THE PURIFICATION AND PROPERTIES OF A
HEXOKINASE FROM THE CORN SCUTELLUM

By

HERBERT CHARLES JONES HI

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF

THE UNIVERSITY OF FLORIDA

IN PARTL^L FULFILLMENT OF THE REQUIREMENTS FOR THE

DEC»EE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
June, 1965

!l!he vriter vlshes to express sincere appreciation to Dr. T. E.
HuEsihreys for his help, guidame, leadership, patience and for the
tise of his laboratory facilities in the course of this rssearchj to
Dr. G. Ray Hbggle who inspired tte vriter to pursue graduate study;
to Drs. D. S. Anthony, R. H. Biggs and T. ¥. Steams for their aid and
service on the committee j and to the Departoent of Botany and the
Departnent of Health, Education and Welfare for financial support
throu^ National Defense Education Act ELtle IV and Hational
Institutes of Health feUxswships.

JTTp

AGRi-
CULTURAl
LIBPA'-V

UNIVERSITY OF FLORIDA

llllilllilllllliilillllillll

3 1262 08552 5615

TABLE OF COOTEHTS

li

Lisa? OP TABLES v

LIST OP FIGfUR^ vi

HJTBCBUCTION 1

BSVim GF LZTSBATORE 3

MATERIALS AHD MEKIODS 36

Plant Materials

Prepai^tion of the Enzyme

Extraction

Annnonimn sulfate fractioiation

Absorption and elution froa alumina C-y gel

Assajf }§3thods

Method 1
Ifethod 2

Assay for phosphofruetokinase, phosphoglucomutsise and
glucose -6-phosphatase activities

Protein Determination

Chemicals and £nz;yines

Page
RESULTS k2

Purification
Substrate Specificity

Nucleoside triphosphates

Metal Activators

Inhiljitozv

Sugaars

Nucleoside di- and triphosphates

Sugar phosphates

Anions

pH and Temperature Optima

DISCUSSION 7^

SUMMARY 80

BIBLIOGRAPHY 8l

BIOGRAPHICAL SKETCH 89

ir

LIST OF TABIDS

1. MtCHAELIS COSBTASITS {Km) AND RSLATIVS J^JAXIMAL

RASES FOR BRAIN AM) YEAST HEXOiCCKASE 7

2. SFFECTS OP GLUC0SE-6-P AMD RELATEID COMPOUNDS OSS

PHOSPHOSSLATIOHS BY BRAIN HSXOKDJASE 9

3. PURIPICATIOI OP ffiiXOKIHASE ^3

k, ATPASE ACnVTIY OP THE HEXDKIMSE PREPARATIONS 2^5

5. SUBSTRAOS SPSCIFIC33?3f OiP CORN SCOTELLUM

EEKOKENABE 49

6. EFFECT OP !3W;LEX)SIDE35U[PH^mTSS AS SilBSTrRHSS

FOR ATP 55

7. ACTIVATION OP HEXOKUmSE BY METAL IONS 60

8. EFFECT OF NUCLEOSIDE DI- AND TRIPHOSPHATBS AS

INHBITOaS OP ATP IS THE HEXOKISASE REACnCU 68

UST QPFICRJRSS

Figuare Page

1. Effect of glucose concentration on the rate of

phosphorylation 52

2. Lii^T?eaver-Burk plot of the effect of glucose

concentration on the rate of phosphorylation » ^

3. Effect of ATP concentration on the rate of

phosphorylation, and coJDpetitive inhibition

by ADP and AMP 57

i^. Linaveaver-^nrk plots of the effect of ATP

concentration on the rate of phosphorylation,

and competitive inhibition by ADP and AMP 59

5. Effects of Co"*"*" and Mn++ concentrations on tl^

rate of phosphorylation fe

6. Effect of Mg"*^ concentration on the rate of

phosphorylation 62

7. Line'v^aver-Burk plots of the effect of l^"^, Co"^

and Ifa"*^ concentration on the rate of

phosphorylation 6h

8. Lineweaver-Burk plots of the competitive inhibition

of glucose phosphorylation by xylose and H*-

acetylglucosamine 6f

9. Effect of pH on the rate of phosphorylation

escpressed as the per ceaat of the aaximuEi rate

obtained 71

10. Effect of tamperature on the rate of

phosphorylation 73

IHTRODUCnON

The properties of plant enzymes, especially those of higher
plants, are less veil known than those of similar enzymes isolated
from animal tissues. It has been assvuoed that the properties
associated vlth the characterized animal and lover plant (yeast)
enzymes are the same for higher plants. This may be the case, but
it is necessary that the plant enzymes be characterized in order to
gain a better understanding of the similarities and differences in
metabolism between plants and animals.

The enzyme, hexokinase, which catalyzes the phosphorylation of
glucose in the presence of adenosine-5' -triphosphate and magnesium ion,
occupies an important position in the metabolism of sugars in both
plants and animals. Humphreys and Garrard {kl) have presented evidence
which suggests that the hexokinase reaction may be important in control-
ling the rate of glucose uptake by the com scutellumj an organ
positioned between the root-shoot axis and endosperm of the com seed,
where glucose absorbed from the endosperm is converted to sucrose which
is subsequently translocated to the developing seedling during gennina-
tion (28). Their data indicates that glucose -6-phosphate competitively
inhibits an enzymatic step associated with net glucose uptake in
scutelluia slices, and they suggest that the step might be the
hexokinase-catalyzed phosphorylation of glucose. Since it has been
demonstrated that brain hexokinase (15, I6) and to a small extent,
yeast hexokinase (33), is inhibited by glucose-6-phosphate, although

1

2

noncompetltlvely. It seemed desirable to investigate the properties
of com scutellum hexokinase.

REVIEW OF LITERATURE

Historical

In 1927, Msyerhof (73) gave the naias, hexoMnase, to an alcohol
precipi table fraction of autolyzed yeast vhich when added to extracts
of aged frog or rabbit Dsusele greatly enhanced glycolysis. Independ-
ently, in 1935^ Euler and Mler (29) and latwak-i'fejin and Mann (66),
Isolated an enzyme from yeast— heterophosphatese— which catalyzed the
following reaction in the presence of Mg "*"•":

Hexose + ATP >Hexo3e-6-phosphate + ADP

tfeyerhof (jk), in a description of the hexokinase reaction the same
year, reported that the enzyme descrihed "by the two groups was the
sazse one responsible for the activity of his original preparation.

Kalckar (51), in 1939* demonstrated that kidney extracts
phosphorylated glucose and fructose amd, in 1^, Gelger (36) obsei'ved
that extracts from brain tissue phosphorylated fructose, mannose and
glucose. Belitzer and Golovskaya (5), the same yeax, showed that
muscle tissue contained hexokinase, which in the presence of glucose
and creatnine catalyzed the phosphorylation of glucose to l«xose-6«
phosphate. In 19*H> Ochoa (78) showed the presence of hexokinase in
acetone powder of rat brain. The acetone partially Inactivated
adenosinetriphosphatase (ATPase). The hexokinase required l^ and
transferred phosphate from A!H> to glucose without the liberation of
free oirthoi^iosphate .

3

k

Colowick et al. (l2), in 19^1, demonstrated that phosphorylation
of glucose preceded the oxidation of glucose in heart muscle and
kidney extracts and that hexokinase catalyzed phosphorylation of
mannose to niannose-6-phosphate (M6P). The M6P was in turn converted
to fructose-6-phosphate (F€P) by an isomerase.

In 19'^6, Berger et ^. (6) and Rinitz and MacDonsuLd (57) succeeded
in crystallizing hexokinase from, ysast, the only organism frcMa which it
has been crystallized. The hexokinases from various sources have been
recently reviewed by Crane (20) .
Hexokinases of Vgarious Animal Tissues

Ifammalian brain hexokinases* Colowick et al. (lU) prepared
purified beef brain hexokinase by eluting acetone powder fran beef
brain with water and f3rau;tional precipitation of the eluate with
anaaonium sulfate. The hexokinase was precipitated between 30 and 50
per cent saturation with a two-fold increase in specific activity.
The 30 to 50 per cent fraction could be purified further by
refractionation between k3 and 50 per cent saturation with annaonium
sulfate. The product of the beef brain hexokinase reaction was shown
to be glucose -6-phosphate (Gr6P).

Jfeyerhof and Wilson (75) observed that brain extracts catalyzed
the phosphorylation of glucose and fructose at about the same rate at
0.020M concentrations of the substrates, while at concentrations of
0.0015M to 0.003M the rate was the san» for glucose and much lower for
fructose. They observed the same difference relative to the AK»
concentiration and there was a greater A2Pase activity in the presence
of fructose than glucose. They suggested that there are actually two
separate enzymes in brain extracts— a glucokinase and a fructokinase .

5

Wiebelhous and Lardy (icA) noted that dialyzed extracts of "beef
brain showed different hexokinase activities with respect to glucose and
fructose in the presence of inhibitory concentrations of sodium salts.
Sodium salts inhibited phosphorylation of glucose^ but not of fructose,
indicating the possibility of two different heMjlsinases. Iheir beef
brain extracts promoted the phosphoxylation of glucose, fructose and to
a lesser extent, mannose, but were not active with L-glucose, galactose,
L-sorbose, D-gluconate, S-keto-O-glucoaate, D-ribose, D-arabinose,
L-rhamnose and D-xylose. Inorganic pyrophosphate inhibited activity
^ to 80 per cent depending on its coTOentration. Magnesium ion, up
to two times the concentration of the pyrophosphate, did jaot reverse
inhibition. Orthophosphate was only slightly inhibitory.

Long (63) found that of five rat tissues— brain, liver, kidney,
steletal muscle, and intestine— brain had the greatest amount of
hexokinase activity, while liver had the least.

Crane and Sols (16, 17, 86) prepared particulate he:a>kinase from
brain, which was free of interfering enzymes, by fractional centrifuga-
tion and solubilization of the enzyme with lipase and/or deoxycholate .
They obtained 35 per cent recovery. With this preparation, it was
found (as V^il-M&Iherbe and Bone (102) had reported in 195X) that g6P
inhibited hexose phosphorylation noncompetitively (15), while ADP
behaved as a competitive inhibitor (85). They (86) examined the
specificity of brain hexokinase toward thirty-five compounds structur-
ally related to glucose in order to deteimine which groups of the
glucose molecule were possibly involved in formation of a glucose-
brain hexokinase complex. Sixteen of the analogs served as substrates

6

(Tab3je l), five behaved as competitive inhibitors, and the remainder
vere inactive. They estimated the relative influence of each hydrojcyl
group of glucose by (^mgaxing the Michaelis constants (Kia) and the
relative maximal rates (VtaMc) of phosphorylation (Table l). The con-
clusion was that the formation of a glucose -enzyme ccxuplex involved
the ring structure and hydroxyl giroups at carbon atoms 1, 3, k and 6,
and that each hydroxyl group had a specific quantitative influence on
enzyme-substrate affinity.

Crane and Sols (l6) investigated the specificity for inhibition
of the brain hexokinase reaction by G6P and related compounds. They
tested some twenty-five compounds and only six were inhibitory
(Table 2). They concluded that the inhibitor complex involved the
pyranose ring structure, the hydroxyl groups at carbon atoms 2 aad ^i-
and the phosphate group at cart>on atom 6. They interpreted the lack
of influence by carbon atoms 1 and 3* and the influence by carbon
atom 2 as indicating that the enzyme possessed a third, specific
binding site for G6P in addition to the two for ATP and glucose and
that the data supported their hypothesis that inhibition by G6P, when
present, was a part of an intrinsic cellular mechanism for the control
of the rate of the hexokinase reaction. They found inhibition by the
hexose phosphates to be reversible, independent of either glucose or
ATP concentration EUid, therefore, nonconqpetitive .

Maley and Lardy (67) tested the effects of a variety of N-substi-
tuted glucosamines on brain hexokinase. All were powerful inhibitors
(Ri»10"3M to 10"^) . Tbey interpreted their data (and from studying
molecular models) as indicating that the substituted glucosamines

7

TABLE 1

MtCHAELIS CONSOMTS (Kn) AND RELATIVE MAXIMAL RATES FOR BRAIH AND

YEAST HEXOKIRASE

Data from references (86) suid (23)

(1)

Modifj

(2)

Led

Coiirpound

1

(3)

Brain

(M

(5) (6)
Yeast

at
Carbor

Kin
(M/L)

Rel.
Max.

Vel.

Km
(M/L)

Rel.
Max.
Vel.

glucose

8X10-^

1.0

1X10-^

1.0

1

1, 5-sorbitan

3X10"2

1.0

3X10-3

0.01

1,2

1, 5-mannitan

2X10-2

0.9

1

glucoheptulose

pxio-'^

0.006

1,2

mamaoheptulose

5x10-5

0.015

2X10-*''*

0.001

1,2

fructose

2X10-3

1.5

7x10-^

1.8

1,2

2,5-sor'bitan

2X10"^

0.08

1,2

arabinose

S

0.1

>ixio-^

0.02

1

methyl- oC-glucoside

+

+

>ixio-^

<0.001

1

methyl- ^-glucoside

+

+

1

1-thloglucose

+

+

1,2

glucal

+

+

1

oC-glucose-1-P

+

+

1

glucono-1, 5-lactone

+

+

2

maimose

5x10-^

o.i^

5X10*5

0.8

2

2-deoxyglucose

3X10*5

1.0

-1*.
3x10^

1.0

2

glucosamine

8X10-5***

0.6

2X10-3

0.7

2 II

glucosone

1X10-5

0.08

2X10-5

0.2

2

N-£w:etylglucosamine

8X10-5**

+

ixio-3*

<0.001

2

N-methylglucosamine

2X10"^**

+

'

(TABLE 1 continued

next page)

8
TABLE 1 (continued)

(1)

Modifi

(2)

.ed

Con5>ound

I

(3)

Brain

ih)

(5) (6)

Yeast

at
Carbon

Km
(M/L)

Rel.
Max.
Vel.

(M/L)

Rel.
Max.

Vel.

