Brain Research, 168 (1979) 393-397 © Elsevier/North-Holland BiomedicalPress
393
Developmental changes in the subcellular distribution of thymidine kinase in the rat cerebellum
NORIKO YAMADA, YOSHIO SAWASAKI and HIROSHI NAKAJIMA Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113 (Japan)
(Accepted January 25th, 1979)
The postnatal neurogenesis of the rat cerebellum is advantageous for the study of the regulatory mechanisms of nerve cell proliferation and differentiation. Thymidine kinase (TK) is an enzyme implicated in mammalian DNA synthesis5,6,1a, and has previously been reported to be localized in the cytosol fraction of proliferative tissuesa,9,10, including rat cerebellum 1~. In the rat cerebellum, cytosol T K activity shows clear a correlation with DNA replicationS, 15, and the activity has been determined in the investigation of the effects of various factorsT, s, such as thyroxineZ, 17 or bilirubin 19 on cerebellar DNA synthesis. However, considerable enzyme activity was observed in adult cerebellum in which nerve cell proliferation has almost ceasedl~,Z0. Recently, TK activity has also been found in mitochondria4,16, and several species of TK have been reported in various tumors and regenerating liverg,11,lL Therefore, the cerebellar T K activity in the adult rat may be due to the liberation of mitochondrial enzymes during homogenization, or may reflect the enzymes which are involved in pyrimidine metabolism, as distinct from DNA replication. The TK in brain mitochondria has not been investigated and its biological significance remains to be elucidated. Reinvestigation of the intracellular distribution of cerebellar TK seems to be necessary in order to clarify these questions. In the present communication, we report the results of an investigation of the subcellular distribution of TK activity in developing rat cerebellum and cerebral cortex. The properties of TK in cerebellar cytosol and mitochondria were also studied in normal rat and Gunn rat in which cerebellar DNA synthesis was impaired due to hereditary hyperbilirubinemiala,19. Adult and 6-day-old Wistar rats were sacrificed by decapitation, and then the cerebral cortex and cerebellum were separated. Heterozygous (Jj) and homozygous (jj) Gunn rat littermates born from Jj mothers were also used at 10 days of age. The activity of TK was determined as described previously 16. Monoamine oxidase (MAO) was assayed as a marker enzyme of mitochondria by the method of Wurtman and Axelrod is. Protein was determined by Biuret reaction with bovine serum albumin as a standard.
394 TABLE I
Subcellular distribution of thyrnidine kinase in developing rat brain Cerebellum and cerebral cortex were homogenized in 10 vols. of A buffer (0.25 M sucrose, 2.5 mM Tris.HCl, pH 7.5, 0.1 m M EDTA, 1 m M mercaptoethanol) and centrifuged at 600g for 10 min. The crude nuclear precipitate was resuspended in A buffer and homogenized in 12 vols. of 2.4 M sucrose, 3 m M MgCI2 and 1 m M mercaptoethanol, and centrifuged at 40,000g for 60 min, thereby sedimenting the purified nuclei. The 600g supernatant was centrifuged at 10,000g for 10 min to precipitate the mitochondrial fraction. The 10,000 g solution was centrifuged at 105,000 g for 60 rain to precipitate the microsomal fraction. The supernatant was pooled. All fractions were suspended in A buffer and used for enzyme assays. MAO is an abbreviation for monoamine oxidase. 6 days (%)
Cerebellum homogenate nuclei (crude) (purified) mitochondria microsome supernatant recovery Cerebral cortex homogenate nuclei (crude) (purified) mitochondria microsome supernatant recovery
100 (11.7) 8.9 8.8 8.2 55.8 84.5 lC0 (8.5) 1.1 64.7 10.5 14.9 97.8
Adult Spec. act. (pmol/ mg protein/min)
38.8 35.7 17.0 20.0 105.1
4.5 1.8 11.2 2.0 2.7
MAO (%)
(%)
Spec. act. (pmol/ mg protein/min)
MAO (5/o)
100 (8.5) 1.1 64.7 10.5 14.9 97.8
I00 (24.4) 0.8 66.2 1.6 4.4 96.6
2.5
100 (15.1 ) 0.1 69.8 I 1,0 0 96.5
100 (22.6) 1.3 45.2 15.4 1.0 83.7
100 (13.6) 0 82.9 3.1 1.6 101.2
1.4 6.2 0.4 0.7
2.8 0 5.3 0.6 0.3
