Group Title: purification and properties of a hexokinase from the corn scutellum
Title: The Purification and properties of a hexokinase from the corn scutellum
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Title: The Purification and properties of a hexokinase from the corn scutellum
Physical Description: vi, 90 leaves : ill, ; 28 cm.
Language: English
Creator: Jones, Herbert Charles, 1936-
Publication Date: 1965
Copyright Date: 1965
 Subjects
Subject: Plant physiology   ( lcsh )
Enzymes   ( lcsh )
Glucokinase   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1965.
Bibliography: Includes bibliographical references (leaves 87-88).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Herbert Charles Jones.
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Bibliographic ID: UF00097904
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000467258
oclc - 37463284
notis - ACN1598

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THE PURIFICATION AND PROPERTIES OF A

HEXOKINASE FROM THE CORN SCUTELLUM



















By

HERBERT CHARLES JONES III


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA

June, 1965














The writer wishes to exiress sincere appreciation to Dr. T. E.

Humphreys for his help, guidance, leadership, patience and for the

use of his laboratory facilities in the course of this research; to

Dr. G. Ray Noggle who inspired the writer to pursue graduate study;

to Drs. D. 8. Anthony, R. H. Biggs and T. W. Stearns for their aid and

service on the ccamittee; and to the Department of Botany and the

Department of Health, Education and Welfare for financial support

through National Defense Education Act Title IV and National

Institutes of Health Fellowships.


ACKIOWLEDGEMENTS














TABLE OF COETLTS


ACI LEDGMEI . . . . . . . . . . . .

LIST OF TABLES ........................

LIST OF FIGURES .......................

I ODUCTION .........................

REEW OF LTERATURE .....................

MATERIALS AND MEHIODS .....................

Plant Materials

Preparation of the Enzyme

Extraction
Ammonium sulfate fractionation
Absorption and elution from alumina C- gel

Assay Methods

method 1
fthod 2
Assay for phosphofructokinase, phosphoglucomutase and
glucose-6-phosphatase activities

Protein Determination

Chemicals and Enzymes








Page

RESULTS ............................ 42

Purification

Substrate Specificity

sugars
Nucleoside triphosphates

Metal Activators

Inhibitors

Sugars
Nucleoside di- and triphosphates
Sugar phosphates
Anions

pH and Temperature Optima

DISCUSSION .......................... 74

SIM4ARY ................... ......... 80

BIBLIOGRAPHY ................. ...... 81

BIOGRAPHICAL SKETCH . . . . . . . . . . 89













LIST OF TABLES

Table Page

1. MICHALIS COSTAiTS (Ki) AMD RELATIVE M:m~ AL
RATES FOR BRAIN AD YEAST EO SE . . . . . 7

2. EFFECTS OF GLU~OE-6-P AND RELAmD COMPOUmDS O
PHOSPHORATIONS BY BRAIN HMONASE . . . .. 9

3. PUR IATION OF HEO~AE . . . . . . . 43

4. ATPASE ACTIVITY OF T~E HEmKNS PREPARATIO . . .. 5

5. suBSTRAM SPECIFICITY OF comN SCTEJIUM
EXOICEtAS ....................... 49

6. EFFECT OF MCL.EOSIDEHRIP OSFEATES AS SSUISPITw ES
FORAT~ ......................... 55

7. ACTIVATION OF EXKIASE BY MAL IOS . . . . . 60

8. EFFECT OF NUCLEOSIDE DI- AND TRIPHOSPHATS AS
INHIBITORS OF ATP IN THE EXDNASE REACT . . . 68













LIST OF FIGURES

Figure Page

1. Effect of glucose concentration on the rate of
phosphorylation ..................... 52

2. Lineveaver-Burk plot of the effect of glucose
concentration on the rate of phosphorylation . . 5

3. Effect of ATP concentration on the rate of
phosphorylation, and competitive inhibition
by ADP and AMP ..................... 57

4. Lineveaver-Burk plots of the effect of ATP
concentration on the rate of phosphorylation,
and competitive inhibition by ADP and AMP . . ... 59

5. Effects of Co e and Mn+H concentrations on the
rate of phosphorylation . . . . . . .... 62

6. Effect of Mg+ concentration on the rate of
phosphorylation ..................... 62

7. Lineveaver-Burk plots of the effect of PM++, Co"-
and Mnf- concentration on the rate of
phosphorylation ..................... 64

8. Lineweaver-Burk plots of the competitive inhibition
of glucose phosphorylation by xylose and D-
acetylglucosamine ................... 67

9. Effect of pH on the rate of phosphorylation
expressed as the per cent of the maximum rate
obtained ........................ 71

10. Effect of temperature on the rate of
phosphorylation ................... .. 73













INTRODUCTION


The properties of plant enzymes, especially those of higher

plants, are less well known than those of similar enzymes isolated

from animal tissues. It has been assumed that the properties

associated with the characterized animal and lower 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 (47) have presented evidence

which suggests that the hexokinase reaction may be important in control-

ling the rate of glucose uptake by the corn scutellum; an organ

positioned between the root-shoot axis and endosperm of the corn seed,

where glucose absorbed from the endosperm is converted to sucrose which

is subsequently translocated to the developing seedling during germina-

tion (28). Their data indicates that glucose-6-phosphate competitively

inhibits an enzymatic step associated with net glucose uptake in

scutellum slices, and they suggest that the step might be the

hexokinase-catalyzed phosphorylation of glucose. Since it has been

demonstrated that brain hexoklnase (15, 16) and to a small extent,

yeast hexokinase (33), is inhibited by glucose-6-phosphate, although








2

noncompetitively, it seemed desirable to investigate the properties

of corn scutellum hexokinase.













REVIEW OF LITERATURE


Historical

In 1927, Meyerhof (73) gave the name, hexokinase, to an alcohol

precipitable fraction of autolyzed yeast which when added to extracts

of aged frog or rabbit muscle greatly enhanced glycolysls. Independ-

ently, in 1935, Euler and Adler (29) and Lutwak-Mann and Mann (66),

isolated an enzyme from yeast--heterophosphatese-which catalyzed the

following reaction in the presence of Mgr-:

Hexose + ATP~e--- -xose-6-phosphate + ADP

Mzyerhof (74), in a description of the hexokinase reaction the same

year, reported that the enzyme described by the two groups was the

same one responsible for the activity of his original preparation.

Kalckar (51), in 1939, demonstrated that kidney extracts

phosphorylated glucose and fructose and, in 1940, Geiger (36) observed

that extracts fran brain tissue phosphorylated fructose, mannose and

glucose. Belitzer and Golovskaya (5), the same year, showed that

muscle tissue contained hexokinase, which in the presence of glucose

and creatnine catalyzed the phosphorylation of glucose to hexose-6-

phosphate. In 1941, Ochoa (78) showed the presence of hexokinase in

acetone powder of rat brain. The acetone partially inactivated

adenosinetriphosphatase (ATPase). The hexokinase required Mt+ and

transferred phosphate from ATP to glucose without the liberation of

free orthophosphate.









Colowick et al. (12), in 1941, demonstrated that phosphorylation

of glucose preceded the oxidation of glucose in heart muscle and

kidney extracts and that hexokinase catalyzed phosphorylation of

mannose to mannose-6-phosphate (M6P). The M6P was in turn converted

to fructose-6-phosphate (F6P) by an isomerase.

In 1946, Berger et al. (6) and Kunitz and MacDonald (57) succeeded

in crystallizing hexokinase from yeast, the only organism from which it

has been crystallized. The hexokinases from various sources have been

recently reviewed by Crane (20).

Hexokinases of Various Animal Tissues

Mammalian brain hexokinases. Colowick et al. (14) prepared

purified beef brain hexokinase by eluting acetone powder front beef

brain with water and fractional precipitation of the eluate with

aimonium 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 45 and 50 per cent saturation with ammonium

sulfate. The product of the beef brain hexokinase reaction was shown

to be glucose-6-phosphate (G6P).

Mayerhof 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 same for glucose and much lower for

fructose. They observed the same difference relative to the ATP

concentration and there was a greater ATPase 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 (10) noted that dialyzed extracts of beef

brain showed different hexokinase activities vith 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 hexokinases. Their beef

brain extracts promoted the phosphorylation of glucose, fructose and to

a lesser extent, mannose, but were not active with L-glucose, galactose,

L-sorbose, D-gluconate, 2-keto-0-gluconate, D-ribose, D-arabinose,

L-rhamnose and D-xylose. Inorganic pyrophosphate inhibited activity

46 to 80 per cent depending on its concentration. Magnesium ion, up

to two times the concentration of the pyrophosphate, did not reverse

inhibition. Orthophosphate was only slightly inhibitory.

Long (63) found that of five rat tissues--brain, liver, kidney,

skeletal muscle, and intestine--brain had the greatest amount of

hexokinase activity, while liver had the least.

Crane and Sole (16, 17, 86) prepared particulate hexokinase 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 Weil-Malherbe and Bone (102) had reported in 1951) 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 determine which groups of the

glucose molecule were possibly involved in formation of a glucose-

brain hexokinase complex. Sixteen of the analogs served as substrates









(Table 1), five behaved as competitive inhibitors, and the remainder

were inactive. They estimated the relative influence of each hydroxyl

group of glucose by comparing the Michaelis constants (Km) and the

relative maximal rates (Vmax) of phosphorylation (Table 1). The con-

clusion was that the formation of a glucose-enzyme complex involved

the ring structure and hydroxyl groups at carbon atoms 1, 3, 4 and 6,

and that each hydroxyl group had a specific quantitative influence on

enzyme-substrate affinity.

