410

Braht Research, 98 (1975) 410-414

t~ Elsevier Scientific Publishing Company, Amsterdam

Printed in The Netherlands

Mitochondrial and cytosolic hexokinases in rat brain, with special reference to the change in experimental diabetes*

MASAKO OUCHI, CHISATO DOHMOTO, TOSHIE KAMIKASHI, KOKO MURAKAMI** AND SADAHIKO 1SHIBASHI Department of Physiological Chemistry, Hiroshima University School of Medicine, Hiroshima 734 (Japan)

(Accepted July 7th, 1975)

It is known that hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) activity in the brain is predominantly localized in mitochondria 1,4,7, whereas almost all of the activity in other tissues is found in cytosol. Hexokinase bound to mitochondria can be released by various methods, and studies have been carried out on the relationship between the cytosolic and mitochondrial hexokinases 1°,11,22,25,29,3°. The significance of the presence of the two types of hexokinases in the brain has not, as yet, been thoroughly elucidated. In the present study, we purified hexokinase type I from both mitochondria and cytosol fractions of the rat brain. The mode of inhibition by p-chloromercuribenzene sulfonate, a sulfhydryl inhibitor, of the two hexokinases was different. On the other hand, experimental diabetes and insulin treatment caused a significant change in the cytosolic hexokinase activity without an appreciable change in the mitochondrial counterpart. Donryu strain rats were used throughout. Experimental diabetes was induced by the intravenous injection of 70 mg/kg of streptozotocin (Upjohn Int. Inc.). On the 4th day, 4 I U of insulin was intraperitoneally injected in some of the diabetic rats. The rats were killed by exsanguination 2-3 h after the insulin injection. The entire brains were taken out and homogenized in 20 m M Tris • HC1 buffer (pH 7.4) containing 250 m M sucrose and 10 m M glucose, with chilling. Mitochondria and cytosol fractions were obtained by the conventional differential centrifugation method 9. Hexokinase activity was measured by the method of Walker 27. For the measurement of the latent hexokinase activity, the method of Wilson 2s was used. The hexokinase isoenzyme pattern was examined by electrophoresis and subsequent development on cellulose acetate membrane 19,23. As the marker enzymes for the subcellular fractions, activities of glucose 6-phosphate dehydrogenase (D-glucose-6-phosphate: N A D P oxidoreductase, EC 1.1.1.49) 12, succinate dehydrogenase (succinate: (acceptor) * A preliminary report of this work was presented at the 1974 meeting of the Japanese Biochemical Society (Seikagaku, 46 (1974) 498). ** Present address: Institute for Cancer Research, Fox Chase, Philadelphia, Pa. 19111, U.S.A.

411 oxidoreductase, EC 1.3.99.1) 3 and NADH-cytochrome c reductase ( N A D H :(acceptor) oxidoreductase, EC 1.6.99.3) 15 were measured. Protein was determined fluorimetrically 6 and blood glucose was determined colorimetrically 15. Hexokinase was released from the mitochondria with 0.9 M KCI in the buffer and purified by DEAE-Sephadex column chromatography 21. Cytosolic hexokinase was purified as reported previously5,8,19. About two-thirds of hexokinase activity in rat brain was found in the mitochondria fraction and the rest was mostly distributed in the cytosol, as reported in previous papers 1,7. Measurement of the marker enzyme activity revealed that the subcellular fractionation was effected satisfactorily. So, purification of hexokinase was undertaken from both mitochondria and cytosol fractions and the preparations with a specific activity of, at least, approximately 40 units (#mole N A D P H formed/min)/mg protein, which was more than 60 times that of the original fraction, were obtained. The electrophoretic mobility of the two hexokinases coincided well with each other and both were judged to be the isoenzyme type I from the mobility. Km values for glucose and ATP of the mitochondrial type I hexokinase were 4.4 × 10 -5 M and 5.2 × 10 -4 ,respectively, while that of the cytosolic type I isoenzyme was 5.9 × 10 -~ M and 5.0 × 10 -4 M, respectively. These values were not only similar to each other but also to the values reported for the type I isoenzymes of various tissues 24. However, a marked difference was found in the response to p-chloromercuribenzene sulfonate between the two type I hexokinases, as shown in Fig. 1. The doublereciprocal plots for the velocity against the concentration of glucose and ATP showed that the sulfhydryl inhibitor acted apparently as a non-competitive inhibitor on the a

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Fig. 1. Double-reciprocal plots for the velocity of hexokinases prepared from rat brain v e r s u s glucose and ATP concentration, Symbols: [2], Control; l , with p-chloromercuribcnzane sulfonate (concentration in parentheses), a: mitochondrial hexokinas¢ (20 nM for glucose; 33 nM for ATP). b: cytosolic hexokinase (33 nM for glucose; 67 nM for ATP).

