hexokinase isoenzymes in tissues of the adult and developing guinea pig

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 175, 477-486 (1976)

Hexokinase lsoenzymes in Tissues of the Adult and Developing Guinea Pig

ANNE FAULKNER AND COLIN T. JONES

Nufield Institute for Medical Research, University of Oxford, Headley Way, Headington, Oxford, OX3 9DS, England

Received November 10, 1975

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.

Four isoenzymes in mammalian tissues phosphorylate glucose. They have been separated by DEAE-cellulose’ chromatography and are labeled hexokinase I-IV de- pending on electrophoretic mobility (l-3). The I-III isoenzymes (EC 2.7.1.1) have a low Km for glucose, are subject to product inhibition to different extents, and are found in various proportions in most tissues (4-8). The fourth isoenzyme (glucokinase, EC 2.7.1.2) has a highK, for glucose, is subject to little product inhibition, and is found in the liver only (9, 10). In some tissue, e.g., brain and muscle, a substantial proportion of hexokinase activity (mainly I and II) appears to be bound to mitochondria (11, 12).

Because both hexokinase and glucokinase activity are normally present in the

’ Abbreviations used: HK, hexokinase isoenzymes; DEAE-, diethylaminoethyl; ATPase, adeno- sinetriphosphatase (EC 3.6.1.3).

477 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

liver the role in glucose uptake of hexokinase as distinct from glucokinase is difficult to establish. Also the presence of glu- coneogenesis and of relatively large amounts of glycogen presents technical problems for the measurement of glucose uptake. The fetal liver can have little glycogen and no glucokinase or gluconeogene- sis (13) and may be suitable for such an analysis. Unfortunately details of isoenzyme distribution in fetal tissues are available for the fetal rat liver only (14-17).

The activities, isoenzyme distribution, and properties of hexokinase in the liver and other tissues have been determined during the latter half of gestation in the fetal guinea pig. The isoenzyme distribution is discussed in relation to the control of glucose utilization. The poor correlation between values for glucose uptake and the extent of product inhibition of the fetal liver enzyme is also discussed.

478 FAULKNER AND JONES

METHODS

Animals Dated pregnant guinea pigs of the Dunkin-Hart-

ley strain were produced and maintained as described by Elvidge (18).

Assay of Hexokinase in Crude Extracts Animals were stunned by a blow to the head.

Fetuses were removed from the uterus, weighed, and bled from the neck, and tissues were isolated and maintained on ice. Homogenates were prepared in 20 volumes of 25 mM Tris-HCl, pH 7.5, containing 100 mM KCl, 2.5 m&i MgCl,, 1 mM EDTA, and 0.1 mM dithiothreitol using a Potter-Elvehjem glass ho- mogenizer fitted with a Teflon pestle. Liver homogenates prepared with one volume instead of 20 volumes of the homogenizing buffer gave similar results.

Hexokinase was assayed spectrophotometrically following NADP reduction in a linked assay system essentially as described by Salas et al. (19). The reaction mixture contained 0.25 pmol of NADP, 10 pmol of MgCl,, 50 wmol of KCl, 0.5 pmol of glucose, 100 milliunits of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 2 pmol of ATP, and extract in a total volume of 1 ml. After preincubation for 2 min at 25°C the reaction was started by the addition of ATP. In crude preparations the presence of 6-phosphogluconate dehydrogenase (EC 1.1.1.44) can result in a NADPH/glucose g-phosphate ratio in excess of 1. To correct for the additional NADPH formed during the reaction, a known activity of purified hexokinase (- 50% of extract activity) was added to each tissue extract after endogenous activity had been determined.

If the NADPH formation in the tissue = x pmol/ min, the activity of the purified hexokinase in the absence of 6-phosphogluconate dehydrogenase = a pmol/min and the NADPH formation after addition of a units of hexokinase t.o the tissue = y pmol/min; then NADPH/glucose 6-phosphate = (y - x)/a.