Crane and Sols (16) investigated the specificity for inhibition

of the brain hexocinase 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 and 4

and the phosphate group at carbon 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 and, therefore, noncompetitive.

Maley and Lardy (67) tested the effects of a variety of N-substi-

tuted glucosamines on brain hexokinase. All were powerful inhibitors

(K-10O'3M to 10HM). They interpreted their data (and from studying

molecular models) as indicating that the substituted glucosamines





7

TABLE 1

MICHAELIS CONSTAITS (Km) ARI RELATIVE MAXIMAL RATES FOR BRAIN AND
YEAST HEXOKINASE

Data from references (86) and (23)


(1)
Modified
at
Carbon


(2)

Compound


glucose

1 1,5-sorbitan

1,2 1,5-mannitan

1 glucoheptulose

1,2 mannoheptulose

1,2 fructose

1,2 2,5-sorbitan

1,2 arabinose

1 methyl- C-glucoside

1 methyl- F-glucoside

1 1-thioglucose

1,2 glucal

1 oc-glucose-l-P

1 glucono-1,5-lactone

2 mannose

2 2-deoxyglucose

2 glucosamine

211 glucosone

2 N-acetylglucosamine

2 N-methylglucosamine


(3) (4)
Brain
Km Rel.
(M/L) Max.
Vel.

8xlo-6 1.0

3X10-2 1.0

2X10o2 0.9

2X10- 0.006

5X10-5 0.015

210-3 1.5

2X10-1 0.08

2 0.1

+ +

+ +

+ +

+ +

+ +

+ +

5x10-6 0.4

3X10' 1.0

8XI-5*** 0.6

1X10-5 0.08

8X10-5** +

2X10'** +

(TABLE 1 continued next page)


(5) (6)
Yeast
Km Rel.
(M/L) Max.
Vel.

iX10-4 1.0

3X10-3 0.01





2X0o-4* 0.001

7X10"4 1.8



>1X10-1 0.02

>1X10o- <0.001










5x10-5 0.8

3X10-4 1.0
3x1o"' l.o

2X103 0.7

2X10-5 0.2

1X10-3* <0.001





8

TABLE 1 (continued)


(1) (2) (3) (4) (5) (6)
Modified Brain Yeast
at Compound Km Rel. Km Rel.
Carbon (M/L) Max. (M/L) Max.
Vel. Vel.


2 2-0-methylglucose

3 allose

3,2 altrose

3 3-deoxyglucose

3 3-0-methylglucose

4 galactose

4,3 gulose

5,4 1,4-sorbitan

5,1 L-sorbose

6 6-deoxyglucose

6 xylose

6,2 lyxose

6,3 ribose

6 6-deoxy-6-fluoroglucose

1,2 1-methylfructose

2 2-C-hydroxymethylglucose

1,2,3 3-O-methylfructose

sorbitol

glucoguloheptose

i-inositol


+ +

X1o-3 0.5

3Xio'3 0.11

2X10-2 0.2

+ +

1X10"1 0.02
I +


+I

2X10'3*

2X10"3"

110-3**
,1


>1X10 1 0.1





>1X10o
5XlO'2 0.002


+ >x10'*
+ >Lvxlo1- <0.001


1X102* <0.001





5X10'3* <0.001

>2X10"3 <0.001

2>X10U 3 <0.001

>1%10':3 <0.001


*Inhibition constant (Ki)
**Determined by competitive inhibition of fructose
***Determined at pH 7.5
+Undetectable
P=phosphate






9
TABLE 2

EFFECT OF GLUCOSE-6-P AND RELATED COMPOUNDS ON PHOSPHORYLATIONS BY
BRAIN HEXOKINASE

Data from reference (16)

(1) (2) (3) (4) (5) (6)
Modified Ester Substrate Ester Per cent Ki
at concen- inhibi- (M/L)
Carbon No. traction tion

1 1,5-Sorbitan-6-P 2-Deoxyglucose 0.67 38 1X10"3

1 oC-Glucose-l,6-diP 2-Deoxyglucose 0.6 47 7X104

1 @-Glucose-1,6-diP 2-Deoxyglucose 1.0 0

1 Glucoheptulose-7-P 2-Deoxyglucose 8.6 0

1 Methyl-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 Glucosamine-6-P Glucose 1.5 0

2,1 Fructose-6-P 2-Deoxyglucose 2.5 0

2,1 2,5-Sorbitan-6-P 2,5-Sorbitan 13 0

2,1 Fructose-1,6-diP 2-Deoxyglucose 1.8 O

2,1 1,5-Mannitan-6-P 1,5-Mannitan 4.0 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

2,3,4,1 Xylose-5-P 2-Deoxyglucose 3.3 0

3 Allose-6-P 2-Deoxyglucose 9.2 58 7X103

3 3-Deoxyglucose-6-P Mannose 3.2 14 2X10-2

4 Galactose-6-P Mannose 20 0

(TABLE 2 continued next page)





10

TABLE 2 (continued)


k1) (2)J
Modified Ester
at
Carbon No.

5,1 L-Sorbose-1-P

6,1 o<-Glucose-l-P

No ring Gluconate-6-P

No P Glucuronate


(3) (4) (5) (6)
Substrate Ester Per cent KI
concen- inhibi- (M/L)
tration tion

2-Deoxyglucose 0.45 40 7X10"4

2-Deoxyglucose 2.5 0

2-Deoxyglucose 1 0

2-Deoxyglucose 2 0









combined with the active enzyme site and that the inhibition might be

through blocking of the site on the enzyne to ATP, since the substi-

tuted groups did not overlap the carbon 6 position on the sugar.

By kinetics studies of the mechanism of the brain hexokinase

reaction, romm (31) and FroMe and Zewe (32) found that 06P acted as

a competitive inhibitor of AIP and as an uncompetitive inhibitor with

respect to glucose. Inhibition by ADP was of a more complex nature,

but appeared to be noncompetitive with respect to ATP and uncompetitive

with respect to glucose. They concluded that their results were

consistent with a mechanism involving either a phospho- or a gluco-

enzyme complex, but were at variance with any mechanism in which both

substrates need to be present simultaneously on the enzyme for the

reaction to occur. They also concluded that there seemed to be no

reason for G6P occupying a third site on the enzyme. They presented

the following compulsory pathway type mechanism for the brain bexo-

kinase reaction:

(1) Enzyme + ATP Enyme-X complex + ADP

(2) Enzym-X complex + glucose E azyme-Y complex

(3) Enzyme-Y complex --- Eaym + G6P

The nature of the Enzyme-X and Enzyme-Y complexes was unknown. The

mechanism is similar to that presented by Hamas and Kochavi (42) for

the yeast hexokinase reaction.

Kerly and Leaback (54) measured the specificity of hexokinaae of

the brain of several nonnamsmlian 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









glucose and fructose. With pigeon brain extract, phosphorylation of

fructose was inhibited by mannose and N-acetylglucosamine, while

phosphorylation of both fructose and glucose was inhibited by G6P and

F6P. The affinity of pigeon brain hexokinase for fructose was similar

to that of the beef brain enzyme, except beef brain hexokinase was not

inhibited by F6P (16).

iMuscle. Crane and Sols (17) 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). The 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 brain hexokinase. Skeletal muscle

hexokinase activity was optimum at pH 8.0.

Crane and Sols (17) partially purified particulate heart muscle

hexokinase by fractional centrifugation and solubilization of the

enzyme from 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, 1,5-sorbitan-6-P, and L-sorbose-l-P for heart

muscle hexokinase were 25 per cent, or less, of those for the brain

enzyme.

Strickland (90) observed that glycolysis by a muscle extract in

the presence of added hexokinase could be inhibited by 0.003M








13

glyceraldehyde, but the inhibition could be reversed by a small excess

of hexokinase. Be concluded that glyearaldehyde inhibition could be

pin-pointed as inhibition of glucose phosphorylation by hexokinase.

Walaas and Walaas (99) extracted an acetone powder of rat skeletal

muscle with tris-EDTA solution and obtained a five-fold increase in

specific activity by fractionating the eluate with cold ethanol. The

ethanol precipitate was dried in vacuo. Fractionation of either the

acetone powder eluate or the ethanol-precipitable fraction with

anmonium sulfate decreased recovery. EDTA, when added to the crude

hcoogenate from which the acetone powder was prepared, increased

recovery. Added glucose or ATP only slightly stabilized the ensyme.

Prolonged contact with 0.05M MgC completely inhibited the enzyme.

Chloride ion was without effect. Orthophosphate provided slight

protection and 0.03M pyrophosphate was very effective in stabilizing

muscle hexokinase. However, 0.005M pyrophosphate strongly inhibited

hexokinase during incubation of the reaction mixture. Potassium

chloride, in a narrow range of 0.02 to 0.05M, slightly activated the

hexokinase during incubation while orthophosphate inhibited. The

hexokinase had a pH optimum at 8.0 to 8.2 and one-third maximum activity

at pH 7. Maximum activity of hexokinase was observed at a molar ratio

Mg/ATP 1 for several concentrations of ATP. Magnesium ion in excess

of ATP, except at relatively high concentrations, was not inhibitory.