2

2-OHBethylglucose

+

+

3

allose

7X10*3

0.5

>ixio*-'-

0.1

3,2

altrose

3X10-3

0.11

3

3-deoxyglucose

2X10-2

0.2

3

3-0-raethylglucose

+

+

>1X10"^

<0.001

k

galactose

UQJO-^

0*02

5x10-2

0.002

h,3

gulose

+

+

5,h

1,4-sorbitan

+

+

>ixio'''"*

<0.001

5,1

L-sorbose

+

+

>ixio-^

<0.001

6

6-deoxyglucose

2X10"3»*

+

6

xylose

2K10-3**

+

1X10-2*

<0.001

6,2

lyxose

2X10" **

+

6,3

ribose

+

+

6

6-deoxy-6-fluorogliicose

5X10-3*

<0.001

1,2

l-methylfructose

>ixio-^

< 0.001

2

2-C-hyaroxyinethylglucos

e

2X10*3*

<0.001

1,2,3

S-O-methylfructose

>ixio"^

<0.001

sortitol

+

+

glucoguloheptose

+

+

i-inositol

+

+

^Inhibition constant (Ki)
**Itetermined by competitive inhibition of fructose
***Detenained at pH 7*5
+Undetectable
Psphosphate

9

TABLE 2

EFFECT OF (a.UCOSE-6-P AND RELATED COMPOUHDS ON PHOSPHORYLATIONS SI
BRAIN HEXOKENASE

Data from reference (l6)

(1)

(2)

(3)

(M (5)

(6)

Modified

Ester

Substrate

Ester Per cent

Ki

at

concen- inhibi-

(M/L)

Carbon No.

tration tion

1

l,5-Sorbitan-6-P

2-Deoxyglucose

0.67

38

ixio'3

1

oC-Glucose-l,6-diP

2-Deoxyglucose

0.6

hi

7X10-^

1

^-Glueose-l,6-diP

2-DeoxyElucose

1.0

0

1

Glucoheptulose -7-P

2-Deoxyglucose

8.6

0

1

tfethyl-glucoside -6-P

2-Deoxyglucose

3.8

0

2

Mannose-6-P

Mannose

8.5

0

2

2-Deoxyglucose -6-P

Glucose

1.0

0

2

Glucosaiaine-6-P

Glucose

1.5

0

2,1

Fructose-6-P

2-Deoxyglucose

2.5

0

2,1

2,5-Sorbltan-6-P

2,5-Sorbitan

13

0

2,1

Fructose-l,6-diP

2-DeoaQrglucose

1.8

0

2,1

l,5-Mannitan-6-P

1,5-Manm"tan

k.O

0

2,1

Mannoheptulose-7-P

Mannoheptulose

2.0

0

2,1

Arabinose-5-P

Arabinose

5.5

0

2,3

Altrose-6-P

2-Deoxyglucose

7.5

0

2,3,1

Ribose-5-P

2-Deoxyglucose

2.0

0

2,3,1^,1

Xylose-5-P

2-Deoxyglucose

3.3

0

3

Allose-6-P

2-Deoxyglucose

9.2

58

7X10-3

3

3-Deoxyglucose-6-P

Mannose

3.2

Ik

2X10-2

k

Galactose-6-P

Mannose

20

0

(TABLE

2 continued next

page)

10
TABLE 2 (continued)

(1)

(2)

(3)

ih)

(5)

(^)

Modified

Ester

Substrate

Ester

Per cent

Ki

at

concen-

inhibi-

(M/L)

Carbon Kb.

tration

tion

5,1 L-Sorbose-l-P

6,1 oC-Glucose-1-P

No ring Gluconate -6-P

No P Glucuronate

2-Deoxyglucose 0.if5 ^0

2-Deoxyslucose 2.5 0

2«Deoxyglucose 1 0

2-DeQxyglucose 2 0

7X10'

ccmtoined vlth the active enzyme site aaad that the inhibition ffli^t "be
through blocking of the site on the enzyme to ATP, since the substi-
tvrted groups did not overlap the csaiAion 6 position on the sugar.

By kinetics studies of the msehaaism of the brain hexokinase
reaction, Fromm (31) and Eroran and Zswe (32) found that g6P acted as
a conrpetitive inhibitor of ASP and as an uncompetitive iiihibitor vith
respect to glucose. Inhibition by ADP was of a more ccaaplfix nature,
but appeared to be noncon^etitive with respect to A1P and uncoB5>etitive
vith respect to glucose. They concluded iSmt their results vere
consistent with a nechanism involving either a phospho- or a gluco-
enayce cooplex, but were at variance with any laechanism in which both
substrates need to be present sitaultaneously on the enzyiae for the
reaction to occur. OSiey also concluded that there seemed to be no
reason for G6P occvipying a third site on the enzyms. They presented
the following coc^julsory pathway type mechsmlsia for the brain hexo-
kinase reaction:

(1) EnzyE» + A!EP > Enzyaie-X coiaplex + ADP

(2) Enzyne-X congjlex + glucose > Enzyne-Y coogplfix

(3) Enzytne-y complex ^Enzyae + g6P

The nature of the Enzyme-X and Enzyme-Y con^lexes was unknown. The
mechanism is similar to that presented by Hamnes and Kbchavi (lf2) for
the yeast hexofcLnause reaction,

Kerly and Leaback (5^*) aaasured the specificity of hexokinase of
the brain of several nonmammallan animals. They found that extracts
of brain from pigeon, four-day-old chick, two elasmobranchs, two
teleost fishes, frog and squid catalyzed the phosphorylation of

12
glucose and fructose. With pigeon train extract, phosphorylation of
fructose was inhibited by mannose and K-acetylglucosamine, vhile
phosphorylation of both fructose and glucose was inhibited by GoP and
F6P. The affinity of pigeon brain hexo kinase for fructose was similar
to that of the beef brain enzyme, except beef brain hexokinase was not
inhibited by F6P (i6) .

Muscle . Crane and Sols (iT) partially purified soluble skeletal
muscle hexokinase by fractional precipitation with cold acetone and
drying in vacuo. The enzyme was precipitated by 33 per cent acetone
(v/v) with 50 per cent recovery and a specific activity of one.
Skeletal muscle hexokinase catalyzed the phosphorylation of glucose,
mannose, fructose, glucosamine and 2-deoxy-D-glucose (2 DOG). Kie Km
for glucose and mannose of the muscle enzyme was about ten times
higher than that for the brain enzyme. The Km for fructose was only
slightly higher than that of bi^n hexokinase. Skeletal muscle
hexokinase activity was optimum at pH 8.0.

Crane and Sols (l?) partially purified particulate heart muscle
hexokinase by fractional centrifugation and solubilization of the
enzyme frcai the particulate fraction with 0.1 per cent Triton X-100.
ADP inhibited heart muscle and skeletal muscle hexokinase competitively
(KiADP » KmATP) and G6P inhibited noncompetitively. The inhibition
constants (Ki) of G6P, l,5-sorbitan-6-P, and L-sorbose-1-P for heart
muscle hexokinase were 25 per cent, or less, of those for the brain
enzyme.

Strickland (90) obsei^red that glycolysis by a muscle extract in
the presence of added hexokinase could be inhibited by O.OO3M

13
glyceraldehyde, but the inhibition could be reversed by a small excess
of TaexoMxiase, He concluded that glyceraldehyde Inhibition could be
pin-pointed as inhibition of glucose phosphorylation by hexokinase.

Walaas and Valaas (99) extracted an acetone powder of rat sksletal
naxBcle vith trls-EDTA solution and obtained a five-foM increase in
specific activity by firaetlonating the eliiate vith cold ethanol. The
ethanol precipitate vas dried in vacuo* Fractionation of either the
acetone powder eluate or the ethanol-precipitable fx-action with
aBBoonium sulfate decreased recovery. EDSA, when added to the crude
hosQOgenate from which the acetone powder was prepared, increased
recovery. Added glucose or A3P only slightly stabilized the enzyme.
Prolonged contact with 0.05M %Clg completely inhibited the enzyme.
Chloride ion was without effect. Orthophosphate provided slight
protection ajid O.03M pyrophosphate was very effective in stabilizing
muscls he»)kinase. However, O.OO^M pyrophosphate strongly inhibited
hexokinase during irwtJbatlon of the reaction mixture. Potassium
chloride, in a narrow range of 0.02 to O.O5M, slightly activated the
hexokinase during Incubation while orthophosphate inhibited. Tbs
hexokinase had a pS optimum at 8.0 to 8.2 and one-third maximum activity
at pB 7* Maximum activity of hexokinase was observed at a molar ratio
%/ATP ■ 1 for several concentrations of ATP. Magnesltm ion in excess
of ATP, except at relatively high concentrations, was not inhibitory.
At a molar ratio ATP/Mg greater than k, ATP inhibited the reaction.
Tbe !Qa for both ATP and Vlg was 1.7X10'3m. Inoarganic pyrophosphate
ixihlbited the hexokinase reaction cooqpetitively with respect to VqATP
when the molar ratio -was 1. At inhibitory concentrations of Mg"*^,

Ik

pyrophosphate had a small activating effect on the enzyme, which they
inteirpreted as a release of Mg"^ inhibition. They suggested that
inorganic pyrophosphate inhibited hexoMnase by forming a Mg "'^-pyrophos-
phate complex vhich excluded MgATP from the enzyme -substrate complex
and furthennore, that the MjgATP complex was the actual substrate (other
than glucose) for muscle hexoklnase. Muscle hexoklnase was also
activated by Ca++, Co "*"•■, Mn"*^ and, to a very small extent by Zn"*"^. The
maximum activities for these activators were lower than that for Mg''^
euid the IQn for eswh was lower than that for Ife"*^. The molar ratio
(metal/ ATP) for maximum activation by the ions was less than 1. Strong
inhibition occurred as the concentrations of the metals were increased
above those giving maximum activtition. Hiey explained the differences
between these activators and Mg"*^ as being due to the different metal
ions combining with different ligand centers on the enzyme.

Walaas and Walaas (99) also reported that inosinetriphosphate (ITP),
guanidine triphosphate (GTP) and uridinetriphosphate (UTP) would not
substitute for ATP in the muscle hexoklnase reaction.

Griffiths (l»-0) fovmd that O.OO5M alloxan inhibited muscle hexoklnase
completely smd O.OO5M ninhydrin inhibited the enzyme 80 per cent.
Inhibition by O.OO3M alloxan was reversed by O.OO5M cysteine, but O.OIM
cysteine only partially reversed inhibition by 0.0025M ninhydrin. He
interpreted the results as indicating that the inhibition probably
amounted to more than Just thiol destruction since reversal required a
molar ratio of cysteine to alloxan that was greater than 1.

Liver hexoklnase. Long (63) measured levels of hexoklnase in
several rat tissues and found that liver had the lowest level of hexoklnase.

15

Crane and Sols (l?) described a purification procedure for the
soluble hexokinase of rat liver. They concentrated a 100,000XG super-
natant fraction of liver extract vith ammonium sulfate up to 50 per cent
saturation. Their liver preparation catalyzed phosphorylation of
glucose, mannose, fructose, glucosamine and 2D0G. There was some
evidence that fructose and glucose might have been phosphorylated by
separate enzymes. Lange and Kohn (58) found that allose, talose and
gulose vere also substrates of rat liver heaKJkinase . They studied
hexokinase in rat intestine, Mdiwy and liver extracts and their data
indicated that while the hexokinases from the three sources were similar,
they were not identical. The Sn for glucose, 2DCXj and glucosamine were
4X10"5, 9x10*5 and 3.7X10"^, respectively, and the relative maximal
velocities were in the same order.

Vinuela et al. (98) detected two enzymes in rat liver extracts
which catalyzed phosphorylation of glucose. One enzyme precipitated
between 20 and 50 per cent saturation with amnonium sulfate, while the
second enzyne precipitated between 60 and TO per cent saturation. The
first enzyme had a low Kn for glucose, vas inhibited by g6P, and only
moderately by N-acetylglucosamine . The latter enzyme had a higher IQn
for glucose, was not inhibited by G6P, was strongly inhibited by
N-acetylglucosamine, and had a low Km for mannose. Tbey interpreted
the characteristics of the first enzyme as being those of a typical
animal hexokinase. It was relatively stable and was present in small
amounts in normal liver. They designated the second enzyme, a gluco-
kinase. It showed a maximal rate for glucose phosphorylation of
approximately 1.0 mlcron*ole per minute per gram liver at 21 to 23°C or

16
2 micromoles at 37°C vhich vas comjaiuble to the rate of glycogen
synthesis from glucose in rat liver iO,k to 1 micromole per minute
per g liver) . Tt^ activity also apparoached the maximum rate of
glycogen synthetase (3 micromoles per minute per g at 3T^C). Kie
glucokinase disappeared in stairved and diabetic animals. They sug-
gested that the physiological instability of the glucokinase accounted
for the inability of liver to synthesize glycogen from glucose and
mannose in diabetic animals, while fructose and galactose, vhich vere
incoirporated into glycogen, vers phosphorylated by other hexokinases
specific for those sugars* The Ka for glucose of the hexoMnase and
of the glucokinase were found to be IXIC^m and JX10"%, respectively.
The glucokinase was not active on fructose to any extent. Their con-
clusion was that the glucokinase was associated with the glycogen
synthesizing system of the liver since: (l) its activity was compa-
rable with the rate of glycogen synttesis, (2) it was not inhibited
by its product and (3) UDPG-glycogen glucosyltransf erase is dependent
on high levels of G6p for maximum activity.

Walker and Rao (lOO) examined the effects of SDOG, glucosamine
and N-acetylglucosamine on the hexokinase and glucokinase of rat liver.
Hhe three compounds inhibited both enzymes competitively, but the Ki
for 2D0G was much higher for the glucokinase than for the other two
compounds. The Ki for 2D0G was comparable to the Kin for glucose for
the enzyme.

Kidney hexokinase. Kalckar (51) shovred that kidney extracts
catalyzed phosphorylation of glucose and fructose. Colowick et al.
(l^) demonstrated phosphorylation of mannose by kidney extracts.
Maxmose isomerase in the extract convearted MSp to p6p.

17

Laoge and Kbhn (58) fouiid the Kin for glucose^ 2D0G and glucosamine
to be k.SXM)'^, 9X10*5m and O.IM, respectively. Allose and talose vere
also phosphorylated, while altrose and N-acetylglucosamine were not. It
appeared that kidney hexokinase differed from hexokinases of other rat
tissues (and other animal and plant hexokinases) in its specificity
towards modification at carbon atcsi 2 since glucosamine was such a
poor substrate.

Intestine hexokinase. Sols (Qj) examined the hexoldLnase of rat
intestinal mucosa, and found fructose was phosphorylated at rates less
than glucose, while galactose, 3^iethylglucose , L-sorbose, manno-
heptulose, N-acetylglucosamine, xylose, rihose and L-arabinose were
not idiosphorylated. G6P at 6X10"3m inhibited hexokinase activity by
50 per cent, which was ten times the concentration required to inhibit
the brain enzyme to the same extent. All the sugars were phosphorylated
by the same enzyme. The Kn for glucose and fructose were 2X10 M and
l<0aO"3M, respectively. These results failed to support the hypothesis
that the rate of phosphorylation of sugars limits the rate of s\agar
absoirption by intestinal mucosa since galactose and 3-methyl glucose,
which were not phosphorylated, were absorbed at faster rates than other
sugars (except glucose). Fructose, whose rate of absorption vas inter-
mediate, was phosphorylated at a rate greater thsm any of the other
sugars.