In liver and kidney extracts most of the ratios were in the range 1.3-1.6 and hexokinase activities were appropriately corrected. In the other tissue extracts the ratio was about 1 and thus no correction to the hexokinase activity was necessary. Assay of liver and kidney hexokinase with NAD+-linked glucose- B-phosphate dehydrogenase (Leuconostoc mes- enteroides) gave activities similar to the corrected values for the assay described above.

Glucokinase activity was assayed as the increase in reaction rate when the glucose concentration was increased 200-fold (20). Because of the presence of hexokinase III (this is inhibited by high glucose) and a high-K% glucose dehydrogenase the limit of glucokinase detection was about 0.2 pmol min-’ (g wet wt)-‘.

Total hexokinase activity is defined as the rate of glucose g-phosphate formation using the crude homogenate. Soluble hexokinase is the activity obtained using the supernatant fraction after centri- fuging the crude homogenate of 100,OOOg for 1 h. Particulate-bound hexokinase is the difference between the total and soluble activities. Latent hexokinase is defined as the difference in activities of homogenates prepared in the presence and absence of 0.1% (v/v) Triton X-100. Triton X-100 had no effect on soluble hexokinase activity.

DEAE-Cellulose Chromatography of Hex- okinase Isoenzymes Isoenzymes of hexokinase were separated by

DEAE-cellulose chromatography (Whatman DE52) on a 20 x l-cm column equilibrated with 20 mM Tris-HCl, pH 7.5, containing 100 mM KCl, 1 mM MgSO,, 0.1 mM EDTA, and 0.1 mM dithiothreitol (1). Aliquots (l-2 ml) of the 100,OOOg supernatant fraction from a 50% (w/v) homogenate were applied to the column followed by 30 ml of equilibrating buffer. Hexokinase isoenzymes were eluted with a linear 0.1-0.25 M KC1 gradient at a flow rate of 40 ml/h.

Kinetic Studies Km’s for glucose and ATP. After DEAE-cellulose

chromatography fractions with maximum hexokinase activity were used for kinetic analyses. Incuba- tion mixtures were as described above but the glucose and ATP concentrations were varied as described in Results. The reactions were performed at 37°C. Kinetic constants were determined from weighted Lineweaver-Burk plots as described by Wilkinson (21).

Glucose 6-phosphate inhibition. Inhibition of hexokinase activity by glucose 6-phosphate was studied using a modification of an isotopic assay as described by Gots and Bessman (22). Incubation mixtures contained in 1 ml: 25 pm01 of glycylglycine- KOH, pH 7.5,lO pmol of MgCl*, 50 pmol of KCl, 0.5 pmol of [U-‘4Clglucose (0.25 j&i), extract, and glucose 6-phosphate as indicated. After equilibration at 37°C the reaction was started by the addition of 2 pmol of ATP and proceeded for 10 min before the addition of 1 ml of 0.2 M N&OH containing 500 pmol of glucose. The radioactive glucose 6-phosphate was isolated using a batchwise extracting procedure with dried Dowex 2-X8-100. The resin Dowex (0.2 g) was shaken with the incubation mixture for 15 min then washed with 6 x 5 ml of di5

tilled water. Glucose g-phosphate was eluted from the resin with 1 ml of M HCl. Aliquots of the eluant were counted using a dioxane-based scintillation fluid in a Philips liquid scintillation counter. Incorporation of glucose into glucose g-phosphate

HEXOKINASE ISOENZYMES AND DEVELOPMENT 479

was linear for up to 20 min. When ATPase activity was low glucose 6-phosphate inhibition of hexokinase was determined in a continuous spectrophotome- tric assay following ADP production (6). There was essentially no difference in the results obtained from the two assay systems. Controls in which no ATP was added were run.

ATP, glucose, and glucose 6-phosphate concentrations were determined enzymatically using glucose-6-phosphate dehydrogenase and hexokinase.

Units A unit of activity is defined as that which cata-

lyzes the formation of 1 pmol of glucose 6-phosphate/ min.

Values are expressed as means &SD, the number of observations being given in parentheses.