100 (17,4) 0,4 69.0 10,6 0 97.0
Table I shows the intracellular distribution of TK in cerebellum and cerebral cortex of 6-day-old and adult rats. The distribution of MAO activity predominantly in the mitochondrial fraction seems to imply the specificity of separation. The purity of the mitochondrial fraction was also studied by electron microscopy and found to be little contaminated by microsomal membrane. In 6-day-old rats, brain DNA synthesis is highly active in the cerebellum, but already decreased in the cerebral cortex TM, and cerebellar TK shows consistently high activity throughout development 15,1a. At this stage, the predominant distribution and the highest specific activity of cerebellar TK was found in the supernatant fraction, whereas in the cerebral cortex 65 ~o of the TK activity was recovered in the mitochondrial fraction, in which TK showed the highest specific activity. A similar selective distribution of enzyme in mitochondria was also observed in both the cerebellum and cerebral cortex of the adult rat. The specific activity of cytosol TK in both regions was less than one-hundredth that of the cerebellum at 6 days of age. Yamagami et al. reported that in whole brain of 6day-old rats the majority of TK activity was recovered in cytosol 2°. However, the localization of the enzyme differed clearly between cerebellum and cortex even in the
395 TABLE II Effect o f nucleotides on thymidine kinase in developing rat cerebellum and liver % inhibition of original activity dI'TP (mM)
dCTP (mM)
0.01
0.1
1.0
0.01
0.1
1.0
Cerebellum 6 days supernatant mitochondria adult supernatant mitochondria
97 74 54 60
100 95 96 83
100 98 100 94
0 1 19 14
0 16 47 37
5 21 84 76
Liver fetal supernatant mitochondria adult supernatant mitochondria
98 97 91 39
99 99 98 68
100 100 100 97
0 0 13 7
0 6 49 27
2 24 75 65
same brain, showing a dependence on the stage of development. These findings suggest that T K in the cytosol fraction correlates closely with D N A synthesis of nerve cells, as is found in several proliferative tissuesa,11,16. The slight enzyme activity in the cytosol fraction of the adult brain may not be the released enzyme of mitochondria, since the homogenization with hypotonic buffer showed no change in enzyme activity in this fraction. Therefore, such activity seems to reflect the T K activity of proliferative cells other than nerve cells or the existence of T K species not involved in D N A synthesis. Taylor et al. have reported two species of T K in liver cytosol le. The enzyme in fetal liver is not inhibited by dCTP, but the activity in adult liver was nearly completely inhibited. Such a development-dependent change of susceptibility of T K to dCTP was also found in liver mitochondria and cerebellum (Table II). At a concentration of 1 mM, dCTP inhibited both supernatant and mitochondrial T K of adult cerebellum by 70-80 ~ , whereas in 6-day-old rats no and only slight inhibition, respectively, were observed in the supernatant and mitochondrial fractions. From these results, it appears that several forms of T K seem to exist in the cerebellum, and certain molecular forms of the enzyme in cytosol may be involved in D N A synthesis. Similar heterogeneity of T K was also suggested in cerebellar mitochondria. Therefore, the T K activity in cytosol of adult cerebellum seems predominantly due to the enzyme form not involved in D N A synthesis. In a previous study la, we reported that cerebellar hypoplasia of the Gunn rat seems to be due to the impaired D N A synthesis of external granular cells possibly due to selective damage in Purkinje cells. The T K activity of cerebellar cytosol in the jj Gunn rat decreased from a very early stage, being 80 ~o of normal Jj rat at 6 days and 50 ~o at 10 days. This decrease in enzyme activity preceded a decrease in D N A content by at least 4 days, and no change of D N A synthesis was observed in non-cerebellar parts of the jj brain. Therefore, it seemed of interest to study the subcellular distribution of cerebellar T K in Jj and jj Gunn rats.
