyl- 
choline 
(e) 

= -4.58  T  log -^ 

(b-d) 

5-0 
5-8 

41 
39 

1.2 
1-45 

1-25 

1-5 

0.52 
0.15 

2.6- 10-^ 
1.26- 10-^ 

0.17 
0.23 

—  3350 

—  3170 

References  p.  321. 


3l6  S.  HESTRIN  VOL.  4  (1950) 

increase  of  ester  concentration  at  equilibrium  may  be  expected.  The  values  found  for 
the  K  of  the  choline  esterifications  approximated  0.2  within  the  limits  of  the  experi- 
mental error*.  The  reasonably  good  constancy  of  the  values  for  K  despite  the  large 
variation  of  the  absolute  concentration  of  ester  at  equilibrium  in  the  investigated  pfj 
range  supports  the  suggestion  that  undissociated  acid  rather  than  the  anion  enters  into 
the  equilibrium  of  the  esterification. 

A  value  for  the  A  F  oi  choline  ester  hydrolysis  may  be  calculated  from  K  with 
the  aid  of  the  relationship 

55-5 


-ZIF  =  RTln 


K 


whose  derivation  has  been  discussed  recently  by  Meyerhof  and  Green^*.  —  Zl  F  calcu- 
lated in  this  manner  was  found  to  approximate  3200  cals.  Although  molarities  rather 
than  activities  are  used  above  to  calculate  K,  it  is  believed  likely  that  error  from  this; 
cause  in  the  value  for  A  F  does  not  exceed  10%**.  It  is  noteworthy  that  the  value  for 
A  F  oi  hydrolysis  of  two  choline  esters  is  of  an  order  similar  to  the  observed  in  the  case 
of  several  anionic  esters^'*. 

The  amount  of  the  acetylcholine  at  equilibrium  is  minute  in  comparison  to  the 
concentration  of  the  other  participants  of  the  system.  However,  it  seems  desirable  in 
view  of  the  great  biological  potency  of  acetylcholine  to  consider  the  possibility  that 
esterase  functions  as  an  agent  of  acetylcholine  synthesis  in  vivo,  supplementing  in  this 
respect  the  role  of  choline  acetylase.  It  has  been  demonstrated  that  acetylcholine 
esterase  in  the  nerve  axon  is  localized  in  the  neuronal  surface  membranes^^.  The  con- 
centration of  esterase  substrates  and  the  pn  prevailing  in  the  membrane  are  unknown, 
but  there  is  reason  to  believe  that  H+  and  choline+  may  be  significantly  higher  at  the 
membrane  interface  than  in  the  surrounding  milieu^^.  Specific  binding  of  ester  and 
sudden  variation  in  pf£  at  the  membrane  with  resulting  shift  of  equilibrium  are  con- 
ceivable. For  a  local  choline  concentration  of  o.oi  M  and  a  similar  concentration  of 
undissociated  acetic  acid,  the  value  0.2  for  K  leads  to  an  equilibrium  acetylcholine 
concentration  of  0.06  micrograms  per  ml.  An  ester  concentration  of  this  order  would 
be  sufficient  to  produce  major  biological  effects. 

C.    FORMATION    OF   HYDROXAMIC   ACIDS 

The  ability  of  proteolytic  enzymes  to  catalyse  ester  hydrolyses  has  been  demon- 
strated by  Neurath  and  his  coworkers^'.  The  ability  of  0-acyl  hydrolases-lipase^^  and 
esterase^  to  form  hydroxamic  acids  by  the  condensation  of  fatty  acid  with  hydroxyl- 
amine  is  an  interesting  counterpart  to  this  situation  in  which  a  group  of  hydrolases 
catalyses  both  O-  and  N-acylation. 

The  effect  of  reactant  concentrations  on  the  rate  of  the  formation  of  hydroxamic 
acid  in  the  presence  of  the  electric  tissue  esterase  is  shown  by  experiments  summarized 
in  Fig.  5.  Within  a  wide  range  of  reactant  concentration  the  relation  between  reaction 
rate  and  reactant  concentration  remains  almost  linear.  Reactant  concentrations  up  to 
0.75  M  or  higher  failed  to  saturate  the  enzyme.  Its  affinity  for  acetate,  propionate,  and 

*  Inaccuracy  in  the  measurement  of  p^  would  exert  a  relatively  large  effect  on  the  value  of  K. 
The  computation  of  K  for  pn  above  6  suffers  from  an  additional  inaccuracy  because  the  concentration 
of  ester  approached  the  limit  of  the  ester  determination  as  the  pn  increased  above  6. 

**  I  am  much  indebted  to  Professor  O.  Meyerhof  for  the  discussion  of  this  question. 

References  p.  321. 


VOL.  4  (1950) 


ACYLATIONS   BY   ACETYLCHOLINE    ESTERASE  II 


317 


hydroxylamine  may  be  concluded,  therefore,  to 
be  of  a  much  lower  order  than  the  afifinity  of 
the  enzyme  for  acetylcholine.  This  conclusion 
has  been  further  supported  by  the  demonstra- 
tion that  neither  acetate  nor  hydroxylamine 
significantly  affect  the  rate  of  acetylcholine 
hydrolysis  by  the  esterase.  The  substrate  con- 
centration-activity relationship  observed  in 
hydroxylamine  acylation  resembles  that  of 
neutral  ester  hydrolysis  by  the  enzyme^'  ^. 

The  rate  of  reaction  of  acetate  with  hydr- 
oxylamine in  the  presence  of  esterase  is  very 
small  as  compared  to  the  rate  of  hydrolysis  of 
acetylcholine  by  a  similar  concentration  of  the 
enzyme,  the  relative  magnitude  of  the  rates 
being  in  the  proportion  of  one  to  one  or  two 
thousand.  The  rate  of  hydroxamic  acid  forma- 
tion, like  the  hydrolysis  of  acetylcholine,  varied 
in  a  direct  manner  with  the  esterase  concentra- 
tion (see  Fig.  6). 

The  specificity  of  electric  tissue  esterase  in 
regard  to  the  fatty  acids  which  it  can  cause  to 
condense  with  hydroxylamine  is  rather  sharply 
defined  (see  Table  IV).  As  in  choline  ester 
hydrolysis^,  a  maximum  is  observed  with 
acetic  acid.  A  lower  rate  is  found  with  propionic 
acid.  The  enzyme-catalyzed  reaction  observed 
with  butyric  acid  was  almost  negligible.  The 
findings  with  formic  acid  reveal  a  relatively 
large  spontaneous  reaction  between  formate  and 


Fig.  5.  Formation  of  hydroxamic  acid  as 
a  function  of  reactant  concentration.  The 
reaction  mixtures  are  0.5  M  as  to  sodium 
acetate  and  i.o  M  as  to  sodium  chloride. 
Ph  6.8.  37°  C.  n ,  ▼ ,  X ,  O ,  —  correspond 
to  mixtures  with  o.i,  0.2,  0.5,  and  i.o  M 
hydroxylamine  respectively.  Curves  i  to  3 
of  the  inset  are  not  mutually  comparable 
since  they  were  obtained  with  different 
batches  of  the  enzyme.  Relative  reaction 
rates  are  plotted  on  the  ordinates  and 
reactant  concentrations  in  molarity  on  the 
abscissae.  Curve  i  summarizes  the  detail 
of  the  main  part  of  the  figure  showing  the 
effect  of  variation  of  hydroxylamine  con- 
centration. Curves  2  and  3  show  the  effect 
of  variation  of  acetate  and  propionate  con- 
centration respectively  in  the  presence  of 
1.0  M  hydroxylamine. 


TABLE  IV 

SUBSTRATE   SPECIFICITY   OF   ELECTRIC   TISSUE   ESTERASE   IN    FORMATION   OF   HYDROX.\MIC   ACID 

The  reaction  mixtures  are  1.0  M  as  to  hydroxylamine  and  0.75  M  as  to  the  sodium  salt  of  the  fatty 

acid  in  0.9  M  solution  of  sodium  chloride  at  pn  6.2-6.4.  Temperature  37°  C.  Propionhj'droxamic, 

butyrhydroxamic,  and  acethydroxamic  acid  yield  equivalent  amounts  of  colour  per  mole  with  ferric 

chloride.  The  amount  of  the  formhydroxamic  acid  is  calculated  on  the  same  basis. 


Hydroxamic  acid,  /tM/ml, 

Enzyme  addition 

Fatty  acid 

at  times  in  minutes 

50 

100 

200 

+ 

formate 

1-3 

2.4 

formate 

0.7 

1-3 

+ 

acetate 

30 

6.0 

9-9 

— 

acetate 

0.0 

0.0 

0.1 

+ 

propionate 

i.r 

2-3 

— 

propionate 

0.0 

0.0 

+ 

butyrate 

0-3 

0.6 

— 

butyrate 

0.2 

0-5 

References  p.  321. 


3i8 


S.  HESTRIN 


VOL.  4  (1950) 


,6 

1.0 

P^ 

/• 

■^••6 

/ 

0 

0.5 

./ 

A 

/X 

0 

/ 

/ 

A 

0 

/ 

y 

/ 

t 

05 

1.L 

•C4 

/ 

/ 

/ 

/ 

^^ 

4 

/ 

^^^ 

2 

f     , 

/ 

^^ 

/ 

▼-^ 

/ 

/^ 

1 

60 


120 


180 


240 

Minufes 


hydroxylamine,  and  some  enzymatic  catalysis 
of  this  reaction.  Substitution  of  an  a-amino 
group  into  acetate  or  propionate  caused  com- 
plete loss  of  their  ability  to  condense  with 
hydroxylamine  in  the  presence  of  the  enzyme. 
The  ability  of  the  esterase  to  effect  hydro- 
lysis of  acethydroxamic  acid  was  examined  at  a 
substrate  concentration  of  3  /^M/ml  in  phosphate 
buffer  at  pn  74.  Even  with  a  great  concentration 
of  enzyme  no  hydrolysis  of  acethydroxamic 
acid  was  found,  although  acetylcholine  added  to 
the  same  reaction  mixture  was  hydrolysed 
rap'dly.  An  acyl  transfer  reaction  between  acet- 
hydroxamic acid  and  chohne  with  resultant 
intermediary  formation  of  hydrotysable  acetyl- 
choline could  be  excluded,  since  addition  of 
choline  to  the  same  reaction  mixture  failed  to 
evoke  a  disappearance  of  acethydroxamic  ac'd. 
The  Ph  dependence  of  hydroxylamine 
acylation  by  electric  tissue  esterase  is  illus- 
trated by  the  experiment  g'ven  in  Table  V. 
The  reaction  between  acetate  and  hydroxyl- 
amine showed  a  peak  in  a  range  near  pfj  6.3. 
The  Ph  function  of  hydroxylamine  acylation 
by  the  esterase  is  thus  very  different  from  the  pn  function  of  acetylchohne  h5^drolysis 
by  the  enzyme.  The  finding  that  the  pn  dependence  of  hyd'-oxylamine  acylation  and 
choline  ester  hydrolysis  are  quite  different  is  consistent  with  an  assumption,  discussed 
later,  concerning  the  mechanism  of  these  two  reactions. 

TABLE  V 

FORMATION    OF    HYDROXAMIC    ACID    IN    PRESENCE    AND    ABSENCE    OF    CHOLINE    AT    DIFFERENT    pjj 

Reaction  mixtures  are  0.5  M  as  to  acetate  and  i.o  M  as  to  hydroxylamine  in  0.9  M  solutiDn  of  sodium 
chloride  at  pn  specified  with  or  without  addition  of  0.5  M  choline  chloride.  In  absence  of  choline 
addition,  an  equivalent  amount  of  sodium  chloride  was  added.  The  pn  was  determined  in  aliquots 
with  a  glass  electrode  after  four-fold  dilution  with  water.  Temperature  37°  C.  The  formation  of 
hydroxamic  acid  in  absence  of  enzyme  was  «negligible  at  pH  6.3  and  5.3  and  none  was  detected  at 
Ph  7  and  higher.  The  reaction  time  was  4  hours. 


Fig.  6.  Formation  of  hydroxamic  acid  as 
a  function  of  esterase  concentration.  Re- 
action mixtures  were  i.o  M  as  to  hydroxyl- 
amine and  sodium  acetate  in  1.0  molar 
sodium  chloride,  pn  6.8.  37°  C.  Curves 
I  to  3  correspond  to  relative  enzyme 
concentrations  10,  6,  and  3.  In  the  inset 
the  relative  reaction  rate  is  plotted  on 
the  ordinate  and  the  relative  enzyme 
concentration  on  the  abscissa. 


PH 

Chohne 

Hydroxamic 
acid,  /<M/ml 

7-9 

+ 

0.2 

7-5 

— 

1-5 

7-1 

+ 

0.2 

7-1 

— 

1.6 

6-3 

+ 

0.7 

6.3 

— 

2.4 

5-3 

+ 

0.4 

5-3 

0.2 

In  the  presence  of  choline,  the  rate  of  the  acetylation  of  hydroxylamine  by  esterase 
References  p.  321. 


VOL.  4  (1950) 


ACYLATIONS   BY   ACETYLCHOLINE    ESTERASE  II 


319 


acting  at  pn  7.1  was  reduced  markedly  (Fig.  7).  The  _  '^ 
effect  of  Ph  on  the  choline  inhibition  is  illustrated   g- 
by  the  experiment  shown  in  Table  V.  As  in  the  .^ 
case  of  acetylcholine  hydrolysis,  the  lowering  of  p^   ° 
reduced  the  inhibitory  effect  of  choline.  At  pn  5.3,    I 
a  reg'on  in  wh'ch  the  enzyme  activity  was  rather  | 
low  but  still  measurable,  an  activating  effect  by  ^ 
choline  on  hydroxamic  acid  formation  was  observed. 
The  inhibitory  effect  of  choline  can  be  ascribed  to 
its  ability  to  combine  with  the  enzyme  at  an  active       ^ 
site^".  An  explanation  of  activation  by  choline  may 
be  found  in  the  fact  that  at  acid  p^  the  concentra- 
tion of  acetylcholine  in  the  system  is  increased. 
It  has  been  shown"  that  acetylcholine  acetylates 
hydroxylamine  rap'dly  at  alkaline  pn  and  slowly 
at  acid  pn.  the  rate  being  dependent  on  the  concen- 
tration of  the  acetylcholine  at  constant  hydroxyl- 
amine concentration.  z-\t  pn,  7  the  concentration 
of    acetylcholine   in   the  acetate-hydroxylamine- 
choline-system  is  neglig'ble.  The  ability  of  choline 
to  serve  as  an  acetyl  carrier  at  this  pn  must  therefore 
become  very  small. 

The  inh'bito^y  effect  of  choline  on  hydroxyl- 
amine acylation  and  the  finding^  that  incubation  of 
the  enzyme  with  specific  inhibitors — prost'gmine 
and  tetraethylpyrophosphate — abolishes  the  abi- 
lity to  catalyse  hydT'oxamic  acid  formation  support  the  view  that  the  same  enzyme  ,and 
possibly  the  same  p-osthetic  group,  effects  both  acetylcholine  hydrolysis  and  hydrox- 
amic acid  formation.  But  the  reaction  of  hydrolysis  of  ac^  tylcholine  is  reversible,  while 
that  of  hydroxylamine  acylation  appea^^s  to  be  irreversible.  Choline  shows  a  fairly 
marked  affinity  for  the  enzyme,  whereas  hydroxylamine  shows  little  or  no  affinity.  The 
possibility  has  therefore  to  be  considered  that  the  role  of  esterase  in  hydroxylamine 
acylation  is  confined  to  the  activation  of  the  carboxylic  acid  reactant,  and  that  a 
terminal  reaction  between  activated  carboxylic  acid  and  hydroxylamine  is  spontaneous 
and  irreversible.  In  the  case  of  choline  acylation  it  is  assumed  that  the  esterase  may 
activate  the  two  reactants. 