Lange and Kbhn (^) found the Kin for glucose, 2D0G and glucosamine
of rat intestine hexokinase to be 6.5X10*5, 9X10"^ and 3.3X10"^,
respectively. Allose and talose, as with kidney hexokinase, were also
active. Changes at carbon atom 3 of glucose (allose) had considerably

18

greater effect on activity than changes at carbon atom 2(2D0G or
glucosamine). Changes at either carbon atoms 2 and 3(altrose) or 3
and U(gulose) resulted in loss of all activity. They concluded that
all the active substrates of kidney, liver and intestine l^xokinase
were similar in at least two respects: (l) They had an available
hydroxyl at carbon atom 6 and (2) they had a hydroxyl available at the
anomeric position (carbon atom l).

Erythrocyte hexokinase. Christensen et al. (U) found that the
hexokinase of rat erythrocyte phosphorylated glucose, nannose and
fructose at the relative rates 1.0:0.77:0.36, respectively. Galactose
was not active. The activity of normal or diabetic rats was not
affected by insulin or adrenal cortical extracts.

Retina hexokinase. Hoare and Kerly (Uh) showed that extracts of
rat retina phosphorylated glucose, fructose, mannose and glucosamine.
Magnesium ion, manganese ion and to a lesser extent, cobalt ion
activated dialyzed extracts. Glucose and glucosamine inhibited
phosphorylation of fructose.

Krebs - 2 ascites tumor hexokinase. McCcanb and Yushok (69)
partially purified the hexokinase of Krebs -2 ascites tumor. The hexo-
kinase had a pH maximum between 5 '6 and 7'8' The Kin for glucose and
I4g++ were 1.7X10*^ and IXIO'^M, respectively. The Ki for G6P inhibition
was 1*X10"^, while V^, SD0G6P and F6P were not inhibitory. Adenosine
diphosphate was inhibitory. Glucosone (Mm » 8X10"%) was also phos-
phorylated by the tumor enzyme. The hexokinase activity was localized
in the mitochondria of both tumor and normal tissues.

Dcaaestle fowl hexokinasea . llslsata et ^* (71) examined tissue
extracts from several organs of domestic fowl (vhite leghorn hen) and
found that the hea3rt and gizzard hexokinases vere active vith both
glucose and fructose, vhile the enzyoe of liver phosphorylated fructose
hut not glucose.

Honey bee hexokinase* Ruiz-Amil (8l) partially pixrified hexokinase
from hcmey bee by fractional precipitation of the enzyme between 50 and
70 per cent saturation vith aoDionium sulfate ai^L column'^haramatography
on DEAE-cellulose. The yield vas 38 per cent of the original hea»kinase
in the crude extract. The thorax contained the largest amount of hexo-
kinase activity (75 per cent). She enzyme showed optimum activity
between pH 7*5 ^ad 8.8. Glucose -6-ph0sphate behaved as a cai3>etitive
inhibitor of glucose, while 2!iX)G6P and L-glycerophospliate did not
inhibit the enzyme to any extent. The Ksa for MP and the KL for its
coaigetitive inhibitor, ADP, were 7.5X10'^M and 9K10"^, respectively.
05^ KJQ for fructose (SXOX)"^) was similar to that of the mamnalian
brain enzyme, while those for glucose (UXIC^m) and mannose (IXIO*^)
Mere similar to those of yeast hexokinase. Ruiz-Amil concluded that
Idle enzyme from honey bee vas similar to the hexokinase of other
animals in most respects.

Locust hexokinase. Ksrly and Leaback (53) studied the hexokinase
in the thorax muscle and the salivary gland of locust (Locus ta
migratoria) . Ihe properties of the enzyme were similar to those of
the iMWtwHan brain enzyme vith respect to substrates and inhibitors*
Glucose had a Km of 5.5X10"3m. Both G6P and P6P, at a concentration
of 5X10**3m, coinplBtely inhibited hexokinase activity.

20

House fly hexokinase. Chefurlsa (lO) observed hexo kinase activity
in a soluble extract of house fly (Musca domestic L.)«

Sea urchin egg hexokinase. Krahl et al. (56) studied hexokinase
in egg and enibryo homogenates of sea urchin (Arbacia punctulata) . The
enzyme was shown to have a pH optimum of 7« The substrate specificity
of the enzyine was similar to that of the other animal hexokinases. The
Kk for the substrates that were examined fell in about the same range
(2 to 8x10-5m).

Bivalve hexokinase. Mekata et al. (7I) observed that the
hexokinase of the fresh-water mussel (l^rropsis schlegeli) phosphorylated
fructose but not glucose.

Worm hexokinase. Bueding and MacKinnon (7) purified the hexokinase
of the parasitic worm. Schistosoma mansoni, 15- to 20-fold with the aid
of calcium phosphate and alumina Cy gels. They found that the worm
contained four distinct hexokinases: (l) glucokinase, (2) fructokinase,
(3) mannokinase and (k) glucosamine kixiase . The glucokinase was
inhibited by ADP, G6P, sorbose -1-phosphate (SIP), and glucosamine -6-
phosphate. The inhibition of the glucokinase by ADP was noncompetitive
with respect to ATP. The Km for glucose, Bfe++ and ATP were higher for
worm glucokinase than for those of mammalian brain hexokinase. Worm
fructokinase did not phosphorylate L-sorbose in contrast to fructo-
kinase of mammalian liver and muscle. Worm glucokinase was inhibited
by sulfhydryl inhibitors and such inhibition was reversed by glutathione.
Bacterial Hexokinases

Echinococcus hexokinases . Agosin and Aravena (l) partially
purified the hexokinases of hydrated cyst scolices of Echinococcus

21
granulosus ♦ They isolated four separate hexoklnases: (l) glucokinase,
(2) fructokinase, (3) maiuiokinase and (h) glucosamineklnase .
2-Deoxy-D-glucose was not phosphorylated by any of the hexoklnases.
Gluco-, inanno- and fructokinase vere inhibited by g6P, while M6P
inhibited gluco- and mannokinase. Phosphorylation of fructose by
fructokinase was competitively inhibited by ADP. GlucokLnase was
inhibited by p-chloromercuric benzoate (P(3MB) and the inhibition was
reversed by cysteine.

Spirochaeta hexokinase. Smith (84) examined a hexokinase of the
particulate system of Spirochaeta recurrentis ♦ !I5ie enzyv^ showed
maximum specificity toward glucose and mannose, while fructose and
L-sorbose were phosphorylated to a lesser extent. Galactose was not
active. Glucosamine and N-acetyl^lucosamine inhibited glucose phos-
phorylation. The enzyme was quite sensitive to low concentrations of
ADP.

Pseudomonas hexoklnases. Klein (55) observed that extracts of
Pseudomonas putrefaciens catalyzed phosphorylation of glucose and
glucosamine. The hexokinase of the extracts slmwed a vei^ low affinity
for fructose (O to 10 per cent relative to glucose) and mannose (O to 5
per cent). Phosphorylation was not inhibited by 0.012M g6p. Sulfhydryl
(SH) poisons inhibited the hexokinase and the inhibition was reversed by
cysteine .

Hochster and Watson (45) reported that extracts of Pseudomonas
hydrophila had a distinct pentokinase (a xylokinase) that catalyzed
phosphorylation of xylose (iQa = 2.3X10"3m).

22

Escherichia hexokinase. Cardini (8) partially purified the
hexokinases of Escherichia coll. Extracts of E. coll phosphorylated
glucose, fructose and mannose at relative rates of 1.00:0.14:0.29,
respectively. He observed that acetone or alcohol precipitation
removed the phosphorylating ability of the extracts for fructose,
while that for glucose and mannose remained constant, indicating that
a separate kinase was responsible for fructose phosphorylation in the
extracts.

Staphylococcus hexokinase. Cardini (8) found the hexokinase of
Staphylococcus aurens was active only with glucose as the substrate.
He observed that normal cells of E. coll and S. aurens could be induced
to phosphorylate galactose in the carbon 1 position by growing the
cells on lactose.

%cobacterlum phlei inorganic polyphosphate glucokinase. Szymona
and Ostrowskl (92) isolated an inorganic polyphosphate glvicoklnase
from Mbrcobacterium phlei which appeared to phosphorylate glucose by a
direct transfer of phosphate from inorganic polyphosphates containing
more than four phosphate residues. The apparent Km for inorganic poly-
phosphate and glucose were l.T5X10~^ and 2.8XloAl, reppectively.
Plant Hexokinases

Yeast hexokinase. Berger et al. (6) and Kunitz and I'facDonald
(5?) reported methods for crystallizing hexokinase of yeast at the
same time. Both methods were based mainly on fractional precipitation
of the enzyme with ammonium sulfate and ethanol from a toluene treated
extract. The enzyme was crystallized from an ammonium sulfate solution
and recrystalllzed up to six times.

23

Darrow and Colowick (23) reported a simpler method that gave a
good yield and high purity. Their method employed fractional precipi-
tation of the enzyme vith ammonium sulfate^ absorption and elutlon
from hentonite gel and repeated crystallization from ammonium sulfate.
The method gave about 150-fold increase in specific activity over the
crude homogenate.

The Km for substrates and Ki for inhibitors, as well as relative
maximum velocities (compared to glucose) reported by Darrow and
Colowick are given in Table 1. The specificity of the enzyme for
substrates and inhibitors, except for G6P, was similar to that of
brain hexokinase. However, the iOn's were a magnitude smaller and
yeast hexokinase failed to phosphorylate mannoheptulose . Gottschalk
(39) presented evidence that indicated that the furanose ring con-
figuration of fructose might be the form which was active with yeast
hexokinase .

The Km for ATP and Mg"*^ were found to be 10X10-5 (68) and
260X10-5m (6), respectively. The enzyme was specific for ATP. Deoxy-
ATP, ITP, UTP, CTP(cytosinetriphosphate), CTP, deoxy-CTP, deoxy-GTP
and adenosine tetraphosphate were ineffective as substitutes for ATP
(23) in the reaction. The requirement for Mg'*~'"was not replaced Or
antagonized by Ca (2).

Kaji et al. (50) showed that crystalline hexokinase, which was
prepared by the method of Darrow and Colowick (23), had a weak ATPase
activity which paralleled the hexokinase activity through six
crystallizations. Hexokinase inhibitors such as N-acetylglucosamine

2fo
and sorbose -l-phosphate inhibited the A!EPase activity of the
hexokinase preparatioi^* They observed parallel inactivation of the
A^EPase and hexokinase activities by various amounts of silver nitrate.
Chromatography of the crystalline hexoMnase on DEAE-cellulose gave
fractions in which the ATPase activity/iffixokinase activity was
identical. 55ie Kia of the ATPase for ATP was 5X10"3m coc^>ared to
2X10"t;4 for that of the hexokinase. Ths hexokinase and AOPase were
found to have distinctly different specificity towards nucleotides.

Berger et al. (6) reported that thiol compounds pxvvided no
protective action for their crystalline enzyme, while Bailey and Vfebb
(3) reported that aH SH poisons, including Lewisite, were powerful
inhibitors of crystalline yeast hexokinase. Dixon and Needham (2^)
reported inhibition of yeast he«)kinase by mustard gas, while Stroninie
(91) demonstrated that disulfiram was a potent inhibitor of the
enzyme, as was diethyldithiocarbaiaate when oxidized to disulfiram by
cyctochrode C. Baamard and Ramel (k) obtained results that indicated
one to four -SH groups per molecvile were required for the active
center (s) of yeast hexokinase and that these groups were not normally
available for reaction with -SH reagents, but probably became available
after a time-depeiident structural change occurring at 30°C. Berger
et al. (6) found that fliroride at O.I25M did not inhibit crystauLline
hexokinase when Mg^^ and phosphate were present in concentrations of
6.5 and 1X10-3M, respectively, but Bailey and Vfebb (3) observed that
0.(AM NaF inhibited hexokinase activity h6 per cent.

Glucose -6-phosphate has been reported to be both inhibitory to
(101) and without effect (l3, 102) on yeast hexokinase. Fromm and

25

Zewe (33) presented data that was interpreted as indicating that G6iP
acts as a cisnpetitive inhibitor of both foirward reaction substii^tes.
Tbey suggested that the concentrations of ASF used by other investi-
gators aay have been too high to see inhibition. Trayser and Colowick
(96) reported the dissociation constant of G^ to be itXlO'^M, vhile
Haranes and Kbchavi (k2) found it to be 1X10"%.

Stzmiss and Moat (89) reported that biotin stiioulEEted fermentation
of glucose and fructose by air-dried yeast grown in a medium deficient
in the vitatain. Biotin also stimulated hexokLnasa activity in extracts
frcxa ceUs grown on deficient medium. Trayser and Colowick (9'<-) found
fully active yeast hexokinase did not contain biotin. The results
indicated that the enz^ne was not a metaUoprotein and did not contain
a readily demonstrable prosthetic group.

Crystalline yeest hexokinase has been reported to be a j?roibe±a
of the a31)umin type with a maximian molecular weight (MW) of 9^,000
(3, 57) and a minimum MW of 30,000 (57)* Its isoelectric point and
greatest stability is at pH 4.8 (3, 57). It has a pH optimum at
pH 7.5 (88). It has a turnover number (TN) of 13,000 moles per 10^ g
protein per minute at 30*^ and pH 7.5 (6). The Q.^q of the reaction
between 0 and 30°C has been reported to be appsroximately 1.9 (6) and
is rapidly inactivated at temperatures above 55-60°C (88).

Six-times crystallized l^xokinase was separated into at least two
major cca^nents by either starch gel electrophoresis or DEAE-cellulose
column chromatography (23) . The forms, A and B, obtained by column
chromatography were reported by Darrow and Colowick (22) to be
indistinguishable in specific activity, in Kn values for siibstrateB

26

and in sensitivity to Inhibition by various substrates. However,
Trayser and Colowick (97) reported that the crystalline enzyme vas
separated into six molecular fonns (isozymes) of equal specific
activity by DEAE chromatography. The isozymes showed different
catalytic properties with respect to Km, %jax, pH optima, ATPase
activity and sensitivity to inhibitors.

Berger et al. (6) found that the crystalline enzyme showed a
loss of activity when highly dilute, which could be prevented by
diluting the enzyms in the presence of small amounts of insulin
(6 micrograms per ml) or serum albumin (60 micrograms per ml).
Glucose, fructose and to some extent mannose, prevented inactivation
of the enzyme in crude preparations and by trypsin. Insulin also
protected against inactivation by dilute alkali.

Several investigators (35* 52, 8o), have denranstrated the
reversibility of the hexokinase reaction by measuring exchange of
either C -labeled glucose between G6P and glucose or P-^ -labeled ADP
between ADP and AOP. However, the equilibrium greatly favored G6P
synthesis.

