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Isoenzymes of hexokinase in the developing, normal and neoplastic human brain
Europ. ,7. CancerVol. 14, pp. 189-193. Pergamon Press 1978. Printed in Great Britain
Isoenzymes of Hexokinase in the Developing, Normal and Neoplastic Human Brain* M. J. BENNETT,I W. R. TIMPERLEY,t++ C. B. TAYLOR§ and A. SHIRLEY HILL[ [ ~ Department of ?Teuropathology, The Royal Infirmary, Sheffield $6 3DA §Department of Biochemistry, University of Sheffield, Sheffield IIDepartment of Gynaecological Pathology, Jessop Hospital for Women, Sheffield, United Kingdom A b s t r a c t - - T h e activities and isoenzyme distributions of soluble hexokinase were
investigated spectrophotometrically and by cellulose acetate membrane electrophoresis in extracts of normal human brain, a series of fetal brains ranging in gestational age between 6 and 42 weeks and in 27 gliomas. The en:yme wasfirst detectedat 14 weeks gestational age, at a level approximately half of the normal adult level. Only one isoen:yme (type I) was present. The activity gradually increased until similar levels to thosefound in the adult were reached at term. The rise in enzyme levels was particularly rapid between 35 and 40 weeks. This" rapid rise was associated with the appearance of a second isoenzyme of hexokinase (type I I ),first detectedat the age of 36 weeks gestation. Normal adult brain contained type I hexokinase only. Further work needs to be done to determine when the type II en:yme disappears. Types I and II isoenzymes were detected in fourteen of the gliomas studied, with a high incidence in the poorly differentiated tumours.
INTRODUCTION
have been observed in human uterine tumours [17, 18]. The present paper is a study of the activities and isoenzyme patterns of soluble hexokinase in normal adult human brain, in a series of 31 fetal brains ranging in gestational age from 6 to 42 weeks and in 27 gliomas.
HEXOKINASE (ATP : D-Hexose-6-phosphotransferase. EC 2.7.1.1.) has been shown to exist in multiple molecular forms in mammalian tissues. Four isoenzymes have been identified by chromatographic [1] and electrophoretic [2] techniques. They have been numbered from I - I V in order of increasing mobility towards the anode. The distribution of types I - I I I has been shown to vary from tissue to tissue, at different ages and with the nutritional status of the animal [3-7]. Type IV hexokinase is the specific glucokinase [8,9] and has been found only in liver and kidney [3-7, 10]. Mammalian brain has been shown to contain mainly type I hexokinase with a trace of the type II enzyme [5-7]. Studies of hexokinase in neoplasia have been mainly confined to experimental rat hepatomas. Several groups have demonstrated a progressive loss ofglucokinase and an increase in hexokinase with increasing tumour malignancy [11-16]. Recently variations in the isoenzyme pattern
MATERIAL AND METHODS Glucose-6-phosphate dehydrogenase (G-6P D H ) and disodium nicotinamide adenine dinucleotide phosphate (NADP) were purchased from Boehringer Mannheim, West Germany; dithiothreitol (DTT), and phenazine methosulphate (PMS) from the Sigma Chemical Company, London; and all other reagents from British Drug Houses, United Kingdom. All reagents were of the purest grade available. "Celagram" cellulose acetate membranes (78 x 150mm) were obtained from Shandon Scientific Company, United Kingdom. Normal human cerebral tissues were either obtained at autopsy within 15hr of death, or directly after surgical removal, The cases analysed ranged in age from 17 to 50 yr. Fetal brains up to 28 weeks gestation were obtained from spontaneous or induced abortions and fetal brains between 28 and 42 weeks
Accepted 15 September 1977. *We are indebted to the endowment funds of the Trent Region Health Authority (Teaching) tbr the provision of financial support. Grant Code No. 212. +.To whom requests for reprints should be addressed. 189
190
M . J . Bennett, W. R. Timperley, C. B. Taylor and A. Shirley Hill