The  writer  is  deeply  indebted  to  Professor  D.  Nachmansohx  for  encouragement 
and  for  many  suggestions.  Thanks  are  expressed  to  Mrs  Emily  Feld  Hedal  and  Miss 
Louise  d'Alessio  for  their  assistance  in  the  performance  of  the  experiments. 


'20         Ao          60          60         100 

Minutes 
Fig.  7.  Effect  of  choline  on  formation 
of  hydroxamic  acid.  The  reaction  mix- 
tures are  i.o  M  as  to  hydroxylamine 
and  0.75  M  as  to  sodium  acetate  in 
0.9  M  solution  of  sodium  chloride  at 
Pjj  7.1.  Temperature  37°  C.  O,  mixture 
without  choline;  X,  mixture  with  0.9M 
choline  chloride.  The  reaction  in  ab- 
sence of  enzyme  is  negligible  in  both 
cases.  The  inhibitory  effect  of  choline 
was  unaffected  by  the  choline  concen- 
tration in  the  range  of  o.i  to  0.9  M. 


SUMMARY 

I.  Some  general  properties  of  ester  hydrolysis  and  synthesis  by  the  purified  acetylcholine 
esterase  of  the  electric  tissue  of  Electrophorus  electricus  have  been  investigated  with  the  aid  of  a 
simple  colorimetric  technique  for  the  determination  of  an  ester  in  the  presence  of  its  hydrolysis 
products. 

References  p.  321. 


320  S.  HESTRIN  VOL.  4  (1950) 

2.  The  hydrolysis  of  acetyl-  and  propionylcholine  by  the  esterase  have  been  shown  to  be  rever- 
sible. The  equilibrium  of  the  reaction  was  found  to  be  characterized  by  the  ratio: 

[acetylcholine]   [water] 


[chohne]   [RCOOH] 

where  RCOOH  represents  the  undissociated  form  of  the  carboxylic  acid. 

3.  The  possibility  that  esterase  plays  a  part  in  synthesis  of  acetylcholine  at  the  neuronal  mem- 
brane surface  has  been  discussed. 

4.  The  condensation  of  fatty  acids  with  hydroxylamine  by  the  action  of  the  esterase  has  been 
investigated  in  respect  to  its  dependence  on  reactant  concentration,  enzyme  concentration,  carboxylic 
acid  structure,  and  pn- 

5.  Acethydroxamic  acid  was  not  hydrolysed  by  the  esterase  either  in  the  presence  or  absence 
of  choline.  The  reaction  of  hydroxamic  acid  formation,  unlike  ester  hydroh'sis  by  the  enzyme,  thus 
appear  to  be  irreversible. 

6.  Condensation  of  acetate  with  hydroxylamine  in  the  presence  of  esterase  acting  at  pjj  6.3 
and  above  was  markedly  inhibited  by  choline. 

7.  A  reaction  mechanism  which  could  explain  some  of  the  differences  observed  between  the 
catalysis  of  choline  ester  hydrolysis  and  that  of  hydroxamic  acid  formation  by  the  same  esterase 
has  been  discussed. 

r£sum£ 

1.  Quelques  proprietes  generales  de  I'hydrolyse  et  de  la  synthese  des  esters  par  I'acetylcholine 
esterase  purifie  du  tissu  electrique  de  Electrophorus  electricus  ont  ete  etudiees  a  I'aided'une technique 
colorimetrique  pour  la  determination  d'un  ester  en  presence  de  ses  produits  d'hydrolyse. 

2.  On  a  montre  que  I'hydrolyse  de  I'acetylcholine  et  de  la  propionylcholine  par  I'esterase  est 
reversible.  L'equilibre  de  la  reaction  est  caracterise  par  le  quotient: 

[acetylcholine]   [eau] 
[chohne]   [RCOOH]       ^ 

oil  K  represente  la  forme  non  dissociee  de  I'acide  carboxylique. 

3.  La  possibilite  que  I'esterase  joue  un  role  dans  la  synthese  de  I'acetylcholine  a  la  surface  de  la 
membrane  neuronale  a  ete  discutee. 

4.  La  condensation  des  acides  gras  avec  I'hydroxylamine  sous  Taction  de  I'esterase  a  ete  etudiee 
en  ce  qui  concerne  sa  dependance  de  la  concentration  de  la  substance  reagissante  et  de  I'enzyme,  de 
la  structure  de  I'acide  carboxylique  et  du  pn- 

5.  L'acide  acetylhydroxamique  n'a  pas  ete  hydrolyse  par  I'esterase  ni  en  presence  ni  en  absence 
de  choline.  Ainsi  la  formation  de  I'acide  hydroxamique,  contrairement  a  I'hydrolyse  d'un  ester  par 
I'enzyme,  semble  etre  irreversible. 

6.  La  condensation  d'acetate  avec  I'hydroxylamine  en  presence  d'esterase  a  un  pH  de  6.3,  est 
considerablement  inhibee  par  la  choline. 

7.  Un  mecanisme  de  reaction  a  ete  discute  qui  pourrait  expliquer  certaines  differences  observees 
entre  I'hydrolyse  d'un  ester  cholinique  et  la  formation  d'acide  hydroxamique  catalysees  par  la  meme 
esterase. 

ZUSAMMENFASSUNG 

1.  Einige  allgemeine  Eigenschaften  der  Esterhydrolyse  und  -synthese  durch  gereinigte  Acetyl- 
cholinesterase aus  dem  elektrischen  Gewebe  von  Electrophorus  electricus  wurden  untersucht  und 
zwar  mit  Hilfe  einer  einfachen  kolorimetrischen  Arbeitstechnik  zur  Bestimmung  eines  Esters  in 
Gegenwart  seiner  Hydrolyseprodukte. 

2.  Es  wurde  gezeigt  dass  die  Hydrolyse  von  Acetyl-  und  Propionylcholin  durch  die  Esterase 
reversibel  ist  und  dass  das  Reaktionsgleichgewicht  durch  den  Quotienten 

[Acetylcholin]  [Wasser] 
[Cholin]   [RCOOH]""  ^ 

charakterisiert  ist,  wo  RCOOH  die  nicht  dissoziierte  Form  der  Carbonsaure  darstellt. 

3.  Die  Moglichkeit  wurde  erortert,  dass  Esterase  bei  der  Acetylcholin-Synthese  an  der  Ober- 
fliiche  der  Neuronmembrane  eine  RoUe  spielen  konnte. 

4.  Die  Kondensation  von  Fettsauren  mit  Hydroxylamin  unter  der  Einwirkung  der  Esterase 
wurde  in  Bezug  auf  die  Abhangigkeit  dieser  Reaktion  von  der  Konzentration  der  reagierenden  Sub- 
stanz  und  des  Enzyms;  sowie  von  der  Struktur  der  Carbonsaure  und  dem  pn  untersucht. 

References  p.  321. 


VOL.  4  (1950)  ACYLATIONS   BY   ACETYLCHOLINE   ESTERASE  II  32I 

5.  Acetylhj^droxamsaure  wurde  durch  die  Esterase  weder  in  Gegenwart  noch  in  Abwesenheit 
von  Cholin  hydrolysiert.  Es  scheint  also,  dass  die  durch  das  Enzym  katalysierte  Hydroxamsaure- 
bildung  zum  Unterschied  von  der  Esterhydrolyse  irreversibel  sei. 

6.  Die  Kondensation  von  Acetat  mit  Hydroxylamin  in  Gegenwart  von  Esterase  bei  pjj  6.3  wurde 
durch  ChoHn  stark  gehemmt. 

7.  Ein  Reaktionsmechanismus,  welcher  einige  Unterschiede  zwischen  der  katalytischen  ChoUn- 
esterhydrolyse  und  der  Hydroxamsaurebildung  unter  Einwirkung  derselben  Esterase  erklaren 
konnte,  wurde  erortert. 

REFERENCES 

^  D.  Nachmansohn,  Bull.  Johns  Hopkins  Hosp.,  83  (1948)  463. 

-  S.  Hestrin,  /.  Biol.  Chem.,  in  press. 

*  D.  Nachmansohn  and  M.  A.  Rothenberg,  /.  Biol.  Chem.,  158  (1945)  653. 

*  D.  Nachmansohn  and  M.  A.  Rothenberg,  E.  A.  Feld,  J .  Biol.  Chem.,  174  (1948)  247. 
^  K.  AuGUSTiNSSON,  Avch.  Biochem.,  in  press. 

*  K.  AuGUSTiNSSON  AND  D.  Nachmansohn,  /.  Biol.  Chem.,  179  (1949)  543. 
^  S.  Hestrin,  /.  Biol.  Chem.,  in  press. 

*  M.  A.  Rothenberg  and  D.  Nachmansohn,  /.  Biol.  Chem.,  168  (1947)  223. 

*  D.  Glick,  J.  Gen.  Physiol.,  21  (1938)  289. 
K.  AUGUSTINSSON,  Acta  Physiol.  Scand.,  15  (1948)  SuppL  52. 

Methoden  der  Fermentforschung,  Georg  Thieme  Verlag,  Leipzig,  Vol.  i  (1941)  783. 
R.  Ammon,  Handbuch  d.  Enzymologie,  Akademische  Verlagsgesellschaft,  Leipzig  1940,  p.  350. 
R.  Ammon  and  H.  Kwiatkowski,  PflUgers  Arch.  ges.  Physiol.,  234  (1934)  269. 
O.  Meyerhof  and  H.  Green,  /.  Biol.  Chem.,  178  (1949)  655. 

E.  J.  Boell  and  D.  Nachm.\nsohn,  Science,  92  (1940)  513. 
J.  Danielli,  Proc.  Roy.  Soc,  122  B  (1937)  i55- 
S.  Kaufman,  H.  Neurath,  and  G.  Schwert,  /.  Biol.  Chem.,  177  (1949)  793. 

F.  Lipmann,  Advances  in  Enzymol.  Vol  VI,  Interscience,  New  York  1946,  p.  257. 

Received  June  28th,  1949 


322  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


OBSERVATIONS  ON  A  FACTOR  DETERMINING  THE 
METABOLIC  RATE  OF  THE  LIVER 

by 

EINAR  LUNDSGAARD 

Institute  of  Medical  Physiology,  University  of  Copenhagen  {Denmark) 


In  a  paper  published  some  years  ago^  brief  mention  was  made  of  experiments  on 
isolated,  artificially  perfused  livers  in  which  the  rate  of  oxygen  uptake  in  the  liver  was 
consistently  found  to  decrease  during  the  first  30-45  minutes  after  the  liver  had  been 
isolated.  This  phenomenon  has  intrigued  me  ever  since,  and  although  the  cause  of  this 
drop  in  metabolic  rate  in  a  liver  isolated  from  the  "periphery"  is  not  ascertained  a  short 
appraisal  of  the  experience  gained  so  far  may  be  presented. 

Most  of  the  experiments  have  been  carried  out  on  cat  livers.  The  metabolism  of 
the  isolated  cat  liver  is  peculiar  in  that  carbohydrates  are  not  metabolized^.  The  respira- 
tory quotient  of  the  isolated  cat  liver  is  always  very  low  —  generally  below  0.7.  The 
blood  sugar  concentration  never  decreases.  Irrespectively  of  the  blood  sugar  level  a 
steady  increase  in  blood  sugar  concentration  is  observed.  This  increase  must  be  due  to 
a  gluconeogenesis  as  it  is  observed  also  in  livers  in  which  the  glycogen  store  has  been 
exhausted  by  starvation.  It  appears  most  likely  that  the  lack  of  carbohydrate  metabo- 
lism in  the  isolated  cat  liver  is  not  an  artefact  but  a  characteristic  feature  in  the  liver 
metabolism  of  this  species.  Nevertheless  one  might  claim  that  a  liver  which  does  not 
metabolize  carbohydrate  must  be  in  an  abnormal  state  and  that  the  drop  in  metabolic 
rate  might  have  some  connection  with  this  abnormal  state.  Contrary  to  the  cat  liver 
the  isolated  rabbit  liver,  however,  stores  glucose  as  glykogen  and  oxidizes  carbohydrate 
and  although  my  experience  with  the  rate  of  oxygen  consumption  in  the  isolated  rabbit 
liver  is  far  more  limited  than  my  expeiience  with  cat  livers  it  can  safely  be  stated  that 
in  the  isolated  rabbit  liver  also  a  drop  in  metabolic  rate  is  encountered  immediately 
after  isolation. 

It  might  well  be  questioned  whether  any  importance  can  be  attached  to  a  drop  in 
metabolic  rate  in  an  organ  kept  alive  by  artificial  perfusion.  Such  a  view  appears  justi- 
fied, however,  since  such  a  decline  in  oxygen  uptake  is  observed  in  experiments  on  livers 
only  and  not  in  experiments  on  other  organs.  In  the — unfortunately  unsuccessful — 
endeavour  to  make  preparations  of  isolated  cat  intestines  function  normally  with  respect 
to  absorption  a  considerable  number  of  experiments  have  been  carried  out  in  which 
the  oxygen  uptake  of  the  isolated  cat  intestine  was  determined.  The  oxygen  consumption 
of  such  a  preparation  always  remains  constant.  In  perfusing  experiments  on  hind  limb 
preparations  the  oxygen  uptake  always  increases  markedly.  This  increase  generally 
continues  for  the  entire  experimental  period  of  two  hours  which  is  the  time  most  often 
used  in  my  experiments.  The  marked  difference  between  the  changes  in  oxygen  uptake 
in  a  typical  experiment  on  a  liver  preparation  as  compared  with  a  hind  limb  preparation 

References  p.  32 g. 


VOL.  4  (1950) 


METABOLIC    RATE    OF   THE    LIVER 


323 


\ 

\ 

> 

\ 

'^*\[_ 

• 

30 


60 


90  120 

time  in  win 


Fig.  I.  Spontaneous  changes  in  oxygen 
consumption  during  artificial  perfusion 

of  a  cat  liver  (• •)  and  a  hind  limb 

preparation  (x x). 


i 

6 
E  4 

.S 

5  3 


is  shown  in  Fig.  i.  The  oxygen  uptake  has  been  followed  by  frequent  photoelectric 
determinations  of  the  oxygen  content  in  the  venous  blood.  The  galvanometer  readings 
in  each  experiment  have  been  standardized  by  .s  5 
at  least  4  determinations  of  the  venous  oxygen  ^ 
content  by  the  Van  Slyke  technique.  Care  has  ^  4 
been  taken  to  obtain,  as  great  differences  be-  5 
tween  the  oxygen  content  in  the  samples  used  g  3 
for  the  standardization  as  possible.  The  oxygen  g" 
content  in  the  arterial  blood  was  determined  2 
with  the  Van  Slyke  technique  at  the  beginning 
and  at  the  end  of  the  experimental  period  and  in  ' 
some  experiments  also  in  the  middle  of  this 
period.  Though  the  initial  pronounced  decrease  in 
oxygen  consumption  is  only  observed  in  experi- 
ments with  isolated  livers  it  can  not  of  course  be 
ruled  out  that  this  decrease  might  be  due  to  an 
impairment  of  the  circulation  in  the  liver  or 
some  other  damage  developing  during  the  first 

period  after  the  isolation  of  the  organ.  The  question  whether  it  is  possible  to  restore  the 
oxygen  uptake  after  it  has  attained  its  low  and  rather  constant  level  must  be  of 
decisive  importance  for  the  evaluation  of  the  phenomenon. 