Agren and Engstrom (2) isolated phosphoserine from an acid
hydrolyzate of purified yeast hexokinase incubated in P^^.iabeled
ATP or G6P. They suggested that the hexokinase reaction involves the
formation of a stable phosphoenzyme intermediate. Najjar and McCoy
(77) ruled out the phosphoenayme hypothesis for yeast hexokinase when
they found no exchange of phosphorus between C-'-^-labeled glucose aiid
C-'^- labeled Gk5P (or vice versa) in the presence of the enzyme. They
then postulated the following mechemism for the reaction:

m

(1) Glucose-enzyioe +AtIlP^-> 6-phospho6lucose-enzyaie + ADP

(2) 6-Phosphoglucose-en3z;yiae + glucose — ^glucose -enzyme + GQ?
They argued that the formulation accounted for: (a) tte lack of
exchange of phosphoroi^ between glucose and G6P, (b) laSseling of the
enzyiae vith g6p32 or ATp32 on the assuanption that labeling of
phosphoserine might hscve been due to the transfer of phosphorous
during acid hjrdrolysis of the protein, (c) the marked inhibition of
the enzyjae by G6P, and (d) C-'-^-glucose labeled the enzyne. They con-
tended that vhen G6P accianulates, the greater part of the enzyiae
■irould exist in the phosphoglucose -enzyme fonu, thereby bringing about
a corresponding areduction in the glucose-enzyme concentration vhleh.
vould retard tte forward rate of the first reaction and cwisequently
inhibit the overall reaction. They reported that C-'-^-labeled glucose
labeled 3O-6O per cent of -ttie enzysne. One flav in their foranLLation
is that there is no report in -tias literature that G6p laarbedly
inhibits yeast hexokinase.

Froosn and Zeve (33) examined the kinetics of the yeast hexokinase
reaction and they interpreted their results as being consistent vith
the "classical" mechanisn in which glucose and ASP add randomly to
the enzyne and equilibrium kinetics prevail. Their data indicated
that participation of either a phosphoenzyme con^plex or a glucoenzyme
complex was unlikely. Their results showed that nannose and PSXP
behaved as coinpetitive inhibitors of glucose and ATP, respectively.
Inhibition by AMP was competitive with respect to ATP and noncompeti-
tive with respect to glucose.

Trayser and Colowiek (95> 9^) come to essentially the
conclusions as Fronaa and Zewe (33) sirwe they coiOd not detect either
a ^osphoenzyae conrplex or a glucoenayiue coagplex in their kinetic
studies of the yeast hexokinase reaction.

Hanmes and Kbchavi (4l, k2, k3) did a detailed study of the
kinetics of the yeast hexokinase reaction. They derived from -ttieir
data what they considered to be the most probable mechanism for the
reaction. It involved the ccaabination of HgAlEP and a glucoenzyme
coniplex to foira two quaternary intenaediates which in turn decomposed
to MgADP and a dissociable 6-ph03phoglucoenzyiae ccxaplex. Ihey pire-
sented the following compulsory pathway mechanism which is similar to
that postulated for brain hexokinase:

(1) Jfe-H- + AIP yMfeATP

(2) E + G — ^-E - G coj^plex

(3) E - G + ffeAaSP yXj^ yXg — >E - G6B + JfeAKP

(k) E - G6P >-E +G6P

(5) %ADP — >-Ms++ + ADP
where E, G, Xi, Xg and g6P are enzyme, glucose, first quaternary com-
plex, secoQd quaternary coniplex, and glucose -6-phosphate, respectively.
They did point out, however, that it is possible that both substrates
may have to be present at the same time in order for the enzyme to
have the correct conformation for phosphate transfer. Their data
revealed that lfe++may not be a very ingportant factor for binding of
the substrate to the enzyne, but that the primary rele of Mg++most
likely is to polarize the oxygen-phosphate bond that is being broken,
while anchoring the phosiftiate group of A3P to the enzyme. Kuclear

29
aagnetie resonance studies indicated that binding of IfeAlEP to the
enzyme might occur through the adenine and/or rihose portion of A3P.

Haajncs and Kbchavi (^2) observed a large difference in the
binding constants ft>r glucose and G6P by the enzyme and interpreted
it as indicating that either the hydroxyl group at casrbon atom 6 of
glucose VBS in5»ortant in the binding process or that the electrostatic
effect of the charged phosphate group inhibited binding of g6P to the
enzyme. They further concludfid that g6P brolse binding of %A!EP very
effectively. It can be seen from the foregoing that the sechanism of
the yeast hexoMnase reaction is unclear, and Frtxm and Zeve (33) point
out that the differences may be in the assun^tions made and in the
interpretation of data.

Aspergillus bexokinase. Davidson {2k) purified the hexoklnase of
Aspergillus parasiticus 225-fold. He employed fractional precipitation
with aanonium sulfate (50-70^), followed by fractionation tilth cold
acetone (38-U6?6) , negative absorption to alumina C-^ gel, and refractiona-
tion with ammoniun sulfate (60-80^) . The purified enzyme phosphorylated
D-glucose, D-galactose, D-glucosamine, D-galactosamius, D-mannose and
D-fructose at the relative rates (based on glucose) of 1.00:0.86:0.58:
0.1tO:0.67:0.36, respectively. L-sorbose and L-arabinose were active
to a very small extent. The enzyme phosphorylated galactose and
galactosamine to yield the respective 6-phosphates . The iOn for glucose
and galactose were almost identical (l.6X10-^M con^ared to 4.3X10-%!).
They interpreted their data as indicating that all the substrates were
phosphorylated by the saias enzyne and that the substrate specificity
suggested that the hexolsinase was different from that of either the
yeast or brain enzyme.

30

Neurospora hexoldnase. Msdina aad Nicholas (70) purified the
hexokinase of Neurospora craasa 60-fold. The enzyme phosphorylated
glucose and at a lower rate, mannose, fructose and glucosamine. The
enzyme vas noncompetitively inhibited by G6P and was competitively
inhibited by N-acetylglucosamine. lodoacetate, EDTA and PCMB vere also
inhibitors of the enzyme.

Higher plants. Saltman (82) demonstrated the occurrence of
hexokinase in several plants and purified the soluble hexokLnase of
wheat germ 5-fold. The hexokinases of the plants that were examined
were distributed between insoluble and soluble fractions. The distri-
bution depended on the tissue and the method of preparation of the
tissue.

An insoluble hexokinase preparation was used for chaiwsterization
of the enzyme. Saltman 's results, however, showed that the soluble and
insoluble enzymes vere almost identical in their properties.

The enzyn^ phosphorylated glucose, fructose, mannose and
glucosamine, in the presence of ATP and Mg++, at the relative rates
1.00:0.62:0.68:0.52, respectively. The Kia for glucose was U.^XlO'Sl
and that for ATP was 8.7X10"'^M, while the Km for glucose by the soluble
enzynte was U.6X10"^. Galactose, ribose, arabinose, ribulose, adenosine,
glyceraldehyde, dihydroxyacetone , mannitol and glucose -1-phosphate (GIP)
were inactive as substrates. Inosinetriphosphate (ITP) was 35 per cent
as effective as ATP. Magnesium ion activation was optimum at a concen-
tration of the ion equal to the concentration of ATP. Activation by
Mn''"'' was 80 per cent as effective as Mg++ at O.OIM, while Co++ was
ineffective. Cupric, zinc and mercuric ions strongly inhibited the

31
enzyme. Potassium, sodium and afflmonium ions neither activated nor
inhibited the enzyme. Substaiices vbich influence -SH groups had little
efftect on either the soluble or insoluble hezokinase. Zinc ion
inhibited the enzyme nonc(a5)etitively. Dinitrofphenol (5X10"3m) inhibi-
ted phosphorylation 70 per cent, while G6P (5X10"%) only inhibited 17
per cent. !Ehe vheat germ hexokinase was found to be similar to yeast
hexDkinase.

Hekata et al. (71) demonstrated the occurrences of heasoldnase
in a variety of higher plscits.

Itoh (48) partially purified hexokinase from homogsnates of
soybeans, Ozuki beans and mung bean by frswitional p2?ecipitation of the
ciTide homogenates with ammonium sulfate. The hexokinase of soybean
precipitated at about 60 per cent saturation. The highest activity of
soybean J^xokinase appeared ttiree days after germination at 26®C.

Itoh and Znouye (4-9) sepaxuted three different hexokinases of
soybean by fractional precipitation with ammcmium sulfate. Ihey
identified: (l) a glucokinase, specific for glucose and glucosamine,
(2) a fmictokinase and (3) a galactokinass, specific for galactose and
galactosamine.
Comparison of Animal and Plant Hexokinases

It appeal^ that aniaal and plant hexokinases are similar in
substrate specificity and, in many cases, Rn. The principal differences
a^ppear to be tte following:

(1) Hexose-6-ph08phates produce a more marked inhibition of
aTTtnt^] than of plant hexokinases.

32

(2) ADP inhibition of plant hsarokinases appears to be
coanpetitive with respect to ATP, while in animals it
appears to be of a more con^ilex nature.

(3) The hexokinase reaction catalyzed by brain hexokinase from
animals seems to follow a corapulsoiy pathway mechanism
involving a stable phospho- or glucoenzyme coniplex(es) .
Although there is disagreement, it appears that the enzyme
in plants might pixnaote a random interaction of glucose and
ATP in the presence of Mg''"'", without the formation of stable
phospho- or glucoenzyne congolexes (33, 95) •

Hexokinase and Sugar Uptate

The subject of sugar uptalfie by various animal tissues has been
thoroughly reviewed (18, 72) and will not be gone into in detail,
except to point out a few findings conceiTiing the role of hexokinase
in sugar t5>takB.

Lundsgaard (65) first proposed that the active absorption of
sugars depended upon the sequential phosphorylation and dephosphoryla-
tion of the actively absorbed sugar. He later abandoned the hypothesis.
Drabkin (26) revived the hypothesis and proposed that the driving force
of active absorption involved the phosphorylation of the sugar outside
the cell by hexokinase and dephosphorylation inside the cell by glucose-
6-phosphatase .

Sols (87) and Crane and Krane (22) have studied the specificity
of intestinal hexokinase and sugar absorption, respectively, and have
demonstrated the specificity of the enzyme to be contrary to that of
absorption. Dratz and Handler (27) have reported that the labeling of

33

the sia^cr-phosphate pool vith p32 is inconsistent with the
phosphorylation-dephosphorylation hypothesis. Landau and Wilson
(61) concluded from their data that absorbed glucose does not pass
through the G6P pool of hamster intestine.

Crane (18) points out that, in the intestine, aH actively
abso3rbed sugars have the ooaama stxw:ture:

DH
Suga3rs that are not actively absorbed have a large (3-0-butyl-D-glucose)

or ionized substituent on some part of the structure or lack one of the
essential features of the glucose molecule (2-deoxy-D-glucose or
fructose). It also appears that the pyranose form is essential;
specifically the CI chair foim. fe also points out -ttiat the configura-
tion specificity at caaA>on atoia 2 doesn't vBcessaxlXy hold for other
aniinal tissues. He obsesrved that the process of sugar ahsorption hy
the intestine follows Mtchaelis-Jfenten kinetics, requires the presence
of the free sugar, and doesn't depend on the gross sastabolism of the
substrate since 3-0-Eethyl-D-gl'acose, which is absorbed at a great rate,
is not netabolized.

CraiK et al. (19) and Crai^ (21) observed that sugar uptalae by
the intestine depends vqsod. the presence of Ma+ ions and that the extent
of accumulation depends on the external concentration of Na+. Eiey
proposed that glucose, on entering the brush border cells of the intes-
tine, combines with a carrier-Wa+ complex. The coc5>lfix moves through
the diffusion barrier to the interior of the cell ^&ere the Jfe+ portion

31^

of the complex is extruded "back through the diffusion barrier in the
reverse dii«ctlon "by an energy requiring process, vhich is inhibited
by strophanthidin, leaving the glucose "trapped" in the cytoplasm
(e.g. the so-called sodium "pump" involving the menibrane ATPase system).

Cirillo (9) has shown that the yeast cell transports various
nonfermentable sugars across the cell membrane and that both nonfer-
mentable and fermentable sizgars appear to share a common membrane
transport mechanism.

Morgan et ^. (76) have demonstrated, from their studies of glucose
transport and phosphorylation in the perfused heatrt of normal aiid
diabetic rats, that glucose uptaloe is controlled by the combined opera-
tion of two sequential steps, nembrane transport and intracellular
phosphorylation, and that in the steady state the net rate of membrane
transport and intracellular phosphorylation ai^e equal. In the absence
of insulin, glucose uptaJse is limited by membrane transport. In the
presence of insulin, glucose uptake is accelerated and glucose phos-
phorylation is increased. Phosphorylation becomes increasingly limiting
as the external concentration of glucose is raised and provides the
major limitation to glucose uptake. They point out that the apparent
iOa for glucose phosphorylation by hexokinase in diabetic heart muscle
is at least 7 times higher than in normal tissvie. 03iey found insulin
in vitro to have no large, immediate effect on glucose phosphorylation.

Randle (79) reported that the uptake of glucose and the
accxanulation of D-xylose in isolated rat diaphragm sire accelerated by
inorganic phosphate, G6P, GIF, F6P, FDP, AMP and ATP. He points out
that the effect is not marked and that perhaps it is the phosphate

35
group which is responsible for the acceleration. Anoxia and
2,l»-dinitrophenol were also found to accelei^te glucose uptalse, but
such treatment also increased the rate of uptaise of substances
(soAitol) which are not ordinarily absorbed by diaphragm tissue. He
suggests that uptake might be regulated by phosphorylation and dephos-
phorylation of a mentorane carrier.

Horecker et al. (k6) observed that a mutant strain of E. coli
()0i6), which utilized galactose much more rapidly than glucose, in
contrast to the wild type, has a galactokinase that has a much higher
affinity for ATP than the glucokinase of the cell. Bie galactokinase
is easo inducible in the wild type cells, \lhen AW is limiting in
extracts of either strain, galactose is a strong inhibitor of glucose
phosphorylation. The inhibition also occurs in_ vivo in the mutant,
but doesn't in the wild type.

The findings of Humphreys and Garrard (kj) with respect to
glucose t5>take by com scutellum slices are reviewed in the introducti<»i
and discussion sections of this dissertation.

Glasziou (37, 38) has reported detailed studies of sugar uptake
and transfonnation in immature sugar cane intemodal disks. His
results do not indicate a role for hexokinase in sugar uptake by that
tissue.