Electrophoresis Horizontal starch-gel electrophoresis was per-

formed on crude supernatant fractions using a gel composed of 10 mM Tris-HCl, pH 8.5,5 mM MgCl*, 5 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10 mM glucose, 10% sucrose, and 10% starch. For the detection of HK III, ] glucose was omitted from gels. The reservoirs contained 250 mM Tris-HCl, pH 7.5, and 100 mM KCl. Electrophoreses were run for 8 h at 14 V/cm at 0°C (Pherograph Mini 68, Hormuth-Vetter, Wieslock). Sliced gels were stained for hexokinase activity at 37°C in the absence of light by modilica- tion of the method of Katzen and Schimke (3). To one volume of 1% agarose solution was added two volumes of 100 mM Tris-HCl, pH 7.5, containing 2.5 mM M&l,, 2 mM KCN, 0.1 mM EDTA, 1 mM ATP, 0.15 mM NADP, 1 mM glucose, 0.5% bovine serum albumin, 100 milliunits of glucose-6-phosphate dehydrogenase/ml, 40 pg of phenazine methosulfate/ ml and 400 kg of nitro blue tetrazolium/ml. This was rapidly mixed and poured onto the cut surface of the gel. After 1-2 h, hexokinase activity was detected as blue bands on a pale background. No bands were observed in the absence of ATP.

Materials Glucose-g-phosphate dehydrogenase and hexoki-

nase were obtained from the Boehringer Corpora- tion (London) Ltd., Ealing, London, W5 2T2. ATP, NADP, glucose 6-phosphate, Triton X-100, Tris, nitro blue tetrazolium, phenazine methosulfate and Dowex 2-X8-100 were obtained from Sigma (London) Chemical Co., Kingston-upon-Thames, Surrey. Fatty acid free bovine serum albumin fraction V was obtained from Miles Laboratories Ltd., Slough. n-[U-14Clglucose was obtained from the Radi- ochemical Centre, Amersham, Bucks. All other chemicals were obtained from British Drug Houses Ltd., Poole, Dorset.

RESULTS

Isoenzymes in Adult Tissues

The activities of hexokinase in crude homogenates of the adult tissues are given in Table I. Hexokinase activity in the liver and hindlimb skeletal muscle was substantially lower than in the other tissues. In the liver all the activity was recovered in the 100,OOOg supernatant while in the other tissues various proportions were particulate bound (Table II). Up to three isoenzymes were consistently seen after electrophoresis of 100,OOOg supernatants from adult tissues (Fig. 1.) All tissues studied contained a slow-moving isoenzyme (HK I). A fast-moving isoenzyme (HK III) was detected in significant activity in the liver and lung with trace amounts in the skeletal and cardiac muscle and brain. A third isoenzyme (HK II) with intermediate mobility appeared in the lung and skeletal muscle and in trace amounts in the cardiac muscle.

DEAE-cellulose chromatography of extracts from cardiac muscle, lung, and kidney confirmed that HK I was the predomi- nant isoenzyme in those tissues. It was eluted at 0.11 M KC1 (Fig. 2). Chromatog- raphy of liver extracts from male guinea pigs (-300 g) confirmed that HK III was the major isoenzyme (Fig. 3~). It was eluted at 0.13-0.14 M KCl.

No significant glucokinase was detected in extracts from the livers of adult male or maternal guinea pigs. After intravenous administration of 1 g of glucose every 4 h for 14 h to 300-g male guinea pigs with access to normal diet, glucokinase activity

TABLE I

THE ACTIVITY OF HEXOKINASE IN THE TISSUES OF THE ADULT GUINEA PIG

Tissue Activity (pm01 min-’ g-l)

Liver 0.2 e 0.06 (13) Kidney 1.34 + 0.64 (18) Brain 3.2 -t 0.6 (16) Cardiac muscle 2.4 + 0.6 (18) Skeletal muscle 0.31 2 0.15 (15)

n Activity was determined on crude homogenates at 25°C as described in Methods. The values are means + SD.