396 TABLE III Subcellular distribution of thymidine kinase in lO-day-oM Gunn rat cerebellum* Heterozygote
Homogenate Mitochondria Supernatant Recovery
Homozygote
Distribution o~ (Jo)
Spec. act. (pmol/ mg protein/min)
Distribution (%)
Spec. act. (pmol/ mg protein/min)
100 3.4 57.3 60.7
22.4 5.8 47.0
100 6.4 54.0 60.4
14.5 4.7 26.2
* 20 cerebella of both heterozygous and homozygous Gunn rats were used.
A s shown in Table III, the specific activity o f T K in jj cytosol is a b o u t h a l f o f t h a t in Jj, t h o u g h n o difference was observed between Jj a n d jj m i t o c h o n d r i a l activity, indicating that T K in cytosol was selectively decreased in jj cerebellum. In addition, these results also show t h a t the m i t o c h o n d r i a l d a m a g e m a y n o t be the p r i m a r y lesion in the bilirubin e n c e p h a l o p a t h y , as r e p o r t e d previously la,19. In p r e l i m i n a r y experiments, the cerebellar T K activity has been s e p a r a t e d into at least 3 species by D E A E - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y , a n d n e a r l y all o f the activity in the a d u l t cerebellum was f o u n d to be d i s t r i b u t e d in the fractions which were n o t present in y o u n g c e r e b e l l u m in the proliferative stage. Their purification a n d c h a r a c t e r i z a t i o n are in progress in o u r l a b o r a t o r y .
1 Altman, J. and Das, G. D., Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration, and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions, J. comp. Neurol., 126 (1966) 337-390. 2 Bal~zs, R., Biochemical effects of thyroid hormones in the developing brain. In D. C. Pease (Ed.), Cellular Aspect of Neural Growth and Differentiation, University of California Press, Los Angeles, 1971, pp. 273-320. 3 Baugnet-Mahieu, L., Goutier, R. and Semal, M., Studies on the intracellular distribution. Thymidine phosphorylating kinase activities in regenerating rat liver, Europ. J. Biochem., 4 (1968) 323-328. 4 Berk, A. J. and Clayton, D. A., A genetically distinct thymidine kinase in mammalian mitochondria. Exclusive labeling of mitochondrial deoxyribonucleic acid, J. biol. Chem., 248 (1973) 2722-2729. 5 Bollum, F. J. and Potter, V. R., Nucleic acid metabolism in regenerating rat liver. VI. Soluble enzyme which convert thymidine to thymidine phosphate and DNA, Cancer Res., 19 (1959) 561-565. 6 Bresnick, E., Mainigi, K. D., Buccino, R. and Burleson, S. S., Studies on deoxythymidine kinase of regenerating rat liver and Escherichia coli, Cancer Res., 30 (1970) 2502-2506. 7 Bronzert, D. A., Monaco, M. E. and Lippman, M. E., Purification and general properties of estrogen responsive cytoplasmic thymidine kinase from human breast-cancer, Fed. Proc., 37 (1978) 1780. 8 Epstein, S., Esanu, C. and Raben, M. S., The effect of growth hormone and cortisone on thymidine kinase activity in rat adipose tissue, Biochim. biophys. Acta (Amst.), 186 (1969) 280-285. 9 Lee, L.-S. and Cheng, Y.-C., Human deoxythymidine kinase. I. Purification and general proper-
397 ties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia, J. biol. Chem., 251 (1976) 2600-2604. 10 Masui, H. and Garren, L. D., On the mechanism of action of adrenocorticotropic hormone. The stimulation of thymidine kinase activity with altered properties and changed subcellular distribution, J. biol. Chem., 246 (1971) 5407-5413. 11 Nawata, H. and Kamiya, T., Two molecular forms of thymidine kinase in the cytosol of regenerating rat liver, J. Biochem. (Tokyo), 78 (1975) 1215-1224. 12 Okuda, H., Arima, T., Hashimoto, T. and Fujii, S., Multiple forms of deoxythymidine kinase in various tissues, Cancer Res., 32 (1972) 791-794. 