gestation were obtained from post mortem specimens.. None of these post mortems showed any evidence of cerebral abnormalities. Samples of tumours of the nervous system were obtained directly after surgical removal. All tissues were either used immediately or stored at - 2 0 ° C until used. No changes were observed in "enzyme activity or in isoenzyme patterns after storage of whole tissues or soluble cell fractions at - 2 0 ° C for up to one month. Samples were homogenised over ice in a bench homogeniser (Silverson U.K.) for two periods of 30see with a 1 min interval. The tissues were homogenised in 100 m M potassium phosphate buffer pH7.4 containing 2 m M EDTA and 0 . 2 m M DTT. The crude homogenates were centrifuged at 2 0 , 0 0 0 x g for 45 min and the clear supernatant containing the soluble cell fraction filtered through gauze. Part of this fraction was made up to a final dilution of 1 in 10 with homogenizing buffer for enzyme assay. The remainder was made up to a final dilution of 1 in 4 prior to electrophoresis. Hexokinase was assayed by coupling the enzyme reaction to the reduction of NADP by the G-6-PDH reaction and following the change in absorption at 340 nm in an SP1800A recording spectrophotometer (Pye Unicam, U.K.) with a constant cell temperature of 37°C. The assay system was essentially that of Beutler [ 19] and consisted of a 10 min preincubation of the enzyme preparations in 0.1 M Tris/HC1 buffer pH8.0 containing 2 . 0 m M D-glucose, 0.2raM NADP, 0.2 units/ml of G-6-PDH and 3.0 m M magnesium chloride. The reaction was initiated by the addition of ATP at a final concentration of 3.0mM. Addition of 0.2 units per ml of 6phospho-gluconate dehydrogenase caused no further increase in enzyme activity. Total protein concentrations were measured by the automated modification of the biuret reaction [20]. A unit of enzyme activity is defined as the amount of enzyme which will convert 1/~mole of D-glucose to glucose-6-phosphate/min/mg protein under the assay conditions at 37°C. Electrophoretic procedure was adapted from a previously reported method [18]. Eight samples of approximately 0.1--0.2 munits of enzyme activity were run side by side on each cellulose acetate membrane using a buffer concentration of 5 0 m M barbitone, 5 . 0 m M EDTA, 1.0 m M D T T and 10.0 m M D-glucose at pH8.6. A total potential of 28.2V/cm was applied for l hr by which time a marker of albumen-bound bromophenol blue had migrated 4.8 cm towards the anode. The isoenzymes were localised in situ by placing the membrane in
contact with a 0.5% agarose gel containing 5 0 m M Tris, 5 . 0 m M ATP, 5 . 0 m M MgC12, 2.0 m M KCN, 0.5 m M NADP, 25 ~ug/ml PMS, 0.4mg/ml nitro blue tetrazolium, 0.2 units/ml G-6-PDH and 10.0 m M D-glucose at a final pH of 8.2. Incubation was carried out at 37°C for 1 hr in the dark after which time the insoluble blue bands associated with enzyme activity could be seen. The membranes were fixed in 5% acetic acid for 15min and scanned at 578nm with a vitatron densitometer (Fisons, U.K.). Controls were set up using identical electrophoretic conditions but in this case the membranes were stained in a medium lacking substrate (D-glucose) and no bands of enzyme activity were seen on any of these membranes. RESULTS
The pattern of development of hexokinase activity in fetal brain is shown in Fig. 1. The enzyme was first detected at the gestational age of 14 weeks and at this time the level was approximately half that found in the normal human adult brain. This level showed a gradual increase throughout gestation reaching the adult level at term. There was no significant difference in the activity of hexokinase in adult grey and white matter in material obtained at autopsy, but the activity in grey and white matter was slightly higher in fresh surgical specimens than in grey and white matter obtained at autopsy. Mean hexokinase activity in autopsy specimens was 0.0030 pmole/min/mg protein ( N = 1 0 , S.D. =0.0018) whilst that for surgically removed specimens was 0.0038 pmole/min/mg protein (N = 28, S.D. =0.0016). This indicates a slight loss of activity during autolysis. Electrophoresis of fetal and adult brain homogenates showed that during the first 36 0.00'.
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Fig. 1. The development of hexokinase activity in fetal human brain. Extracts of fetal brain were prepared and assayed spectrophotometrically by the method described in the text. The correspondingadultmeanvaluesareO.OO30#mole/min/mg protein (units/min/mg protein) for autopsy specimens and 0.0038 llmole/min/mg proteinfor surgicalspecimens. The horizontal axis is gestational age in weeks.
Isoenzymes of Hexokinase in the Developing, Normal and Neoplastic Human Brain 70
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Fig. 2. Scan profiles at 578 nm of fetal and normal adult cerebral tissues subjected to cellulose acetate .membraneelectrophoresisfor one hour by the method described in the text, and stainedfor hexokinase activity in situ.