412 TABLE I C H A N G E IN M I T O C H O N D R I A L A N D CYTOSOLIC HEXOKINASE ACTIVITIES IN DIABETES A N D AFTER INSULIN ADMINISTRATION

Experimental diabetes was induced by the intravenous injection of streptozotocin (70 ms/ks). In some of the diabetic rats, 4 IU of insulin was intraperitoneally injected 2-3 h prior to the sacrifice, The values are means &: S.D, Group

Control Diabetic ~- insulin

No. of rat

16 14 6

Blood glucose (mg/dl)

117 ± 12 293 - 67 98 J: 33

ttexokinase activity mitochondrial

cytosolic (units)

5.94 A=1.11 6.24 _i= 1.34 6.39 ~ 1.40

3.36 ± 0.73 5.24 z~: 1.15 3.82 ~ 0.37

P value ]br cytosolic activity

< 0.001 N.S.

mitochondrial hexokinase with respect to both substrates (Fig. I a), whereas the mode of the inhibition of the cytosolic counterpart was competitive, as reported previously s (Fig. lb). Thus, some difference in the structure was suggested between the two hexokinases in rat brain, though both were judged as the isoenzyme type I from the above criteria. To elucidate the relation between and role of the mitochondrial and cytosolic hexokinases in the brain, changes in the activity of the two enzymes were examined under the experimentally induced diabetic condition. As shown in Table I, cytosolic hexokinase activity was significantly increased in accordance with the increase in the blood glucose level, whereas the change in the activity of the mitochondrial enzyme was slight. No appearance of the other types of hexokinase isoenzyme was detected in the cytosol. Furthermore, such a change in the hexokinase activity in the cytosol was restored by the injection of insulin in the diabetic animals in concurrence with the decrease in the blood glucose level. The result seems to indicate that the cytosolic hexokinase in the brain responds specifically to the changes in the glucose supply to the organ. To examine whether the change in the hexokinase activity in the cytosol was due to the release and/or activation of the latent type in the mitochondria 16,~s, the latent hexokinase activity was measured. By the addition of Triton X-100 to a concentration of 0.5 %, hexokinase activity in the mitochondria was increased 2.9-fold in the controls and 3.0-fold in the diabetic rats. Though marked activity of the latent type was demonstrated, the result failed to explain the specific change in hexokinase activity in the cytosol mentioned above. The cytosolic activity itself was little changed by the addition of Triton X-100 to the same concentration. In the brain, hexokinase plays a regulatory role in glycolysis 13,t4. The extent of involvement of the mitochondrial and cytosolic hexokinases in this regulation, however, is still open to debate. From molecular considerations it has been speculated that the two hexokinases are in equilibrium, depending mainly on the concentration of glucose-6-phosphate and A D P - A T P balance 2,~°,z2,29,3°. Recent findings, that changes

413 i n the activity o f the two hexokinases were c o m p l e m e n t a r y u n d e r various experimental conditions, are in line with this conceptl0,11. However, there are a n u m b e r o f papers rep o r t i n g enzymological differences between the soluble a n d particulate hexokinases18, 26. It m a y be inferred from our results that the equilibrium, t h o u g h it m a y exist, is n o t so simple a n d there m a y be an activation m e c h a n i s m specific to the cytosolic hexokinase. Such a m e c h a n i s m m a y be of interest as an a d a p t a t i o n of the b r a i n to the necessity to m a i n t a i n c o n s t a n t glucose metabolism.