13 Sawasaki, Y., Yamada, N. and Nakajima, H., Developmental features of cerebellar hypoplasia and brain bilirubin levels in a mutant (Gunn) rat with hereditary hyperbilirubinaemia,J. Neurochem., 27 (1976) 577-583. 14 Sung, S. C., DNA synthesis in the developing rat brain, Canad. J. Biochem., 47 (1969) 47-50. 15 Sung, S. C., Thymidine kinase in the developing rat brain, Brain Research, 35 (1971) 268-271. 16 Taylor, A. T., Stafford, M. A. and Jones, O. W., Properties of thymidine kinase partially purified from human fetal and adult tissue, J. biol. Chem., 247 (1972) 1930-1935. 17 Weichsel, M. E., Jr. and Dawson, L., Effects of hypothyroidism and undernutrition on DNA content and thymidine kinase activity during cerebellar development in the rat, J. Neurochem., 26 (1976) 675-681. 18 Wurtman, R. J. and Axelrod, J., A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol., 12 (1963) 1439-1440. 19 Yamada, N., Sawasaki, Y. and Nakajima, H., Impairment of DNA synthesis in Gunn rat cerel:ellure, Brain Research, 126 (1977) 295-307. 20 Yamagami, S., Mori, K. and Kawakita, Y., Changes of thymidine kinase in the developing rat brain, J. Neurochem., 19 (1972) 369-376.
393
Developmental changes in the subcellular distribution of thymidine kinase in the rat cerebellum
NORIKO YAMADA, YOSHIO SAWASAKI and HIROSHI NAKAJIMA Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113 (Japan)
(Accepted January 25th, 1979)
The postnatal neurogenesis of the rat cerebellum is advantageous for the study of the regulatory mechanisms of nerve cell proliferation and differentiation. Thymidine kinase (TK) is an enzyme implicated in mammalian DNA synthesis5,6,1a, and has previously been reported to be localized in the cytosol fraction of proliferative tissuesa,9,10, including rat cerebellum 1~. In the rat cerebellum, cytosol T K activity shows clear a correlation with DNA replicationS, 15, and the activity has been determined in the investigation of the effects of various factorsT, s, such as thyroxineZ, 17 or bilirubin 19 on cerebellar DNA synthesis. However, considerable enzyme activity was observed in adult cerebellum in which nerve cell proliferation has almost ceasedl~,Z0. Recently, TK activity has also been found in mitochondria4,16, and several species of TK have been reported in various tumors and regenerating liverg,11,lL Therefore, the cerebellar T K activity in the adult rat may be due to the liberation of mitochondrial enzymes during homogenization, or may reflect the enzymes which are involved in pyrimidine metabolism, as distinct from DNA replication. The TK in brain mitochondria has not been investigated and its biological significance remains to be elucidated. Reinvestigation of the intracellular distribution of cerebellar TK seems to be necessary in order to clarify these questions. In the present communication, we report the results of an investigation of the subcellular distribution of TK activity in developing rat cerebellum and cerebral cortex. The properties of TK in cerebellar cytosol and mitochondria were also studied in normal rat and Gunn rat in which cerebellar DNA synthesis was impaired due to hereditary hyperbilirubinemiala,19. Adult and 6-day-old Wistar rats were sacrificed by decapitation, and then the cerebral cortex and cerebellum were separated. Heterozygous (Jj) and homozygous (jj) Gunn rat littermates born from Jj mothers were also used at 10 days of age. The activity of TK was determined as described previously 16. Monoamine oxidase (MAO) was assayed as a marker enzyme of mitochondria by the method of Wurtman and Axelrod is. Protein was determined by Biuret reaction with bovine serum albumin as a standard.