Table 1. of tumours with type II isoenzyme detected
No.
Tumour type
Percentage of activity defined by relative peak area
Enzyme activity
Type I
#mole/rain/rag
Type II
Astrocytoma grade IV N=15
10
87___14%
13___14%
0.0035_+0.0014"
Astrocytoma grade I I I N=6
1
98-t- 4%
2_+ 4%
0.0029_+0.0018"
Astrocytoma grade II N=4
2
98_+ 3%
2_+ 3%
0.0022_+0.003 *
Ganglioglioma N= 1
0
100
0
0.0072
Ependymoma
0
100
0
0.0023
*Standard deviation.
weeks of gestation and in adult specimens, only one band of hexokinase, type I, was present, whilst during the last 4-6 weeks of gestation two isoenzymes, types I and II, were consistently observed (Fig. 2). In an electrophoretic survey of 25 astrocytomas (15 grade IV, 6 grade III and 4 grade II), type II hexokinase was seen in 10 of the grade IV astrocytomas, in one of the grade III astrocytomas and in 2 of the grade II astro-
cytomas. The more benign gliomas, including two of the grade II tumours, a single ganglioglioma, and a well-differentiated ependymoma, showed an identical pattern to that of normal adult brain. Electrophoresis was carried out on samples showing approximately similar activities of enzyme and the relative proportions of each isoenzyme present were calculated by integration of the respective peak areas after scanning (Table 1 ).
192
M. J. Bennett, W. R. Timperley, C. B. Taylor and A. Shirley Hill DISCUSSION
Between 15 and 44% of hexokinase in the mammalian brain has been shown to be mitochondrial-bound [21,22]. It has also been suggested that the soluble hexokinase in brain is largely ofglial cell origin and that the particulate fraction is neuronal [23]. In the present study only soluble hexokinase was measured and the results obtained could bear a direct relationship to the glial cell content. All tumours studied were glial cell in origin. The study has shown that the soluble fraction of hexokinase, the major glycolytic rate limiting enzyme in brain [24] appeared in signiticant quantities at about 14 weeks gestational age. Other glycolytic enzymes including aldolase, pyruvate kinase, phosphofructokinase and phosphoglucomutase have also been shown to appear at this age in man [25]. Since soluble hexokinase may be a marker ofglial cells in brain this period may coincide with an important period of glial cell development. Electrophoretic studies show that only type I hexokinase was present in fetal brain up to 36 weeks gestational age when the type II isoenzyme appeared. This enzyme is not present in the normal adult human brain and the significance of its appearance late in fetal development is unclear. There is clearly a need for fhrther work on this enzyme.
Type II hexokinase was also found in many of the gliomas studied. The highest incidence was seen in the more malignant tumours (grade IV). The presence of this enzyme in neoplastic and fetal brain and its absence in adult brain implies that the isoenzyme is a carcino-embryonic antigen, one of a large number that have now been described [26, 27]. The appearance of the enzyme does not signify a dedifferentiation towards primitive undifferentiated forms since the isoenzyme was only observed in the later stages of development when the tissues are showing evidence of differentiation. It seems that this reversion is towards a highly specialised period of cerebral development implying an ordered alteration in genetic activity in this particular case. The fact that the isoenzyme form appeared predominantly in the more malignant tumours implies that this particular misprogramming of genetical information is a consequence of neoplasia and not a cause of it.
Acknowledgements--The authors would like to thank Mr. J. Hardman, Mr. A. A. Jefferson and Mr. D. M. C. Forster, Cori'sultant Neurosurgeons at the Royal Infirmary, Sheffieldfor providing surgical material; Mr. K. A. Horton for technical assistance; and Mrs. Pamela Kirk for typing the manuscript. They are also indebted to the endowment funds of the Trent Region Health Authority (Teaching) for financial support (Grant Code No. 212).
REFERENCES 1. 2.
3. 4. 5. 6. 7.
8. 9. 10.