On  the  assumption  that  the  decrease  in  oxygen  consumption  is  due  to  a  disappear- 
ance of  some  substance  present  in  fresh  blood  but  gradually  used  up  by  the  liver  the 
simplest  way  to  try  to  restore  the  oxygen  uptake  would  be  to  renew  the  blood  after 
the  drop  in  oxygen  uptake  has  developed.  The  result  of  such  a  simple  experiment  is 
shown  in  Fig.  2.  As  is  seen  the  addition  of  fresh  blood  to  the  perfusion  apparatus  causes 
a  marked  but  transitory  increase  in  the  oxygen  uptake.  A  quantitative  comparison 
between  the  increase  obtained  by  adding  fresh  blood  and  the  initial  drop  in  oxygen 
uptake  is  difficult  since  it  is  not  possible  to  renew  the  blood  in  the  apparatus  completely. 
It  is  only  possible  to  remove  some  of  the  blood  and  add  some  fresh  blood.  In  this  way 

not  more  than  about  50%  renewal  of  the  blood 
is  obtained.  As  some  change  in  the  cell  volume 
of  the  perfusion  blood  resulting  from  the  addi- 
tion of  fresh  blood  cannot  be  avoided,  and  as 
this  alters  the  standardization  of  the  galvano- 
meter readings  care  has  been  taken  to  draw 
simultaneously  a  sample  of  arterial  and  venous 
blood  for  Van  Slyke  determinations  as  near  as 
possible  to  the  "peak"  as  judged  from  the  gal- 
vanometer readings.  In  this  way  the  magnitude 
of  the  increase  in  the  neighbourhood  of  the  maxi- 
mum is  ascertained  by  the  Van  Slyke  technique. 
In  some  experiments  blood  used  for  the  per- 
fusion of  a  liver  for  one  to  one  and  a  half  hours 
has  been  used  for  perfusion  of  an  other  freshly 
prepared  liver.  In  these  experiments  the  oxygen  uptake  of  the  second  liver  was  low 
from  the  start  of  the  perfusion  and  remained  low. 
References  p.  329. 


Sr 

N 

\ 

A 

^ 

\_^ 

1    \ 

) 

-< 

30 


60 


90  120 

time  in  min 


Fig.  2.  Oxygen  uptake  of  isolated  cat 

liver,  between  >  and  <  perfusion  blood 

partly  exchanged  with  fresh  blood. 


324 


E.  LUNDSGAARD 


VOL.  4  (1950) 


S  5 

i 


From  these  simple  observations  it  seems  safe  to  conclude  that  the  observed  drop 
in  oxygen  uptake  in  an  isolated  liver  is  due  to  changes  in  the  blood  and  not  to  changes 
in  the  liver  tissue  as  such. 

Though  as  mentioned  the  most  probable  assumption  is  that  the  decline  in  oxygen 
uptake  is  due  to  the  disappearance  of  some  substance  from  the  blood  the  possibility 
remains  that  it  is  due  to  accumulation  of  some  inhibitory  substance.  Also  in  that  case 
addition  of  fresh  blood  might  be  expected  to  cause  an  increase  by  dilution  of  the  inhi- 
bitory agent.  Though  the  course  of  the  fall  in  oxygen  uptake  appears  incompatible  with 
such  an  assumption  an  attempt  has  been  made  to  elucidate  this  possibility  experimentally. 
Some  livers  were  perfused  with  washed  red,  blood  corpuscles  suspended  in  an  arti- 
ficial plasma.  Dextran,  a  polysaccharide  preparation,  was  added  to  the  artificial  plasma 
to  secure  a  normal  colloid  osmotic  pressure.  Though  the  result  of  these  experiments  was 
not  quite  clearcut  due  to  technical  difficulties  which  need  not  be  mentioned  here  it  can 
safely  be  stated  that  only  a  very  slight  initial  fall  in  oxygen  uptake  was  observed  in 

these  experiments. 

The  observations  so  far 
mentioned  support  the  assump- 
tion that  the  liver  normally  is 
supplied  by  the  blood  with  a 
substance  which  affects  its  me- 
tabolic rate. 

That  this  hypothetic  sub- 
stance probably  is  not  a  specific 
hormone  formed  in  one  of  the 
endocrine  glands  is  indicated  by 
experiments  carried  out  in  the 
following  way. 

A  perfusion  apparatus  with 
a  double  pump  and  two  circuits 
but  with  a  common  oxygenator  and  blood  reservoir  was  used.  A  liver  was  isolated  and 
attached  to  one  of  the  circuits,  the  other  being  short  circuited.  The  oxygen  uptake  of  the 
liver  was  followed  in  the  usual  way  and  when  the  oxygen  uptake  had  dropped  a  hind 
limb  preparation  was  attached  to  the  previously  short  circuited  circuit.  The  venous  blood 
returning  from  the  liver  and  the  hind  limb  preparation  in  this  way  is  mixed  in  the  oxy- 
genator and  the  blood  reservoir  and  the  liver  is  supplied  with  a  mixture  of  blood  retur- 
ning from  the  liver  and  the  hind  limb  preparation.  As  seen  from  Fig.  3  the  oxygen 
uptake  of  the  liver  starts  to  increase  as  soon  as  the  hind  limb  preparation  is  shunted 
in.  In  about  15  minutes  it  reaches  a  fairly  constant  level  which  is  maintained  until  the 
hind  limb  preparation  is  shunted  out.  The  shunting  out  of  the  hind  limb  preparation 
is  followed  by  a  gradual  decline  in  the  oxygen  uptake  following  a  course  similar  to  that 
of  the  initial  fall.  The  increase  is  marked  though  the  initial  high  oxygen  uptake  is  not 
restored.  In  the  experiment  presented  in  Fig.  3  the  hind  limb  preparation  after  having 
been  left  without  circulation  for  35  minutes  again  was  shunted  in  for  20  minutes.  The 
response  was  practically  identical  with  the  first  response.  The  correspondence  between 
the  two  response  must  be  emphasized  inasmuch  as  it  speaks  strongly  against  the  possi- 
bility that  lactic  acid  may  be  responsible  for  the  increase  in  oxygen  uptake.  This  point 
will  be  discussed  later ;  it  may  be  only  mentioned  that  the  lactic  acid  concentration  in 

References  p.  32g. 


\ 

\ 

/'•" 

A 

r"^ 

V 

— .- 

.y 

^N.^ 

/ 

X. 

t 

1 

\ 

30 


60 


90 


120 


150  180 

time  in  min. 


Fig.   3.  Oxygen  consumption  of  isolated  cat  liver.   Hind 
limb  preparation  shunted  in  at  f   and  out  at   |  . 


VOL.  4  (1950)  METABOLIC    RATE   OF   THE    LIVER  325 

the  blood  at  the  start  of  the  experiment  (oxygen  uptake  4.08  ml/min)  was  20  mg%,  at 
the  maximum  of  the  first  response  (oxygen  uptake  3.30  ml/min)  7  mg%  and  at  the 
maximum  of  the  second  response  (oxygen  uptake  3.25  ml/min)  26  mg%. 

These  observations  on  the  rate  of  oxygen  consumption  in  the  isolated  liver  would 
probably  not  have  been  published  if  the  effect  of  the  periphery  on  the  metaboHc  rate  of 
the  liver  had  not  been  revealed  in  a  much  more  striking  manner  in  some  other  experi- 
ments performed  for  quite  a  different  purpose. 

A  cat  was  hepatectomized  by  connecting  the  protal  vein  with  the  right  renal  vein 
through  a  cannula  of  suitable  shape  and  ligating  the  hepatic  vessels.  Heparin  had  been 
injected  to  prevent  clotting.  The  blood  sugar  concentration  of  the  animal  was  kept  as 
constant  as  possible  by  continuous  intravenous  injection  of  glucose.  In  some  experi- 
ments in  which  the  hepatectomy  was  not  successful  the  cat  was  eviscerated.  No  dif- 
ference has  been  observed  in  the  results  obtained  in  experiments  on  hepatectomized 
and  eviscerated  animals.  As  soon  as  the  operation  was  finished  a  cat  liver  was  isolated 
and  run  with  artificial  perfusion  for  35  to  50  minutes.  After  this  period  of  time,  the  oxygen 
uptake  of  the  liver  has  fallen  to  a  constant  low  level.  The  glucose  concentration  in  the 
perfusion  blood  was  followed.  From  these  determinations  the  glucose  output  of  the  iso- 
lated liver  can  be  computed  with  fair  accuracy  as  the  blood  volume  is  known.  35  to  50 
minutes  after  the  start  of  the  artificial  perfusion  the  oxygen  uptake  of  the  liver  was 
determined  by  means  of  the  Van  Slyke  technique. 

The  isolated  liver  was  then  connected  with  the  hepatectomized  cat  in  the  following 
way.  The  venous  outflow  from  the  liver  was  connected  with  the  jugular  vein  of  the  he- 
patectomized cat  which  henceforward  shall  be  denoted  the  "donor".  From  the  carotic 
artery  of  the  donor,  blood  was  allowed  to  run  into  a  100  ml  cylinder  containing  about 
50  ml  of  blood.  Simultaneously  the  pump  was  shifted  from  the  blood  reservoir  connected 
with  the  oxygenator  to  the  100  ml  cylinder  cutting  out  the  oxygenator  and  reservoir 
from  the  circuit.  The  blood  which  flowed  from  the  donor  into  the  cylinder  was  then  taken 
up  by  the  pump  and  sent  through  the  liver  at  a  constant  rate  determined  by  the  pump. 
From  the  liver  the  blood  returned  to  the  donor.  By  means  of  a  clamp  on  the  outflow 
from  the  carotic  artery  of  the  donor  it  was  fairly  easy  to  manage  to  keep  the  blood  volume 
in  the  cylinder  constant,  z.^.,  to  secure  that  the  amount  of  blood  leaving  equalled  the 
amount  of  blood  entering  the  donor. 

When  the  liver  was  connected  with  the  donor  the  glucose  infusion  was  stopped. 
At  suitable  intervals  samples  were  drawn  simultaneously  from  the  blood  entering  and 
leaving  the  liver.  Oxygen,  carbon  dioxide,  glucose,  and  lactic  acid  determinations  have 
been  performed  on  these  samples.  Oxygen  and  carbon  dioxide  were  determined  with 
the  Van  Slyke  technique,  glucose  according  to  Hagedorn- Jensen,  and  lactic  acid 
according  to  Barker  and  Summerson  modified  by  LePage. 

The  results  related  to  our  problem  are  presented  in  Table  L  It  is  seen  that  within 
10  minutes  after  the  connection  of  the  liver  with  the  donor  the  rate  of  oxygen  consump- 
tion in  the  liver  has  increased  100%  or  even  more.  One  hour  after  the  connection  the 
oxygen  uptake  of  the  liver  in  most  experiments  shows  a  slight  further  increase.  In  other 
words  the  connection  with  a  donor  of  a  liver  run  with  artificial  perfusion  until  the  oxygen 
uptake  has  dropped  to  a  low  level  increases  the  rate  of  oxygen  uptake  to  a  rate  similar 
to  that  observed  immediately  after  isolation  of  the  liver,  i.e.,  presumably  to  the  normal 
rate.  It  may  be  mentioned  that  this  very  considerable  change  in  rate  of  oxidations  is 
not  accompanied  by  any  change  in  the  respiratory  quotient. 
References  p.  32g. 


326 


E.  LUNDSGAARD 


VOL.  4  (1950) 


TABLE  I 

OXYGEN   CONSUMPTION   OF  CAT  LIVERS  BEFORE,    IO-I5  MINUTES  AND  60  MINUTES  AFTER  CONNECTION 

WITH  A  "donor"  ml/min 


Before 

10-15  niin  after 

60  min  after 

1.8 

3-8 

3-9 

2.6 

50 

5-2 

2.4 

5-1 

4.8 

2.2 

4.0 

4-7 

1-7 

4.6 

50 

2.2 

5-4 

4-7 

2.0 

4.4 

4-9 

Average  2.14 

4.61 

4-74 

As  the  high  rate  of  oxygen  consumption  in  the  liver  after  connection  with  the  donor 
is  maintained  or  even  increases  slightly  during  the  entire  experimental  period  though 
the  liver  is  still  artificially  perfused  the  possibility  that  the  decline  in  oxygen  uptake 
might  be  a  direct  consequence  of  the  artificial  perfusion  is  ruled  out.  The  conditions 
before  and  after  connection  with  the  donor  differ  in  only  one  respect.  Before  the  connec- 
tion when  the  blood  is  oxygenated  in  the  oxygenator  the  oxygen  tension  is  higher  in 
the  blood  entering  the  liver  than  after  the  connection  when  the  blood  is  oxygenated  in 
the  lungs  of  the  donor.  Though  it  is  most  improbable  that  the  oxygen  tension  of  the 
blood  entering  the  liver  is  of  any  significance  a  few  experiments  have  been  carried  out 
in  which  the  perfusion  blood  was  oxygenated  with  alveolar  air  collected  in  a  Douglas 
bag  instead  of  the  ordinary  mixture  of  oxygen  and  4%  carbon  dioxide.  The  oxygen 
uptake  of  the  liver  in  these  experiments  showed  exactly  the  same  variations  as  in  experi- 
ments carried  out  with  the  usual  technique. 

The  glucose  output  from  an  isolated  cat  liver  averages  about  2  mg  per  minute. 
The  glucose  output  from  a  liver  after  connection  with  a  donor  averages  about  9  mg  per 
minute.  The  extra  amount  of  glucose  given  off  by  a  liver  after  connection  with  a  donor 
undoubtedly  originates  from  lactic  acid. 

In  ordinary  perfusion  experiments  on  cat  livers  the  lactic  acid  concentration 
rapidly  falls  to  very  low  levels  (3  to  5  mg%).  In  experiments  in  which  the  artificially 
perfused  liver  is  connected  with  a  donor  the  lactic  acid  concentration  in  the  blood  with 
which  the  liver  is  supplied  is  as  high  as  30  to  50  mg%.  A  definite  drop  in  lactic  acid 
concentration  from  ingoing  to  outgoing  blood  corresponding  roughly  to  the  increase  in 
glucose  concentration  is  demonstrable. 

As  the  lactic  acid  concentration  declines  during  the  first  period  of  a  liver  perfusion 
experiment  during  which  the  oxygen  uptake  falls  off  also  and  as  the  lactic  acid  concen- 
tration is  markedly  increased  after  connection  with  a  donor  when  the  oxygen  consump- 
tion increases  strongly  one  might  think  that  the  concentration  of  lactic  acid  in  the  blood 
is  responsible  for  the  changes  in  the  oxygen  uptake  of  the  liver.  Observations  have 
previously  been  mentioned  however  which  do  not  agree  with  such  an  assumption. 
Furthermore  a  number  of  experiments  have  been  carried  out  in  which  l  (  +  )  lactic  acid 
was  added  to  the  perfusion  blood.  If  at  the  start  of  the  perfusion  l  (  +  )  lactic  acid  is 
added  to  the  blood  in  amounts  increasing  the  concentration  to  well  above  100  mg% 
the  decline  in  oxygen  uptake  proceeds  as  usual  and  if  lactic  acid  in  varying  amounts  is 
References  p.  329. 