At a molar ratio ATP/Mg greater than 4, ATP inhibited the reaction.

The Km for both ATP and Mg was 1.7X10"3M. Inorganic pyrophosphate

inhibited the hexokinase reaction competitively with respect to MgATP

when the molar ratio was 1. At inhibitory concentrations of Mg+,







14

pyrophosphate had a small activating effect on the enzyme, which they

interpreted as a release of Mgs inhibition. They suggested that

inorganic pyrophosphate inhibited hexokinase by forming a Mg++-pyropbos-

phate complex which excluded MgATP from the enzyme-substrate complex

and furthermore, that the MgATP complex was the actual substrate (other

than glucose) for muscle hexokinase. Muscle hexokinase was also

activated by Cat", Co+,, Mn"+ and, to a very small extent by Zn'-. The

maximum activities for these activators were lover than that for Mg+

and the Km for each was lower than that for Mgt. 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 activation. They 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),

guanidinetriphosphate (GTP) and uridinetriphosphate (UTP) would not

substitute for ATP in the muscle hexokinase reaction.

Griffiths (40) found that 0.005 alloxan inhibited muscle hexokinase

completely and 0.005M ninhydrin inhibited the enzyme 80 per cent.

Inhibition by 0.003M alloxan was reversed by 0.005M cysteine, but 0.01M

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 hexokinase. Long (63) measured levels of hexokinase in

several rat tissues and found that liver had the lowest level of hexokinase.









Crane and Sols (17) described a purification procedure for the

soluble hexokinase of rat liver. They concentrated a 100,000XG super-

natant fraction of liver extract with ammonium sulfate up to 50 per cent

saturation. Their liver preparation catalyzed phosphorylation of

glucose, mannose, fructose, glucosamine and 2DOG. 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 were also substrates of rat liver hexokinase. They studied

hexokinase in rat intestine, kidney and liver extracts and their data

indicated that while the hexokinases from the three sources were similar,

they were not identical. The Km for glucose, 2DOG and glucosamine were

4X10-5, 9X10-5 and 3.7XI04M, 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 ammonium sulfate, while the

second enzyme precipitated between 60 and 70 per cent saturation. The

first enzyme had a low Km for glucose, was inhibited by G6P, and only

moderately by N-acetylglucosamine. The latter enzyme had a higher Km

for glucose, was not inhibited by G6P, was strongly inhibited by

N-acetylglucosamine, and had a low Km for mannose. They 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 micramole per minute per gram liver at 21 to 230C or








16

2 micromoles at 37C which was comparable to the rate of glycogen

synthesis from glucose in rat liver (0.4 to 1 micromole per minute

per g liver). The activity also approached the maximum rate of

glycogen synthetase (3 micromoles per minute per g at 37C). The

glucokinase disappeared in starved 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, which were

incorporated into glycogen, were phosphorylated by other hexokinases

specific for those sugars. The Km for glucose of the hexokinase and

of the glucokinase were found to be X105M and 1X0I2M, 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: (1) its activity was compa-

rable with the rate of glycogen synthesis, (2) it was not inhibited

by its product and (3) UDPG-glycogen glucosyltransferase is dependent

on high levels of G6P for maximum activity.

Walker and Rao (100) examined the effects of 2DOG, glucosamine

and N-acetylglucosamine on the hexokinase and glucokinase of rat liver.

The three compounds inhibited both enzymes competitively, but the Ki

for 2DOG was much higher for the glucokinase than for the other two

compounds. The Ki for 2DOG was comparable to the In for glucose for

the enzyme.

Kidney hexokinase. Kalckar (51) showed that kidney extracts

catalyzed phosphorylation of glucose and fructose. Colovick et al.

(14) demonstrated phosphorylation of mannose by kidney extracts.

Mannose isomerase in the extract converted M6P to F6P.









Lange and Kohn (58) found the Km for glucose, 2D00 and glucosamine

to be 4.8X10"5M, 9X1O'5M and 0.1M, respectively. Allose and talose were

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 atom 2 since glucosamine was such a

poor substrate.

Intestine hexokinase. Sols (87) examined the hexokinase of rat

intestinal mucosa, and found fructose was phosphorylated at rates less

than glucose, while galactose, 3-methylglucose, L-sorbose, manno-

heptulose, N-acetylglucosamine, xylose, ribose and L-arabinose were

not phosphorylated. G6P at 6X10"fI 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 Km for glucose and fructose were 2XIOT0 and

&X10-3M, respectively. These results failed to support the hypothesis

that the rate of phosphorylation of sugars limits the rate of sugar

absorption 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 was inter-

mediate, was phosphorylated at a rate greater than any of the other

sugars.

Lange and Kohn (58) found the Km for glucose, 2DOG and glucosamine

of rat intestine hexokinase to be 6.5X10o5, 9X10-5 and 3.3X101M,

respectively. Allose and talose, as with kidney hexoklnase, were also

active. Changes at carbon atom 3 of glucose (allose) had considerably









greater effect on activity than changes at carbon atom 2(2DOG or

glucosamine). Changes at either carbon atoms 2 and 3(altrose) or 3

and 4(gulose) resulted in loss of all activity. They concluded that

all the active substrates of kidney, liver and intestine hexokinase

were similar in at least two respects: (1) They had an available

hydroxyl at carbon atom 6 and (2) they had a hydroxyl available at the

anomeric position (carbon atom i).

Erythrocyte hexokinase. Christensen et al. (1l) found that the

hexokinase of rat erythrocyte phosphorylated glucose, mannose and

fructose at the relative rates 1.0:0.77:0.34 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 (44) 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. McComb 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 Km for glucose and

wtM vere 1.TX104 and 1X10'3M, respectively. The Ki for G6P inhibition

was 4XIO, while M6P, 2DOG6P and F6P were not inhibitory. Adenosine

diphosphate was inhibitory. Glucosone (Km 8X10'6M) was also phos-

phorylated by the tumor enzyme. The hexokinase activity was localized

in the mitochondria of both tumor and normal tissues.









Domestic fowl hexokinases. Mskata et al. (71) examined tissue

extracts from several organs of domestic fowl (white leghorn hen) and

found that the heart and gizzard hexokinases were active with both

glucose and fructose, while the enzyme of liver phosphorylated fructose

but not glucose.

Honey bee hexokinase. Ruiz-Amil (81) partially purified hexokinase

from honey bee by fractional precipitation of the enzyme between 50 and

70 per cent saturation with ammonium sulfate and column-chromatography

on DEAE-cellulose. The yield was 28 per cent of the original hexokinase

in the crude extract. The thorax contained the largest amount of hexo-

kinase activity (75 per cent). The enzyme showed optimum activity

between pH 7.5 and 8.8. Glacose-6-phosphate behaved as a competitive

inhibitor of glucose, while 2DOG6P and L-glycerophosphate did not

inhibit the enzyme to any extent. The KI for ATP and the Ki for its

competitive inhibitor, ADP, were 7.5X10'4M and SX10 M, respectively.

The Km for fructose (3X103SM) was similar to that of the mammalian

brain enzyme, while those for glucose (4X10"5M) and mannose (II0"-M)

were similar to those of yeast hexokinase. Ruiz-Anil concluded that

the enzyme from honey bee was similar to the hexokinase of other

animals in most respects.

Locust hexokinase. Kerly and Leaback (53) studied the hexokinase

in the thorax muscle and the salivary gland of locust (Locusta
migratoria). The properties of the enzyme were similar to those of

the n mmallan brain enzyme with respect to substrates and inhibitors.

Glucose had a Km of 5.5X10"3M. Both 06P and "6P, at a concentration

of 5i10'3M, completely inhibited hexokinase activity.








20

House fly hexokinase. Chefurka (10) observed hexokinase activity

in a soluble extract of house fly (Musca domestic L.).

Sea urchin egg hexokinase. Krahl et al. (56) studied hexokinase

in egg and embryo homogenates of sea urchin (Arbacia punctulata). The

enzyme was shown to have a pH optimum of 7. The substrate specificity

of the enzyme was similar to that of the other animal hexokinases. The

Km for the substrates that were examined fell in about the same range

(2 to 8X10-SM).

Bivalve hexokinase. Mekata et al. (71) observed that the

hexokinase of the fresh-water mussel (Hyrpsis schleeli) 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: (1) glucokinase, (2) fructokinase,

(3) mannokinase and (4) glucosaminekinase. The glucokinase was

inhibited by ADP, G6P, sorbose-l-phosphate (SIP), and glucosamine-6-

phosphate. The inhibition of the glucokinase by ADP was noncompetitive

with respect to ATP. The Km for glucose, Mg++ 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 (1) partially

purified the hexokinases of hydrated cyst scolices of Echinococcus








21

granulosus. They isolated four separate hexokinases: (1) glucokinase,

(2) fructokinase, (3) mannokinase and (4) glucosaminekinase.

2-Deoxy-D-glucose was not phosphorylated by any of the hexokinases.

Gluco-, manno- and fructokinase were inhibited by G6P, while M6P

inhibited gluco- and mannokinase. Phosphorylation of fructose by

fructokinase was competitively inhibited by ADP. Glucokinase was

inhibited by p-chloromercuric benzoate (PCMB) and the inhibition was

reversed by cysteine.