C. GONZALEZ,T. URETA, R. SANCHEZand H. NIEMEYER,Multiple molecular forms of ATP: hexose-6-phosphotransferase from rat liver. Biochem. biophys. Res. Commun. 16, 347 (1964). H. M. KATZEN, D. D. SODERMAN and H. M. NITOWSKY, Kinetic and electrophoretic evidence for multiple forms of glucose-ATP phosphotransferase activity from human cell cultures and rat liver. Biochem. biophys. Res. Commun. 19, 377 (1965). H.M. KATZENand R. T. ScraMS:E, Multiple forms ofhexokinase in the rat; tissue distribution, age dependence and properties. Proc. nat. Acad. Sci. (Wash.) 549 1218 (1965). L. GROSSBARDand R. T. SCmMKE, Multiple fbrms of rat tissue hexokinase. Purification and properties of soluble forms. J. biol. Chem. 241, 3546 (1966). H. M. KATZEN, The multiple forms of mammalian hexokinase and their significance to the action of insulin. Advanc. Enzyme Regul. 5, 335 (1967). R . T . ScmugE and L. GROSSBARD,Studies on isozymes of hexokinase in animal tissues. Ann. N.Y. Acad. Sci. 151, (1), 332 (1968). H. M . KATZEN, D. D. SODERMAN and V. J. CIRILLO, Tissue distribution physiological significance ofmuhiple forms ofhexokinase. Ann. N. Y. Acad. Sci. 151, (I), 351 (1968). E. VINUELA,M. SALAS and A. SOLS, Glucokinase and hexokinase in liver in relation to glycogen synthesis. J. biol. Chem. 238, 1175 (1963). A. SOLS,M. SALASand E. VINUELA,Induced biosynthesis of liver glucokinase. Advanc. Enzyme Regul. 2, 177 (1964). S.J. PILKIS and R. J. HANSEN, Resolution of two high-Kin ATP: n-hexose-6phosphotransferase bands by starch-gel electrophoresis. Biochem. biophys. Aeta (Amst.) 159, 189 (1968).
Isoenzymes of Hexokinase in the Developing, Normal and Neoplastic Human Brain 11. J . B . SHATTON,H. P. MORRIS and S. WEINHOUSE,Kinetic electrophoretic and chromatographic studies on glucose-ATP phosphotransferase in rat liver and hepatomas. Cancer Res. 29, 1161 (1969). 12. S. WEINHOUSE,V.J. CRISTOFALO,C. SHARMAand H. P. MORRIS,Some properties ofglucokinase in normal and neoplastic liver. Advanc. Enzyme Regul. 1~ 363 (1963). 13. F.A. FARINA,R. C. ADELMAN,C. H. Lo, H. P. MORRIS and S. WEINHOUSE, Metabolic regulation and enzyme alterations in Morris hepatomas. CancerRes. 28, 1897 (1968). 14. S. WEINHOUSE, Glycolysis, respiration and anomolous gene expression in experimental hepatomas: GHA Clowes memorial lecture. Cancer Res. 32, 2007 (1972). 15. R.M. SHARMA,C. SHARMA,A. J. DONNELLY,H. P. MORRISand S. WEINHOUSE, Glucose-ATP phosphotransferases during hepatocarcinogenesis. Cancer Res. 25, 193 (1965). 16. F. FARRON,The isoenzymes ofhexokinase in normal and neoplastic tissues of the rat. Enzyme 13, 233 (1972). 17. Y. KmECHI,S. SATOand T. SUGIMURA,Hexokinase isoenzyme patterns of human uterine tumours. Cancer (Philad.) 30~ 444 (1972). 18. S. SATO,Y. KIKUCHI,K. TAKAKURA,T. C. CHIEN and T. SUGIMURA,Diagnostic value ofaldolase and hexokinase isozymes for human brain and uterine tumours. Gann, Monograph on Cancer Research 13, 279 (1972). 19. E. BEETLER,Red Cell Metabolism. p. 38. Grune and Stratton, New York (1971). 20. J.F. FAILING,JR.,M. W. BUCKLEYand B. ZAK,Automatic determination of serum proteins. Amer. J. clin. Path. 33, 83 (1960). 21. H.S. BACHELARD,The subcellular distribution and properties of hexokinases in the guinea pig cerebral cortex. Biochem. J. 104, 286 (1967). 22. E.A. NEWSHOLME,F. S. ROLLESTONand K. TAYLOR,Factors effecting the glucose6-phosphate inhibition ofhexokinase from cerebral cortex tissues of the guinea pig. Biochem. J. 106, 193 (1968). 23. V. BIOL,D. BIESOLD,E. L. DOWEDOWAand S. PIGAREWA,Die hexokinaseaktivifftt verschiedener hinregionen der katze. Acta biol. rned. germ. 26, 27 (1971). 24. O . H . LOWRYand J. V. PASSANNEAE,The relationships between substrates and enzymes of glycolysis in brain. J. biol. Chem. 239, 31 (1964). 25. M.J. BENNETT, Ph.D. thesis. University of Sheffield, England (1975). 26. W.H. FISHMAN,Carcinoplacental isoenzyme antigens. Advanc. Enzyme Regul. 11, 293 (1973). 27. F. SCHAPIRA,Isozymes and cancer. Advanc. Cancer Res. 18, 77 (1973).