TABLE II PROPORTIONS OF S~LIJBLE HEXOKINASE IN THE DEVELOPING TISSUES OF THE GUINEA PIG

Tissue 8 of total activity

Fetal Neonatal Maternal

Brain 50.0 zt 18.6 (38) 27.7 r 17.4 (16) 44.5 k 10.6 (12) Cardiac muscle 47.3 + 19.3 (38) 28.8 -t 17.4 (16) 52.9 -c 19.8 (14) Kidney 78.2 f 22.0 (38) 38.7 2 18.1 (16) 55.3 t 6.6 (12) Muscle 88.0 2 15.2 (25) 61.1 + 16.4 (16) 77.0 2 22.2 (12) Placenta 73.5 + 19.7 (36)

a Activities were determined in crude homogenates and 100,OOOg supernatants at 25°C as described in Methods. Fetal age, 30 days to term; neonatal age, birth to 14 days. The values are means ‘- SD.

m- -

i E H K LI L” PI s

FIG. 1. Starch-gel electrophoresis of hexokinase isoenzymes from tissues of the adult guinea pig. Horizontal electrophoresis of lOO,OO@ supernatants from adult tissue homogenates was performed at 14 V/cm (10 n&cm*) and 0-2°C for 8 h as described in the experimental section. B, brain; H, cardiac muscle; K, kidney; Li, liver; Lu, lung; Pl, placenta, S, skeletal muscle.

was present at 0.88 + 0.44 (5) unit/g in the crude 100,OOOg supernatant fraction. This glucokinase activity was also detected after DEAE-cellulose chromatography, appearing after HK III at about 0.2 M KC1 (Fig. 41.

Isoenzymes in the Developing Liver Total hexokinase activity in the fetal

liver fell progressively from a value of about 1 unit/g at day 30 to 0.2 unit/g just before term (Fig. 5a). No change was observed in maternal liver activity during pregnancy nor in the activity of the neonatal liver. Between 30 and 40 days of gestation fetal liver activity was detected in both particulate and soluble fractions (Fig. 5b). In addition to the isoenzymes found in the adult tissues the liver and blood f%om the fetal guinea pigs had two further isoenzymes with electrophoretic mobilities between HK I and HK II (Figs.

la)

8

Fraction Number

FIG. 2. DEAE-cellulose chromatography of hexokinase from heart, lung, and kidney of adult guinea pig. Chromatography of 100,OOOg supernatants of tissue homogenates was performed as described in the methods section, and 3-ml fractions were col- lected. (al, Cardiac muscle; (b), lung; (c), kidney.

6 and ‘7). These were also occasionally detected in trace amounts in maternal blood. Chromatography of the lOO,OO@ supernatants from developing liver showed that, early in gestation, activity eluted largely in the region of HK I and the relative proportion of HK III increased during development (Figs. 3 and 8).

The kinetic properties of HK I and HK III obtained aRer chromatography of extracts from fetal, neonatal, and adult livers are given in Table III. No differences in properties were observed during develop-


ment. Inhibition of HK III (but not HK I) the early fetal liver, all fractions of activ- occurred above 200 PM glucose. Inhibition ity other than those corresponding to HK of HK III by glucose 6-phosphate was com- III taken after chromatography exhibited petitive with ATP, complex kinetics were kinetic properties similar to HK I. observed for HK I. The apparent Ki for Glucokinase activity was not detected glucose 6-phosphate inhibition of HK I was (~0.2 units/g) in the fetal liver nor was it 22.5 PM (ATP, 2 mrq glucose, 0.5 mM). At consistently seen in the neonatal liver of 150 PM glucose 6-phosphate, 5 or 10 mM guinea pigs fed on the normal diet. No phosphate gave only a small reversal of evidence for glucokinase activity was ob- this inhibition. Despite the presence of tained after electrophoresis or chromatog- several isoenxymes in the region of HK I in raphy.

10

8

0 20 40 60 80 Fraction Number

FIG. 3. DEAE-cellulose chromatography of hexokinase from the liver of male guinea pigs. Details as for Fig. 2. (a), 3-Day neonate; (b), lo-day neonate; (c), 2-3 months old.

0.25

i + 40 So 80 ’ b io io ’ M

fetal neonatal AGE (days)

Isoenzymes in Other Tissues during De- velopment

Total hexokinase activity in the whole placenta fell progressively during gestation from about 3 to 1 unit/g (Fig. 9). The

1.0 -0.25

f _.’ 0.8 0.2

x- -6

$ 0.6. <’ . .