Spirochaeta hexokinase. Smith (84) examined a hexokinase of the

particulate system of Spirochaeta recurrentis. The enzyme showed

maximum specificity toward glucose and mannose, while fructose and

L-sorbose were phosphorylated to a lesser extent. Galactose was not

active. Glueosamine and N-acetylglucosamine inhibited glucose phos-

phorylation. The enzyme was quite sensitive to low concentrations of

ADP.

Pseudomonas hexokinases. Klein (55) observed that extracts of

Pseudomonas putrefaciens catalyzed phosphorylation of glucose and

glucosamine. The hexokinase of the extracts showed a very low affinity

for fructose (0 to 10 per cent relative to glucose) and mannose (0 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 (Km = 2.3X103M).







22

Escherichia hexokinase. Cardini (8) partially purified the

hexokinases of Escherichia coll. Extracts of E. coli 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. coli and S. aurens could be induced

to phosphorylate galactose in the carbon 1 position by growing the

cells on lactose.

Mycobacterium phlei inorganic polyphosphate glucokinase. Szymona

and Ostrowski (92) isolated an inorganic polyphosphate glucokinase

from Mycobacterium 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 1.75X0In M and 2.8XI0 lM, respectively.

Plant Hexokinases

Yeast hexokinase. Berger et al. (6) and Kunitz and MacDonald

(57) 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 recrystallized up to six times.









Darrow and Colovick (23) reported a simpler method that gave a

good yield and high purity. Their method employed fractional precipi-

tation of the enzyme with ammonium sulfate, absorption and elution

from bentonite 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

Colovick 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 Km'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 ATF

(23) in the reaction. The requirement for Mg was not replaced or

antagonized by Ca+ (2).

Kaji at 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









and sorbose-l-phosphate inhibited the ATPase activity of the

hexokinase preparations. They observed parallel inactivation of the

ATPase and hexokinase activities by various amounts of silver nitrate.

Chromatography of the crystalline hexokinase on DEAE-cellulose gave

fractions in which the ATPase activity/hexokinase activity was

identical. The Em of the ATPase for ATP was 5X10'3M compared to

IX10'4M for that of the hexokinase. The hexokinase and ATPase were

found to have distinctly different specificity towards nucleotides.

Berger et al. (6) reported that thiol compounds provided no

protective action for their crystalline enzyme, while Bailey and Webb

(3) reported that all SH poisons, including Lewisite, were powerful
inhibitors of crystalline yeast hexokinase. Dixon and Needham (25)

reported inhibition of yeast hexokinase by mustard gas, while Stromme

(91) demonstrated that disulfiram was a potent inhibitor of the

enzyme, as was diethyldithiocarbamate when oxidized to disulfiram by

cyctochrome C. Barnard and Raael (4) obtained results that indicated

one to four -SH groups per molecule were required for the active

centers) of yeast hexokinase and that these groups ware not normally

available for reaction with -SH reagents, but probably became available

after a time-dependent structural change occurring at 300C. Berger

et al. (6) found that fluoride at 0.1254 did not inhibit crystalline

hexokinase when )Mg+ and phosphate were present in concentrations of

6.5 and X10-3M, respectively, but Bailey and Webb (3) observed that

0.04M NaF inhibited hexokinase activity 46 per cent.

Glucose-6-phosphate has been reported to be both inhibitory to

(101) and without effect (13, 102) on yeast hexokinase. From and








Zere (33) presented data that was interpreted as indicating that G6P

acts as a competitive inhibitor of both forward reaction substrates.
They suggested that the concentrations of A P used by other investi-

gators may have been too high to see inhibition. Trayser and Colowick

(96) reported the dissociation constant of G6P to be 4X10-3M, while
a ,nces and Kochavi (42) found it to be Xo10"'.
Strauss and Moat (89) reported that biotin stimulated fermentation

of glucose and fructose by air-dried yeast grown in a medium deficient

in the vitamin. Biotin also stimulated hexokinase activity in extracts
from cells grown on deficient medium. Trayser and Colovick (94) found

fully active yeast hexokinase did not contain biotin. The results

indicated that the enzyme was not a metalloprotein and did not contain

a readily demonstrable prosthetic group.

Crystalline yeast hexokinase has been reported to be a protein

of the albumin type with a manima molecular eight (MW) of 96,000

(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 105 g
protein per minute at 300C and pH 7.5 (6). The Q10 of the reaction

between 0 and 300C has been reported to be approximately 1.9 (6) and

is rapidly inactivated at temperatures above 55-600C (88).

Six-times crystallized hexokinase was separated into at least two

major components 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 Colovick (22) to be

indistinguishable in specific activity, in Km values for substrates









and in sensitivity to inhibition by various substrates. However,

Trayser and Colowick (97) reported that the crystalline enzyme was

separated into six molecular forms (isozymes) of equal specific

activity by DEAE chromatography. The isozymes showed different

catalytic properties with respect to Km, Vmax, pH optima, ATPase

activity and sensitivity to inhibitors.

Berger et al. (6) found that the crystalline enzyme shoved a

loss of activity when highly dilute, which could be prevented by

diluting the enzyme 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, 80), have demonstrated the

reversibility of the hexokinase reaction by measuring exchange of

either C14-labeled glucose between G6P and glucose or P32-labeled ADP

between ADP and ATP. However, the equilibrium greatly favored G6P

synthesis.

Agren and Engstrom (2) isolated phosphoserine from an acid

hydrolyzate of purified yeast hexokinase incubated in P32-labeled

ATP or G6P. They suggested that the hexokinase reaction involves the

formation of a stable phosphoenzyme intermediate. NaJJar and McCoy

(77) ruled out the phosphoenzyme hypothesis for yeast hexokinase when

they found no exchange of phosphorus between C1l-labeled glucose and

C2-labeled G6P (or vice versa) in the presence of the enzyme. They

then postulated the following mechanism for the reaction:








(1) Glucose-enzyme +ATP.-> 6-phosphoglucose-enzyme + ADP

(2) 6-Phosphoglucose-enzyme + glucose ->.glucose-enzyme + G6P

They argued that the formulation accounted for: (a) the lack of

exchange of phosphorous between glucose and G6P, (b) labeling of the
enzyme with G6P32 or ATP32 on the assumption that labeling of

phosphoserine might have been due to the transfer of phosphorous

during acid hydrolysis of the protein, (c) the marked inhibition of

the enzyme by G6P, and (d) C14-glucose labeled the enzyme. They con-

tended that when G6P accumulates, the greater part of the enzyme

would exist in the phosphoglucose-enzyme form, thereby bringing about

a corresponding reduction in the glucose-enzyme concentration which

would retard the forward rate of the first reaction and consequently

inhibit the overall reaction. They reported that C1-labeled glucose

labeled 30-60 per cent of the enzyme. One flaw in their formulation

is that there is no report in the literature that G6P markedly

inhibits yeast hexoklnase.

From and Zewe (33) examined the kinetics of the yeast hexokinase

reaction and they interpreted their results as being consistent with

the "classical" mechanism in which glucose and ATP add randomly to

the enzyme and equilibrium kinetics prevail. Their data indicated

that participation of either a phosphoenzyme complex or a glucoenzyme

complex was unlikely. Their results showed that mannose and ADP

behaved as competitive inhibitors of glucose and ATP, respectively.

Inhibition by AMP was competitive with respect to ATP and noncompeti-

tive with respect to glucose.







28

Trayeer and Colowick (95, 96) come to essentially the same

conclusions as Froma and Zewe (33) since they could not detect either

a phosphoenzyme complex or a glucoenzyme complex in their kinetic

studies of the yeast hexokinese reaction.

Hamess and Koehavl (41, 42, 43) did a detailed study of the

kinetics of the yeast hexokinase reaction. They derived from their

data what they considered to be the most probable mechanism for the

reaction. It involved the combination of MgATP and a glucoenzyme

complex to form two quaternary intermediates which in turn decomposed

to MgADP and a dissociable 6-phoaphoglucoenzyme complex. They pre-

sented the following compulsory pathway mechanism which is similar to

that postulated for brain bexokinase:

(1) Mg- + ATP-g-- ATP

(2) E + G- G complex

(3) E G + EMgAP X-- XA E 0 + GADP

(4) E 06P E + G6P

(5) MSADP -- .++ + ADP

here E, G, X1, X2 and G6P are enzyme, glucose, first quaternary com-

plex, second quaternary complex, 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 g + may not be a very important factor for binding of

the substrate to the enzyme, but that the primary role of Mg++most

likely is to polarize the oxygen-phosphate bond that is being broken,

while anchoring the phosphate group of ATP to the enzyme. Nuclear








29

magnetic resonance studies indicated that binding of MgATP to the

enzyme might occur through the adenine and/or ribose portion of ATP.
Hales and Kochavi (42) observed a large difference in the

binding constants for glucose and GP by the enzyme and interpreted

it as indicating that either the hydroxyl group at carbon atom 6 of

glucose was important in the binding process or that the electrostatic

effect of the charged phosphate group inhibited binding of 06P to the

enzyme. They further concluded that G6P broke binding of 4gATP very

effectively. It can be seen from the foregoing that the mechanism of

the yeast hexokinase reaction is unclear, and Fromn and Zeve (33) point

out that the differences may be in the assumptions made and in the

interpretation of data.