193
Isoenzymes of Hexokinase in the Developing, Normal and Neoplastic Human Brain* M. J. BENNETT,I W. R. TIMPERLEY,t++ C. B. TAYLOR§ and A. SHIRLEY HILL[ [ ~ Department of ?Teuropathology, The Royal Infirmary, Sheffield $6 3DA §Department of Biochemistry, University of Sheffield, Sheffield IIDepartment of Gynaecological Pathology, Jessop Hospital for Women, Sheffield, United Kingdom A b s t r a c t - - T h e activities and isoenzyme distributions of soluble hexokinase were
investigated spectrophotometrically and by cellulose acetate membrane electrophoresis in extracts of normal human brain, a series of fetal brains ranging in gestational age between 6 and 42 weeks and in 27 gliomas. The en:yme wasfirst detectedat 14 weeks gestational age, at a level approximately half of the normal adult level. Only one isoen:yme (type I) was present. The activity gradually increased until similar levels to thosefound in the adult were reached at term. The rise in enzyme levels was particularly rapid between 35 and 40 weeks. This" rapid rise was associated with the appearance of a second isoenzyme of hexokinase (type I I ),first detectedat the age of 36 weeks gestation. Normal adult brain contained type I hexokinase only. Further work needs to be done to determine when the type II en:yme disappears. Types I and II isoenzymes were detected in fourteen of the gliomas studied, with a high incidence in the poorly differentiated tumours.
INTRODUCTION
have been observed in human uterine tumours [17, 18]. The present paper is a study of the activities and isoenzyme patterns of soluble hexokinase in normal adult human brain, in a series of 31 fetal brains ranging in gestational age from 6 to 42 weeks and in 27 gliomas.
HEXOKINASE (ATP : D-Hexose-6-phosphotransferase. EC 2.7.1.1.) has been shown to exist in multiple molecular forms in mammalian tissues. Four isoenzymes have been identified by chromatographic [1] and electrophoretic [2] techniques. They have been numbered from I - I V in order of increasing mobility towards the anode. The distribution of types I - I I I has been shown to vary from tissue to tissue, at different ages and with the nutritional status of the animal [3-7]. Type IV hexokinase is the specific glucokinase [8,9] and has been found only in liver and kidney [3-7, 10]. Mammalian brain has been shown to contain mainly type I hexokinase with a trace of the type II enzyme [5-7]. Studies of hexokinase in neoplasia have been mainly confined to experimental rat hepatomas. Several groups have demonstrated a progressive loss ofglucokinase and an increase in hexokinase with increasing tumour malignancy [11-16]. Recently variations in the isoenzyme pattern
MATERIAL AND METHODS Glucose-6-phosphate dehydrogenase (G-6P D H ) and disodium nicotinamide adenine dinucleotide phosphate (NADP) were purchased from Boehringer Mannheim, West Germany; dithiothreitol (DTT), and phenazine methosulphate (PMS) from the Sigma Chemical Company, London; and all other reagents from British Drug Houses, United Kingdom. All reagents were of the purest grade available. "Celagram" cellulose acetate membranes (78 x 150mm) were obtained from Shandon Scientific Company, United Kingdom. Normal human cerebral tissues were either obtained at autopsy within 15hr of death, or directly after surgical removal, The cases analysed ranged in age from 17 to 50 yr. Fetal brains up to 28 weeks gestation were obtained from spontaneous or induced abortions and fetal brains between 28 and 42 weeks
Accepted 15 September 1977. *We are indebted to the endowment funds of the Trent Region Health Authority (Teaching) tbr the provision of financial support. Grant Code No. 212. +.To whom requests for reprints should be addressed. 189
190
M . J . Bennett, W. R. Timperley, C. B. Taylor and A. Shirley Hill