394 TABLE I
Subcellular distribution of thyrnidine kinase in developing rat brain Cerebellum and cerebral cortex were homogenized in 10 vols. of A buffer (0.25 M sucrose, 2.5 mM Tris.HCl, pH 7.5, 0.1 m M EDTA, 1 m M mercaptoethanol) and centrifuged at 600g for 10 min. The crude nuclear precipitate was resuspended in A buffer and homogenized in 12 vols. of 2.4 M sucrose, 3 m M MgCI2 and 1 m M mercaptoethanol, and centrifuged at 40,000g for 60 min, thereby sedimenting the purified nuclei. The 600g supernatant was centrifuged at 10,000g for 10 min to precipitate the mitochondrial fraction. The 10,000 g solution was centrifuged at 105,000 g for 60 rain to precipitate the microsomal fraction. The supernatant was pooled. All fractions were suspended in A buffer and used for enzyme assays. MAO is an abbreviation for monoamine oxidase. 6 days (%)
Cerebellum homogenate nuclei (crude) (purified) mitochondria microsome supernatant recovery Cerebral cortex homogenate nuclei (crude) (purified) mitochondria microsome supernatant recovery
100 (11.7) 8.9 8.8 8.2 55.8 84.5 lC0 (8.5) 1.1 64.7 10.5 14.9 97.8
Adult Spec. act. (pmol/ mg protein/min)
38.8 35.7 17.0 20.0 105.1
4.5 1.8 11.2 2.0 2.7
MAO (%)
(%)
Spec. act. (pmol/ mg protein/min)
MAO (5/o)
100 (8.5) 1.1 64.7 10.5 14.9 97.8
I00 (24.4) 0.8 66.2 1.6 4.4 96.6
2.5
100 (15.1 ) 0.1 69.8 I 1,0 0 96.5
100 (22.6) 1.3 45.2 15.4 1.0 83.7
100 (13.6) 0 82.9 3.1 1.6 101.2
1.4 6.2 0.4 0.7
2.8 0 5.3 0.6 0.3
100 (17,4) 0,4 69.0 10,6 0 97.0
Table I shows the intracellular distribution of TK in cerebellum and cerebral cortex of 6-day-old and adult rats. The distribution of MAO activity predominantly in the mitochondrial fraction seems to imply the specificity of separation. The purity of the mitochondrial fraction was also studied by electron microscopy and found to be little contaminated by microsomal membrane. In 6-day-old rats, brain DNA synthesis is highly active in the cerebellum, but already decreased in the cerebral cortex TM, and cerebellar TK shows consistently high activity throughout development 15,1a. At this stage, the predominant distribution and the highest specific activity of cerebellar TK was found in the supernatant fraction, whereas in the cerebral cortex 65 ~o of the TK activity was recovered in the mitochondrial fraction, in which TK showed the highest specific activity. A similar selective distribution of enzyme in mitochondria was also observed in both the cerebellum and cerebral cortex of the adult rat. The specific activity of cytosol TK in both regions was less than one-hundredth that of the cerebellum at 6 days of age. Yamagami et al. reported that in whole brain of 6day-old rats the majority of TK activity was recovered in cytosol 2°. However, the localization of the enzyme differed clearly between cerebellum and cortex even in the
395 TABLE II Effect o f nucleotides on thymidine kinase in developing rat cerebellum and liver % inhibition of original activity dI'TP (mM)
dCTP (mM)
0.01
0.1
1.0
0.01
0.1
1.0
Cerebellum 6 days supernatant mitochondria adult supernatant mitochondria
97 74 54 60
100 95 96 83
100 98 100 94
0 1 19 14
0 16 47 37
5 21 84 76
Liver fetal supernatant mitochondria adult supernatant mitochondria
98 97 91 39
99 99 98 68
100 100 100 97
0 0 13 7
0 6 49 27
2 24 75 65