:

I Y I 1 0 20 40 60 80 lco 120 140

Fraction No.

FIG. 4. DEAE-cellulose chromatography of hexokinase and glucokinase of adult guinea pig liver after glucose treatment. Chromatography of 100,OOOg supernatants of tissue homogenates from glucose-treated animals was performed as described in the methods section. Assay was performed at 0.5 (-0-) and 100 mM glucose (-•-). KC1 concentration (- -).

30 40 50 60 0 neo”a, 20 M fetal

AGE (days1

FIG. 5. The activity and distribution of hexokinase in the liver of the developing guinea pig. Activity was determined in crude homogenates and 100,OOOg supernatants at 25°C as described in the experimental section. (a), Total activity in crude homogenate; (b), percentage of total activity recovered in 100,OOQg supematant. M, maternal liver activity *SD.


activity was predominantly in the soluble fraction (Table II).

Between 30 and 65 days, fetal kidney activity was low and did not change, the mean activity was 0.55 + 0.17 (50) unit/g. After 65 days there was a significant increase (P < 0.001) to a mean value of 1.05

I -----I-

FIG. 6. Starch-gel electrophoresis of hexokinase isoenzymes in the liver of the developing guinea pig. Horizontal electrophoresis of 100,OOOg supernatants from fetal, neonatal, and adult liver homogenates was performed at 14 V/cm (10 mA/cm2) and O-2% for 8 h as described in the methods section. A, adult; FBl, fetal blood.

*t(e) n

\ i L . ~I~ Jo --- 0 20 40 60 80

Fraction Number

FIG. 8. DEAE-cellulose chromatography of hexokinase from the fetal liver. Details as for Fig. 2. (a), 35-Day fetus; (b), 45day fetus; (c), &day fetus; (d), 65-day fetus; (e), maternal liver.

MLi MBI FLi Lu FBI S FIG. 7. Starch-gel electrophoresis of hexokinase isoenzymes from tissues of the adult and

fetal guinea pig. Details as for Figs. 1 and 6. Tissues were taken from a guinea pig 45 days pregnant. Two maternal tissues, MLi-liver and MBl-blood, and four fetal tissues, Fli-liver, Lu- lung, FBl-blood, and S-skeletal muscle, were subjected to electrophoresis.

Isoenzyme


TABLE III KINETIC CONSTANTS FOR HEXOKINASE ISOENZYMES

Glucose (K,,J (UM)

Glucose 6-fL;phate fKJ

HKI 55.8 f 16.1 (10) 0.21 * 0.1 (9) -a

HK III 18.6 f 15.6 (10) 1.3 + 1.1 (7) 48.7 f 25.4 (6)

(2 Complex kinetics.

+ 0.22 (7) units/g. In the first 2 weeks after birth the mean activity of 1.7 + 0.95 (10) units/g was higher (P < 0.002) than before birth.

The total hexokinase activity was low and constant in fetal skeletal muscle and brain; both were significantly higher (P < 0.001) in the first week after birth then fell (Table IV). In fetal cardiac muscle the activity was significantly higher (P < 0.001) than in the adult and did not fall until the second week of neonatal life (Ta- ble IV).

r’ 2 . .

2

: * ’ -. . . t . .

F . c ; 1.

. * . e. l . : . . . . .

. . -*: . .

/ . . .

I I

The placenta and all fetal tissues except the liver had predominantly HK I with only traces of the other isoenzymes, and no developmental changes were observed. Particulate-bound hexokinase was present in various proportions in all tissues and its contribution to the total activity rose significantly (P < 0.01) after birth (Table II).

30 40 50 60

Fetal Age (days)

Term

FIG. 9. The activity of hexokinase in the guinea pig placenta. Activity was determined in crude homogenates at 25°C as described in the methods section.

Particulate Hexokinase Activity in the Brain

Homogenization of tissues with 0.1% Triton X-100 had no consistent effect on tissue activity except in the brain. The increases in activity for the fetal, neonatal, and adult brains were 64.5 + 29.1% (30), 50.8 + 16.6% (14), and36.7 + 8.7% (3), respectively. The increase in fetal brain activity was significantly higher (P < 0.02) than in the adult. The presence of particulate-bound activity that is only lib- erated on disruption of subcellular parti- cles has been reported previously (51-53).