VOL.  4  (1950)  METABOLIC    RATE   OF   THE    LIVER  32? 

added  to  the  blood  after  the  oxygen  uptake  has  reached  its  constant  low  level  only 
a  very  slight  increase  in  the  oxygen  uptake  or  no  increase  at  all  is  observed.  Consequently 
the  possibihty  that  the  lactic  acid  concentration  in  the  blood  is  responsible  for  the 
changes  in  oxygen  consumption  observed  in  these  experiments  can  be  definitely  ruled  out. 

If  lactic  acid  is  added  to  the  blood  after  the  oxygen  uptake  of  an  isolated  liver  has 
been  allowed  to  drop  off  the  rate  of  disappearance  of  lactic  acid  amounts  to  only  one 
fourth  to  one  third  of  the  rate  observed  in  a  liver  connected  with  a  donor.  Thus  not  only 
the  rate  of  oxygen  consumption  but  also  the  rate  of  a  reaction  such  as  conversion  of 
lactic  acid  to  glucose  or  glycogen  is  influenced  by  the  hypothetical  substance  present  in 
fresh  blood.  The  statement  appears  justified  that  this  substance  influences  the  "meta- 
bolic rate"  of  the  liver. 

The  nature  of  the  substance  influencing  the  metabolic  rate  of  the  liver  has  not  been 
elucidated;  accordingly,  this  paper  can  be  considered  only  as  a  preliminary  note.  A  series 
of  substances  however  can  be  ruled  out  since  they  have  no  effect  on  the  rate  of  oxygen 
uptake  in  the  liver  when  added  to  the  blood  about  one  hour  after  the  start  of  the  per- 
fusion. Some  of  these  substances  have  been  added  to  the  blood  in  a  single  dose,  others 
have  been  added  continuously  at  a  rate  giving  concentrations  in  the  blood  comparable 
with  the  normal  concentrations.  Without  going  into  details  a  few  of  the  substances 
tested  so  far  are  listed  (Table  II). 

TABLE  II 


"Kochsaft"  of  muscle  Choline 

Fresh  muscle  extract  Methionine 

ATP  Tyrosine 

Creatine  Tryptophan 

Cytochrom  C  Arginine 

Glutathione  Threonine 

Citric  acid  Ascorbic  acid 

Oxalo-acetic  acid  AdrenaUne 

Fu  marie  acid  nor- Adrenaline 

Succinic  acid  Desoxycorticosterone  glycoside  (Ciba) 

Pyruvic  acid  "Corsunal"* 

Lactic  acid  Insulin 

Acetic  acid  Fresh  crude  extract  of  anterior  pituitary 


*  Extract  of  ox-adrenals  prepared  by  Nordisk  Insulin  Laboratory  according  to  Grollman 

AND  FiROR 

Among  the  substances  listed  in  Table  II  only  adrenaline  and  nor-adrenaline  had 
a  definite  but  quite  transitory  effect  of  increasing  oxygen  uptake.  This  effect,  however, 
could  not  be  maintained  by  continuous  addition  of  the  substances. 

It  must  be  mentioned  that  pyruvic  acid  and  the  aminoacids  glycine  and  alanine  in 
large  doses  (300  mg)  have  a  marked  effect  on  the  oxygen  uptake  in  the  isolated  liverV 
As  continuous  addition  of  pyruvic  acid  at  a  rate  of  2  mg  per  minute  (blood  flow  50  to 
60  ml/min)  has  no  effect  on  the  oxygen  uptake  and  as  the  amino  acid  content  in  blood 
perfused  through  a  liver  does  not  decrease  as  does  the  oxygen  uptake  during  the  first 
period  of  the  experiment  it  appears  that  pyruvic  acid  and  amino  acids  can  safely  be 
ruled  out  as  factors  responsible  for  the  changes  in  oxygen  uptake  in  the  Hver  observed 
in  these  experiments. 

The  problem  to  which  attention  is  directed  in  the  present  paper  undoubtedly  is 

References  p.  32 g. 


328  E.  LUNDSGAARD  VOL.  4  (1950) 

related  to  the  observation  made  by  many  investigators^'  *»  ^'  «» '  that  the  respiration  of 
tissue  slices  is  higher  and  more  stable  in  serum  than  in  Ringer  solution.  Though  this 
observation  is  not  absolutely  identical  with  those  of  the  writer,  it  appears  most  probable 
that  the  substance  (or  substances)  in  serum  which  enchances  tissue  respiration  is  the 
same  as  the  substance  (or  substances)  which  is  gradually  removed  from  the  blood  by  an 
isolated  liver  causing  a  decline  in  the  rate  of  oxidations.  The  question  of  the  nature  of 
the  serum  constituents  which  enchance  tissue  respiration  has  been  delt  with  in  a  rather 
explicit  manner  by  Warren  in  two  publications.  In  the  first  of  these^  it  has  been  demon- 
strated that  the  stimulating  effect  of  serum  on  tissue  respiration  partly  can  be  attributed 
to  its  bicarbonate  content.  According  to  Warren  the  maximal  effect  of  adding  bicarbon- 
ate to  a  Ringer-phosphate  medium  is  obtained  at  a  concentration  of  only  3  mM  per  liter. 
Variations  in  the  bicarbonate  concentration  at  higher  levels  are  without  any  influence 
on  the  rate  of  oxidations.  Since  whole  blood  under  constant  and  fairly  high  carbon 
dioxide  pressure  has  been  used  in  the  experiments  described  one  can  certainly  rule  out 
changes  in  bicarbonate  content  as  being  responsible  for  the  observed  changes  in  oxygen 
uptake  in  the  isolated  liver. 

In  accordance  with  Canzanelli  et  al.^,  Warren  finds  substances  capable  of 
enchancing  the  respiration  of  tissue  slices  in  the  ultrafiltrate  of  serum.  Only  about  50% 
of  the  effect  can  be  attributed  to  bicarbonate.  In  his  second  paper  Warren^  reports 
attempts  to  fractionate  serum  with  respect  to  its  action  in  enchancing  tissue  respiration. 
From  his  elaborate  experiments  Warren  concludes  that  lactic  acid  and  amino  acids 
are  not  involved  in  the  stimulating  effect  of  serum  on  tissue  respiration.  I  draw  the 
same  conclusion  from  my  observations.  Warren  further  suggests  that  the  active 
substance  is  a  dicarboxylic  acid,  but  he  has  not  put  this  assumption  on  a  direct  trial  by 
adding  dicarboxylic  acids  to  the  Ringer-phosphate  medium  used  in  his  experiments. 
In  my  experiments  I  have  tested  different  organic  acids  assumed  to  be  formed  as  inter- 
mediates in  tissue  metabolism.  However,  no  effect  on  the  low  oxygen  uptake  of  the 
isolated  liver  was  observed. 


SUMMARY 

Observations  are  presented  indicating  that  the  normal  metabolic  rate  of  the  liver  is  dependent 
on  a  substance  (or  substances)  formed  in  the  extrahepatic  tissues  and  carried  to  the  hver  through 
the  blood.  This  still  unidentified  substance  is  used  or  destroyed  in  the  hver  tissue. 

RfiSUMfi 

L'auteur  prdsente  des  observations  indiquant  que  la  vitesse  normale  du  metabolisme  du  foie 
depend  d'une  substance  (ou  de  substances)  form^e  dans  les  tissus  extrah^patiques  et  qui  est  amenee 
au  foie  par  le  sang.  Cette  substance  non  encore  identifi(5e  est  utilisee  ou  detruite  dans  le  tissu  h6pa- 
tique. 

ZUSAMMENFASSUNG 

Beobachtungen  werden  beschrieben  die  darauf  hinweisen,  dass  die  Normalgeschwindigkeit  des 
Lebermetabolismus  von  einer  Substanz  (oder  von  Substanzen)  abhangt,  die  in  ausserhalb  der  Leber 
gelegenen  Geweben  gebildet  und  durch  das  Blut  der  Leber  zugefuhrt  wird.  Diese  noch  nicht  identifi- 
zierte  Substanz  wird  im  Lebergewebe  verbraucht  oder  zerstort. 

References  p.  32^. 


VOL.  4  (1950)  METABOLIC    RATE    OF   THE    LIVER  329 

REFERENCES 

^  E.  LuNDSGAARD,  Actu  Physiol.  Scand.,  4  (1942)  330. 

2  E.  LuNDSGAARD,  NiELS  A.  NiELSEN,  AND  S.  L.  0RSKOV,  Skund.  Avch.  PhysioL,  76  (1936)  296. 

3  A.  Canzanelli,  G.  Rogers,  C.  Dwyer,  and  D.  Rapport,  Am.  J.  Physiol.,  135  (1942)  316. 

*  D.  Friend  and  A.  B.  Hastings,  Proc.  Soc.  Exptl  Biol.  Med.,  45  (1940)  137. 
5  H.  Laser,  Nature,  136  (1936)  184. 

8  M.  Schaffer,  T.  Chang,  and  R.  Gerard,  Am.  J.  Physiol.,  iii  (1935)  697. 

^  B.  Walthard,  Z.  ges.  exptl  Med.,  94  (1934)  45. 

^  C.  O.  Warren,  /.  Biol.  Chem.,  156  (1944)  559. 

*  C.  O.  Warren,  /.  Biol.  Chem.,  167  (1947)  543. 

Received  March  25th,  1949 


33Q  BIOCHIMICA   ET  BIOPHYSICA   ACTA  VOL.  4  (1950) 


IS  ACETALDEHYDE  AN  INTERMEDIARY  PRODUCT  IN 
NORMAL  METABOLISM? 

by 

ERIK  JACOBSEN 

Biological  Laboratories  of  Medicinalco,  Copenhagen  S.  {Denmark) 


Mainly  through  the  work  of  Meyerhof,  Parnas,  Embden,  and  Cori,  their  collabo- 
rators and  pupils,  the  intermediary  products  of  the  first  part  of  carbohydrate  metabolism 
are  well  known.  The  intermediary  products  have  been  isolated  and  the  enzymes 
involved  thoroughly  studied.  It  is  now  generally  accepted  that  glycogen  or  glucose  is 
broken  down  to  pyruvate  through  a  series  of  phosphorylated  compounds.  Pyruvate 
forms  a  "natural  dividing  point"  between  the  anaerobic  and  the  aerobic  phases  of 
carbohydrate  metabolism.  It  has,  however,  been  extremely  dilhcult  to  study  the  inter- 
mediary products  and  the  corresponding  enzymes  in  volved  in  the  further  oxidation  of 
this  substance.  Several  hypotheses  concerning  this  part  of  carbohydrate  metabolism 
have  been  proposed.  The  experimental  facts  hitherto  obtained  seem  to  be  best  explained 
by  Krebs'  citric  acid  cycle-theory.  The  individual  processes  are  well  known  and  need 
no  further  description  (Krebs,  1943).  Nevertheless  it  is  not  known  whether  other 
processes  are  also  involved  in  the  oxidation  of  pyruvate  and  alternative  schemes  have 
been  proposed.  The  early  theory  of  Thunberg  (1920)  and  Knoop  (1923)  suggests  that 
pyruvic  acid  is  decarboxylated  to  acetaldehyde  which  is  then  oxidized  to  acetic  acid. 
This  compound  is  in  turn  condensed  to  succinic  acid.  Their  theory  has  now  been  aban- 
doned, mainly  because  it  has  been  impossible  to  demonstrate  any  formation  of  succinic 
acid  from  acetic  acid  in  living  cells  or  cell  extracts.  It  has,  however,  been  shown  by 
several  authors  that  acetaldehyde  can  be  formed  during  tissue  metabolism.  In  in  vitro 
experiments  with  minced  tissues  acetaldehyde  has  been  trapped  by  means  of  aldehyde 
fixatures  following  the  technique  of  Neuberg.  Hirsch  (1923)  identified  acetaldehyde 
formed  in  muscles  of  frogs  or  fishes.  Neuberg  and  Gottschalk  (1924)  showed  the 
formation  of  acetaldehyde  in  different  tissues  of  warm-blooded  animals  and  their  results 
have  been  confirmed  and  enlarged  by  Palladin  and  Utevvski  (1929),  Gorr  (1932), 
Tanko,  Munk,  and  Abonyi  (1940)  and  others.  Addition  of  pyruvate  to  the  minced 
muscles  increases  the  yield  of  acetaldehyde  (Utewski,  1929)  and  the  formation  of 
acetoin,  a  condensation  product  of  acetaldehyde  and  pyruvic  acid,  from  pyruvate  has 
been  shown  by  Green  et  al.  (1941)  and  by  Stotz,  Westerfeld,  and  Berg  (1944).  In 
animal  tissues  acetate  was  identified  as  an  oxidation  product  of  pyruvic  acid  by  Krebs 
and  Johnson  (1937),  Weil-Malherbe  (1937)  and  Long  (1938).  It  was  shown  that 
pyruvate  anaerobically  dismutes  into  lactate  +  acetate  +  carbon  dioxide.  Although 
Krebs  and  Johnson  emphasize  that  this  process  in  animal  tissues  differs  from  that  of 
decarboxylation  of  pyruvic  acid  in  microorganisms,  it  cannot  be  excluded  with 
certainty  that  acetaldehyde  even  in  this  process  acts  as  an  intermediary  product. 
References  p.  334. 


VOL.  4  (1950)  ACETALDEHYDE  IN  NORMAL  METABOLISM  33I 

Acetaldehyde  is  oxidized  very  rapidly  in  vivo  (Lubin  and  Westerfeld,  1945) :  and  the 
acetoin  formed  in  vivo  also  appears  to  be  very  rapidly  metabolized. 

Even  if  the  citric  acid  cycle  is  the  main  path  of  the  normal  metabolism  of  pyruvic 
acid  and  some  of  the  results  showing  a  possible  formation  of  acetaldehyde  from  pyruvic 
acid  are  due  to  artefacts  in  the  sense  that  the  biochemical  processes  only  occur  under 
more  or  less  abnormal  conditions,  it  is  still  possible  that  pyruvate  in  normal  metabolism 
is  partly  broken  down  with  acetaldehyde  serving  as  an  intermediary  product.  Hitherto 
no  means  have  been  available  to  decide  to  what  extent  this  secondary  path  plays  a  role 
in  the  normal  metabolic  processes  of  the  organism. 

At  the  present  experiments  performed  in  this  laboratory  are  able  to  throw  a  light 
on  the  question. 