Aspergllus hexokinase. Davidson (2e) purified the hexokinase of

Aspergillus parasiticu 225-fold. He employed fractional precipitation

with amonium sulfate (50-70%), followed by fractionation with cold

acetone (38-46%), negative absorption to alumina Cy gel, and refractiona-

tion vith ammonium sulfate (60-80%). The purified enzyme phosphorylated

D-glucose, D-galactose, D-glucosamine, D-galactosamine, D-mannose and

D-fructose at the relative rates (based on glucose) of 1.00:0.86:0.58:

0.40:0.67:0.36, 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 Em for glucose

and galactose were almost identical (l.6X10 M compared to 4.3XIOp- ).

They interpreted their data as indicating that all the substrates were

phosphorylated by the same enzyme and that the substrate specificity

suggested that the hexolinese was different from that of either the

yeast or brain enzyme.







30
Neurospora hexokinase. Medina and Nicholas (70) purified the

hexokinase of Neurospora crassa 60-fold. The enzyme phosphorylated

glucose and at a lower rate, mannose, fructose and glucosamine. The

enzyme was noncompetitively inhibited by G6P and was competitively

inhibited by N-acetylglucosamine. Iodoacetate, EDTA and PCMB were also

inhibitors of the enzyme.

Higher plants. Saltman (82) demonstrated the occurrence of

hexokinase in several plants and purified the soluble hexokinase 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 characterization

of the enzyme. Saltman's results, however, showed that the soluble and

insoluble enzymes were almost identical in their properties.

The enzyme 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 Km for glucose was 4.4X10'A

and that for ATP was 8.7X104M, while the Km for glucose by the soluble

enzyme was 4.6X10'4. Galactose, ribose, arabinose, ribulose, adenosine,

glyceraldehyde, dihydroxyacetone, mannitol and glucose-l-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 0.01M, while Co++ was

ineffective. Cupric, zinc and mercuric ions strongly inhibited the







31

enzyme. Potassium, sodium and ammonimu ions neither activated nor

inhibited the enzyme. Substances which influence -SH groups had little

effect on either the soluble or insoluble hexokinase. Zinc ion

inhibited the enzyme noncompetitively. Dinitrophenol (5X10"3M) inhibi-

ted phosphorylation 70 per cent, while G6P (5XIO'3M) only inhibited 17

per cent. The wheat germ hexokinase was found to be similar to yeast

hexokinase.

Mekata et al. (71) demonstrated the occurrences of hexokinase

in a variety of higher plants.

Itoh (48) partially purified hexokinase from homogenates of

soybeans, Ozuki beans and mung bean by fractional precipitation of the

crude homogenates with ammonium sulfate. The hexokinase of soybean

precipitated at about 60 per cent saturation. The highest activity of

soybean hexokinase appeared three days after germination at 260C.

Itoh and Inouye (49) separated three different hexokinases of

soybean by fractional precipitation with ammonium sulfate. They

identified: (1) a glucokinase, specific for glucose and glucosamine,

(2) a fructokinase and (3) a galactokinase, specific for galactose and

galactosamine.

Comparison of Animal and Plant Hexokinases

It appears that animal and plant hexokinases are similar in

substrate specificity and, in many cases, Km. The principal differences

appear to be the following:

(1) Hexose-6-phosphates produce a more marked inhibition of

animal than of plant hezokinases.









(2) ADP inhibition of plant hexokinases appears to be

competitive with respect to ATP, while in animals it

appears to be of a more complex nature.

(3) The hexokinase reaction catalyzed by brain hexokinase from

animals seems to follow a compulsory pathway mechanism

involving a stable phospho- or glucoenzyme complex(es).

Although there is disagreement, it appears that the enzyme

in plants might promote a random interaction of glucose and

ATP in the presence of Mg", without the formation of stable

phospho- or glucoenzyme complexes (33, 95).

Hexokinase and Sugar Uptake

The subject of sugar uptake 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 concerning the role of hexokinase

in sugar uptake.

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 sugar-phosphate pool with p32 is inconsistent with the

phosphorylation-dephophohrylation 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, all actively

absorbed sugars have the common structure: -




H
Sugars 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 Cl chair form. SH also points out that the configura-

tion specificity at carbon atom 2 doesn't necessarily hold for other

animal tissues. He observed that the process of sugar absorption by

the intestine follows Michaells-Mfnten kinetics, requires the presence

of the free sugar, and doesn't depend on the gross metabolism of the

substrate since 3-0-mnthyl-D-glucose, which is absorbed at a great rate,

is not metabolized.

Crane et al. (19) and Crane (21) observed that sugar uptake by

the intestine depends upon the presence of Na+ ions and that the extent

of accumulation depends on the external concentration of Na+. They

proposed that glucose, on entering the brush border cells of the intes-

tine, combines with a carrier-Na+ complex. The complex moves through

the diffusion barrier to the interior of the cell where the Na+ portion









of the complex is extruded back through the diffusion barrier in the

reverse direction by an energy requiring process, which is inhibited

by strophanthidin, leaving the glucose "trapped" in the cytoplasm

(e.g. the so-called sodium "pump" involving the membrane 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 sugars appear to share a common membrane

transport mechanism.

Morgan et al. (76) have demonstrated, from their studies of glucose

transport and phosphorylation in the perfused heart of normal and

diabetic rats, that glucose uptake is controlled by the combined opera-

tion of two sequential steps, membrane transport and intracellular

phosphorylation, and that in the steady state the net rate of membrane

transport and intracellular phosphorylation are equal. In the absence

of insulin, glucose uptake 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

Km for glucose phosphorylation by hexokinase in diabetic heart muscle

is at least 7 times higher than in normal tissue. They found insulin

in vitro to have no large, immediate effect on glucose phosphorylation.

Randle (79) reported that the uptake of glucose and the

accumulation of D-xylose in isolated rat diaphragm are accelerated by

inorganic phosphate, G6P, GIP, 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

2,4-dinitrophenol were also found to accelerate glucose uptake, but

such treatment also increased the rate of uptake of substances

(sorbitol) which are not ordinarily absorbed by diaphragm tissue. He

suggests that uptake might be regulated by phosphorylation and dephos-

phorylation of a membrane carrier.

Horecker et al. (46) observed that a mutant strain of E. coli

(Kn6), 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. The galactokinase

is also inducible in the wild type cells. When ATP 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 (47) with respect to

glucose uptake by corn scutellum slices are reviewed in the introduction

and discussion sections of this dissertation.

Glasziou (37, 38) has reported detailed studies of sugar uptake

and transformation in immature sugar cane internodal disks. His

results do not indicate a role for hexokinase in sugar uptake by that

tissue.













MATERIALS AND MBBODS


Plant Materials

Corn grains (Zea mays L., var. Funk's G-76) were soaked

twenty-four hours in running tap water and planted, scutellum-side up,

on four layers of moist filter paper on trays. The trays were covered

with aluminum foil and placed in the dark at 23C for five days. The

scutella were removed from the germinated grains and placed in ice-cold

glass distilled water until all the scutella were harvested. All sub-

sequent steps were carried out in the cold or in an ice-water bath.

Preparation of the Enzyme

Extraction. The scutella were weighed and then ground in a

chilled Waring Blendor for one and a half minutes in four volumes (v/v)

of ice-cold 0.005M ethylenediaminetetraacetate (EDTA), 0.005M magnesium

chloride (MgC12) and 0.01M potassium chloride (KCl), pH 7.0. The use

of either two volumes or six volumes of extracting solution decreased

recovery of the hexokinase. Cysteine (0.005M) or glutathione (0.01M)

did not increase recovery.

The homogenate was squeezed through two layers of cheesecloth

and the filtrate was centrifuged at OC and 2,000XG for one hour. The

supernatant fraction was filtered through glass wool to remove the

fatty layer and was then centrifuged at 0C and 32,00XG for one hour.

The supernatant fraction was filtered through glass wool and saved.

This fraction was designated "crude homogenate."







37

Ammonium sulfate fractionation. The crude homogenate was made

50 per cent saturated in ammonium sulfate ((lii)2SO04) by slow addition

of the salt to the magnetically stirred solution. The solution was

allowed to equilibrate, with stirring, for fifteen minutes after the

addition of the salt. The solution was centrifuged at 12,000XG for

twenty minutes at OOC. The precipitate was dissolved in a minimum

amount of 0.05M potassium phosphate buffer, pH 7.0, 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

handled 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 treatments

with alumina Cr gel. The first two treatments absorbed substantial

amounts of the ATPase (Table 4) and the final treatment absorbed the

hexokinase which was used for this research. The following paragraph

describes the procedure that was used to purify the enzyme with

alumina Cy gel.

Absorption and elution from alumina Cy gel. The F-2 fraction

was treated with solid alumina Cy gel (7.4 per cent solids, Sigma

Chemical Company) as follows: (1) The F-2 fraction was treated with

0.0137g gel per ml (equivalent to 2.4 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









mixture was centrifuged at 0C and 12,000XG for twenty minutes. The

supernatant fraction was decanted and saved for further treatment.

The precipitate was dispersed and eluted for four hours with a volume

of 0.2M ammonium sulfate equal to one-third the volume of the F-2

fraction. The mixture was centrifuged at 00C and 12,00OXG 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 -200C until

needed. This adjustment was necessary because the enzyme was inacti-

vated during storage at pH's below approximately 7. This eluate was

designated "A". The precipitate was eluted a second time with the

same volume of 0.4M ammonium 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 to 4.7 mg 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-l".