1 BACHELARD,H. S., The subceilular distribution and properties of hexokinase in the guinea-pig cerebral cortex, Biochem. J., 104 (1967) 286-292. 2 BACHELARD,H. S., ANDGOLDFARB,P. S. G., Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase, Biochem. J., 112 (1969) 579-586. 3 BONNER,W. D., Succinic dehydrogenase. In S. P. COLOWICANDN. O. KAPLAN(Eds.), Methods in Enzymology, Vol. 1, Academic Press, New York, 1955, pp. 722-729. 4 CRANE, R. K., AND SOLS, A., The association of hexokinase with particulate fractions of brain and other tissue homogenates, 3". biol. Chem., 203 0953) 273-292. 5 GROSSaARD,L., AND SCHIMKE, R. T., Multiple hexokinases of rat tissues. Purification and comparison of soluble forms, J. biol. Chem., 241 (1966) 3546-3560. 6 HIRAOKA, Y., AND GLICK, D., Studies in histochemistry. LXXI. Measurement of protein in millimicrogram amount by quenching of dye fluorescence, Analyt. Biochem., 5 (1963) 497-504. 7 JOHNSON,M. K., The intracellular distribution of glycolytic and other enzymes in rat-brain homogenates and mitochondrial preparations, Biochem. J., 77 (1960) 610-618. 8 KAMIKASHI,T., KIZAKI,H., MURAKAMI,K., AND ]SHIBASHI,S., Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect ofa thiol inhibitor, Biochem. J., 137 (1974) 139-142. 9 KATZEN,H. i . , SODERMAN,D. D., AND WILEY, C. E., Multiple forms of hexokinase. Activities associated with subcellular particulate and soluble fractions of normal and streptozotocin diabetic rat tissues, J. biol. Chem., 245 (1970) 4081-4096. 10 KNULL, S. R., TAYLOR, W. F., AND WELLS, W. W., Effect of energy metabolism on in vivo distribution of hexokinase in brain, J. biol. Chem., 248 (1973) 5414-5417. 11 KNULL,H. R., TAYLOR,W. F., ANDWELLS,W. W., Insulin effect on brain energy metabolism and the related hexokinase distribution, J. biol. Chem., 249 (1974) 6930-6935. 12 LANGDON,R. G., Glucose 6-phosphate dehydrogenase from erythrocytes. In W. A. WOOD(Ed.), Methods in Enzymology, Vol. 9, Academic Press, New York, 1966, pp. 126-131. 13 LOWRY, O. H., AND PASSONNEAU,J. V., The relationships between substrates and enzymes of glycolysis in brain, J. biol. Chem., 239 (1964) 312~.2. 14 LOWRY,O. H., PASSONNEAU,J. V., HASSELBERGER,F. X., AND SCHULZ,D. W., Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain, J. biol. Chem., 239 (1964) 18-30.

15 MAULER,H. R., DPNH cytochrome c reductase (animal). In S. P. COLOWICKAND N. O. KAPLAN (Eds.), Methods in Enzymology, Vol. 2, Academic Press, New York, 1955, pp. 688-693. 16 MAYER,R. J., AND Hi~BSCHER,G., Mitochondrial bexokinase from small-intestinal mucosa and brain, Bioehem. J., 124 (1971) 491-500. 17 MOMOSE,T., INADA,A., MUKAI,Y., ANDWATANABE,M.,Organicanalysis. XXIII.Determination of blood sugar and urine sugar with 3,6-dinitrophthalic acid, Talanta, 4 (1960) 33-37. 18 MOORE,C. L., Purification and properties of two hexokinases from beef brain, Arch. Bioehem., 128 (1968) 734-744. 19 MORAKAMI,K., ANDIsnm~,sm, S., Regulation of hexokinase isozymes: Apparent interconversion between type II and type III or inactive enzyme complex formation in rat tissues, J. Biochem., 71 (1972) 675-684. 20 PURICH, D. L., AND FROMM,H. J., The kinetics and regulation of rat brain hexokinase, J. biol. Chem., 246 (1971) 3456-3463. 21 REt)KAR,V. D., ANt) KENKARE,U. W., Bovine brain mitochondrial hexokinase. Solubilization, purification, and role of sulflaydryl residues, J. biol. Chem., 247 (1972) 7576-7584.

414 22 ROSE, 1. A., AND WARMS, J. V. B., Mitochondrial hexokinase. Release, rebinding and location, J. biol. Chem., 242 (1967) 1635-1645. 23 SATO, S., MATSUSHIMA, T., AND SUGIMURA, T., Hexokinase isozyme patterns of experimental hepatomas of rats, Cancer Res., 29 (1969) 1437-1446. 24 SCHIMKE, R. T., AND GROSSBARD, L., Studies on isozymes of bexokinase in animal tissues, Ann. N. Y. Acad. Sci., 151 (1968) 332-350. 25 SCHWARTZ, G. P., AND BASEORD, R. E., The isolation and purification of solubilized hexokinase from bovine brain, Biochemistry, 6 (1967) 1070-1079. 26 TUTTLE, J. P., AND WILSON, J. E., Rat brain hexokinase: a kinetic comparison of soluble and particulate forms, Biochim. biophys. Acta (Amst.), 212 (1970) 185-188. 27 WALKER, D. G., On the presence of two soluble glucose-phosphorylating enzymes in adult liver and the development of one of these after birth, Biochim. biophys. Acta (Amst.), 77 (1963) 209226. 28 WILSON, J. E., The latent bexokinase activity of rat brain mitochondria, Biochem. biophys. Res. Commun., 28 0967) 123-127. 29 W~LSON,J. E., Brain hexokinase. A proposed relation between soluble-particulate distribution and activity in vivo, J. biol. Chem., 243 (1968) 3640-3647. 30 WILSON, J. E., Studies on the molecular weight and lipoprotein nature of glucose-6-phospbatesolubilized rat brain hexokinase, Arch. Biochem., 154 (1973) 332-340.