Hald,  Jacobsen,  and  Larsen  (1948)  have  shown  that  individuals  given  tetra- 
ethylthiuramdisulphide  (Antabuse)  will  give  a  series  of  symptoms  after  ingestion  of 
minute  amounts  of  alcohol.  The  occurrence  of  these  symptoms  is  due  to  an  increased 
formation  of  acetaldehyde  from  alcohol,  resulting  in  an  increased  concentration  of 
acetaldehyde  in  the  blood  (Hald  and  Jacobsen,  1948;  Asmussen,  Hald,  and  Larsen, 
1948;  and  Larsen,  1948).  If  the  metabolic  rate  of  acetaldehyde  is  slowed  after  ingestion 
of  Antabuse,  the  increased  concentration  of  this  substance  in  the  organism  is  easily 
explained.  Preliminary  experiments  in  this  laboratory  showed,  however,  that  no  differ- 
ence in  the  rate  of  acetaldehyde  elimination  in  normal  and  Antabuse-treated  animals 
could  be  seen  when  acetaldehyde  was  given  during  short  periods  and  in  such  an  amount 
that  the  final  concentration  of  acetaldehyde  in  the  blood  was  20-25  mg/%.  In  collabo- 
ration with  Dr.'s  Jens  Hald  and  Valdemar  Larsen  I  have  made  a  series  of  further 
experiments  showing  that  the  metabolic  rate  of  small  concentrations  of  acetaldehyde 
is  decreased  in  animals  treated  with  Antabuse.  These  experiments  will  be  published  in 
detail  by  Hald,  Jacobsen,  and  Larsen. 

A  series  of  rabbits  weighing  from  2.0-2.5  kg  were  given  0.50  g  Antabuse  48,  24 
and  16  hours  prior  to  the  experiment.  The  animals  were  anesthetized  with  urethan. 
Blood  samples  were  taken  from  a  cannula  inserted  in  the  carotid  artery.  Coagulation 
was  prevented  by  the  injection  of  1500  units  of  heparin  intravenously.  Acetaldehyde 
determinations  were  made  by  Stotz's  method.  A  cannula  was  inserted  into  the  jugular 
vein.  Two  to  ten  per  cent  solutions  of  acetaldehyde  in  Tyrode's  solution  were  infused 
through  the  cannula  at  a  known  constant  rate.  The  infusing  apparatus  consisted  of 
a  10-30  ml  syringe,  the  piston  of  which  was  controlled  by  a  screw  driven  mechanically 
by  a  gramophone  motor.  The  experiments  generally  lasted  i  ^-2  l^  hours.  During  this 
period  the  infusion  rate  was  maintained  at  a  constant  level  which  did  not  exceed  the 
capacity  of  the  rabbits  to  metabolize  acetaldehyde.  There  was  no  accumulation  of 
acetaldehyde  in  the  tissues  during  the  experiment. 

An  average  sized  rabbit  is  usually  capable  of  eliminating  7-8  mg  acetaldehyde  per 
minute.  The  concentration  of  acetaldehyde  in  the  blood  was  determined  30  minutes  after 
the  beginning  of  the  infusion  and  at  intervals  of  '^U-^U  hours.  The  levels  of  acetaldehyde 
in  blood  corresponding  to  a  fixed  infusion  rate  of  acetaldehyde  varying  between  0.75  mg 
and  9  mg  per  minute  were  determined  in  two  series  of  rabbits :  one  normal  series,  and 
one  consisting  of  rabbits  treated  with  Antabuse  in  the  manner  described  above.  A  con- 
siderable variation  of  the  blood  acetaldehyde  is  noted  from  time  to  time  although  the 
infusion  rate  was  kept  as  constant  as  possible.  The  results  of  the  experiments  are 
tabulated  in  Fig.  i.  A  clear  difference  between  the  concentration  of  acetaldehyde  in 
References  p.  334. 


332 


E.  JACOBSEN 


VOL.  4  (1950) 


blood  in  the  two  series  is  shown.  When  the  same  amount  of  acetaldehyde  is  metabolized, 
the  level  of  acetaldehyde  in  blood  is  higher  in  the  Antabuse-treated  animals  than  in  the 
untreated  ones.  The  smaller  the  amounts  of  acetaldehyde  metabolized  per  minute,  the 
greater  is  the  relative  difference  between  the  two  groups.  When  0.75-2.0  mg  is  infused 
per  minute,  the  acetaldehyde  level  in  blood  of  the  Antabuse-treated  rabbits  is  5-10  times 
that  of  the  normal  animals,  whereas  it  is  less  than  twice  when  8-9  mg  are  infused  per 
minute.  The  same  results  are  obtained  in  perfusion  experiments  with  isolated  liver  and 
hind  limbs.  An  account  of  these  experiments  will  be  published  at  a  later  date. 

If  acetaldehyde  is  found  as  a  normal  split  product  in  metabolism,  the  experiments 
described  here  show  that  this  will  result  in  an  increased  concentration  of  acetaldehyde 
in  the  blood  of  rabbits  treated  with  Antabuse.  Acetaldehyde  in  blood  was  determined 
in  normal  and  Antabuse-treated  rabbits.  The  results  are  given  in  Table  I.  No  significant 
statistical  difference  between  the  two  groups  is  seen. 


0 

c 

> 

y- 

•• 

^ 

y 

y 

1 

Antabuse  -  treated — 

^v^ 

/ 

y 

y 

^Normal 

^5 

y^ 

/ 

E 
4 

y 

^ 

/ 

0 

D 

-^ 

3 

:/ 

• 

y 

2 
1 

y 

^o 

,    ^ 

X 

y 

0 

0  ^ 

0 

0 

0 

^' 

>^ 

• 

"    0 

1         2  3         ^         5  6  7         8         9         10        11        12        13 

mg  ocefaldehyde  infused  per    minute 

Fig.  I.  Correlation  between  infusion  rate  of  acetaldehyde  into  the  jugular  vein  and  mg  acetaldehyde 
per  100  ml  blood  in  normal  rabbits  and  rabbits  treated  with  Antabuse  (tetraethylthiuramdisulphide) 


Similar  results  are  obtained  in  perfusion  experiments.  A  series  of  livers  and  hind 
limbs  from  normal  rabbits  and  rabbits  treated  with  Antabuse  were  artificially  perfused 
with  blood  as  described  by  Nielsen  (1933).  On  an  average  the  livers  weighed  about 
80  g,  and  the  muscles  of  the  hind  limbs  430  g.  The  average  oxygen  uptake  per  minute 
was  1-3  ml  per  minute  in  the  livers  and  2-4  ml  per  minute  in  the  hind  limbs.  When 
acetaldehyde  was  added  to  the  perfusion  blood,  the  blood  which  passed  through  the 
livers  or  muscles  from  Antabuse-treated  animals  showed  a  considerably  higher  concen- 
tration of  acetaldehyde  than  blood  that  passed  through  organs  of  normal  animals. 
From  the  amount  of  blood  perfused  per  minute  and  the  difference  in  acetaldehyde 
concentrations  in  the  blood  before  and  after  the  perfusion  it  is  possible  to  calculate  the 
amount  of  acetaldehyde  passing  into  the  perfusion  blood  per  minute.  If  any  substantial 
quantity  of  acetaldehyde  is  formed  during  normal  metabolism,  a  difference  should  be 
seen  between  the  perfusion  experiments  made  with  normal  animals  and  with  Antabuse- 
treated  animals.  As  seen  in  Table  II  this  is  not  the  case.  At  times  the  acetaldehyde 
References  p.  334. 


VOL.  4  (1950) 


ACETALDEHYDE  IN  NORMAL  METABOLISM 
TABLE  I 


333 


Antabuse  treated 
rabbits 

Normal  rabbits 

Number  of  animals 

28 

19 

Range  of  acetaldehyde  concentration  in  blood 

o.oi  to  0.25  mg  % 

0.00  to  0.30  mg  % 

Average  and  standard  deviation  of  average 

0.104  i  0.012  mg  % 

0.085  ±  0-017  mg  % 

a  =  Standard  deviation  of  single  determinations 

0.021  mg  % 

0.023  mg  % 

Acetaldehyde  in  mg  100  ml  blood  in  rabbits  treated  with  Antabuse   (tetraethylthiuramdisulphide) 
and  in  normal  rabbits 

TABLE  II 


Antabuse 
treated  rabbits 

Normal  rabbits 

Number  of  experiments 

16 

17 

Range  of  mg  acetaldehyde  formed  per  minute 

—  0.04  to  +  0.15 

— -  0.02  ±  O.II 

>    liver 

Average  and  standard  deviation  of  average 

0.032  ±  0.013 

0.030  J;    O.OI  I 

a  =  Standard  deviation  of  single  determinations 

0.053 

0.047 

Number  of  experiments 

9 

12 

Range  of  mg  acetaldehyde  formed  per  minute 

—  0.02  to  +  0.12 

—  0.07  to  +  0.07 

).  hind  limbs 

Average  and  standard  deviation  of  average 

0.012  ±^  0.006 

0.008  J^  O.OOI 

0  =  Standard  deviation  of  single  determinations 

0.017 

0.030 

Acetaldehyde  formation  per  minute  in  isolated  organs  from  rabbits  treated  with  Antabuse  and  from 
normal  rabbits 

formation  is  negative.  This  indicates  that  the  concentration  of  acetaldehyde  is  lower 
in  the  blood  which  has  been  perfused  through  the  organ  than  in  the  blood  which  enters 
the  organ.  Of  course  the  analytical  error  is  rather  high  when  determining  small  concen- 
trations of  acetaldehyde  and  so  will  influence  the  results  considerably.  Furthermore 
substances  other  than  acetaldehyde  may  give  reactions  which  influence  the  determina- 
tions to  a  considerable  degree  when  small  concentrations  of  acetaldehyde  are  found  in 
the  blood.  Nevertheless  the  production  of  acetaldehyde  under  the  above  mentioned 
conditions  appears  to  be  of  very  little  importance. 
References  p.  334. 


334  E.  JACOBSEN  VOL.  4  (195O) 

Thus  it  may  be  concluded  that  very  httle,  if  any,  acetaldehyde  can  be  formed  during 
normal  metabolism  and  that  the  alternative  paths  in  metabolism  in  which  acetaldehyde 
is  supposed  to  be  an  intermediary  product,  do  not  play  a  significant  role. 

SUMMARY 

It  has  been  shown  that  acetaldehyde  metabohsm  is  delayed  in  animals  treated  with  tetra- 
ethylthiuramdisulphide  (Antabuse) . 

No  increase  of  acetaldehyde  formation  can  be  seen  in  total  organisms  and  in  isolated  livers 
and  muscles  from  rabbits  treated  with  Antabuse. 

From  these  observations  it  is  concluded  that  acetaldehyde  plays  a  very  insignificant  role  as  an 
intermediary  product  in  normal  metabolic  processes. 

r£sum£ 

On  montre  que  le  metabolisme  de  I'acetaldehyde  est  retarde  dans  les  animaux  traites  au  tetra- 
ethylthiuramdisulfide  (Antabuse). 

Aucune  augmentation  de  la  formation  d'acetaldehyde  n'a  pu  etre  observ^e  dans  les  organismes 
entiers  et  dans  les  foies  et  les  muscles  de  lapins  traites  a  I'Antabuse. 

De  ces  observations  nous  concluons  que  I'acetaldehyde  joue  un  role  tres  peu  important  dans  les 
processus  metaboliques  normaux. 

ZUSAMMENFASSUNG 

Es  wird  gezeigt,  dass  der  Metabolismus  des  Acetaldehyds  in  mit  Tetraathylthiuramdisulfid 
(Antabuse)  behandelten  Tieren  verzogert  ist. 

Eine  Zunahme  der  Acetaldehydbildung  in  ganzen  Organismen  oder  in  isolierten  Lebern  und 
Muskeln  von  mit  "Antabuse"  behandelten  Kaninchen  wurde  nicht  beobachtet. 

Aus  diesen  Beobachtungen  wird  geschlossen,  dass  das  Acetaldehyd  eine  sehr  unbedeutende 
Rolle  als  Zwischenprodukt  der  normalen  metabolischen  Prozesse  spielt. 

REFERENCES 

E.  AsMUSSEN,  J.  Hald,  and  v.  Larsen,  Acta  Pharmacol.  Toxicol.,  4  (1948)  311. 
G.  GoRR,  Biochem.  Z.,  254  (1932)  12. 

D.  E.  Green,  W.  W.  Westerfeld,  B.  Vennesland,  and  W.  E.  Knox,/.  Biol.  Cheni.,  140  (1941)  683. 
J.  Hald  and  E.  Jacobsen,  Acta  Pharmacol.  Toxicol.,  4  (1948)  305. 

J.  Hald,  E.  Jacobsen,  and  V.  Larsen,  Acta  Pharmacol.  Toxicol.,  4  (1948)  285. 
J.  Hirsch,  Biochem.  Z.,  134  (1923)  415. 

F.  Knoop,  Klin.  Wochschr.,  2  (1923)  60. 

H.  A.  Krebs,  Advances  in  Enzymol.,  3  (1943)  191. 

H.  A.  Krebs  and  W.  A.  Johnson,  Biochem.  J.,  31  (1937)  645. 

V.  Larsen,  Acta  Pharmacol.  Toxicol.,  4  (1948)  321. 

C.  Long,  Biochem.  J.,  32  (1938)  171 1. 

M.  LuBiN  and  W.  W.  Westerfeld,  /.  Biol.  Chem.,  161  (1945)  503. 

C.  Neuberg  and  a.  Gottschalk,  Biochem.  Z.,  146  (1924)  164,  185. 

N.  A.  Nielsen,  Skand.  Arch.  Physiol.,  66  (1933)  19. 

A.  Palladin  and  A.  Utewski,  Biochem.  Z.,  200  (1928)  108. 

E.  Stotz,  /.  Biol.  Chem. ,1^8  (1943)  585. 

E.  Stotz,  W.  W.  Westerfeld,  and  R.  L.  Berg,  /.  Biol.  Chem.,  152  (1944)  41. 

B.  Tank6,  L.  Munk,  and  J.  Abonvi,  Z.  physiol.  Chem.,  264  (1940)  91. 
T.  Thunberg,  Skand.  Arch.  Physiol.,  40  (1920)  i. 

H.  Weil-Malherbe,  Biochem.  J .,  31  (1937)  2202. 
A.  Utewski,  Biochem.  Z.,  204  (1929)  81. 

Received  April  14th,  1949 


VOL.  4  (1950)  BIOCHIMICA   ET  BIOPHYSICA   ACTA  335 


THE  QUANTUM  EFFICIENCY  OF  PHOTOSYNTHESIS 

by 

OTTO  WARBURG,  DEAN  BURK  and  VICTOR  SCHOCKEN 
National  Cancer  Institute,  National  Institute  of  Health,  United  States  Public  Health  Service, 

Bethesda,  Maryland 

and 

STERLING  B.  HENDRICKS 

Plant  Industry  Station,  United  States  Department  of  Agriculture,  Beltsville, 
Maryland  ( U.  S.  A .) 


Photosynthesis  is  a  unique  endothei  mic  photochemical  reaction  in  which  chemical 
energy  is  gained  from  visible  light  energy  by  the  combined  action  of  several  quanta. 
Nothing  similar  is  known  in  the  nonliving  world.  It  was  first  reported  a  quarter  of 
a  century  ago^  that  in  photosjmthesis  the  greater  part  of  the  absorbed  visible  light 
energy  could  be  converted  into  chemical  energy  under  optimum  conditions.  Indeed,  no 
more  than  four  quanta  of  red  light  seemed  to  be  necessary  to  produce  one  molecule  of 
oxygen  gas,  which  is  close  to  the  thermodynamic  requirement  of  three  quanta.  It  is 
easy  to  understand  that  this  result,  lacking  any  analogy,  has  sometimes  been  doubted 
by  theoreticians,  and  it  is  a  fact  that  certain  investigators  have  raised  methodological 
objections^.  For  this  reason  we  have  reinvestigated  the  question  of  the  minimum  quan- 
tum requirement  of  photosynthesis  as  measured  by  oxygen  and  carbon  dioxide  gas 
exchange.  The  present  paper  is  a  short  summary  of  our  findings  by  new  and  simplified 
methods. 