(3) To the supernatant fraction of the second gel treatment was added

0.0951 g per ml of gel (equivalent to 7.1 mg solids per ml) and the mix-

ture centrifuged and eluted as before. The two eluates were designated

"C" and "C-l". Fractions C and C-1 were the preparations used to

obtain the results reported in this paper. Both preparations had

identical Km for glucose, ATP and Mg"e and were considered to be the

same enzyme. They contained some adenosinetriphosphatase (ATPase) and

phosphoenolpyruvic phosphatase (PEPase) activity. Sodium molybdate

(Na2Mo207), 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 concentrating them along with the hexokinase.

Assay Methods

Two methods were used for the assay of hexokinase activity and

for characterizing the enzyme.

Method 1. The rate of glucose-6-phosphate (G6P) formation at

2500 was measured by following nicotinamide adenine dinucleotide

phosphate (NADP) reduction in the presence of excess glucose-6-phosphate

dehydrogenase (G6PD) spectrophotometrically at 340 millimicrons. The

standard reaction cuvette contained the following: glucose, 20 micro-

moles; MgC12, 20 micromoles; Adenosine-5'-triphosphate, 20 micro-

moles; NADP, 1 micromole; Tris buffer, pH 8.0, 180 micromoles; G6PD,

1EU; hexokinase preparation and water to 3.2 or 3.3 ml. The blank

cuvette did not contain G6PD. This method was used to measure: (1)

the rate of phosphorylation of glucose and fructose (coupled to

phosphoglueoisomerase), (2) competitive inhibition by nons.abstrates

and glucose-l-phosphate (GIP), (3) nucleotide activation and inhibi-

tion, (4) metal activation, and (5) pH and temperature optima, which

were examined by running the hexokinase reaction at the various pH's

and temperatures in a total volume of 6 ml with twice the above

ingredients, except for HADP and 06PD. 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 NADP and G6PD.

Method 2. The rate of adenosine-5'-diphosphate (ADP) production

at 250C by hexokinase was determined by measuring the oxidation of

reduced nicotinamide adenine dinucleotide (HADH) in the presence of







40

excess phosphoenolpyruvate (PEP), pyruvic kinase (PK) and lactic

dehydrogenase (LD) spectrophotometrically at 340 millimicrons. The

standard reaction cuvette contained the following: Tris buffer, pH

8.0, 180 micromoles; NADE, 0.282 micromoles; PEP, 10 micromoles;

Na2Mo207, 6 micromoles; MgC12, 20 micromoles; 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 NADH and a control was included, which

contained no sugar, to measure ATPase activity. This method was used

to measure: (1) the rate of phosphorylation of mannose, 2-deoxy-D-

glucose, L-glucose, L-mannose, N-acetyl-glucosamine, ribose, xylose,

and galactose, and (2) inhibition by nonsubstrates and the hexose-6-

phosphates, namely, G6P, 2-deoxy-D-glucose-6-phosphate (2DOG6P) and

mannose-6-phosphate (l6P). Mannose-6-phosphate inhibition could not

be measured by the G6PD reaction because it contained fructose-6-

phosphate (F6P) which was converted to G6P by phosphoglueoisomerase

in the scutellum hexokinase preparation.

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-6-

phosphatase activities. Phosphofructokinase was assayed by coupling

the phosphorylation of F6P to NADH oxidation in the presence of excess

aldolase, triosephosphate isomerase (TPI), and C-glycerophosphate

dehydrogenase (c-GPD). Phosphoglucomutase activity was determined

by Method 1 above, using GIP as the substrate. Glucose-6-phosphatase








41

was measured by the same method after incubation of the hexokinase

preparation with a Imown amount of G6P for ten minutes.

Protein Determination

Protein was estimated using the Folin-Ciocalteu reagent method

of Lowery et al. (64) as described by Layne (59). The colorimetric

readings were made on the Beclman DU Spectrophotometer at 720 milli-

microns. Crystalline bovine serum albumin was used as the standard.

Chemicals and Enzymes

All the chemicals and enzymes used in this investigation were of

the highest purity obtainable commercially. The enzymes were pur-

chased from the California Foundation for Biochemical Research. The

nucleotides, IADP, NADH, PEP, G6P, 1sP, F6F, 2DOG, and 2DOG6P were

obtained from Sigma Chemical Company. The other chemicals were

obtained either from the above firms, or from Nutritional Biochemicals

Company or Fisher Scientific Company.













RESULTS


Purification

The hexokinase in the crude homgenate could not be assayed

satisfactorily because of interfering enzyme activities.

Table 3 outlines the results of a typical purification of the

hexokinase from the scutellum. Although the amount of hexokinase in

the crude homogenate could not be estimated accurately, Table 3 shows

an "apparent" 30-fold increase in specific activity of the 50 to 75

per cent saturated amonium 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 between 50 and 75 per cent saturation

with ammonium sulfate. The F-2 fraction contained the following

enzymes which might interfere with either one or the other assay:

glucose-6-phosphatase (G6Pase), phosphoenolpyruvic phosphatase

(PEPase), phosphoglucoisomerase (PGI), and adenosinetriphosphatase

(ATPase), but did not contain phosphoglucomutase (PGM), phosphofructo-

kinase (PFK), 6-phosphogluconic dehydrogenase and enzymes that destroy

reduced HAD or NADP. The G6Pase activity should not have interfered

seriously with the G6PD 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 Cy gel resulted in a

2.3-fold increase in specific activity of the C-l fraction over the

42













TABLE 3
PURIFICATION OF HEOKIXNASE


Specific
activity Total % Recovery
Fraction (micromoles/min/mg activity from
protein) (units*) F-2 fraction

Crude homogenate 0.002 2.80 --
F-I (0-50%(Nm( )2804) 0.002 0.57 ---
F-2 (50-75%(NHB)gSo0 ) 0.060 12.60 ---
A (1st alumina C7 ) 0.026 0.64 5.1
A-l Igel treatment 0.012 0.11 0.9
B (2nd alumina ca-) 0.070 1.83 14.5

B-1 (gel treatment ) 0.049 0.81 6.4

C (3rd alumina C ) 0.102 3.45 27.4
( )
C-1 (gel treatment ) 0.141 3.91 31.0


*One unit amount of enzyme that will phosphorylate 1 micromole of
glucose per minute at 250C.







44

F-2 fraction. This amounted to an "apparent" 70-fold increase over

the crude homogenate. When the F-2 fraction was dialyzed against the

extraction solution (EDTA-Mg-ECl) the APase 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 the ATPase activity could be

satisfactorily separated from the hexokinase activity as is shown in

Table 4. Buffers such as potassium phosphate, glycylglycine and tris

at several concentrations and several pH values were ineffective in

elating the hexokinase from the gel. Tris buffer in concentrations

above 0.1M inhibited the hexokinase activity irreversibly. The C and

C-l fractions, which were used as the enzyme preparations in these

investigations, contained PEPase activity which was completely

inhibited by 0.0012 sodium molybdate, a small amount of ATPase activ-

ity (Table 4) for which a correction was applied, and PGI activity.

The G6Pase activity of the F-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 105,000XG for one and

one-quarter hours nor was it inhibited by ouabain (0.0005M to 0.005M)

or by fluoride (0.002M to 1.2M). The addition of Mg+ 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 F-2 fraction separated into 11 or 12 bands













TABLE 4

ATPASE ACTIVITY OF E ASE HEX E PEPAERATIICIS


Specific
activity Total % Recovery
Fraction (micromoles/min/mg activity from
protein) (units*) F-2 fraction

F-1 0.002 0.51 -

F-2 0.096 20.10

A 0.076 1.33 6.6

A-1 0.233 1.79 8.9

B 0.058 1.o4 5.2

B-1 0.134 1.55 7.7

C 0.025 0.58 2.9

C-i 0.020 0.38 1.9



*One unit amount of enzyme that will produce 1 micromole of
ADP per minute at 250C.









in the positive direction, while the C and C-i fractions separated

into four bands--two large bands between two smaller bands. The four

bands had Rf's corresponding to four similar bands separated from the

F-2 fraction. Hexokinase activity could be detected in one of the

bands, with Rf of 0.45-0.60, 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. The activity, 0.160

micromoles/cuvette/hour, was small and attempts to elute a significant

amount of the hexokinase from whole gel slices or homogenized slices

with several concentrations of glucose, phosphate buffer, ammonium

sulfate or glycylglycine buffer were unsuccessful. Most of the ATPase

activity in the gel was localized in a band with Rf of 0.77-1.0(calcu-

lated with reference to the distance traveled by the salt front). Such

a band had an activity of 0.037 micrcmoles/cuvette/hour. The other

bands also had some ATPase activity.

Acrylamide gel electrophoresis of commercial crystalline hexo-

kinase (CalBiochem) also gave four bands that had a separation pattern

similar to that of the C and C-1 fractions with the two large bands

having Rf's almost identical to those of the C or C-i 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 eluted with 1 ml of 0.04M glycylglycine buffer, pH 7.5, for thirty

minutes. The resulting eluate had an activity of 0.224 units/mi or

about 0.9 per cent recovery.

Attempts to purify the enzyme by absorption and elution from

bentonite by the method described by Darrow and Colowick (23) were







47

unsuccessful. The hexokinase and ATase activities were absorbed

but could not be eluted.