MATERIALS AND METHODS

Plant Materials

Com grains (Zea mays L., var. Funk's G-76) vers soaked
tventy-four hours in running tap vater and planted, scutellum-side i^,
on four layers of moist filter paper on trays. The trays -were covered
with alvmiinum foil and placed in the dark at 23°C for five days. The
scutella vere removed from the germinated grains and placed in ice-cold
glass distilled vater until all the scutella vere harvested. All sub-
sequent steps vere carried out in the cold or in an ice -vater bath.
Preparation of the Enzyme

Extraction. The scutella vere veighed and then ground in a
chilled Waring Blendor for one and a half minutes in four volumes (v/v)
of ice-cold O.OO5M ethylenediaminetetraacetate (EDTA), O.CXJJM magnesium
chloride (MgCl2) and O.OIM potassium chloride (KCl), pH 7.0. The use
of either tvo volumes or six volumes of extracting solution decireased
recovery of the hexokLnase. Cysteine (O.OO5M) or glutathione (O.OlM)
did not inc3rease recovery.

The homogenate vas scLueezed through tvo layers of cheesecloth
and the filtrate vas centrifuged at 0°C and 2,000XG for one hour. The
supernatant fraction vas filtered thixjugh glass wool to remove the
fatty layer and was then centrifuged at 0°C and 32,0OOXG for one hour.
The supernatant fraction vas filtered through glass vool and saved.
This fraction vas designated "crude homogenate."

36

37

AmiK3nlum sulfate fractionation. The crude homogenate vas made
50 per cent saturated in ammoniuin sulfate ((HHi^)2S0l^) "by slov addition
of the salt to the magnetically stirred solution. The solution was
allowed to equilibrate, with stirring, for fifteen tainutes after the
addition of the salt. The solution was centrifuged at 12,000XG for
twenty minutes at 0°C. The precipitate was dissolved in a minimum
amount of O.O5M potassium phosphate buffer, pH 7.O, and was dialyzed
against several four liter changes of the same buffer for forty-eight
hours. This fraction was designated "F-l".

The 50 per cent saturated supernatant fraction from the above
centrifugation was made 75 per cent saturated in ammonium sulfate and
hand]£d in the same manner as F-l. After dialysis the dissolved
protein fraction was designated "F-2" and the supernatant fraction,
"F-3" . The F-2 fraction contained most of the recoverable activity
(Table 3) and was further purified by three successive trea-baents
with alumina Cy gel. The first two treaianents absorbed substantial
amounts of the ATPase (Table h) and the final treatment absorbed the
hexoklnase which was used for this research. The following paragraph
describes the procedure that was used to purify the enzyme with
alumina Cr gel.

Absorption and elution from alumina Cy^ gel. The F-2 fraction
was treated with solid alumina C^ gel (7.^ per cent solids, Sigma
Chemical Company) as follows: (l) The F-2 fraction was treated with
0.0137g gel per ml (equivalent to 2.U mg solids per ml). The gel was
dispersed by stirring with a ground-glass homogenizer and was allowed
to stand, with occasional stirring, for about thirty minutes. The

38

mixture vas centrifuged at 0°C and 12,000XG for tventy minutes. The
supernatant fraction was decanted and saved for further treatment.
The precipitate was dispersed and eluted for four hours with a volume
of O.SfL ammoniiim sulfate equal to one-third the volume of the F-2
fraction. The mixture was centrifuged at 0°C and 12,000XG for twenty-
minutes. The supernatant fraction was decanted, adjusted to pH 7-7 '5
with solid tris(hydroxymethyl)aminomethane base (tris base), portioned
among several plastic centrifuge tubes and frozen at -20°C until
needed. 15iis adjustment was necessary because the enzyme was inacti-
vated during storage at pH's below approximately 7* ^is eluate was
designated "A". The precipitate was eluted a second time with the
same volune of O.l^M aimaonium sulfate and treated in the same manner
as the previous eluate. It was designated "A-l". (2) More gel
(0.063 g per ml, equivalent ix> h.J ros solids per ml) was added to the
supernatant fraction from the prior gel treatment. The mixture was
handled as before and the two eluates designated "B" and "B-1".
(3) To the supernatant fraction of the second gel treatment was added
0.0951 g per ml of gel (equivalent to 7*1 njg solids per ml) and the mix-
tare centrifuged and eluted as before. The two eluates were designated
"C" and "C-1". Fractions C and C-1 were the preparations used to
obtain the results reported in this paper. Both preparations had
identical Kn for glucose, ATP and Mg"*^ and were considered to be the
same enzyme. They contained some adenosinetriphosphatase (ATPase) and
phosphoenolpyruvic phosphatase (PEPase) activity. Sodium molybdate
(Na2Mo20Y), 0.002M, completely inhibited the PEPase. Fractionation
of C and C-1 with solid ammonium sulfate, which resulted in 80 to

39

90 per cent recovery, did not remove the two interfering enzymes and
only succeeded in concentirating them along vith the hexokinase.
Assay Methods

Two methods were used for the assay of hexokinase activity and
fbr characterizing the enzyme.

Method 1. The rate of glucose -6-phosphate (g6p) formation at
25°C was measured by following nicotinamide adenine dinucleotide
phosphate (KADP) reduction in the presence of excess glucose -6-phosphate
dehydrogenase (G^»D) spectrophotometrically at 3^ millimicrons. The
standard reaction cuvette contained the following: glucose, 20 micro-
moles; ffeCl2, 20 micromoles; Adenosine-5' -triphosphate, 20 micro-
nKJles; NADP, 1 micromole; Tris buffer, pH 8.0, l8o micromoles; G6PD,
lEU; hexokinase preparation and water to 3*2 or 3.3 ml. The blank
cuvette did not contain G6pd. This method wsis used to measure: (l)
the rate of phosphorylation of glucose and fructose (coupled to
phosphoglucoiscanerase), (2) competitive inhibition by nons'ibstrates
and glucose-1-phosphate (GIP), (3) nucleotide activation and inhibi-
tion, (k) metal sictivation, and (5) pH and teaiperatui^ optima, which
were examined by running the hexokinase reaction at the various pH's
and tempeiratures in a total volume of 6 ml with twice the above
ingredients, except for KADP and C16PD. The reaction was stopped at
the end of 10 min by placing the reaction tube in a boiling water bath
for 2 min. A 3 ml aliquot was assayed for G6P with KADP and G6PD.

Method 2. The rate of adenosine-5' -diphosphate (ADP) production
at 25°C by hexokinase was determined by measuring the oxidation of
reduced nicotinamide adenine dinucleotide (NADH) in the presence of

ko

excess phosphoenolpyruvate (PEP), pyruvic kinase (PK) and lactic
dehydrogensise (LD) spectarophotometrically at 3^*0 miHifflicrons . The
standard inaction cuvette contained the following: Tris buffer, pH
8.0, 180 micromoles; NADH, 0.282 micromolesi PEP, 10 micromoles;
Na2Mo20Y, 6 micronrales; MSCI2, 20 micrranolesj ATP, 10 micromoles;
PK, 1 EU; LD, 1 EU; hexokinase preparation and water to 3»2 or 3*3 ml.
The blank cuvette contained no KADH and a control was included, which
contained no sugar, to measure ATPase activity. This method was used
to measure: (l) the rate of phosphorylation of mannose, 2-deoxy-D-
glucose, L-glucose, L-mannose, N-acetyl-glucosamine, ribose, xylose,
emd galactose, and (2) inhibition by nonsubstrates and the hexose-6-
phosphates, namely, G6P, 2-deoxy-D-gluco3e-6-phosphate (2D0G6P) and
mannose -6-phosphate (M5p). Mannose -6-phosphate inhibition could not
be measured by the G6PD areaction because it contained fructose -6-
phosphate (f6p) which was converted to G6P by phosphoglucoisoaerase
in the scutellum hexokinase pareparation.

Activity determinations by the two methods gave identical rates
for glucose after correcting for ATPase activity. With both methods
the observed rates were constant over at least sixteen minutes after
an initial one or two minute lag period.

Assay for phosphofructokinase, phosphoglucomutase and glucose-S-
phosphatase activities. Phosphofructokinase was assayed by coupling
the phosphorylation of F6P to NADH oxidation in the presence of excess
aldolase, triosephosphate isoinerase (TPl), and oC -glycerophosphate
dehydix)genase (<c-GPD). Phosphoglucomutase activity was determined
by Method 1 above, using GIP as the substrate. Glucose-6-phoBphatase

in

was measured by the same method after incubation of the hexokinase
preparation vith a known amount of G6P for ten minutes.
Protein Determination

Protein vas estimated using the Folin-Ciocalteu reagent method
of Lowery et al. {6k) as described by Layne (59). "She colorimetric
readings were made on the Beckman DU Spectraphotometer at 720 milli-
microns. Crystalline bovine serum albumin was used as the standard.
Chemicals and Enzymes

AH the chemicals and enzymes used in this investigation were of
the highest purity obtainable commercially. The enzymes were pur-
chsised from the California Foundation for Biochemical Research. The
nucleotides, NADP, NADH, PEP, G6p, t^, P6p, 2D0G^ and 2D0G6P were
obtaired frcwa Sigma Chemical Cou^jany. The other chemicals were
obtained either from the above firms, or fron Ifcctritional Biochemicals
Company or Fisher Scientific Coapany.

Pvirlfication

The hexoklnase in the exude homgenate could not be assayed
satisfactorily because of interfering enzyme activities.

Table 3 outlines tbe results of a typical purification of the
hexokinase from the scutellum. Although the amount of hexokinase in
the crude hoinogenate could not be estimated accurately. Table 3 shovs
an "apparent" 30-fold increase in specific activity of the 50 to 75
per cent saturated ammonium sulfate fraction (F-2) over the crude
homogenate. From Table 3 it can be seen that almost all of the hexo-
kinase activity was precipitated betxreen 50 and 75 per cent saturation
with ammonium sulfate. The F-2 frswtion contained the following
enzymes which might interfere with either one or the other assay:
glucose-6-phosphatase (G6Pase), phosphoenolpyruvic phosphatase
(PEPase), phosphoglucoisomerase (PGI), and adenosine triphosphatase
(ATPase), but did not contain phosphoglucomutase (PGM), phosphofructo-
kinase (PFK), 6-phosphogluconic dehydrogenase and enzymes that destroy
reduced KAD or NADP. The G6Pase activity should not have interfered
seriously with the G6FD assay method because the excess G6PD in the
reaction mixture should act as a trap for the G6P produced by the
hexokinase .

Treatment of the F-2 fraction with alumina C-^ gel resulted in a
2.3-fold increase in specific activity of the C-1 fraction over the

k2

i^3

a»BLB 3
PURIFICATION OF HEXOKERASE

Fraction

Specific
activity
(nieroiHOlfis/min/
protein)

Total
activity
(units*)

it Recovery

from

F-2 fraction

Crude homogenate

0.002

2.80

«..

F-1 iO-50$iW^)^SOi^)

0.002

0.57

•^

F-2 i30'l5%{m^)^Oi^)

0.060

12.60

*•*

A (1st alumina Cy )
A-1 (gel treatment )

0.026

0.64

$.1

0.012

0.11

0.9

B (2nd alumina C^ )
B-1 (gel treatment )

0.070

1.83

1*^.5

O.OH9

0.81

6.4

C (3rd alumina Cr )
C-1 (gel treatment )

0.102

3M

27.4

O.HH

3.91

31.0

One unit a amount of enzyme that will phosphorylate 1 micromole of
glucose per minute at 25°C.

F-2 fraction. Thia amounted to an "apparent" 70-fold increase over
the crude homogenate. When the F-2 fraction was dialyzed against the
extraction aoXution (EDTA-Mg-KCl) the ATPase activity paralleled the
absorption and the elution of the hexokinase activity from the gel,
but dialysis of the fraction against 0.05M potassium phosphate "buffer,
pH 7.0, changed the absorption and elution characteristics of the pro-
tein with respect to the gel so that "Uie AQPase activity could be
satisfactorily separated frrai the hexokinase activity as is shown in
Table k. Buffers such as potassium phosphate, glycylglycine and tris
at several concentrations and several pH values were ineffective in
e luting the l^xo kinase from the gel. Tris buffer in concentrations
above 0.1»M inhibited the hexokinase activity irreversibly. The C and
C-1 fractions, which were used as the enzyme preparations in these
investigations, contained PEPase activity which was completely
inhibited by 0.00^ sodium molybdate, a small sanoiint of ATPase activ-
ity (iSable k) for which a correction was applied, and PGI activity.
The G6Pase activity of the P-2 fraction was not absorbed onto the gel.

The ATPase activity of the F-2 fraction was neither decreased by
centrifuging the crude homogenate fraction at 105,O0OXG for one and
ons-quarter houirs nor was it inhibited by ouabain (O.OOO5M to O.OO5M)
or by fluoride (0.002M to 1.34). The addition of M^ but not Na+or
K*" was required for ATPase activity. Since the content of ADP in the
reaction mixture was increased by the addition of AMP, part of the
"apparent ATPase" activity was due to adenylic kinase .

Further purification of the enzyme by acrylamide gel electro-
phoresis was attempted. The P-2 fraction sepaarated into 11 or 12 bands

h3

TABLE k
AOJPASE ACTIVrPy OF THE HEXOKEMSE PREPARATiaHS

Fraction

Specific

activity

(micrcanole s /laln/mg

protein)

Total
activity
(units*)

$ Recovery

from

F-2 fraction

F-1

0.002

0.51

...

F-2

0.096

20.10

##»

«

0.076

1.33

M

A-1

0.233

1.T9

t*5

B

0.058

i.oi*

|.t

B-1

0.134

1.55

%1

i •

0.025

0.58

Z.$

C-l

0.020

0.38

1.9

*0ne unit « amount of enzyme that will produce 1 micromole of
ADP per minute at 25°C.

k6
in the positive direction, while the C and C-1 fractions separated
into four bands— -tvo large bands betveen tvo smaller bands. The four
baMs had Rf 's corresponding to four similar bands separated frcan the
F-2 fraction. Hexo kinase activity could be detected in one of the
bands, with Rf of 0,k5'0*60, by slicing out the band, placing it in a
reaction cuvette and following the reduction of NADP at fifteen-
minute intervals up to one and one-half hours. ^Rie activity, O.I60
micromoles/cuvette/hour, was small and attenqpts to elute a significant
amount of the hexokinase from i^ole gel slices or homogenized slices
with several concentrations of glucose, phosphate buffer, ammonium
sulfate or gjycylglycine buffer were unsuccessful. Most of the ATPase
activity in the gel was localized in a band with Rf of O.77-I.O (calcu-
lated with reference to the distance traveled by the salt front). Such
a band had an activity of 0.037 micromoles/cuvette/hour. The other
bands also had some ATPase activity.