I.  CULTIVATION    OF   CELLS 

A  strain  of  Chlorella  pyrenoidosa,  isolated  in  New  England  and  identified  by 
Dr.  Florence  Meier  of  the  Smithsonian  Institution,  and  for  many  years  in  laboratory 
use,  was  cultivated  in  tall  Drechsel  gas  washing  bottles  containing  200  ml  of  the  following 
salt  solution:  5  g  MgS04-7H20,  2.5  g  KNO3,  2.5  g  KH2PO4,  2  g  NaCl,  and  5  mg  FeS04- 
7H2O,  in  I  liter  of  filtered,  unsterilized  well  water  (pn  4-5-5) •  The  cultures  were  main- 
tained at  a  room  temperature  of  25-30°  C,  and  were  aerated  with  5%  COg  in  air  at  a 
rate  {r^  500  ml  per  minute)  rapid  enough  to  prevent  cell  settling,  and  were  constantly 
illuminated  with  a  lOO-watt  incandescent  lamp  at  a  distance  of  about  30  cm.  Cells 
cultivated  by  this  method  gave  more  uniform  material  and  more  regular  manometric 
results  than  when  cultivated  by  the  older  method  (i,  p.  427)  in  which  slowly  aerated 
cells  settled  down  in  Erlenmeyer-shaped  flasks  and  became  partially  anaerobic  until 
reshaken  up,  and  in  which  lowered  light  intensities  were  employed  for  the  terminal 
cultivation  phase. 

References  p.  346. 


336  o.  WARBURG  et  al.,  s.  b.  hendricks  vol.  4  (1950) 

The  cultures  were  used  for  the  experiments  in  the  present  work  after  2-10  days 
growth,  when  they  contained  200-1000  fA  cells,  depending  upon  the  amount  of  initial 
inoculation.  Usually  50-100  [x\  cells  per  200  ml  medium  were  employed  as  inoculum, 
grown  as  just  indicated.  Bacterial  growth  during  either  cell  culturing  or  manometric 
experiments  was  found  with  a  haemocytometer  to  be  negligible,  due  to  the  low  pn,  the 
lack  of  added  organic  matter  in  the  synthetic  medium,  and  possible  antibiotics  produced 
by  the  Chlorella. 

The  cells  for  experimental  use  were  centrifuged  in  an  International  No.  2  Centrifuge 
at  the  lowest  possible  speed  giving  nearly  complete  settling  in  10  minutes  and  were 
taken  up,  with  or  without  further  washing,  in  fresh  nutrient  medium  at  a  concentration 
of  30-50  /u,\  cells  per  ml. 

II.  MONOCHROMATOR 

A  Steinheil  glass  3-prism  spectrograph  operated  with  a  focal  length  of  195  mm  at 
F  3.5  for  the  collimator  and  a  focal  length  of  710  mm  for  the  telescope  was  used  as  a 
monochromator.  The  slit  was  illuminated  with  a  750-watt  projection  lamp.  The  image 
of  the  coiled  filament  at  about  20°  to  its  plane  was  projected  onto  the  slit  with  an 
auxiliary  lens.  A  looo-watt  voltage  regulator  was  used  to  supply  power  to  the  lamp 
which  operated  at  constant  current. 

The  width  of  the  entrance  slit  was  about  2  mm,  corresponding  to  about  20  m/ti  in 
the  red.  A  slit  was  placed  in  the  focal  plane  of  the  telescope  and  was  adjusted  to  have 
a  width  of  about  30  m/t  covering  the  region  630  to  660  m/i.  A  lens  was  placed  behind 
this  slit  to  throw,  in  a  weakly  convergent  beam,  an  image  of  the  exit  prism  face  on  the 
bottom  of  the  manometer  vessel. 

The  area  of  the  beam  at  the  vessel  was  about  3  cm^  and  the  energy  flux  was  about 
0.6  micro  einsteins/min.  This  intensity  was  decreased  when  desired  by  placing  in  the 
light  beam,  just  before  the  exit  slit,  blackened  wire  screens  calibrated  by  the  National 
Bureau  of  Standards. 

III.  MEASUREMENT   OF   LIGHT   ENERGY 

The  energy  of  the  light  beam  was  measured  by  the  recently  developed  chemical 
actinometer^  whereby  for  each  quantum  of  visible  light  absorbed  one  molecule  of  Og 
is  consumed.  In  the  same  or  similar  rectangular  vessel  as  used  for  theyield determinations 
were  placed  2  mg  ethyl  chlorophyllide,  200  mg  thiourea,  7  ml  pyridine,  and  O2  gas. 
The  actinometer  vessel  was  shaken  in  the  thermostat  at  20°  C  in  the  same  manner  and 
in  the  same  cross-section  of  the  light  beam  as  the  vessels  with  the  cell  suspensions  were 
shaken  during  the  yield  determination.  The  total  intensity  of  light,  absorbed  by  the 
actinometer,  should  not  exceed  0.3  microeinsteins  per  minute  under  our  working  con- 
ditions. Higher  intensities,  as  used  for  the  yield  determinations,  were  diminished  for 
this  purpose  by  the  calibrated  screens.  Several  10  minute  periods  were  observed  for 
every  actinometer  determination.  When  in  t  minutes  the  pressure  change  in  the  actino- 
meter vessel  is  hog  mm,  the  total  energy  flux  in  the  light  beam  in  t  minutes  is  — 

or  — -  microeinsteins  (micromole  quanta),  where  the  vessel  constant  kog  is  expressed 
References  p.  346. 


VOL.  4  (1950)  QUANTUM    EFFICIENCY    OF    PHOTOSYNTHESIS  337 

in  mm^.  Then,  when  the  oxygen  developed  by  ilhiminating  the  green  algae  is  n  id 
and  the  oxygen  absorbed  in  the  actinometer  for  the  same  time  and  beam  of  light  is 
»'//l,  the  quantum  requirement  per  mol  of  O.^  developed  in  photosynthesis  is  simph' 

lj(f'  =  n':}i. 

IV.  COMMENTS    ON    THE    2-VESSEL    MANOMETKIC    METHOD 

—  CO., 

If  the  vield  q?  and  the  assimilatorv  quotient,  y  = ,  are  to  be  determined 

simultaneouslv,  two  vessels  must  be  employed.  If  H  be  the  pressure  change  in  vessel 
I  and  H'  that  in  vessel  II,  the  x^g  and  Xco2  values  can  be  calculated  by  well  known 
equations  (see  ^  and  section  8). 

The  2- vessel  method,  simple  when  the  gas-exchanges  in  the  dark  are  determined, 
recjuires  special  attention  when  applied  to  illuminated  cells.  As  will  be  shown  later,  the 
illumination  of  the  cells  is  an  illumination  with  intermittent  light.  This  intermittency 
should  be  equal  in  the  two  vessels,  and  this  is  attainable  if  the  liquid  volumes  are  equal 
in  both  vessels.  Furthermore,  the  respiration  in  most  cell  suspensions  gradually  changes 
with  time,  so  that  the  pressure  changes  in  light  will  also  change  with  time.  Thus  the 
two  vessels  should  be  darkened  and  illuminated  simultaneously  so  that  the  conditions 
of  the  aforementioned  equations  are  fulfilled,  namely 

•^02  ^  -^  02 

^C02   =  ^  CO2 

where  the  primed  magnitudes  refer  to  one  vessel  and  the  non-primed  to  the  other. 

These  conditions  may  be  satisfactorily  met  by  the  method  of  alternately  shifting 
the  mirror  under  the  two  vessels  at  periods  of,  e.g.,  10  minutes,  as  indicated  in  Fig.  i, 
and  discussed  in  the  next  section.  After  two  or  more  cycles,  the  pressure  readings  for 
each  vessel  for  light  and  dark  periods  mav  be  averaged  and  the  light  action  calculated 
from  the  differences  between  the  pressure  changes  in  light  and  dark.  A  possible  error 
involving  noncomparability  of  time  periods  is  thus  eliminated.  This  error  has  been  one 
of  the  main  sources  of  difticulty  in  r///o;'t'//cf-photosynthesis  experiments  with  the  2- 
\-essel  method. 

V.  PROCEDURE 

Simple  H.\ldane-Bakcroft  constant-volume  manometers  with  small  capillaries 
(0.8  mm  diameter)  with  rectangular  vessels  attached  were  shaken  horizontally  (not  by 
arc  motion)  at  140-180  (usually  150)  cycles  per  minute  at  an  amplitude  of  2.0  cm  in  a 
water  bath  at  20°  C.  The  two  rectangular  vessels  of  about  2.2  <3.8  td  inside  width  and 
length  and  13-14  and  i8-ig  ml  volume  respectively,  were  filled  with  200-400  /d  cells  in 
7  ml,  thus  the  liquid  volumes  were  identical  and  the  gas  spaces  differed.  The  vessels 
(with  capillary  sidearm  vents)  were  gassed  on  the  bath,  simultaneously  with  aid  of  a 
manifold,  and  with  shaking.  The  horizontal  (not  arc)  shaking  was  so  effective  that 
physical  after-effects  of  gas  equilibration  in  the  transition  periods  of  dark  to  light  and 
vice  versa  were  not  appreciable  even  when  the  illumination  produced  photosynthesis 
far  above  the  compensation  point  and  pressure  changes  of  5-10  mm  per  minute  were 
involved.  The  manometers  were  usually  read  without  stopping.  The  end  of  the  mano- 
Rejerences  p.  346. 


33S 


O.  WARBURG  et  ah,  S.  B.  HENDRICKS 


VOL.  4  (1950) 


meter  male  joint  was  not  flat  rough  but  concave  and  polished,  so  that  bubble  formation 
in  the  capillary  did  not  occur ;  nor  did  foaming. 

As  indicated  in  Fig.  i  a  beam  of  red  light  (b30-6bo  mu)  of  about  3-4  cm^  area, 
produced  by  means  of  the  Steinheil  monochromator,  entered  the  side  of  the  thermostat 


Window  of  thermostat 


Red  light  of         _ 
measured  intensify 


100  Watt  incandescent 
(White  ligltt) 


FiK-   I 


Fig.  2 


Rejerences  p.  $46. 


VOL.  4  (1950)  QUANTUM   EFFICIENCY   OF   PHOTOSYNTHESIS  339 

through  a  two  walled  window  and  was  reflected  by  a  mirror  onto  the  bottom  of  a  vessel, 
alternately  in  the  one  or  the  other  by  either  shifting  the  mirror  or  the  manometers, 
depending  on  the  design  of  the  experiment.  The  red  light  entering  the  vessel  was  com- 
pletely absorbed.  To  accompUsh  this,  the  amount  of  cells  must  be  sufficiently  great. 
The  amount  depends  upon  the  chlorophyll  content  of  the  cells.  It  was  found  safe,  to 
avoid  loss  of  light,  to  have  300  /J  of  cells  in  each  vessel.  No  influence  of  the  cell  con- 
centration on  the  yield  was  observed  when  Hght  absorption  was  complete  and  shaking 
adequate.  By  this  method,  both  O2  and  COg  exchanges  were  obtained  simultaneously 
and  independently  for  any  and  every  desired  period  of  measurement,  and  every  yield 
determination  was  connected  with  an  experimental  determination  of  the  relationship 
CO2/O2,  so  that  earlier  uncertainties  concerning  this  ratio  (y)  were  eliminated. 

VI.  INTERMITTENCY   OF   ILLUMINATION 

The  cross-section  of  the  light  beam  entering  the  vessels  was  about  3  cm^,  that  is, 
3/8  of  the  bottom  area,  of  the  vessel.  It  can  be  calculated,  if  we  disregard  the  scattering 
of  light,  that  the  major  part  of  the  red  light  (75%)  is  absorbed  within  a  distance  of  about 
I  mm  from  the  bottom  of  the  vessel.  This  means  that  the  light  absorbing  volume  is 
only  about  1/20  of  the  7  ml  of  the  cell-suspension. 

Let  now  the  intensity  of  the  red  light  be  so  strong,  that  the  oxygen  consumption 
of  the  whole  cell  suspension  is  compensated  by  the  oxygen  evolution  (compensation 
point  for  Og).  Then  the  oxygen  development  in  the  absorbing  volume  of  the  cell  sus- 
pension may  approach  20  times  the  point  where  the  cells  become  saturated  with  light 
and  the  increment  yield  zero  (with  our  cell  conditions  the  saturation  intensity  is  about 
30-40  times  the  compensation  intensity) .  But  we  obtain  maximum  or  high  yields  when 
the  vessels  are  shaken  as  described  at  not  only  compensating  but  even  considerably 
higher  intensities,  when  the  latter  are  provided  by  white  light.  This  proves  that  under 
our  shaking  conditions  the  cells  alternate  so  frequently  between  darkness  and  illumina- 
tion that  the  concentrations  of  the  participants  of  all  dark  reactions  virtually  retain 
their  dark  values  —  a  consideration  which  shows  the  methodological  importance  of 
the  kind  and  rate  of  shaking. 

VII.  YIELD    DETERMINATIONS   ABOVE   THE    COMPENSATION    POINT 

A  limiting  feature  of  most  earlier  yield  determinations  was  the  low  total  light 
intensity,  so  low  that  only  a  fraction  of  the  respiration  was  compensated  for  by  the  Hght 
action.  Thus  the  yield  determinations  were  in  a  sense  determinations  of  inhibited  or 
diminished  respiration.  We  have  changed  this  situation  by  illuminating  the  vessels  from 
above  the  thermostat  by  a  lOO-watt  constant-voltage  incandescent  lamp  (as  diagrammed 
in  Fig.  i),  at  such  a  distance  that  the  pressure  changes  in  the  vessels  become  zero  or 
positive;  yield  determinations  were  then  made  with  measured  amounts  of  red  Hght 
added  in  the  usual  manner  from  below  the  vessel.  The  intensity  of  the  white  Hght 
at  the  vessel  surface  was  considerably  smaller  per  unit  area  than  that  of  the  red  light 
but  covered  a  many  fold  greater  area  and  hence  provided  much  more  total  effective 
light  than  did  the  red  beam.  Owing  to  this  relationship  of  intensities  it  was  possible 
to  eliminate  respiration  as  an  experimental  quantity,  and  to  start  the  yield  experiments 
at  positive  rather  than  negative  pressures,  and  yet  still  obtain  (as  experience  showed) 
References  p.  346. 


340  O.  WARBURG    Ct  ul.,  S.  B.  HENDRICKS  VOL.  4  (1950) 

virtually  as  good  yields  from  the  red  light,  whether  the  base  line  were  darkness  or  the 
white  light. 

Another  limiting  feature  of  the  earlier  yield  experiments  was  the  short  duration 
of  not  only  the  periods  of  illumination  (lo  minutes)  but  also  the  total  length  of  the 
experiment  (commonly  less  than  one  hour).  By  the  use  of  white  light  we  have  now 
succeeded  in  extending  the  duration  of  the  manometric  yield  experiments  up  to  at  least 
10  hours,  if  not  indefinitely.  The  effects  of  this  important  advance  are  several.  In  general, 
the  yields  may  now  be  determined  under  nearly  the  same  conditions  as  obtain  during 
the  growth  and  cultivation  of  the  cells,  since  the  light  intensity,  temperature,  medium, 
and  gas  phase  during  the  growth  and  manometry  are  essentially  the  same,  and  further- 
more we  have  found  that  the  shaking  does  not  change  the  cells  under  these  conditions. 