Fractionation of the enzyme preparations at any step in the

outlined procedure with cold acetone or ethanol (-70C) resulted in a

complete loss of activity.

Humphreys and Garrard (47) found that slices of the corn scutellum

take up glucose at the rate of 70-80 micromoles/g fresh weight/hour

(approximately 1 unit per g of tissue) but do not accumulate it.

Therefore, if it is assumed that all the glucose entering the slices

is phosphorylated, the hexokinase content of the tissue should be much

higher than that recovered. Since the F-l and F-2 fractions would

account for only about one-fifth of the total apparent hexokinase

activity of scutellum, attempts were made to increase the yield.

Reduced glutathione or cysteine in concentrations of 0.005M and 0.01M

in the extracting solution did not 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 (0.1 per cent) not only failed to increase the yield, but

also increased the solubility of lipid in the crude homogenates so that

it could 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 material was lower than that obtained by the method described in







48

the materials and methods section. Dialysis of the floating layer

did not solubilize the enzyme and treatment with cold acetone or

ethanol at -70C to remove the lipid destroyed the hexokinase activity.

Itoh (48) was able to use the floating layer to study the hexokinase

of soybean. Dimethylsulfoxide (DMSO) at concentrations of 0.1 and 1.0

per cent in the extracting solution did not affect the yield. Extracts

of acetone powders prepared from scutella yielded smaller amounts of

hexokinase activity.

The endosperm of the geminated corn grains did not contain

detectable hexokinase activity.

Substrate Specificity

Sugars. The specificity of scutellum hexokinase for sixteen

sugars was examined. The Michaelis constants (Io), maximum velocities

(Vmax) and relative Vmax with respect to glucose are given in Table 5.

The enzyme phosphorylated D-glucose, D-mannose, D-fructose, 2-deoxy-D-

glucose and D-glucosamine. Glucose had the lowest Km (6.X10"'5S) and

the greatest Vmax. The substrate concentration versus rate curve for

glucose and the corresponding Linewever-Burk plot (62) are presented

in Figures 1 and 2, respectively.

Nucleoside triphosphates. The Km for ATP was found to be 8X10'M.

Figures 3 and 4 show the curves from which this value was calculated.

Commercial preparations of UTP (uridinetriphosphate), GTP(guanosine-

triphosphate), CTP (cytidinetriphosphate) and TTP (thymidinetriphos-

phate) were found to be poor substitutes for ATP at the concentrations

that were examined (Table 6). The best substitute for ATP was TP which

gave 28 per cent of the rate of 0.0015M ATP at a concentration of 0.003M.










TABLE 5

SUBSTRATE SPECIFICITY OF CORN SCUTELLUM HEXOINASE


Km Relative
Substrate (MX105) Vmax"* Vax


D-glucose 6.4 0.047 1.00

D-mannose 9.3 0.028 0.59

D-fructose 168 0.046 0.97

2-deoxy-D-glucose 30 0.044 0.94

D-glucosamine 37 0.030 0.63

N-acetyl-D-glucosamine 55*

D-xylose 870*

c-methyl-D-glucoside --

3-O-methyl-D-glucose

L-glucose

L-mannose -

6-deoxy-L-mannose

D-galactose

D-galactosamine

D-ribose --

cC-D-glucose-l-phosphate --



*Inhibition constant (Ki)
*O.D./2 min/cuvette at 250C
"No detectable activity or inhibition at 0.03M







50

The low rates obtained by these compounds could have been due to a

small amount of nucleoside diphosphokinase activity for which the

preparations were not assayed. Saltman (82) observed that inosine-

triphosphate (ITP) was 35 per cent as effective as ATP as a phosphate

donor in the hexokinase reaction catalyzed by an insoluble hexokinase

from wheat. Walas and Walaas (99) could not detect hexokinase

activity with ITP, GTP or UTP with their preparations from muscle

tissue.

Matal Activators

Divalent metal cations were required for scutellum hexokinase

activity. Table 7 gives the NI and a comparison of the activation of

scutellum hexokinase by M C+, Co+ and Mn-. Figures 5, 6 and 7 show

the metal concentration versus rate curves and the Lineweaver-Burk

plots for the three metal cations. Magnesium ion was by far the best

activator. An inhibition of magnesium activation was observed when

the molar ratio of ATP/Mg exceeded approximately four. When the ratio

was 7.5 micromoles/1 micronole or 7.5/2 micromoles the rate, compared

to 2 micromoles/l micromole or 2 micromoles/2 micromoles, was 20 and

10 per cent lower, respectively, and caused the LIneweaver-Burk plot

for MgS 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 ratio was maintained. Such

inhibition is similar to that observed by Walaas and Walaas (99).



























80 to





. A
40 Ol
o *p.









H 14
t+ e




















































r) ( J-


Ol x u1'WF Ja9d '70V


P.r)










w
Lj~
0



C-)






























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
































0 2 4 6 8 10 12 14 16
I/(Glucose)













TABLE 6

EFFECT OF NUCLEOSIDEIPHOSPHATFS
AS SUBSRTIRTES FOR AP



Concentration % of Rate Obtained
Nucleosidetriphosphate (M3103) vith 1.510"-3M
AP*

UTP 1.5 19
3.1 28

GTP 1.5 14
3.1 17

CIP 1.5 14
3.1 19

TIP 1.5 15
3.1 19



*Mg+ concentration constant at 6X10'3M


























Figure 3. Effect of ATP concentration
on the rate of phosphorylation, and competitive
inhibition by ADP and AMP. Reaction cuvettes
contained: 180 micramoles Tris buffer, pH 8.0;
20 micromoles glucose; MC12 in amounts equal
to, or greater than, the concentration of
inhibitor plus ATP; 1 micromole of NADP; 1 EU
G6PD; 10 miromoles of ADP or 20 micromoles of
AM; 0.1 ml enzyme preparation and water to
3.3 ml.






























0 2 4 6
ATP Mx I03








iiH

1 04













































0
CM

4/1







60




TABLE 7

ACTIVATION OF HEXOKINASE BY M!AL IONS*


Kn Vmax % VMax
Ion (Mno05) ( O.D./2 min/cuvette) Relative
to M


M914 20 0.040 100
Co+" 70 0.025 63

Mn++ 100 0.020** 50**



%Ratio of ATP/metal ion was always maintained at less than 4.
"Calculated frcn extrapolated Lineveaver-Burk plot. Actual
observed relative VWas was 38 per cent.
















Figure 5. Effects of Co+- and Mn"
concentrations on the rate of phosphorylation.
The standard reaction mixture was used, except
the concentration of ATP was 10 micromoles
per cuvette.


















Figure 6. Effect of Mg++ concentration
on the rate of phosphorylation. The standard
reaction mixture was 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 ATP were
used.

















<1
0
^ 2



0N


Metal Mx103


Mg++ MX 103





























Figure 7. Lineveaver-Burk plots of the
effect of Mg~, Co++ and Mh++ concentration
on the rate of phosphorylation.

































2 3


S/(Metal)







65

No inhibition by Mg++ and Co++ was seen at the concentrations employed

in these experiments.

The addition of potassium ion or sodium ion did not cause activa-

tion of the enzyme.

Inhibitors

Sugars. Of the nonsubstrates listed in Table 5, N-acetylglucosa-

mine and D-xylose were found to be competitive inhibitors of glucose

with Ki of 55X10'5M and 87fOXO-M5, respectively. Figure 8 shows the

Lineveaver-Burk plots from which the Ki in Table 8 were calculated.

The other nonsubstrates had no inhibitory activity.

Nucleoside di- and triphosphates. ADP and AMP were found to be

competitive inhibitors of ATP with Ki of 14X10"5M and IXO'-3, respec-

tively. Figures 3 and 4 show the curves for inhibition by 0.003M ADP

and O.006M AMP. UTP, GI, IP, CTP, UDP and GDP were found to have

little inhibitory activity with respect to ATP at the concentrations

that were examined (Table 8). The slight inhibition of hexokinase by

these six compounds was not increased by increasing the concentration

of the nucleotides.

Sugar phosphates. No inhibition of glucose phosphorylation was

observed with G6P, F6P, MSP, GIP, galactose-6-phosphate (Gal6P) or

ribose-5-phosphate (R5P) at concentrations of the sugar phosphate up to

0.033M. The concentration of glucose in these experiments was that

which resulted in about half maimal velocity (6X10'SM).

Anions. Fluoride in concentrations of 0.002 to 1.2M did not

inhibit scutellum hexokinase. MgCl2 and gSO4 were equally effective

in activating the enzyme.














IB
4 1

4- 1,J
*IT^8 ^c

l ~I aI











































0 0 0 0 0
(-)- in re N -


rrr)








CCj
rO








(3












TABLE 8

EFFECT OF N~CLEOSIDE DI- AMD TRIPHOSPHATES AS INHIBITORS
OF ATP IN THE HEXOKIASE REACTION*



% of Rate
Nucleotide Concentration** Relative to
(MX0o3) 1.5Xo-3M ATP Alone


UTP -- -
3.1 95

GTP 1.5 91
3.1 89

lTP ---- --
3.1 80


3.1 96

ADP --
3.1 42

UDP 1.5 80
3.1 80

GDP 1.5 90
3.1 90



*M+ concentration constant at 6Xfl03M
c*ATP concentration vas 1.5X103M










pH and Temperature Optima

A pH optimum (Figure 9) was observed at pH 8.0, while the optimum

temperature for scutellum hexokinase was found to be 490C (Figure 10).