DISCUSSION

similar to the ruminant or starved or dia- betic rat in having no detectable liver glucokinase or HK II (12,24,50). Glucokinase appearance after glucose injection suggests that in the guinea pig as in the rat glucokinase is under dietary control. The absence or low activity of HK II is more difficult to explain, although it may be a consequence of its known instability (25). At times when activity was consistently found in lung and, to a lesser extent, skeletal muscle little activity was found in the other tissues. This implies that even if some loss of HK II occurred the initial activity in liver, cardiac muscle, kidney, and brain was very low. Problems in consistently detecting HK II and IV have been reported for human tissues (26).

Hexokinase I, III, and IV and smaller The fetal liver is the only tissue where activities of II are present in adult rat detailed information on the developmental liver. During diabetes or starvation the changes in hexokinase activity is available activities of HK II and IV decline then in a number of species. Total activity is reappear on insulin treatment of refeeding usually higher than in the adult liver and (l-3, 23). The adult guinea pig fed the declines with increasing gestational age normal diet supplemented with hay was (16, 19, 2733). No significant glucokinase


TABLE IV

TOTAL HEXOKINASE ACTIVITY IN GUINEA PIG TISSUES DURING DEVELOPMENT

Tissue Activity (pm01 min-l g-l)”

Fetal Neonatal

Week 1 Week 2

Skeletal muscle 0.33 + 0.22 (34) 0.65 k 0.18 (10) 0.41 ? 0.04 (6) Cardiac 4.07 5 1.26 (53) 4.47 + 0.52 (10) 2.26 k 0.34 (6) Brain 1.91 % 0.69 (48) 3.89 2 0.95 (10) 2.16 f 0.43 (6)

a Activity was determined on crude homogenates at 25°C as described in Methods. Fetal age, 30 days to term. Values are means ? SD.

activity has been detected in the fetal liver of the rat, guinea pig, or sheep (19, 22,34) although low activity has been reported in the liver of the pig (32). In the fetal rat liver, varying proportions of HK I, II, and III with traces of IV just before birth have been reported (14-17). In the guinea pig liver, HK I is the major isoenzyme but HK III increases during gestation; HK II was found in trace amounts only. Between 35 and 55 days two additional forms with electrophoretic mobilities between HK I and II were detected. These probably origi- nate from hemopoietic tissue as they were also observed in fetal blood. Their absence from the 65day fetal liver is consistent with the reported decline in hemopoietic tissue that occurs after 50 days (35). These two additional hexokinase isoenzymes have not been reported previously, although a single band of activity between HK I and II has been reported in the fetal rat liver (17). Studies on pyruvate kinase isoenzymes in the liver of the fetal rat, guinea pig, and human also indicate the presence of an isoenzyme, pyruvate kinase type 2, which is probably associated with hemopoietic tissue (36, 37).

Little information is available on the rate of glucose uptake in the adult rat liver. At physiological glucose concentrations it is probably controlled by the activity and kinetic properties of glucokinase (9, 3941) rather than by hexokinase. In contrast, measurements of intracellular metabolites have indicated that the rate of glycolysis is largely controlled by hexokinase in the aerobic and anaerobic fetal rat liver (42). In the fetal guinea pig liver under a variety of conditions the measured intracellular glucose g-phosphate concen-

tration ranges from at least 120 to 170 nmol/ml of intracellular water (43). How- ever glucose uptake by sliced or perfused fetal liver can be of the order of 0.2-0.5 pmol min-’ g-l at 45-55 days of gestation (unpublished observations). On the basis of the in vitro kinetic information, at these high glucose 6-phosphate concentrations guinea pig liver HK I activity in uiuo should be substantially inhibited. Thus either the kinetic properties of hexokinase in uiuo differ from those observed in vitro or there is substantial intracellular com- partmentation of glucose 6-phosphate. HK III which is less sensitive to glucose 6- phosphate inhibition does not represent a major proportion of total liver hexokinase activity until after 65 days. The available evidence suggests that the kinetic properties of the additional hexokinase forms are similar to those of HK I.