same brain, showing a dependence on the stage of development. These findings suggest that T K in the cytosol fraction correlates closely with D N A synthesis of nerve cells, as is found in several proliferative tissuesa,11,16. The slight enzyme activity in the cytosol fraction of the adult brain may not be the released enzyme of mitochondria, since the homogenization with hypotonic buffer showed no change in enzyme activity in this fraction. Therefore, such activity seems to reflect the T K activity of proliferative cells other than nerve cells or the existence of T K species not involved in D N A synthesis. Taylor et al. have reported two species of T K in liver cytosol le. The enzyme in fetal liver is not inhibited by dCTP, but the activity in adult liver was nearly completely inhibited. Such a development-dependent change of susceptibility of T K to dCTP was also found in liver mitochondria and cerebellum (Table II). At a concentration of 1 mM, dCTP inhibited both supernatant and mitochondrial T K of adult cerebellum by 70-80 ~ , whereas in 6-day-old rats no and only slight inhibition, respectively, were observed in the supernatant and mitochondrial fractions. From these results, it appears that several forms of T K seem to exist in the cerebellum, and certain molecular forms of the enzyme in cytosol may be involved in D N A synthesis. Similar heterogeneity of T K was also suggested in cerebellar mitochondria. Therefore, the T K activity in cytosol of adult cerebellum seems predominantly due to the enzyme form not involved in D N A synthesis. In a previous study la, we reported that cerebellar hypoplasia of the Gunn rat seems to be due to the impaired D N A synthesis of external granular cells possibly due to selective damage in Purkinje cells. The T K activity of cerebellar cytosol in the jj Gunn rat decreased from a very early stage, being 80 ~o of normal Jj rat at 6 days and 50 ~o at 10 days. This decrease in enzyme activity preceded a decrease in D N A content by at least 4 days, and no change of D N A synthesis was observed in non-cerebellar parts of the jj brain. Therefore, it seemed of interest to study the subcellular distribution of cerebellar T K in Jj and jj Gunn rats.
396 TABLE III Subcellular distribution of thymidine kinase in lO-day-oM Gunn rat cerebellum* Heterozygote
Homogenate Mitochondria Supernatant Recovery
Homozygote
Distribution o~ (Jo)
Spec. act. (pmol/ mg protein/min)
Distribution (%)
Spec. act. (pmol/ mg protein/min)
100 3.4 57.3 60.7
22.4 5.8 47.0
100 6.4 54.0 60.4
14.5 4.7 26.2
* 20 cerebella of both heterozygous and homozygous Gunn rats were used.
A s shown in Table III, the specific activity o f T K in jj cytosol is a b o u t h a l f o f t h a t in Jj, t h o u g h n o difference was observed between Jj a n d jj m i t o c h o n d r i a l activity, indicating that T K in cytosol was selectively decreased in jj cerebellum. In addition, these results also show t h a t the m i t o c h o n d r i a l d a m a g e m a y n o t be the p r i m a r y lesion in the bilirubin e n c e p h a l o p a t h y , as r e p o r t e d previously la,19. In p r e l i m i n a r y experiments, the cerebellar T K activity has been s e p a r a t e d into at least 3 species by D E A E - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y , a n d n e a r l y all o f the activity in the a d u l t cerebellum was f o u n d to be d i s t r i b u t e d in the fractions which were n o t present in y o u n g c e r e b e l l u m in the proliferative stage. Their purification a n d c h a r a c t e r i z a t i o n are in progress in o u r l a b o r a t o r y .