This optimum is similar to that observed for wheat germ hexo-

kinase (82), muscle hexokinase (99) and honey bee hexokinase (81).

The scutellum hexokinase lost activity rapidly when stored at pH's

below 7.

The temperature optimum (Figure 10) is much higher than that

reported for other hexokinases. Hexokinase isolated by Saltman (99)

from wheat germ had a temperature optimum of 3700 but Taha and

El-Towesy (93) reported that phytases from several seeds including bar-

ley and wheat, had temperature optima in the vicinity of 51C. Berger

et al. (6) found that the Q10 for yeast hexokinase was close to 2 over

the range of 0-300C. The Q10 of the hexokinase from the corn scutellum

(Figure 10) is 2 between 20 and 300C but falls off at temperatures above

and below this range. The Q10 might vary with the enzyme, the com-

ponents of the reaction mixture and at higher temperatures can be a

reflection of the rate of denaturation and the rate of the reaction.

















~f~~s
ap
s
I;i
8,
arf
i
1
o
rUD:











































000000 00000
0 o oo r-- o uo re) N -
9/oI lnwL!XOk/ jO /Ua3J9c




















514-*^
84.. C3i




































u! WO/d-9-9 sOaloA












DISCUSSION


The results indicate that the pattern of substrate specificity of

scutellum hexokinase is similar to that of yeast hexokinase. The Km

of the scutellum enzyme for D-glucose, D-cannose, 2-deoxy-D-glucose

and ATP are of the same order of magnitude as those of the yeast

enzyme (Table 1). The Km for D-fructose is an order of magnitude

larger. However, Gottschalk (39) has shown that the furanose ring

configuration of fructose is the true hexokinase substrate and, at

equilibrium, about 20 per cent of a fructose solution is in the furanose

form. If this is the case with the scutellum enzyme, the true Ke would

be about five times smaller than the value given in Table 5. The Km

for glucosamine is an order of magnitude smaller than that of yeast

hexosmnase.

The most significant difference between the hexokinase frcm the

scutellum and that from brain or yeast is the relative rate of phos-

phorylation of the other four substrates caopared to glucose. Glucose

is phosphorylated at a rate greater than any of the other substrates.

Apparently the scutellum enzyme shows a greater specificity with

respect to differences at carbon atom 2 of the glucose molecule than

does the yeast or brain hexokinases.

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

(. Vmax substrate X Km glucose ) is a true measure of the physiological
Vmax glucose Ka substrate
suitability of a substrate for hexokinase. With the scutellum enzyme

the coefficients of phosphorylation for D-glucose, D-mannose,

74









2-deoxy-D-glucose, D-glucosamine and D-fructose are 1.00:0.41:0.30:

0.11:0.04, respectively. These values indicate that D-glucose is the

most suitable physiological substrate for the scutellum hexokinase.

Correcting for the furanose configuration of fructose would place

fructose closer to (about the same as 2-deoxy-D-glucose), but still

lower than, glucose in suitability as a substrate.

The results from the limited studies of the substrate specificity

and of inhibition by the various nonsubstrates are essentially in

agreement with the findings of Sols et al. (88) for yeast hexokinase

and of Sole and Crane (86) for brain hexokinase. 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 competitive inhibitor. The enzyme also shows specificity for

hydroxyls at carbon atoms 1(G1P and %-methyl-D-glucoside) and

3(3-0-methyl-D-glucose) and for inversion of the hydroxyl group at

carbon atom 4 since galactose and galactosamine were not active as sub-

strates or inhibitors. Inversion of the hydroxyl 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 hydroxyla at carbon atom 6 were tested

except for the hexose-6-phosphates and the fact that they were not

inhibitory suggests that scutellum hexokinase requires a byaroxyl at

that position for binding. This is supported by the fact that xylose

is a weak competitive inhibitor.

Activation of the scutellum hexokinase by Mg+, Co++ and Mn++

closely resembles that observed by Walaas and Walaas (99) for muscle









hexokinase, although inhibition by MgH and Co+f was not observed at

the concentrations used in the experiments. Saltman (82) observed that

MnDb was 80 per cent as effective as MgH for activating the insoluble

hexokinase of wheat germ while Co++ was completely ineffective. The Km

(2X10 M) for activation of the seutellum hexokinase by M$g is a magni-

tude smaller than that reported by Walsas and Walaas (99) for the

muscle enzyme and by Berger et al. (6) for the yeast enzyme.

The results show that ADP and AMP appear to act as competitive

inhibitors of ATP. The Km 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 honey bee hexokinase

(81). Inhibition by ADP with respect to ATP for brain hexokinase has

been reported to be noncompetitive (27) or of a complex nature (32).

In the experiments reported in this investigation the magnesium ion

concentration was maintained at levels equal to, or greater than, the

concentration of substrate (ATP) plus 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 Garrard (47) from

their studies of glucose-uptake by scutellum slices, was not observed

in these investigations. The levels of AlT used in these experiments,

0.003 and 0.006M, may have mashed inhibition, as has been suggested by

Prom and Zeve (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 considers that the role of the scutellum in the

germinating seedling is essentially that of a "sucrose factory" and

that it is capable of absorbing and utilizing glucose at a rapid rate

(1 micromole/min/g fresh weight at 300C) it seems reasonable that the

sucrose and energy synthesizing systems of the scutellum should be

efficient ones and may be analogous to the glycogen synthesizing sys-

tem of rat liver studied by Vinuela at al. (98). They demonstrated

that there was a glucokinase in the liver which was distinguishable

from the typical animal bexokinase of that organ. The glucokinase had

a higher IN for glucose, was not inhibited by G6P and phosphorylated

glucose at a rate which was comparable to the rate of glycogen synthesis

in the liver. Leloir et al. (60) reported that uridine diphosphoglucose-

glycogenglucosyltransferase was activated by high levels of G6P. The

system might also be similar to starch synthesizing system in seeds

which appear to be highly specific towards substrates (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).
Humphreys and Garrard (47) observed that preincubation of scutellum

slices in water prior to introduction of glucose into the bathing

medium, increased the rate of glucose uptake by the slices. There 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









mannose from the slices. The residual inhibition was attributed to

the high level of MSP. The results reported in this paper suggest

that G6P and MSP are not inhibitors of scutellum hexokinase, and the

factors controlling glucose uptake in the scutellum must be other than

inhibition of the hexokinase reaction by hexose-6-phosphates. M6P has

not been found to be an inhibitor of brain (16) or yeast hexokinase

(83), but might well be inhibiting some step in the utilization of G6P.

2-Deoxy-D-glucose has been shown to be a competitive inhibitor of G6P

for the phosphoglucoisomerase of rat kidney (103). 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 an increase in

available AIP and removal of inhibitory ADP. At the same time, ATP

availability and nucleosidediphosphate inhibition could be affecting

the utilization of G6P by limiting the rate of uridinediphosphoglucose

pyrophosphorylase through 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, 34) have demonstrated strong 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-

phorylatlon. 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 believed that the results presented in this paper support

the conclusion that the hezokinase of the corn scutellum is more

correctly a glucokinase which is not inhibited by its product, G6P.
















A hexokLnase was extracted from the scutellum of corn and

purified with an "apparent" 70-fold increase in specific activity.

It was free of interfering enzyme activities and appeared to be the

only soluble hexokinase of the scutellum. The enzyme showed greatest

specificity towards glucose as the substrate and the data supports

the conclusion that the hexokinase 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-acetylglucosamine and xylose were competitive inhibitors of

glucose phosphorylation, while ADP and AMP vere competitive inhibitors

of ATP. The divalent cations of magnesium, cobalt and manganese

activated the enzyme with cobalt and manganese ion being 63 and

38 per cent as effective as magnesium, respectively. The hexokinase

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 490C.

The properties of the hexoklnase from the scutellum are compared

with those of the brain and yeast hexokinases and are discussed in

relation to glucose-uptake by the scutellum.













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BIOGRAPHICAL SKETCH


Herbert Charles Jones III was born October 6, 1936, in

Leesburg, Lake County, Florida. He attended public schools in

Ocala, Florida and was graduated from Ocala High School in June,

194.

He attended the Colorado School of Mines and the University of

Florida before entering the United States Air Force in 1956. While

in the service he married the former Winifred Catherine Gassaway of

Jacksonville, Florida. After two years of active duty with the Air

Force he returned to the University of Florida and was graduated

August, 1960, with the Degree of Bachelor of Science in Forestry.

In September, 1960, he began graduate study at the University of

Florida, majoring in Botany. In June, 1965, the Degree of Doctor of

Philosophy was conferred on him.

He is the father of three children: Catherine Anne, Winifred

Gail and Herbert Charles.

He is a member of the following honorary societies: Xi Sigma PI,

Gamma Sigma Delta, Alpha Zeta and Phi Signa.












This dissertation was prepared under the direction of the

chairman of the candidate's supervisory committee and has been

approved by all members of that committee. It was submitted to

the Dean of the College of Agriculture and to the Graduate Council,

and was approved as partial fulfillment of the requirements for

the degree of Doctor of Philosophy.




June 22, 1965





--7 SW ^-^
Dean, College of Agriculture








Dean, Graduate School


Supervisory Committee:




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