Acrylamide gel electrophoresis of ccmnercial crystalline hexo-
kinase (CalBiochera) also gave foxa: bands that had a separation pattern
similar to that of the C and C-1 fraxitions with the two large bands
having Rf 's almost identical to those of the C or C-1 fractions. Again
the recovery was small. Approximately 28 units of activity were added
to each gel and at the end of the run the gel was cut into small pieces
and e luted with 1 ml of O.Ol|-M glycylglycine buffer, pH 7.5, for thirty
minutes. The resulting eluate had an activity of 0.224 units /ml or
about 0.9 per cent recovery.

Attempts to purify the enzyme by absorption and elutlon from
bentonlte by the method described by Darrow and Colowlck (23) were

hi
unsuccessful. The hexoklnase and ATPase activities vere absorbed
but could not be e luted.

Fractionation of the enzyme preparations at any step in the
outlined procedure with eold acetone or ethanol (-7®C) resulted in a
ccMuplete loss of activity.

Humphreys and Garrard (kT) found that slices of the com scutellum
talJB up glucose at the rate of 70-80 micaromoles/g fresh veight/hour
(approximately 1 unit per g of tissue) but do not accumulate it.
Therefore, if it is assumed that all the glucose entering the sUces
is phosphorylated, the hexokinase content of the tissue should be much
higher than that recovered. Since the F-1 and F-2 fractions wouM
account for only about one-fifth of the total aK>arent hexokinase
activity of scutellum, attempts vere made to increase the yield.
Reduced glutathione or cysteine in concentratiorffi of 0.005M and O.OIM
in the extracting solution did i»t increase the yield of hexokinase.
Including glucose in concentrations of either 1 or 10 per cent in the
extracting solution or in the crude homogenate also did not increase
the yield. Detergents such as deoxycholate (0.026 per cent) and
Triton X-100 (O.l per cent) not only failed to increase the yield, but
also increased the solxibility of lipid in the crude hcaaogenates so that
it coxild not be removed by centrifugation followed by filtering through
glass wool. The lipid interfered with protein precipitation in the
subsequent salt fractionations by causing the protein to float to the
top of the centrifuge tubes with the lipid. The floating material was
difficult to collect quantitatively and the hexokinase activity in
this netericLL was lower than that obtained by the method described in

the materials and xnethods section. Dialysis of the floating layer
did not solubilize the enzyme and treatment vith cold acetone or
ethauol at -7°C to remove the lipid destroyed the hexokinase activity.
Itoh {k8) vas able to use the floating layer to study the hexokinase
of soybean. Dime thylsulf oxide (DMSO) at concentrations of 0.1 and 1.0
per cent in the extx-acting solution did not affect the yield. Extracts
of acetone povdei^s prepared fran scutella yielded smaller amounts of
hexokinase activity.

The endosperm of the germinated com grains did not contain
detectable hexokinase activity.
Substrate Specificity

a^gars. The specificity of scuteHtoa hexokinase for sixteen
sugars vas examined. !Ihe Michaelis constants (Ka), maximum velocities
(Vtaiax) wad relative Vhiax vith xespeet to glucose are given in Table ^.
The enzyme phosphorylated D-glucose, D-mannose, D-finctose, 2-deoxy-D-
glucose and D-glucosamine . Glucose had the lowest Km (6.it-X10"5M) and
the greatest Vinax. The substrate concentration versus rate curve for
glucose and the corresponding Lineveaver-Burk plot (62) are presented
in Figures 1 and 2, respectively.

Nucleoside triphosphates. The IQa for ATP vas found to be 8X10"<M.
Figures 3 and k show the cxxrves from which this value was calculated.
Commercial preparations of IfCP (uridinetriphosphate), GTP(guanosine-
triphosphate), CTP (cytidinetriphosphate) and TCP (thymidinetriphos-
phate) vere found to be poor substitutes for ATP at the concentrations
that were examined (Table 6) . The best substitute for ATP vas UTP -vbldh
gave 28 per cent of the rate of 0.00194 ATP at a concentration of 0.003M.

k9

TABLE 5
SUBSOKATE SPECIFICITy OP CORN SCUTELLUM HEXOKINASE

En Relative

Substrate (MXIG^) Vinax** Vinax

D-glucose

6A

D-maniK)se

t*l

D-fructose

168

2-deoxy-D-glttcose

30

D-glucosamine

37

N-acetyl-D-glucosamine

55*

D-xylose

870*

oc-E»ethyl-D-glucoside

~

S-O-methyl-D-glucose

—

L-glucose

-

L-mannose

«•»

6-deoxy-L-naimose

,.

D-galactose

#*

D-galactosandne

*».

D-riboae

•mm

oC -D-glucose -1-phosphate

— —

0.0^^7

1.00

0.028

0.59

0.01*6

0.97

O.oMf

0.9«*

0.030

0.63

*Inhibition constant (Ki)

"^0.D./2 min/cuvette at 25°C

■Tfo detectable activity or inhibition at O.03M

50

The lew rates obtairted by these compounds could have been due to a
small amount of nucleoside dlphospholdnase activity for which the
prepaarations vere not assayed. Saltman (82) observed that inosine-
triphosphate (ITP) was 35 per cent as effective as A2P as a phosphate
donor in the hexoKinase areaction catalyzed by an insoluble hexokinase
from wheat. Walaas and Walaas (99) could not detect hexokinase
activity with I5CP, CffiP or UTP with -tbeir preparations frcsa muscle
tissue.
Mstal Activators

Divalent metal cations were required for scutellum hexokinase
activity. Table 7 gives the Mn and a con5)arison of the activation of
scutellum hexokinase by N(g+^, Co"^ and Ibi^. Figures 3, 6 and 7 show
the metal concentration versus rate cxirves and the Liiteweaver-Burk
plots for the three metal cations. Magnesium ion was by far the best
activator. An inhibition of magnesixaa activation was observed when
the molar ratio of A3P/Mg exceeded approximately four. When the ratio
was 7.5 micrc«aoles/l micromole or 7«5/2 micromoles the rate, compared
to 2 raicromoles/l micromole or 2 micromoleB/2 micixanoles, was 20 and
10 per cent lower, respectively, and caused the Lineweaver-Burk plot
for Mg"^ to deviate from linearity. Therefore, the ratio was always
maintained less than four.

Manganese ion was inhibitory at concentrations greater than the
concentration of ATP (0.003M) (Table 8 and Figure 5) which indicates a
maximal activation by the ion when the molar ratio ATP/Mn is one. The
inhibition did not occur when such a i^tio was maintained. Such
inhibition is similar to that observed by Walaas and Walaas (99) •

Q H

a 0) o
o ft o

52

to
O

0)

o
o

CD

(9/ X uiiAlE Jdd GOV

Figure 2. Lineweaver-Burk plot of the
effect of glucose concentration on the rate
of phosphoiylation.

54

1 \ \ \ \ \ r

0 2 4 6 8 10 12 14 16

l/(Glucose)

55

TABLE 6

EFFECT OF KUCLEOSIDETRIPHOSPHATES
AS SUBSTITUTES FOR ATP

Concentration ^ of Rate Obtained
Nucleosidetriphosphate (MX1o3) vith 1.5X10"3m

AOP*

3.1 28

GTP 1*5 ^

$*l IT

CTP 1*5 3;^

TOP 1.5 3.5

3.1 35

*HgH- concentration constant at 6x10~3m

Figure 3. Effect of ATP concentration
on the rate of phosphorylation, and con^titive
inhittition by ADP and AMP. Beaction cuvettes
contained: I80 microiaoles Tris buffer, j^ 8.0j
20 micromolfis glucose; Hi^l^ in amounts equal
to, or greater than, the concentration of
inhibitor plus ATP; 1 micromole of NADP; 1 EU
G6H); 10 micranoles of ADP or 20 micromoles of
AMP; 0.1 ml enzyme preparation and water to
3.3 ml.

57

' —
<3

2 4 6

ATP MxlO^

8

59

^//

m

TABLE 7
ACTIVATION OP HEXCmMSE BY METAL IONS*

Ion

Ifin ^

(Mao5)

( O.D./2 min/citvette)

9& ^feias

Relative

toMg

1^

ao

O.OltO

300

CO++

TO

0.025

63

Ma++

IXX)

0.020**

50*»

■*Batio of ATP/metal ion vas always maintained at less than k,
"•■Calculated from extrai«>lated Lineweaver-Burk plot. Actual
observed relative Vinax was 38 per cent.

Figure 5. Effects of Co++ and Mn++
concentirations on the rate of phosphorylation.
Ihe standard reaction mixture was used, except
the concentration of ATP vas 10 ndcrcapaoles
per cuvette.

Figure 6. Effect of Mg"^ concentration
on the rate of phosphorylation. The standard
reaction mixture vas used except: for the
first two concentrations of Mg^^, 2.0 micro-
moles ATP were used and for the last four
concentrations, 7*5 micromoles AOP were
used.

62

' — .
<l

1 r

I 2 3 4 5 6
Metal MX 10 3

Figure 7« Lineweaver-Burk plots of "Bie
effect of Mg"*"^, Co++ and Mn++ concentration
on the rate of phosphorylation.

6k

I /{Metal

65

Ko inhibition by ISq^ and Co'*"'' iraa seen at the concent3:«tions eniployed
in these experiments.

The addition of potassium ion or sodiiaa ion did not cause activa-
tion of the enzyine.
Inhibitors

Sugars. Of the nonsuibstrates listed in Table 5j W-acetylglucosa-
mine and D-xylose vere found to be competitive inhibitors of glucose
vith KL of 55X10"5m a»d BjOHD'^, respectively. Figure 8 shonis the
Lineveavsr-BurlE plots fWaa "which the Ki in Table 8 -wei^ calculated.
lEhe other noi^ubstrates had no inhibitory activity.

Nucleoside di- and triphosphates. ADP and MP vere found to be
coi^>etitive inhibitoz^ of ATP with Ki of ltalO"5M and 1X10"^, respec-
tively. Figures 3 and k show the curves for inhibition by 0.003M ADP
and 0.006m A?ffi>. UTP, GTP, TIP, (SEP, UDP and (HJP were found to have
little inhibitory activity with respect to A!SP at the concentrations
that were examined (Table 8). The slight inhibition of hexokinase by
these six ccaiipounds was not increased by increasing the concentration
of the nucleotides.

Sugar phosphates. Ifo inhibition of glucose ^diosphorylation was
dbserved with G6P, F6p, itSP, G3P, galactose-6-phosphate (Gal6P) or
ribose-5-phosphato (R5P) at concentrations of the sugar phosphate up to
0.03324. The concentration of glucose in these e:5>eri3aents was that
which resTilted in about half maviTnal velocity (6O.0"5m).

Anions. Fluoride in concentrations of 0.002 to 1.2M did not
inhibit scutellum hexokinase. MgCl2 and MgSO],. were equally effective
in activating the enzyote.

^^isl

67

rO

- C\J ^

o
o

3

o

^//

TABLE 8

BSfBCT CSF NUCLEOSIDE DI- AND TKEPHOSPHATES AS IKHIBITORS
OF ATP IN THE HEXOKEMSE REACTION*

Nucleotide

Concentration**
(MX103)

i, of Rate
Relative to
1.5X10-3M ATP Alone

3.1

5

1.5
3.1

%

3.1

80

3.1

96

UTP
GTP
TTP
CIP
ASP

3.1

1.5

3.1

1.5
3.1

80
80

90
90

*t^++ concentration constant at 6X10*^
**ATP concentration was 1.5X10"3m

69
pH and Temperature Optiina

A pH optimum (Figure 9) was observed at pH 8.0, vhile the optimum
temperatxire for scutellum hexokinase was found to be k9°C (Figure lO).

TOiis optimxan is similar to that observed for wheat gena hexo-
kinase (82) , muscle hexokinase (99) and honey bee hexokinase (81) .
The scutellum l^xokinase lost activity rapidly when stored at ixH's
below 7.

The temperatvure optimum (Figure lO) is much higher than that
reported for other hexokinases. Hexokinase isolated by Saltman (99)
froo wheat germ had a temperatxare optlxman of 37®C but Steha and
El-Towesy (93) reported that phytases frcxn several seeds including bar-
ley and wheat, had ten^ierature optima in the vicinity of 51°C. Berger
et al. (6) found that the Q^q ^°^ yeast hexokinase was close to 2 over
the range of 0-30°C. The Q;j^q of the hexokinase fraa the com scutellum
(Figure 10) is 2 between 20 and 30°C but falls off at temperatures above
and below this range. The Q^q n>iSh* vary vith the enzyme, the coti-
ponents of the reaction Hdxtxare and at higher temperatures can be a
reflection of the rate of denaturation and the rate of the reaction.

CO g ^

og

III!

•u CO CJ <U rH

71

X

Q.

dioy uLinuujXD^ ;o iUdOJdd

ffl oj 8j E 0 • . a

03b

. 0) O B
. Sj ^ "H -H
O O -P -P

+> $3 S H Q nj

I

O 0) ? «i^

73

O
o

u!l/\i OI/d-9-D Sdjoi^r/

MSCIBSIOH

!Hm results indicate that the pattern of substrate specificity of
Bcutellum hexoMnase is similar to that of yeast hexoMnase. The Km
of the scutellum enzyoe for D-glucose, D-mamiose, 2-deoxy-D-glucose
and ATP are of the same order of laognitiide as those of the yeast
enzyme (Table l) . The Kia for D-f ruetose is an order of Bagnitude
larger. However, (Sottschalk (39) has shown that the furanose ring
configuration of fructose is the true heaookinase 8ubst3?ate and, at
equilibrium, about 20 per cent of a fructose solution is in the furanose
fora. If this is the case with the scutelluzQ enzyme, the tarue fin would
be about five timss smaller than the value given in lEable 5. The Kn
for glucosamine is an order of magnitude smaller than that of yeast

The most significant difference between the hexokinase frcm the
scutellum and tliat froaa brain or yeast is the arelative rate of phos-
phoarylation of the other four substrates ca^pared to glucose. Glucose
is phosphorylated at a rate greater than any of the other substrates,
i^parently the scutellxm enzjnne shows a greater specificity with
respect to differences at carbon atom 2 of the glucose molecule than
does the yeast or biain hexokinases.

Sols and Crane (86) suggest that the coefficient of phosphorylation

(■ Vmax substrate x Sa glucose ) ±q a true measure of the physiological

Vmax glucose IQn substrate
suitability of a substrate for hexokinase. With the scutellum enzyme

the coefficients of phosphorylation for D-glucose, D-manno8«,

7^

75
2-deoxy-D-glucose, D-glucosamine and D-fructose are 1.00:0.iH:0.30:
0.11:0.0U, respectively. !Riese values iiadicate that D-glucose is tl^
nost suitable physiological svOastrate for the scuteUioa hexokinase.
Correcting for the furanose configuration of fructose voalA place
fructose closer to (about the same as 2-deo3Qr-D-glucose ) , but still
lower than, glucose in suitability as a substrate.