VIII.    EXAMPLES   OF   DATA 

Protocols  I,  2,  and  3  provide  examples  of  the  data  obtained. 

PROTOCOL  No.  I 

Experiment  of  V-26-49.  20°  C.  630-660  m/t.  5%  COg  in  air.  260  jn\  of  cells  per  vessel.  Each 
vessel  alternating  10'  in  dark  and  10'  red  light;  thus  when  vessel  No.  5  was  dark,  vessel  No.  3  was 
illuminated,  and  vice  versa. 


Vessel  No.  5 

Vessel  No.  3 

V  =  13-913  ml 
Vf  =     7.000   ,, 
k'oa  =  0.665     k'co2  =  I- 

235 

ko2 

V  =  17-993  ml 
Vf  =     7.000    ,, 
=  1.046    kco2  =  1-634 

80'  dark  — 91.5  mm 
80' light  +    1.5     „ 

80'  dark  — 26.5  mm 
80'  light  +  15.0     „ 

80'  H'      +93-0  mm 

80'  H       +  41-5  mm 

ction  of  light  in  80'  X02  = 

H 

•kco2  - 
kc02 
ko2 

-H'-k'o2 

,  ,              =  +  151  /<1 
k  CO2 

k'02 

XC02  = 

H 

•ko2  — 
ko2 

■^'•>02   _       ^,8^1 
k  O2 

{Equation  2) 

kc02        k'co2 

Actinometer:  — 8.83  f.i\  O^  per  minute 

.      ^      I          80-8.83 
Quantum  eraciency  for  Oj,  —  =  =4-7 

1  80-8.83 
Quantum  efficiency  for  CO,,  —  =  — —  =  4.2 

(p  168  — 

.     .,                     .                    CO,        —168 
Assimilatory  quotient,  y  =  =  =  ^  — i.ii 

02  +151 
CO 

If  y  =  ^    =  — I.II  is  determined  for  a  given  cell  suspension,  then  X02  and  XCO2  can  be 

obtained  by  the  pressure  changes  in  light  and  dark  in  each  single  vessel.  For  example,  in  vessel  No.  5, 
the  following  figures,  taken  immediately  prior  to  the  readings  above,  were  obtained  upon  illumination 
with  light  of  an  actinometer  value  of  — 5.07  /il  Og  per  minute: 

Vessel  No.  5 

10'  dark  — 12.5  mm  \ 

10'  light  —   2.5     ,,      >  10'  H'  =  +  IO-2  mm  ^ 

10' dark — 13.0     ,,     )  I       /  tt/         , 

,  T   ,  .  [  I  20   H    =  +  20.2  mm 

10       light    1-5  ,,  I  /     TTr  , 

/  1     ,  }   10   H    =  +  10. o     ,,     / 

10   dark  — 10. o     ,,     ) 

References  p.  346. 


VOL.  4  (1950) 


QUANTUM    EFFICIENXY   OF   PHOTOSYNTHESIS 


341 


{Equation  3)  Action  of  light  in  20;     X02  =  H'  —;■ 


k', 


C02"k'02 


Quantum  efficiency  for  Og, 
Quantum  efiiciencv  for  CO, 


^C02  = 

20-5.07 


k'c02+  yk'o2 

I.II-X02=   +3 


=   20. 2-  1.62  =    +  32.1 
-I. II   =  —36.4 


32.8  ^ 

2O-5-07 
36.4 


3-1 
2.8 


PROTOCOL  No.  2 


Experiment  of  V-30-4g.  20°  C.  630-660  va.^.  5°o  CO,  in  air.  270  /il  of  cells  per  vessel. 
Experiment  I.  Alternately  dark  and  light  each  10'.  Actinometer  for  the  red  light  (total)  5.4  jil 
O,  per  minute.  \^Tien  vessel  No.  5  was  dark,  No.  3  was  illuminated  and  vice  versa. 


N 

3-   5 

No.  3 

Constants  as 

in  Protocol  i 

Constants  as  in 

Protocol  I 

10' 

dark 

— 10.5  mm 

10' 

light  +  0.5  mm 

10' 

dark 

—  2.0  mm  10' 

light 

+  3.0  mm 

10' 

— 10. 0     ,, 

10' 

,,           0 

10' 

—  3-5     -     10' 

+  2.0     ,, 

10' 

—  9-0    „ 

10' 

„      +1-5     ,. 

10' 

—  2.5     „     10' 

+  3-5     .. 

10' 

—   8.5     „ 

10' 

,,           0 

10' 

—  2.5     ,,     10' 

+  3-5     .. 

10' 

—   9-0     „ 

10' 

„      +1.0     „ 

10' 

— 0        ,,     10' 

+  3-0     >. 

10' 

—   8.0     „ 

10' 

„      +1.0     „ 

10' 

—  i.o     ,,     10' 

+  5-0    „ 

60' 

dark 

—  55.0  mm 

60' 

light  +4.0  mm 

60' 

dark 

—  1 1.5  mm  60' 

light  +  20.0  mm 

60' 

:  H'  = 

=  4  +  55  = 

-  +  59  mm 

60' 

:  H  = 

20  4-  II-5  = 

+  31 

5  mm 

Experiment  II:  Both  vessels  were  now  constantly  illuminated  with  a  loo-watt  incandescent 
lamp  of  nonmeasured*  light  intensity  and  red  light  of  measured  intensity  added  for  alternating 
periods  of  5'.  Actinometer  for  the  red  light  (total)  5.4  /d  O^  per  minute. 

No.  3 
5'  white  +  14.0  mm    5'  white  +  red  +  15.0  mm 


No 

5 

5' 

white  +  18.5  mm 

5'^^ 

hite  +  red 

+  22.0  mm 

5' 

„      +  iS.o     „ 

5' 

,, 

+  22.5     ,, 

5' 

„      +16.5     „ 

5' 

,, 

+  22.0    ,, 

5' 

..      +17-5     ,. 

5' 

,, 

+  20.5    „ 

5' 

„      +17-0    „ 

5' 

" 

+  23.0    ,, 

+ 14-0 
+ 12.5 
+ 14-0 

+  II-5 
+  12.0 


+  16.5 
+  16.5 
+  14.0 

+  15-0 
+  14-5 


25'  white  +  87.5  mm  25'  white  +  red  +  110 
25':  H'  =  no  —  87.5  =  +  22.5  mm 


30'  white  +  78     mm  30'  white  +  red  +91.5  mm 
30':  H    =  91.5  —  78  =  +  13.5  mm 
25':  H   =  II. 3  mm 


Calculation  of  quantum  efficiency  for  experiment  I  (Dark  ±  Red) 

In  60':  H  +  31.5  mm     H'  +  59  mm 
Applying  equations  (i)  and  (2),  protocol  (i) 

In  60'  X02I   =  +  70-4  /^l  \  CO, 

\       y     ^=      ? 

XCO2   =    —56.0  H\    I  °2 

J_    _    60-5.4 

rp  70.4 

T  6n • ^    f 

5-8 


=  —0.8 


Ouantum  efficiencv  for  O^, 


Quantum  efficiency  for  COg,  —  = 


4.6 


S6 


Calculation  of  quantum  efficiency  for  experiment  II  (White 

In  25':  H  +  II-3  mm  H'  +  22.5  mm 

Applying  equations  (i)  and  (2),  protocol  (i) 


Red) 


In  25'  X02    =  +  30-3  fjl 

XC02  =  —27.2  /d 
Quantum  efficiency  for  O. 


fp 


CO, 

"a 


-  =  — 0.90 

25'5-4 


Quantum  efficiency  for  CO,,   —  = 

<P 


30.3 
25-5-4 


=   4-5 


But  kept  constant  by  a  500-watt  voltage  regulator. 
References  p.  346. 


342  O.  WARBURG   et  uL,  S.  B.  HENDRICKS  VOL.  4  (1950) 

Experiment  III,  with  the  same  cells,  was  performed  between  experiments  I  and  II,  the  white 
light  being,  however,  of  somewhat  lower  intensity.  Here  only  one  vessel  (No.  5)  was  used;  but  if  we 
take  as  y  the  average  value  of  experiments  I  and  II,  that  is  — 0.85,  XQg  can  be  calculated  according 
to  equation  (3),  protocol  (i).  The  readings  in  vessel  (5)  were: 

No.  5 

5'  white  +    5.0  mm  5'  white  +  red  -|-  11.5  mm 

5'      ..       4-    6.5     ,,  5'            „            +    9-5     ., 

5'      ..       +    6.5     ,,  5'            „            +    9.5     „ 

5'      ..       +    5-5     ..  5'            ,.            +  13-0     „ 

5'      .,       +    7-0     ..  5'            ..            +15-0     .. 

25'  white  +  30.5  mm  25'  white  +  red  +  58.5  mm 

25':  H'  =  58.5  —  30.5  =  +  28  mm 
and  with  y  =  — 0.85 

25'  X02  =  +  34  /*! 
The  quantum  efficiency  with  the  actinometer  value  of  experiments  I  and  II   (5.4  /xl  O^  per 
minute)  was 

—  =  lllld  =  4.0  for  O, 
•P  34  — 

The  total  duration  of  these  experiments  was  7  hours  from  the  time  of  initial  equilibration  until 
the  last  yield  determination  that  gave  a  value  —  =  4.5  for  oxygen,  which  was  obtained  at  approxi- 
mately  4  times  the  compensation  point.  The  final  pH  in  the  cell  suspensions  was  5.4. 

PROTOCOL  No.  3 

Comparison  of  the  yield  in  carbonate-bicarbonate  mixtures  and  in  culture  medium 

Experiment  of  VI-i-49.  20°  C.  630-660  m/t.  Three  vessels,  in  each  7  ml  cell  suspension,  containing 
200  /il  of  cells.  Cultures  centrifuged,  then  washed  once  in,  and  taken  up  in,  carbonate-bicarbonate 
mixture.  Intensity  5.4  /xl  Og  per  minute. 

I.  Vessel  No.  7. 
V      ==  13.824  ml 
Vf      =     7.00    ml 
ko2  =     0.657 

Gas  space  air.  Solution  85  parts  M/io  NaHC03+  15  parts  M/io  KgCOg;  Ph9-2.  At  compensation 
point  with  white  light. 

15'  white  light  o 

15'      ,,  ,,      +  red  light  +  11.5  mm 

15'      ..  ..  o 

15'      ..  ..      +     ..        ..      +"-5     ,. 

15'       ..  —    0-5     : 

Light  action  30'  +  23  +  0.5  =  +  23.5  mm  =  15.4  ^l 

I  30'5.4  162   __ 

<P  15-4  15-4        — '- 

II.  Vessels  Nos.  3  and  5,  containing  7  ml  culture  medium,  pn  4-9.  with  200  ^1  of  cells  each. 
Cultures  centrifuged,  then  washed  once  in,  and  taken  up  in,  fresh  culture  medium.  Gas  space  5%  CO^ 
in  air.  Mirror  shifted  every  10'  from  one  vessel  to  the  other;  actinometer  5.4  fd  Oj  per  minute  for 
red  light. 

No.  3 

V        =  17993  ml 
Vf       =     7000    ,, 

k02     =     i-°46 

kco2  =      1-634 

15'  white  light  +  ii.o  mm  15'  white  light  +  red  light  +  29.5  mm 

15'      ,,  ,,      +  red  light  -f  17.0     ,,  15'      ,,  ,,  +  i5-5     .. 

15'      ..  ,.  +10.5     .,  15'      ..  ..      +    ..        -.      +29-0     „ 

15'      ..  ..      +     ..        ..      +16.0     „  15'      „  „  +17-5     .. 


No 

•  5 

V 

=  ] 

[3913  ml 

Vf 

= 

7000   ,, 

k'02 

= 

0.665 

k'c02 

= 

1-253 

30'  H  =  +  II. 5  mm  30'  H'  =  +  25.5  mm 

References  p.  346. 


VOL.  4  (1950) 


QUANTUM    EFFICIENXY   OF    PHOTOSYNTHESIS 


343 


X02 
XC02 


+  41.^  mm  1 
'   'i    J  1  y  =  — 1.04 

—  43-0     .,     j   ^ 


30-5-4 


162 
41-3 


3-9 


(p  41-3 

III.  Vessel  No.  7,  with  same  cells  as  before  but  without  white  light  (below  compensation-point). 

PH9-2 

10  dark  — 33.5  mm 
10'  red  light  — 23.5 
10'  dark  — 30.5 
10'  red  light  — 22.5 
10'  dark  — 30.0 
20'  dark         — 60.5 


Light  action  20' 


20'  dark  — 62.7  mm,  20'  red  light  —46.0  mm 
62.7  —  46  =  +  16.7  mm  =  II  ^1 
j_  _  20-5.4 
"P 


=  9.8 


IV.  Again  Nos.  3  and  5,  but  no  white  light  (under  conpensation  point)  pn  4-9 

No.  5 
10'  red  light  ^   5.0  mm 
10'  dark         — 12        ,, 
10'  red  light  —   4.5     ,, 
10'  dark  — 13        ,, 


No.  3 
10'  dark  — 4.0  mm 

10'  red  light    — 1.5     „ 
10'  dark  — 5.0    ,, 

10'  red  light    — 1.5     ,, 


10'  dark 


-5-0 


10'  red  light  —   4.0 


30'  dark         — 14.0  mm 
30'  red  light  —   4.5     ,, 


Light  action  H 
30' 


+  9.5  mm 


X02 
XCO2 


30' 
30' 

dark 
red  light 

—  37-6 

—  13-5 

mm 

H' 

=  + 

24.1 

mm 

-45 
-53 

6  ) 
0 

y 

= 

—  I. 

18 

30- 

5-4 

= 

3 

6 

45-6 

V.  Again  No.  7,  but  with  half  Ught  intensity  (Actinometer,  2.75  /il  Oj  per  minute),  pn  9-2. 

10' red  light — 24 
10'  dark         — 28 
10'  red  light  — 24.5     ,, 
10'  dark         — 27.0     ,, 
10'  red  light  — 23 


Light  action  10'  27.5 
I  10-2.75 


23.8  =  +  3.7  mm  =  2.42  [x\ 


2.42 


II-3 


The  total  duration  of  these  experiments  was  8  hours. 


IX.  SUMMARY   AND    CONCLUSION 

Since  development  of  the  new  methods  and  procedures  described,  in  a  sequence 

of  thirty  experimental  days,  almost  without  exception  quantum  efficiencies  of  3  to  5 

quanta  per  molecule  of  O.,  produced  by  the  action  of  red  light  have  been  obtained.  The 

CO., 
simultaneously  observed  quotients  of   — -^   for  light  action  lay  between  — 0.8  and 

— 1.3,  which  means  that  the  quantum  efficiencies  for  CO.^  consumption  in  red  light  were 
essentially  the  same  as  those  for  Og  production. 

These  results  were  obtained  not  only  with  low  light  intensities  below  the  compen- 

References  p.  346. 


344  o.  WARBURG  et  al.,  s.  b.  hendricks  vol.  4  (1950) 

sation  point  and  for  short  periods  of  time  (minutes),  but  also  with  Hght  intensities  well 
above  the  compensation  point  (several  fold),  and  in  experiments  lasting  many  hours. 
It  is  important  to  emphasize  that  with  the  same  cell  suspension  the  same  quantum 
yields  may  be  obtained  both  below  and  far  above  the  compensation  point. 