gestation were obtained from post mortem specimens.. None of these post mortems showed any evidence of cerebral abnormalities. Samples of tumours of the nervous system were obtained directly after surgical removal. All tissues were either used immediately or stored at - 2 0 ° C until used. No changes were observed in "enzyme activity or in isoenzyme patterns after storage of whole tissues or soluble cell fractions at - 2 0 ° C for up to one month. Samples were homogenised over ice in a bench homogeniser (Silverson U.K.) for two periods of 30see with a 1 min interval. The tissues were homogenised in 100 m M potassium phosphate buffer pH7.4 containing 2 m M EDTA and 0 . 2 m M DTT. The crude homogenates were centrifuged at 2 0 , 0 0 0 x g for 45 min and the clear supernatant containing the soluble cell fraction filtered through gauze. Part of this fraction was made up to a final dilution of 1 in 10 with homogenizing buffer for enzyme assay. The remainder was made up to a final dilution of 1 in 4 prior to electrophoresis. Hexokinase was assayed by coupling the enzyme reaction to the reduction of NADP by the G-6-PDH reaction and following the change in absorption at 340 nm in an SP1800A recording spectrophotometer (Pye Unicam, U.K.) with a constant cell temperature of 37°C. The assay system was essentially that of Beutler [ 19] and consisted of a 10 min preincubation of the enzyme preparations in 0.1 M Tris/HC1 buffer pH8.0 containing 2 . 0 m M D-glucose, 0.2raM NADP, 0.2 units/ml of G-6-PDH and 3.0 m M magnesium chloride. The reaction was initiated by the addition of ATP at a final concentration of 3.0mM. Addition of 0.2 units per ml of 6phospho-gluconate dehydrogenase caused no further increase in enzyme activity. Total protein concentrations were measured by the automated modification of the biuret reaction [20]. A unit of enzyme activity is defined as the amount of enzyme which will convert 1/~mole of D-glucose to glucose-6-phosphate/min/mg protein under the assay conditions at 37°C. Electrophoretic procedure was adapted from a previously reported method [18]. Eight samples of approximately 0.1--0.2 munits of enzyme activity were run side by side on each cellulose acetate membrane using a buffer concentration of 5 0 m M barbitone, 5 . 0 m M EDTA, 1.0 m M D T T and 10.0 m M D-glucose at pH8.6. A total potential of 28.2V/cm was applied for l hr by which time a marker of albumen-bound bromophenol blue had migrated 4.8 cm towards the anode. The isoenzymes were localised in situ by placing the membrane in
contact with a 0.5% agarose gel containing 5 0 m M Tris, 5 . 0 m M ATP, 5 . 0 m M MgC12, 2.0 m M KCN, 0.5 m M NADP, 25 ~ug/ml PMS, 0.4mg/ml nitro blue tetrazolium, 0.2 units/ml G-6-PDH and 10.0 m M D-glucose at a final pH of 8.2. Incubation was carried out at 37°C for 1 hr in the dark after which time the insoluble blue bands associated with enzyme activity could be seen. The membranes were fixed in 5% acetic acid for 15min and scanned at 578nm with a vitatron densitometer (Fisons, U.K.). Controls were set up using identical electrophoretic conditions but in this case the membranes were stained in a medium lacking substrate (D-glucose) and no bands of enzyme activity were seen on any of these membranes. RESULTS
The pattern of development of hexokinase activity in fetal brain is shown in Fig. 1. The enzyme was first detected at the gestational age of 14 weeks and at this time the level was approximately half that found in the normal human adult brain. This level showed a gradual increase throughout gestation reaching the adult level at term. There was no significant difference in the activity of hexokinase in adult grey and white matter in material obtained at autopsy, but the activity in grey and white matter was slightly higher in fresh surgical specimens than in grey and white matter obtained at autopsy. Mean hexokinase activity in autopsy specimens was 0.0030 pmole/min/mg protein ( N = 1 0 , S.D. =0.0018) whilst that for surgically removed specimens was 0.0038 pmole/min/mg protein (N = 28, S.D. =0.0016). This indicates a slight loss of activity during autolysis. Electrophoresis of fetal and adult brain homogenates showed that during the first 36 0.00'.