1 Altman, J. and Das, G. D., Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration, and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions, J. comp. Neurol., 126 (1966) 337-390. 2 Bal~zs, R., Biochemical effects of thyroid hormones in the developing brain. In D. C. Pease (Ed.), Cellular Aspect of Neural Growth and Differentiation, University of California Press, Los Angeles, 1971, pp. 273-320. 3 Baugnet-Mahieu, L., Goutier, R. and Semal, M., Studies on the intracellular distribution. Thymidine phosphorylating kinase activities in regenerating rat liver, Europ. J. Biochem., 4 (1968) 323-328. 4 Berk, A. J. and Clayton, D. A., A genetically distinct thymidine kinase in mammalian mitochondria. Exclusive labeling of mitochondrial deoxyribonucleic acid, J. biol. Chem., 248 (1973) 2722-2729. 5 Bollum, F. J. and Potter, V. R., Nucleic acid metabolism in regenerating rat liver. VI. Soluble enzyme which convert thymidine to thymidine phosphate and DNA, Cancer Res., 19 (1959) 561-565. 6 Bresnick, E., Mainigi, K. D., Buccino, R. and Burleson, S. S., Studies on deoxythymidine kinase of regenerating rat liver and Escherichia coli, Cancer Res., 30 (1970) 2502-2506. 7 Bronzert, D. A., Monaco, M. E. and Lippman, M. E., Purification and general properties of estrogen responsive cytoplasmic thymidine kinase from human breast-cancer, Fed. Proc., 37 (1978) 1780. 8 Epstein, S., Esanu, C. and Raben, M. S., The effect of growth hormone and cortisone on thymidine kinase activity in rat adipose tissue, Biochim. biophys. Acta (Amst.), 186 (1969) 280-285. 9 Lee, L.-S. and Cheng, Y.-C., Human deoxythymidine kinase. I. Purification and general proper-
397 ties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia, J. biol. Chem., 251 (1976) 2600-2604. 10 Masui, H. and Garren, L. D., On the mechanism of action of adrenocorticotropic hormone. The stimulation of thymidine kinase activity with altered properties and changed subcellular distribution, J. biol. Chem., 246 (1971) 5407-5413. 11 Nawata, H. and Kamiya, T., Two molecular forms of thymidine kinase in the cytosol of regenerating rat liver, J. Biochem. (Tokyo), 78 (1975) 1215-1224. 12 Okuda, H., Arima, T., Hashimoto, T. and Fujii, S., Multiple forms of deoxythymidine kinase in various tissues, Cancer Res., 32 (1972) 791-794. 13 Sawasaki, Y., Yamada, N. and Nakajima, H., Developmental features of cerebellar hypoplasia and brain bilirubin levels in a mutant (Gunn) rat with hereditary hyperbilirubinaemia,J. Neurochem., 27 (1976) 577-583. 14 Sung, S. C., DNA synthesis in the developing rat brain, Canad. J. Biochem., 47 (1969) 47-50. 15 Sung, S. C., Thymidine kinase in the developing rat brain, Brain Research, 35 (1971) 268-271. 16 Taylor, A. T., Stafford, M. A. and Jones, O. W., Properties of thymidine kinase partially purified from human fetal and adult tissue, J. biol. Chem., 247 (1972) 1930-1935. 17 Weichsel, M. E., Jr. and Dawson, L., Effects of hypothyroidism and undernutrition on DNA content and thymidine kinase activity during cerebellar development in the rat, J. Neurochem., 26 (1976) 675-681. 18 Wurtman, R. J. and Axelrod, J., A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol., 12 (1963) 1439-1440. 19 Yamada, N., Sawasaki, Y. and Nakajima, H., Impairment of DNA synthesis in Gunn rat cerel:ellure, Brain Research, 126 (1977) 295-307. 20 Yamagami, S., Mori, K. and Kawakita, Y., Changes of thymidine kinase in the developing rat brain, J. Neurochem., 19 (1972) 369-376.