Information on hexokinase isoenzyme distribution in fetal rat tissues other than the liver suggests that, as in the guinea pig, HK I is the major isoenzyme (17). The two species also show similar changes in total hexokinase activity, with the kidney, brain, and skeletal muscle activity increasing and cardiac muscle activity de- creasing during development (29, 44). In the rabbit a different pattern is observed. Kidney activity does not change, skeletal muscle activity falls, and cardiac muscle activity shows a transient rise after birth (45).

After birth substantial increases in the availability of fatty acids and ketone bod- ies for metabolism occur (46) and it is likely that this is associated with a relative reduction in glucose utilization (47). The increase in particulate hexokinase ac-

HEXOKINASE ISOENZYM [ES AND DEVELOPMENT 485

tivity in all the newborn guinea pig tissues investigated except the liver may be associated with the reduction in glucose utilization. An alteration in the distribution of hexokinase between the soluble and mito- chondrial fractions of the cell has been suggested as a mechanism for control of glycolysis in the brain (48, 49).

ACKNOWLEDGMENTS

We are grateful to Professor G. S. Dawes for his interest and encouragement and Mrs. Paula Webb for expert technical assistance. The work was sup- ported by a grant from the Medical Research Coun- cil.

REFERENCES

1. GONZALEZ, CL, URETA, T., SANCHY, R., AND NIE MEYER, H. (1964) Biochem. Bi@hys. Res. Commun. 16, 347-352.

2. KATZEN, H. M., SODERMAN, D. D., AND NI- TOWSKY, H. M. (1965) B&hem. Biophys. Res. Commun. 19,377-382.

3. KATZEN, H. M., AND SCHIMKE, R. T. (1965)Proc. Nat. Acad. Sci. USA 54, 1218-1225.

4. WEIL-MALHERBE, H., AND BONE, A. D. (1951) Biochem. cJ. 49, 339-347.

5. SOLS, A., AND CRANE, R. K. (1954) J. Biol. Chem. 210, 581-595.

6. GROSSBARD, L., AND SCHIMKE, R. T. (1966) J. Biol. Chem. 241, 3546-3460.

7. KATZEN, H. M. (1967) Advan. Enzyme Regulat. 5, 335-355

8. SCHIMKE, R. T., AND GROSSBARD, L. (1968)Ann. N.Y. Acad. Sci. 151, 332-350.

9. VINUELA, E., SALAS, M., AND SOLS, A. (1963) J. Biol. Chem. 238, PC 1175-PC 1177.

10. PARRY, M. J., AND WALKER, D. G. (1966) Bio- them. J. 99, 266-274.

11. CRANE, R. K., AND SOLS, A. (1953) J. Biol. Chem. 203, 273-292.

12. KATZEN, H. M., SODERMAN, D. D., AND WILEY, C. E. (1970) J. Biol. Chem. 245, 4081-4096.

13. BALLARD, F. J. (1970) in Physiology of the Peri- natal Period (Stave, U. ed.), Vol. 1, pp. 417- 440, Appleton-Century-Crofts, Meredith Cor- poration, :New York.

14. SHATTON, J. B., MORRIS, H. P., AND WEINHOUSE, S. (1969) Cancer Res. 29, 1161-1172.

15. SATO, S., MATSUSHIMA, T., AND SUGIMURA, T. (1969) Cancer Res. 29, 1437-1446.

16. TEPPER, T., AND HOMMES, F. A. (1970) FEBS Symp. 18, 209-214.

17. FARRON, F. (1972) Enzyme 13, 233-237. 18. ELVIDGE, H. (1972) J. Inst. Anim. Technic. 23,

111-117.