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Fig. 1. The development of hexokinase activity in fetal human brain. Extracts of fetal brain were prepared and assayed spectrophotometrically by the method described in the text. The correspondingadultmeanvaluesareO.OO30#mole/min/mg protein (units/min/mg protein) for autopsy specimens and 0.0038 llmole/min/mg proteinfor surgicalspecimens. The horizontal axis is gestational age in weeks.
Isoenzymes of Hexokinase in the Developing, Normal and Neoplastic Human Brain 70
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Fig. 2. Scan profiles at 578 nm of fetal and normal adult cerebral tissues subjected to cellulose acetate .membraneelectrophoresisfor one hour by the method described in the text, and stainedfor hexokinase activity in situ.
Table 1. of tumours with type II isoenzyme detected
No.
Tumour type
Percentage of activity defined by relative peak area
Enzyme activity
Type I
#mole/rain/rag
Type II
Astrocytoma grade IV N=15
10
87___14%
13___14%
0.0035_+0.0014"
Astrocytoma grade I I I N=6
1
98-t- 4%
2_+ 4%
0.0029_+0.0018"
Astrocytoma grade II N=4
2
98_+ 3%
2_+ 3%
0.0022_+0.003 *
Ganglioglioma N= 1
0
100
0
0.0072
Ependymoma
0
100
0
0.0023
*Standard deviation.
weeks of gestation and in adult specimens, only one band of hexokinase, type I, was present, whilst during the last 4-6 weeks of gestation two isoenzymes, types I and II, were consistently observed (Fig. 2). In an electrophoretic survey of 25 astrocytomas (15 grade IV, 6 grade III and 4 grade II), type II hexokinase was seen in 10 of the grade IV astrocytomas, in one of the grade III astrocytomas and in 2 of the grade II astro-
cytomas. The more benign gliomas, including two of the grade II tumours, a single ganglioglioma, and a well-differentiated ependymoma, showed an identical pattern to that of normal adult brain. Electrophoresis was carried out on samples showing approximately similar activities of enzyme and the relative proportions of each isoenzyme present were calculated by integration of the respective peak areas after scanning (Table 1 ).
192
M. J. Bennett, W. R. Timperley, C. B. Taylor and A. Shirley Hill DISCUSSION
Between 15 and 44% of hexokinase in the mammalian brain has been shown to be mitochondrial-bound [21,22]. It has also been suggested that the soluble hexokinase in brain is largely ofglial cell origin and that the particulate fraction is neuronal [23]. In the present study only soluble hexokinase was measured and the results obtained could bear a direct relationship to the glial cell content. All tumours studied were glial cell in origin. The study has shown that the soluble fraction of hexokinase, the major glycolytic rate limiting enzyme in brain [24] appeared in signiticant quantities at about 14 weeks gestational age. Other glycolytic enzymes including aldolase, pyruvate kinase, phosphofructokinase and phosphoglucomutase have also been shown to appear at this age in man [25]. Since soluble hexokinase may be a marker ofglial cells in brain this period may coincide with an important period of glial cell development. Electrophoretic studies show that only type I hexokinase was present in fetal brain up to 36 weeks gestational age when the type II isoenzyme appeared. This enzyme is not present in the normal adult human brain and the significance of its appearance late in fetal development is unclear. There is clearly a need for fhrther work on this enzyme.
Type II hexokinase was also found in many of the gliomas studied. The highest incidence was seen in the more malignant tumours (grade IV). The presence of this enzyme in neoplastic and fetal brain and its absence in adult brain implies that the isoenzyme is a carcino-embryonic antigen, one of a large number that have now been described [26, 27]. The appearance of the enzyme does not signify a dedifferentiation towards primitive undifferentiated forms since the isoenzyme was only observed in the later stages of development when the tissues are showing evidence of differentiation. It seems that this reversion is towards a highly specialised period of cerebral development implying an ordered alteration in genetic activity in this particular case. The fact that the isoenzyme form appeared predominantly in the more malignant tumours implies that this particular misprogramming of genetical information is a consequence of neoplasia and not a cause of it.
Acknowledgements--The authors would like to thank Mr. J. Hardman, Mr. A. A. Jefferson and Mr. D. M. C. Forster, Cori'sultant Neurosurgeons at the Royal Infirmary, Sheffieldfor providing surgical material; Mr. K. A. Horton for technical assistance; and Mrs. Pamela Kirk for typing the manuscript. They are also indebted to the endowment funds of the Trent Region Health Authority (Teaching) for financial support (Grant Code No. 212).
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