The results from the limited studies of the si&strate specificity
and of inhibition by the various nonsubstrates are essentially in
agreement vith the findings of Sols et al. (88) for yeast hexokinase
and of Sols and Crane (86) for brain hexoMnase. The scutellum hexo-
kinase is specific for the D-configuration since the L- forms of the
substrates are neither phosphorylated nor are they inhibitory. The
five-carbon sugars (ribose and xylose) are not phosphorylated but xylose
is a ccanpetitive inhibitor. The enzyme also shows specificity for
hydroxyls at caxtoon atoms 1(G1P and oc-methyl-D-glucoside) and
3(3-0-inethyl-D-glucose) and for inversion of the hydroxyl group at
carbon atom k since galactose and galactosamine vere not active as sxjb-
strates or inhibitors. Inversion of the l^droxyl at carbon atom 2
might account for the low rate of phosphorylation of mannose and the
inversion at carbon atom 3 would account for ribose being noninhibitory.
No analogs of glucose which lack hydroxyls at carbon atom 6 were tested
except for the hexose-6-phosphates and the fact that they were not
inhibitory suggests that scute Hum hexokinase requires a hydroxyl at
that position for binding. This is supported by the fact that xylose
is a weak congetitive inhibitor.

Activation of the scutellum hexokinase by Hg"*"*"* Co^ and lfa++
closely resembles that observed by Walaas and Walaas (99) for muscle

76
hexokinaae, althou^ inhibition by Vig^ and Co"++ vas not observed at
the concentrations used in the experiments. Saltoan (82) observed that
Mn++ vas 80 per cent as effective as Mg*^ for activating the insoluble
hexokinase of wheat germ while Co"*^ was coo5>lBtely ineffective. The Kin
(2X10*^) for activation of the scutellum hexokinase by Hg^ is a magni-
tude SBBller than that reported by Walaas and Walaas (99) for the
muscle enzyme and by Berger ejb al. (6) for tte yeast enzyme.

TbB results show that ADP and AIS> appear to act as competitive
inhibitors of AOP. The Ka for ATP and the Ki for ADP suggests that the
enzyme shows similar affinity for both nucleotides. Similar inhibition
has been demonstrated for yeast hexokinase (33) and hoi»y bee hexokinase
(81) . Inhibition by ADP with respect to ATP for brain hexokinase has
been reported to be noncon^etitive (27) or of a coniplex nature (32).
In the e^qperiments reported in this investigation the magnesiim ion
concentration was maintained at levels equal to, or greater than, the
concentration of substrate (ATP) plxis Inhibitor (ADP or AMP) so that
there would be no inhibition caused by competition for magnesium ion.

Inhibition of the scutellum hexokinase reaction by G6P or by other
hexose-6-phosphates, as suggested by Humphreys and Gaarrazd (kf) from
their studies of glucose -uptake by scutellum slices, was not observed
in these investigations. The levels of ATP used in these esqperiments,
0.003 and O.OO6M, may have masked inhibition, as has been suggested by
Promm and Zewe (33) for yeast hexokinase, but such inhibition must not
have been very large. It may be that there is another glucose-
phosphorylating enzyme in the scutellum but the results indicate that
there are none.

When one conBlders that the role of the scutellum in the
germinating seedling is essentially that of a "sucrose factory" and
that it ia capable of absorbing and utilizing glucose at a rapid rate
(l micromole/min/g fresh weight at 30°C) it seems reasonable that the
sucrose and energy synthesizing systems of the scutellum should be
efficient ones and laay be arialogous to the glycogen synthesizing sys-
tem of rat liver studied by Vinuela et al. (98) . Haey demonstrated
that there was a glucokinase in the liver which was distinguishable
froa the typical animal hexokinase of that organ. The glucokinase had
a higher Km for glucose, was not inhibited by g6P and phosphorylated
glucose at a rate which was comparable to tlte rate of glycogen synthesis
in the liver. Leloir et al. (60) repoarted that uridine diphosphoglucose-
glycogenglucosyltransf erase was activated by high levels of G6P. The
system might also be similar to starch synthesizing system in seeds
Tdiich appear to be highly specific towards stibstrates (30) . Also, the
hexokinase of yeast, an organism which utilizes sugars as its principal
source of energy, is not inhibited to any extent by its products (13,
33, 83, 102).

Euniphreys and Garrard (If?) observed that preincubation of scutellum
slices in water prior to introduction of glucose into the bathing
nedium, increased the rate of glucose uptake by the slices. Biere was
a concomitant decrease in G6P in the slices with the length of preincuba-
tion which they interpreted as indicating that g6P was acting as a com-
petitive inhibitor of glucose phosphorylation and thereby limiting
glucose -uptake. They observed similar inhibition with preincubation in
mannose solutions, which could be partially reversed by washing the

78
mannose from the slices. The residual inhibition was attributed to
the high level of M5P. The resvilts reported in this paper suggest
that Gr6P and M6P are not inhibitoirs of scute Hum hexokinase, aiid the
factors controlling glucose uptake in the scute llvm nwst be other than
inhibition of the hexokinase reaction by hexose-6-phosphates. M6P has
not been found to be an inhibitor of brain (l6) or yeast hexokinase
(83), but might veil be inhibiting some step in the utilization of G6P.
2-Deoxy-D-glucose has been shown to be a cooipetitive inhibitor of G6P
for the lihosphoglucoisanerase of rat kidney (IO3). Both the availabil-
ity of ATP at the site of the hexokinase reaction and ADP inhibition
could affect the rate of glucose phosphorylation and consequently the
rate of glucose uptake. Preincubation might cause sa ioeirease in
available AOP and removal of inhibitory ADP. At the sazne time, ASP
availability and nucleosidediphosphate inhibition could be affecting
the utilization of G6P by limiting the rate of uridinediphosphoglucose
pyrophosphorylase tha-ough the nucleoside diphosphokinase reaction, and
preincubation would reflect increases in high-energy phosphate compounds
at these sites with an accompanying decrease in G6P. Various investi-
gators (30, 3^) have demonstrated strcmg interrelationships between
nucleotides and between nucleotide-sugar compounds in starch and sucrose
synthesis. Morgan et al. (76) have shown that glucose -uptake in the
heart of rat is controlled by both transport and intracellular phos-
phorylation. When membrane transport is not limiting (i.e. sufficient
insulin is available) the rate of phosphorylation limits the rate of
glucose -uptake at high levels of exogenous glucose.

79
It is belie-wd that the jresults presented in this paper support
the conclusion that the hearakiixase of the com scutellum is more
correctly a glucokinase vhich is not inhibited by its product, G6P.

SUMMARY

A hexofcLnase was extracted from the scutellum of com and
purified with an "apparent" 70-fold increase in specific activity.
It was free of interfering enzyne activities and appeared to be the
only soluble hexoldnase of the scutellioa. The enzyme showed greatest
specificity towards glucose as the substrate and tlK data supports
the concliision that the hexokLnase is more specifically a glucokinase.
The enzyme phosphorylated D-glucose, D-mannose, D-fructose, D-glucos-
amine and 2"deoxy-D-glucose . It was specific for ATP as the phosphate
donor. N-acetylglucosaaine and xylose were coii5)etitive inhibitors of
glucose phosphorylation, while ADP and AMP were competitive inhibitors
of A3P. The divalent caticais of magnesium, cobalt and msuiganese
activated the enzyme with cobalt and manganese ion being 63 and
38 per cent as effective as magnesium, respectively. The heaojkinase
was not inhibited by hexose-6-phosphates at concentrations up to
500-times the glucose concentration. Optimum activity of the enzyme
was observed at pH 8.0 and lt9°C.

The properties of the hexokLnase firam the scutellum are compared
with those of the brain and ySast hexoki33ases and are discussed in
relation to glucose -uptake by the scutellum.

80

BIBLIOGRAPHy

1. Agosin, Mbises and Luisa Aravena. Studies on the metabolism of
Echinococcus granulosus* II. Glycolysis with special references
to hexokinases and related glycolytic enzymes. Biochim. Bipphys.
Acta 3^: 90. 1959.

2. Agren, G. and L. Engstrcm. Isolation of 2%>-labeled phosphoserine
from yeast hexoklnase preparation, incubated with labeled AOP or
glucose-6-phosphate. Acta. Chem. Scand. 10: ^89. 195^«

3. Bailey, K and E. C. Webb. Purification of yeast hexoklnase and
its reaction with ^^ • -dichlorodiethyl sulphide. Biochem. J.
42: 60. 19*^8.

k, Barnard, E. A. and A. Ramel. Studies on the active center of
yeast hexokinase. Biochem. J. Qh'. 72P. I962.

5. Belitzer, W. A. and K. S. Golovskaya. Phosphate acceptors in
"respiratory phosphozylation" in mt^cle tissue. Science 92: 536.
19^*0.

6. Berger, Louis, M. W. Slein, S. P. Colowick and C. F. Cori.
Isolation of hexokinase from baker's yeast. J. Gen. Physiol. 29:
379. 19i^6.

7. Bueding, E. and Joan A. IfecKinnon. Hexokinases of Schistosoma
3oni. J. Biol. Chem. 215: U95. 1955.

8. Cardini, C. E. The hexokinases of Escherichia coli. Enzymologia
lU: 362. 1951.

9. CiriUo, V. P. The transport of non-fenaentable sugars across the
yeast cell membrane, p. 3^3. In A. Kleinzeller and A. Kbtyk, (eds.),
Ifembi'aae Transport and tfetabolism. Academic Press Inc., New York.

10. Chefurka, V. Oxidative netabolism of carbohydrates in insects. I.
Glycolysis in the housefly Musca domestica L. Enzymologia 17 : 73*
195!^.

U. Christensen, W. R., C. H. Plimpton and E. G. Ball. The hexokinase
of the rat erythrocyte and the influence of hormonal and other
factor on its activity. J. Biol. Chem. I80: 791- 19'<-9.

81

82

12. Colowick, S. P., E. M. Kklckar and C. F. Cori. Glucose
phosphorylation and oxidation in cell-free tissue extracts.
J. Biol. Chem. 137: 3^+3. 19^1.

13. Colowick, S. P. and A. M. Kalclsar. The role of layokinase in
transphosphorylations . I. Ihe enzymatic phosphorylation of
hexoses by adenyl pyrophosphate. J. Biol. Chem. iW: lO.?. 19^3.

Ik. Colowick, S. P., Gerty T. Cori and M. W. Slein. The effect of
adrenal cortex and anterior pituitary extracts and insulin on the
hexokinase reaction. J. Biol. Chem. I68: 583. IS^l'

15. Crane, R. K. and A. Sols. She association of hexokinase with
particulate fractions of hrain and other tissue homogenates.
J. Biol. Chem. 203: 273. 1953.

16. Crane, R, K. and A. Sols. Ihe nonccmipetitive inhibition of brain
hexokinase by glucose -6 -phosjihate and related cooqoounds. J. Biol.
Chem. 210: 597. 195^.

17. Crane, R. K. and A. Sols. Animal tissue hexokinases (soluble and
insoluble forms), p. 277. In S. P. Colowick and N. 0. Kaplan,
(eds.), Msthods in Enzymology I. Academic Press Inc., Ifew York.
1955.

18. Crane, R. K. Intestinal absorption of sugars. Physiol. Rev.
1<0: 789. i960.

19. Crane, R. K., D. Miller and I. Bthler. Ths restrictions on
possible mechanisms of intestinal active transport of sugars,

p. i^39. fc A. Kleinzeller and A. Kotyk, (eds.), Megfl?rane Transport
and Metabolism. Academic Press Inc., Ifew York. 19bO.

20. Crane, R. K. Hexokinases and pntokinases, p. hj. In P. D. Boyer,
H. Lardy and K. ^yrbSck, (eds.j. The Enzyiaes. Academic Press Inc.,
New York. 1962.

21. Crane, R. K. Studies on the mechanism of intestinal absorption of
sxigars. VI. Tixe specificity and other properties of Ka+-dependent
entrance of sugars into intestinal tissue under anaerobic
conditions, in vitTO. Biochim. Biophys. Acta 59: 9'<-. I962.

22. Crane, R. K. and S. M. Krane. On the mechanism of intestinal
absorption of sxigars. Biochim. Biophys. Acta 20: 5^8. 1956,

23. Darrow, R. A. and S. P. Colowick. Hexokinase from baker's yeast,
p. 226. In S. P. Colowick and K. 0. Kaplan, (eds.). Methods in
Enzymology V. Academic Press Inc., New York. I962.

83

2i^. Davidson, E. A. Hexoklnase from AsperglXlus parasiticus*
J. Biol. Chem. 235: 23. i960.

25. Di2»n, N. and D. M. Ifeedham. Biochemical research on chemical
warfare agents. Nature I58: it32. 19h6»

26. DrabMn, D. L. Hyperglycemia, glycosuria and dephosphorylation—
the role of phosphatase. Proc. Amer. Diabetes Assoc. 8: I71. 191^8.

27. Dratz, A. F. and P. Handler. Benal phosphate and carbohydrate
metabolism studied with the aid of radiophosphorus . J. Biol.
Chem. 197: IH9. 1952.

28. Edetoan, J., S. I. Shihlao and A. J. Keys. Eie role of the
scutelltm of cereal seedlings in the synthesis and transport of
glucose. J. Exp't. Botany. 10: 178. 1959.

29. Euler, H. and Adler, E. liber die Kop^ponexrten der Dehydrasesysteme .
VI. Dehydrierung von Hexosen unter Mitwirioing von Adenosintriphos-
phorsaure. Z. phj^iol. Chem. 235: 122. I935.

30. Pelsete, Maria A. Rongine de and C. E. Cardini. Mechanism of
glucose transfer from sucrose into the starch granule of sweet
com. Arch. Biochem. Biophys. lOij-: 173. I964.

31. Froom, H. J. Ths mechanism of brain hexokinase. Fed. Amer. Soc.
Esp't Biol: Fed. Proc. Abst. 21: 2ij-3. 1962.

32. Fromra, H. J. and Virginia Zewe. Kinetic studies of the brain
hexoklnase reaction. J. Biol. Chsm, 237: 1661. 1962.

33. Fronna, H. J. and Virginia Zewe. Kinetic studies of yeast hexo-
kinase. J. Biol. Chem. 237: 3027. I962.

3^. Prydman, Rosalia B. Starch synthetase of potatoes and waxy maize.
Arch. Biochem. Biophys. 102: 242. I963.

35.

Gamble, Jr. J. L. and V. H. Najjar. Hexokinase reversibility
measured by an exchange reaction using C^^-labeled glucose.
Nature 120s 1023 . 195^.