19. SALAS, M., VINUELA, E., AND Sons, A. (1963) J. Biol. Chem. 283, 3535-3538.

20. WALKER, D. G. (1963) B&him. Biophys. Acta 77, 209-216.

21. WILKINSON, G. N. (1961) Biochem. J. 80, 324- 332.

22. GOTS, R. E., AND BESSMAN, S. P. (1973) Anal. B&hem. 52, 272-279.

23. HANSEN, R., PILKIS, S. J., AND KRAHL, M. E. (1967) Endocrinology 81, 1397-1404.

24. BALLARD, F. J., AND OLIVER, I. T. (1964) Bio- them. J. 92, 131-136.

25. MURAKAMI, K., AND F&.E, I. A. (1974) Arch. Biochem. Biophys. 165, 519-523.

26. HEUMANN, S., FALKENBERG, F., AND PFLEI- DERER, G. (1974) Biochim. Biophys. Acta 334, 328-342.

27. BURCH, H. B., LOWRY, 0. H. KUHLMAN, A. M., SKERJANCE, J., DIAMANT, E. J., LOWRY, S. R., AND VON DIPPE, P. (1963) J. Biol. Chem. 238, 2267-2273.

28. BALLARD, F. J., AND OLIVER, I. T. (1965) Bio- them. J. 95, 191-200.

29. HOMMES, F. A., AND WILMINK, C. W. (1968) Biol. Neonate 13, 181-193.

30. SYDOW, V. G. (1969) Hoppe-Seyler’s Z. Physiol. Chem. 350, 263-268.

31. JAMDAR, S. C., AND GREENGARD, 0. (1970) J. Biol. Chem. 245, 2779-2783.

32. MERSMANN, H. J. (1971) Amer. J. Physiol. 220, 1297-1302.

33. SCHAUB, J., GUTMANN, I., AND LIPPERT, H. (1972) Hormone Metab. Res. 4, 110-119.

34. WALKER, D. G., AND HOLLAND, G. (1965) Bio- them. J. 97, 845-854.

35. PARAT, M. (1923) Bull. Biol. Fr. Belg. 57, 364- 399.

36. OSTERMAN, J., FRITZ, P. J., AND WUNTCH, T. (1973) J. Biol. Chem. 248, 1011-1018.

37. FAULKNER, A., AND JONES, C. T. (1975) Arch. B&hem. Biophys. 170, 228-241.

38. FAULKNER, A., AND JONES, C. T. (1975) FEBS Lett. 53, 167-169.

39. CAHILL, G. F., ASHMORE, J., EARLE, A. S., AND ZOTTU, S. (1958) Amer. J. Physiol. 192, 491- 496.

40. BALLARD, F. J. (1965) Comp. Biochem. Physiol. 14,437-443.

41. WV, R. (1965) J. Biol. Chem. 240, 2373-2381. 42. HOMMES, F. A. (1971) in Metabolic Processes in

Foetus and Newborn Infants (Jonxis, J. H. P., Visser, H. K. A., and Troekstra, J. A., eds.), p.3, Kroese, N. V., Leiden.

43. FAULKNER, A., AND JONES, C. T. (1976) Arch. Biochem. Biophys. (in press).

44. WILSON, J. E. (1972) J. Neurochem. 19, 223-227. 45. STAVE, U. (1964) Biol. Neonat. 6, 128-147. 46. HAHN, P., AND KOLD~VSKY, 0. (1966) in Utilisa-


tion of Nutrients during Postnatal Develop- ment, 1st ed., Pergamon Press, Oxford.

47. RANDLE, P. J. (1964) Symp. Sot. Exp. Biol. 18, 129-142.

48. WILSON, J. E. (1968) J. Biol. Chem. 243, 3640- 3647.

49. KNULL, H. R., TAYLOR, W. F., AND WELLB, W. W. (1973) J. Biol. Chem. 248, 5414-5417.

50. LAURIS, V., CAHILL, A. M., AND CAHILL, G. E. (1966) Diabetes 15, 475-479.

51. WILSON, J. E. (1967) B&hem. Biophys. Res. Commun. 28, 123-127.

52. BIESOLD, D., AND TEICHGRABER, P. (1967) Bio- them. J. 103, 13c-14~.

53. BACHELARD, H. S. (1967) Biochem. J. 104, 286- 292.

hexokinase isoenzymes in tissues of the adult and developing guinea pig

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