The  new  results  resolve  several  uncertainties  left  open  by  the  experiments  of  1923. 
At  that  time  the  light  intensities  were  so  low  that  only  a  fraction  of  the  respiration  was 
compensated  by  the  light.  Thus  the  objection  could  never  have  been  refuted  that  light 
inhibited  respiration  anticatalytically,  that  is,  without  expenditure  of  energy.  But  now, 
in  the  experiments  above  the  compensation  point,  this  question  is  eliminated,  and 
chemical  energy,  corresponding  to  positive  O2  production  and  CO2  consumption,  is  in 
fact  clearly  gained. 

It  was  a  further  shortcoming  of  the  experiments  of  1923,  that  the  yields  had  been 
determined  only  for  short  periods  of  time  [e.g.,  10  minutes).  But  now,  in  the  experiments 
above  the  compensation  point,  the  cells  are  so  nearly  under  their  natural  culture  con- 
ditions, that  there  is  no  evident  time  limit  to  yield  determinations.  Thermodynamically 
this  is  a  noteworthy  advance  since  the  longer  the  experiments  the  surer  becomes  the 
necessary  condition  of  all  calculations  of  yield:  that  the  absorbed  light  energy  is  the 
sole  source  of  energy  for  the  photosynthetic  processes. 

Finally,  we  may  point  out  that  the  methodology  has  been  so  simplified  that  effi- 
ciency determinations  can  be  carried  out  wherever  simple  manometric  equipment  and 
a  suitable  light  source  are  available,  without  the  need  of  a  bolometer,  thermopile, 
cathetometer  or  special  differential, manometer.  In  fact,  demonstration  of  the  high 
quantum  efficiencies  reported  in  this  paper  may  readily  be  made  in  the  laboratory 
classroom. 

A  cknowledgements 

Valuable  aid  in  these  experiments  was  provided  by  Mrs  Lois  B.  Macri,  Mrs  Clara 
F.  Smith,  and  C.  R.  Newhouser.  The  culture  of  Chlorella  pyrenoidosa  was  provided 
by  Dr  F.  E.  Allison  of  the  Plant  Industry  Station,  United  States  Department  of  Agricul- 
ture, Beltsville,  Maryland.  We  wish  to  thank  E.  Machlett  and  Son,  New  York  City, 
for  special  facilitation  of  provision  of  the  manometric  glassware,  and  the  American 
Instrument  Company,  Silver  Spring,  Maryland,  for  the  specially  adapted  thermo- 
stat and  shaking  mechanism  employed. 


RfiSUMfi  ET  CONCLUSIONS 

Depuis  le  developpement  des  nouvelles  methodes  et  des  nouveaux  precedes  decrits,  nous  avons 
trouve,  a  peu  pres  sans  exception,  une  efficience  de  3  a  5  quanta  par  molecule  d'oxygene  produite 

CO 

par  raction   de  la  lumiere  rouge.  Les  coefficients  ^    observes  simultanement  pour  Taction  de  la 

lumiere  se  trouvaient  entre  — 0.8  et  — 1.3,  ce  qui  signifie  que  I'efficience  en  quanta  pour  la  lumiere 
rouge  est  a  peu  pres  la  meme  pour  la  consommation  de  CO,  que  pour  la  production  de  Oj. 

Ces  resultats  ont  €te  obtenus  non  seulement  pour  de  faibles  intensites  et  de  courtes  periodes, 
mais  aussi  pour  des  intensites  bien  au-dessus  du  point  de  compensation  (plusieurs  fois)  et  pour  des 
experiences  durant  plusieurs  heures.  II  est  interessant  de  noter  que  Ton  peut  obtenir  les  memes 
rendements  en  quanta  pour  une  meme  suspension  cellulaire  au-dessous  et  au-dessus  du  point  de 
compensation. 

Les  nouveaux  resultats  resolvent  plusieurs  incertitudes  qui  avaient  subsistees  apres  les  expe- 
riences de  1923.  A  cette  epoque,  les  intensites  de  lumiere  6taient  si  faibles  que  seule  une  fraction  de 

References  p.  346. 


VOL.  4  (1950)  QUANTUM   EFFICIEN'CY   OF   PHOTOSYNTHESIS  345 

la  respiration  etait  compensee  par  la  lumiere.  C'est  pourquoi,  I'objection  n'a  jamais  pu  etre  refutee 
selon  laquelle  la  lumiere  empecherait  la  respiration  anticatalytiquement,  c.a.d.  sans  depense  d'ener- 
gie.  Actuellement  cette  question  se  trouve  eliminee  par  les  experiences  au-dessus  du  points  de  com- 
pensation et  on  a  vraiment  un  gain  en  energie  chimique  correspondant  a  una  production  positive  de 
Og  et  une  consommation  de  COj. 

Une  autre  insuffisance  des  experiences  de  1923  est  due  au  fait  que  les  ren  dements  avaient  ete 
determines  seulement  pour  des  periodes  breves  (p.  ex.  10  minutes).  Actuellement,  oil  Ton  travaille 
au  dessus  du  points  de  compensation,  les  cellules  se  trouvent  si  pres  de  leurs  conditions  de  culture 
naturelles  qu'il  n'y  a  pas  de  temps  limite  evident  pour  les  determinations  de  rendement.  C'est  un 
serieux  avantage  du  point  de  vue  thermodynamique,  car  plus  les  experiences  sont  longues,  et  plus 
surement  la  condition  necessaire  pour  toute  determination  de  rendement  sera  remplie,  c.a.d.  que  la 
lumiere  absorbee  soit  la  seule  source  d'energie  pour  le  processus  photos^^nthetique. 

Finalement,  nous  avons,  tellement  simplifie  la  methodologie  que  des  determinations  d'efficience 
simplifiees  peuvent  etre  effectuees  facilement  partout  ou  Ton  dispose  d'un  simple  manometre  et  d'une 
source  de  lumiere  adequate.  On  n'a  pas  besoin  de  bolometre,  de  thermopile,  de  cathetometre,  ni  de 
manometre  differentiel  special.  En  effet,  Ton  pent  demontrer  I'efficience  quantique  elevee,  rapportee 
dans  ce  memoire,  dans  un  laboratoire  de  classe. 


ZUSAMMENFASSUXG  UXD  SCHLUSSFOLGERUNGEN 

Seit  die  hier  beschriebenen  neuen  Methoden  und  Verfahren  entwickelt  worden  sind,  haben  wir 
in  einer  Reihe  von  30  Arbeitstagen  fast  ohne  Ausnahme  Quantumleistungen  von  3  bis  5  Quanta  pro 
Molekiil  Og  (gebildet  unter  der  Einwirkung  von  rotem  Licht)  gefunden.  Gleichzeitig  wurden  Quotien- 

CO. 

ten fiir  die  Lichtwirkung  gefunden,  die  zwischen  — 0.8  und — 1.3  lagen;  dies  bedeutet  dass 

die  Quantumleistung  in  rotem  Licht  fiir  COj-Aufnahme  und  Og-Abgabe  ungefahr  gleich  war. 

Diese  Ergebnisse  wurden  nicht  nur  fiir  niedrige,  unter  dem  Kompensationspunkt  gelegene 
Lichtintensitaten  und  fiir  kurze  Zeitspannen  (Minuten)  gefunden,  sondern  auch  fiir  hohe,  weit  iiber 
dem  Kompensationspunkt  gelegene  Lichtintensitaten  und  fiir  Versuche  von  mehreren  Stunden.  Mit 
der  gleichen  Zellsuspension  kann  man  unter-  und  oberhalb  des  Kompensationspunktes  dieselbe 
Quantumausbeute  erhalten. 

Die  neuen  Ergebnisse  beheben  einige  Unsicherheiten  der  Versuche  von  1923.  Damals  waren  die 
Lichtintensitaten  so  gering,  dass  nur  ein  Teil  der  Atmung  durch  das  Licht  kompensiert  wurde.  Der 
Einwand,  dass  das  Licht  die  Atmung  antikatalytisch,  also  ohne  Energieverbrauch  hemme,  konnte 
daher  nie  widerlegt  werden.  Nun  aber,  in  den  Versuchen  oberhalb  des  Kompensationspunktes,  ist 
diese  Frage  erledigt;  es  wird  wirklich  Energie  entsprechend  der  Abgabe  von  Oj  und  Aufnahme 
von  CO2  gewonnen. 

Ein  anderer  Mangel  der  Versuche  von  1923  bestand  darin,  dass  die  Ausbeuten  nur  iiber  eine 
kurze  Zeitspanne  (z.B.  10  ]\Iinuten)  bestimmt  wurden.  Nun  aber,  in  den  Versuchen  oberhalb  des 
Kompensationspunktes,  befinden  sich  die  Zellen  so  nahe  den  Bedingungen  einer  normalen  Kultur, 
dass  eine  offensichtliche  Zeitgrenze  fiir  Bestimmungen  der  Ausbeute  nicht  besteht.  Thermodyna- 
misch  gesehen  ist  das  ein  wichtiger  Fortschritt,  denn  je  langer  die  Versuchszeit,  desto  sicherer  wird 
die  fiir  alle  Berechnungen  der  Ausbeute  notwendige  Bedingung  erfiillt  sein :  dass  namlich  die  absor- 
bierte  Lichtenergie  die  einzige  Energiequelle  fiir  den  photosynthetischen  Vorgang  sei. 

Endlich  konnen  wir  darauf  hinweisen,  dass  wir  die  Methodologie  so  vereinfacht  haben,  dass 
Leistungsbestimmungen  mit  einem  einfachen  Manometer  und  einer  passenden  Lichtquelle,  ohne 
Bolometer,  Thermoelement,  Cathetometer  und  Spezial-Differentialmanometer  ausgefiihrt  werden 
konnen.  So  konnen  die  hier  mitgeteilten  hohen  Quantumleistungen  im  Schullaboratorium  nachge- 
wiesen  werden. 


APPENDIX 

I.  Emerson  has  objected^- ^  to  the  yield  determinations  of  1923^  and  1948*  on  the  ground  that 
the  assimilatory  y  =  COg/Og  was  not  determined  simultaneously  with  the  yield  q?;  i.e.,  that  the 
value  of  y  employed,  — 0.91,  which  had  been  determined  gas  analytically,  may  not  be  the  y  during 
the  different  ijf-determinations  carried  out  for  different  periods  of  time,  light  intensities,  and  cell 
cultures. 

As  has  been  mentioned,  we  have  observed  experimental  fluctuations  of  y  from  — 0.8  to  — 1.3 
If  we  had  used  these  y-values  in  1923  for  the  computation  of  9?,  let  us  see  what  the  values  of  97  would 
have  been. 

References  p.  346. 


346  O.  WARBURG   et  al.,  S.  B.  HENDRICKS  VOL.  4  (1950) 

The  volume  of  our  vessel  was  37.0  ml  and  the  volume  of  the  liquid  phase  16.53  rnl-  For  10°  C 


Therefore 


kc02  = 

5-67 

ko2=  1.70 

K02  = 

kc02"ko2 

1^C02  +  7  ^'02 

5.67-1.70 

5.67  +  y  1.70 

V 
—0.8 

K02 

2.24 

Quantum  requirement  — 
4.20 

— 0.91 

2.34 

4.00 

—  1.3  2.78  3.40 

where  the  underlined  values  are  the  values  used  and  obtained  in  1923.  This  calculation  shows  that 
Emerson's  objection  was  not  very  signiiicant  and  could  not  explain  the  divergent  quantum  requi- 
rements of  4  against  10  to  12. 

II.  In  an  effort  to  avoid  difficulties  caused  by  fluctuations  of  y.  Emerson  and  Lewis  made 
quantum-efficiency  measurements  in  carbonate-bicarbonate  solutions  at  pfj  9.1,  which  kept  the  COg- 
pressure  constant  instead  of  using  culture  medium  at  pn  4-9-  They  claimed^  that  in  such  alkaline 
solutions  the  quantum-efficiency  was  the  same  as  in  the  acid  culture  medium:  "then  we  find  the  yields 
measured  in  acid  phosphate  culture  medium  are  in  good  agreement  with  those  measured  in  carbonate 
mixture". 

But  the  experimental  data  were  not  presented  to  substantiate  this  important  statement.  We  can 
confirm  Emerson's  finding  that  in  the  carbonate-bicarbonate  mixtures  the  quantum-requirement  is 
10  to  12,  but  we  cannot  confirm  that  the  same  quantum  efficiency  is  obtained  in  the  acid  culture 
medium.  Data  presented  in  protocol  3  show  that  very  different  quantum-efficiencies  are  obtained  if 
we  determine  the  quantum  efficiency  of  aliquot  portions  of  a  cell  suspension  in  carbonate  mixture  at 
PH  9-1  and  in  culture  medium  at  pn  5-  The  quantum  values  observed  in  the  following  time  sequence 
were 

I 

In  carbonate  mixture  at  pn  9 

In  culture  medium  at  pn  5 

In  carbonate  mixture  at  pn  9 

In  culture  medium  at  pn  5 

In  carbonate  mixture  at  pn  9 

where  the  asterisked  values  were  obtained  above  the  compensation  point  and  the  others  below  the 
compensation  point. 

Maximum  yields  should  therefore  not  be  determined  in  the  carbonate  mixture,  as  has  been  done 
frequently  during  the  last  10  years. 

REFERENCES 

^  O.  Warburg,  tjber  die  katalische  Wirkung  der  lebendigen  Substanz,  Julius  Springer,  Berlin  1928. 
^  J.  Franck  and  W.  E.  loomis,  Photosynthesis  in  Plants,   The  Iowa  State  College  Press,  Ames, 

Iowa  1949. 
^  O.  Warburg  and  V.  Schocken,  Arch.  Biochem.,  21  (1949)  363. 
*  O.  Warburg,  Am.  J .  Botany,  35  (1948)  194. 
^  R.  Emerson  and  C.  M.  Lewis,  Am.  J.  Botany,  28  (1941)  789. 

Received  June  nth,  1949 


10 

5 

3 

9* 

9 

8 

3 

6 

II 

3 

VOL.  4  (1950) 


347 


Fig.  I.  Left  to  right;  F.  Lipmann,  1).  Xachmansohn,  S.Ochoa,  F.  O.  Si.!inuu,  K.lwasaki,  P.Rothschild. 
Kaiser  Wilhelm  Institut  fiir  Biologie,  Berlin  Dahlem,  192S. 


Fig.  2.  Left  to  right:  Sitting:  O.  Meyerhof  and  A.  V.  Hill.  Standing:  K.  Lohmann,  A.  v.  Muralt, 

G.  Benetato,  H.  Blaschko,  .\.  Grollman,  H.  Laser,  Miss  Wagner,  W.  Schulz,  E.  Boyland. 

Kaiser  Wilhelm  Institut  fiir  Medizinische  Forschung,  Heidelberg,  193 1. 


348 


VOL.  4  (1950) 


Fig.  3.  Kaiser  Wilhelm  Institut  fiir  :Medizinische  Forschung,  Heidelberg. 


',%"•*"•■  •">»•'       ■''■ 


Fig.  4.  Left  to  right:  S.  Kore\-,  D.  Nachmansohn,  D.  Burk,  .\.  v.  Szent-Gyorgyi,  O.  Warburg, 

O.  Meyerhof,  C.  Xeuberg,  G.  Wald. 
Marine  Biological  Laboratory,  Woods  Hole,  1949.