36. Geiger, A. Glycolysis in cell-free extracts of brain. Biochem.
J. 3^: 1^65. 19^.

37* Glasziou, K. T. Accumulation and transformation of sugars in
sugar cane stalks. Plant Physiol. 35: 895. 196O.

38. Glasziou, K. T. Accumulation and transformation of sugars in

stalks of sugar cane. Origin of glucose and fructose in the inner
space. Plant Physiol. 36: 175. 1961.

39* Gottschalk, A. Further obsersrations on liie non-feimentability
of d-fructopyranose by yeast. Biochem. J. kl: i<-78. 19^7 •

40. G3:lffiths, Jfervyn. Inhibition of enzymatic transphosphorylation
by alloxan and ninhydrin in tissue extracts. Arch. Biochem. 20:
1^51. 19l^9.

kl. Haraaes, G. G. and D. Kochavi. Stvidies of the enzyme hexokinase.

I. Steady state kinetics at pH 8. J. Amer. Chem. Soc. 8*+: 2069,
1962.

k2, HaaBaes, G. G. and D. Kochavi. Studies of the enzyme hexoMnase.

II. Kinetic inhibition by products. J. Amer. Chem. Soc. 8l^:
2073. 1962.

U-S, Bsmaea, G. G. and D. Kochavi. Studies of the enzyme hexokinase.

III. The role of the metal ion. J. Amer. Caiem. Soc. 6h: 2076.
1962.

kk. Hoare, D. S. and Margaret Ksrly. Phosphorylation of carbohydrates
in retinal extracts. Biochem. J. 58: 38. 195^.

k3. Hochster, R. M. and R. Wade Watson. Phosphoiylation of xylose by
extracts of Pseudomonas hydrophila. Nature I7O: 357- 1952.

k6. Horecker, B. L., M, F. Osbom, W, L. McLellan, G. Avigad and

C. Asensio. ©le role of bacterial peiraeases in metabolism, p. 378.
to A. Kleinzeller and A. Kstyk, (eds.). Membrane ^ansport and
Metabolism. Academic Press Inc., New YorkI I960.

k7* aanphreys, T. E. and L. A. Garrard. Glucose uptake by the com
scutellum. Phytochem. 3: 61f7. 196U.

kQ. Itoh, Tatsuro. The preparation of soy bean hexokinase. J. Agr.
Chem. Soc. (Japan) 3**: 275. I960.

49. Itoh, Tatsuro and Yoshiyki Inouye. Soy bean hexokinases and some
of their reaction products. J. Agr. Chem. Soc. (Japan) 3^*. 305.
i960.

50. Kaji, A., K. A. Trayser and S. P. Colowick. AOP-ase activity of
crystalline hexokinase. Ited. Amer. Soc. Ejcp't. Biol: Fed. Proc.
Abst. 20: 233. 1961.

51. Kalckar, Ksrman. The nature of jtoosphoric esters formed in kidney
extracts. Biochem. J. 33: 631. 1939*

52. Kaufman, Seymour. Studies on the mechanism of the reaction
catalyzed by the phosphorylating enzyme. J. Biol. Chem. 2l6: 153.
1955.

85

53. Kerly, &iargaret and D. H. Leaback. The chaiticteristics of

hexokinase from Locusta mlgratorla ntuscle. Biochem. J. 67: 2if5«
19?7.

^, Kerly, Margaret and D. H. Leatoack. The hexose specificity of
hexoklnase in brain from various species. Biochem. J. 6?: 250.
1957-

55. KLein^ H. P. Some properties of the hexokinase of Pseudomonas
putrefaciens. J. Bacteriol. 66: 65O. 1953 •

56. Krahl, M. E., A. K. Keltch, C. P. Walters, and G. H. A. Clowes.
Hexokinase activity from eggs of the sea iirchin Arbacia :punctulata.
J. Gen. Physiol. 38s 31. 195**.

57. Kunitz, M. and I^feirgaret R. I-JacDonald. Crystalline hexokinase
(hetercrphosphatese). Ifethod of isolation and properties. J. Gen.
Physiol. 29: 393. 19^.

58. Lange, Jr. C. F. and Paul Kbhn. Substrate specificity of hexo-
kinase, J. Biol. Chem. 236: 1. I96I.

59. Layne, Ennis. Spectrophotometric and turbidimetric nethods for
measuring proteins, p. W-7. Jn S. P. Cclovick and N. 0. Kaplan,
(eds.), !fethods in Enzymology III. Academic Press Inc., Nev York.
1957.

60. Leloir, L. F., J. M. Olavarria, Sara H. Golderherg and H.
Carminattis. Biosynthesis of glycogen from uridine diphosphate
glucose. Arch. Biochem. Biophys. 8I: 508. 1959.

61. Landau, B. R. and T. H. Wilson. Role of phosphorylation in
glucose absorption from the intestine of the golden hamster.
J. Biol. Chem. 23^: 7^9. 1959'

62. Line-weaver, H. and D. Burk. The determination of enzyoie
dissociation in constants. J. Amer. Chem. Soc. 56: 658. 193^.

63. Long, C. Studies involving enzymatic phosphorylation. I. Ths
hexokinase activity of rat tissues. Biochem. J. 50: U07. 1952.

64. Lowery, 0. H., Nira J. Rosebrou^, A. L. Farrard, Rose J. Randall.
Protein measurement vith the Polin phenol reagent. J. Biol. Cbem.
193: 265. 1951.

65. Lundsgaai^, E. Effect of phlorizin on the isolated kidney and
isolated liver. Skand. Arch. Physiol. 72: 265. 1935.

^, laitwak-Mann, C. and T. Mann. Uber die Verlaettiing der chemischen
tfasetzungen in der allsoholischen G&mng. I. Mitteilung:
Bildung un Spaltung der Adenosintriphosphorsfiure und deren
Zusaiomenhang mit den VorgHngen der Zuclserspaltiang. Biochem. Zeit.

281: i4o. 1935.

67. Maley, Frank and H. A. Lardy. Synthesis of N-substituted
glucosamines and their effect on hexokinase. J. Biol. Chem. 2llt:
765. 1955.

68. MacDonald, Margaret R. Yeast hexokinase, p. 269. In S. P.
Colowick and N. 0. Kaplan, (eds.), Msthods in Enzymology I.
Academic Press Inc., New York. 1955*

69. McCcHiib, R. B. and W. D. Yushok. Properties of particxilate hexo-
kinase of the KreT3S-2 ascites tiiBior. Biochim. Biophys. Acta 3^:
515. 1959.

70. Jfedina, Antonio and D. J. D. Mcliolas. Some properties of a
zinc-dependent hexokinase from Ifeurosy^ra crassa. Biochem. J. 66s

?r3. 1957.

71. Ifekata, G., Y. Nishikava, B. Hisimira, T. Takahashi, Y. Morita,

S. Kakaniura and T. Itoh. Caaftohydrate metabolism in higher plants.
V. Distribution of hexokinase in higher plants and in some
oviparoiis animals. Shiga daigaku Gakugeigalsabu Kiyo 7: 85. 1957*

72. VSembrams Transport gmd Metabolism. Proceedings of a symposium in
Prague. A. Kleinzeller and A. Kotyk, (eds.) Academic Press Inc.,
Mev York. I96O.

It

73. !feyerhof, 0. Uber die enaymatische Milehs&urebildung im
Muskelextrakt. III. Mitteilung: Die MUchsSurebildving aus dsm
gfirfShigen Hexosen. Biochem. Zeit. I83: 176. 1927.

7U. Meyerhof, 0. Uber die Wirkungsweise der Hexokinase.
Katurwissenschaften 23: 850. 1935 •

75. Meygrhof, 0. and Jean R. Wilson. Studies on glycolysis of brain
preparations. V. Affinity of hexokinase for glucose and
fructose. Arch. Biochem. 19: 502. 1^.

76. Morgan, H. E., R. L. Post and C. R. Park. Glucose transport and
phosphorylation in the perfused heart of normal and diabetic rats,
p. It23. 3^ A. KLeinzeller and A. Kbtyk, (eds.), tfembrane Transport
and Ifetabolism. Academic Press Inc., New York. I960.

77. Ifejjar, V. A. and E. E. McCoy. Mechanism of action of imiscle and
yeast phosphoglucomutase and suggested mechanism for yeast hexo-
kinase. Fed. Amer. Soc. Exp't. Biol: Fed. Proc. Abst. 17: ll^i-l.
1958.

87

78. Ochoa, Severe. Glycolysis and phosphorylation in brain extracts.
J. Biol. Chem. l4l: 2^5. 19*H.

79* Handle, P. J. Monosaccharide transport in muscle and its

regulation, p. 1*31. In A. KLeinzeller and A. Ifotyk, (eds.),
Msnfljrane Transport and Metabolism. Academic Press Inc., New York.

80. Bobbins, E. A. and P. D. Boyer. Determination of the equilibrium
of the hexokinaee reaction and the free-energy of hydrolysis of
ATP. J. Biol. Chem. 2^: 121. 19^.

81, Ruiz-Amil, M. The hexoMnase of the honey bee. J. Ins. Physiol.
8: 259. 1962.

82. Saltman, P. HexofcLnase in higher plants. J. Biol. Chem. 200:
1^5. 1953.

83, Slein, M. W., Gerty T. Cori.and C. F. Cori. A comparative study
of hexokinase from yeast and animal tissues. J. Biol. Chem. I86:
763. 1950.

8h, Smith, P. J. C. Carbohydrate metstbolism in Spirochaeta

recurrentis . k. Some properties of hexokinase and lactic
dehydrogenase in Spirochaetes . Biochem. J. 76; 51^ ♦ I96O.

85. Sols, A. and R. K. Crane. OSie Inhibition of brain hexokinase by
adenosiiKdiphosphate and sulfhydryl reagents. J. Biol. Cbem. 206:
925. 195it.

86. Sols, A. and R. K. Crane. Substrate specificity of brain hexo-
kinase. J. Biol. Chem. 210: 581. 1951!..

87. Sols, A. The hexokinase activity of the intestinal mucosa.
Biochim. Biophys. Acta 19: lkk» I956.

88. Sols, A., Gertrude de la Fuente, Carlos Villar-Palasi' and
Carlos A^ensio. Substrate specificity and sosie other properties
of baker's yeast hexokinase. Biochim. Biophys. Acta 30: 92. I958.

89. Strauss, R. R. and A. G. Moat. A role for biotin in yeast
glycolysis. J. Biol. Chem. 233: 765- 1958.

90. Strickland, L. H. Kie inhibition of glucolysis by glyceraldehyde.
Biochem. J. 35: 859. 19^1.

91. Stromme, J. H. Inhibition of hexokinase by disiOfiram and diethyl
dithiocarbamate. Biochem. Pharmacol. 12: 157 . 19^3 .

92. Szymona, M. and W. Ostrowski. Inorganic polyphosphate glucokinase
of Mycobacterium phlei. Biochim. Biophys. Acta 85: 283. 196^*.

93« Taha, M. M. and Y. M. A. El-Towesy. Phytase enzyms. J. Egypt.
Jfed. Assoc, kit 113. 1958«

Sk. Trayser, K. A. and S. P. Colowick. Properties of crystalline

hexokinase from yeast. I. Analyses for possible cof actors. Arch.
Biochem. Biophys. 9l<-: I56. I96I.

95* Trayser, K. A. and S. P. Colowick. Properties of crystalline
hexokinase from yeast. II. Stiidies on ATP-enzyme interaction.
Arch. Biochem. Biophys. Ski I61. I96I.

96. Ttayser, K. A. and S. P. Colowick. Properties of crystalline

hexokinase from yeast. III. Studies on glucose -enzyme interaction.
Arch. Biochem. Biophys. Skt I69. I96I.

ST* Trayser, K. A. and S. P. Colowick. Properties of crystalline

hexokinase from ysast. IV. Multiple forms of the enzyme. Arch.
Biochem. Biophys. 9I1.: I77. 196I.

98. Vinuela, Eladin, Ifergarita Salas and A. Sols. Glucokinase and
hexokinase in liver in relation to glycogen synthesis. J. Biol.
Chem. 238: PC11T5. I963.

^. Walaas, Eva and Otto Walaas. lbs activation of muscle hexokinase
by divalent metal ions. Acta Chem. Scand. 16: 1682. I962.

100. Walker, D. G. and Shyamala Rao. The effects of glucose analogs
on the hepatic glucose-phosphorylating enzymes. Biochim. Biophys.
Acta 77: 662. 1963.

101. Wajzer, M. J. Cinltique de I'hexokinase. Comp. rend. (Paris)
236: 2116. 1953.

102. Weil-Malherbe, H. and A. D. Bone. Studies on hexokinase. 1.
The hexokiiiase activity of rat-brain extracts. Biochem. J. If9:
339. 1951.

103. Wick, A. H., D. R. Drury, H. I. Nakada and J. B. Wolfe.
Localization of the primary metabolic block produced by 2-deoxy-
glucose. J. Biol. Chem. 22J*: 963. 1957.

lOlj-. Wiebelhaus, V. D. and H. A. Lardy. Phosphorylation of hexoses by
brain hexokinase. Arch. Biochem. 21: 321. 19'»-9»

BIOGRAPHICAL SKEZICH

Hei^ert Charlss Jones III vas bom October 6, I936, in
LeesbiH^, LalsE County, Florida. He attended public schools in
Ocala^ Flozlda and vas graduated from Ocala High School in June,

le attei^ed the Coloarado School of Mines and -ttie tfedvei^ity of
Floilda before entering the United States Air Force in 1956. While
in the service he carried the forsaer Wiaaifred Catheriiae Gassavay of
Jacksonville, Florida. After tw3 yeaars of active duty vith the Air
Force he returned to the Ifaiversity of Florida and was graduated
August, i960, vith the Degree of Bachelor of Science in Forestxy.

In Septeaiber, 196O, he be^ua graduate study at the University of
Florida, majoring in Botany. In June, 1965* the Degree of Doctor of
Philosophy vas conferred on him.

He is the father of three children: Catherine Anne, Winifred
Gail and Herbert Charles.
. Be is a member of tte f&llowing honoiury societies: Xi Sigma Pi,
Sigma Delta, Alpha Zeta and Phi Siesta.

This dissertation vas prepared under the direction of the
chainoan of the candidate's supervisory caamittee and has been
approved by all laembers of that coBanittee. It vsis submitted to
the IJean of the C!ollege of Agriculture aad to the Graduate Council^
and was approved as partial fulfil2meafc of tbe requirements fov
the degree of Doctor of Philosophy.

June 22, 1965

Supervisory Ctonmittees

^A.

Chairman

Dean> College of Agriculture

Dean, Gi-aduate School

80 23 t