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Age-dependent induction of isoenzymes of tyrosine aminotransferase of the liver of the rat
Mechanisms of Ageing and Development, 3 (1974) 187-190
187
© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
A G E - D E P E N D E N T I N D U C T I O N OF I S O E N Z Y M E S OF T Y R O S I N E A M I N O T R A N S F E R A S E OF T H E L I V E R OF T H E R A T
B. K. RATHA and M. S. KANUNGO Biochemistry Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)
(Received May 7, 1974)
SUMMARY The effects of adrenalectomy, hydrocortisone and actinomycin D on the cytoplasmic (c-), mitochondrial (m-) and nuclear (n-) tyrosine aminotransferase (TAT) isoenzymes of the liver of immature (6-) and adult (35-week) male albino rats were studied. Adrenalectomy stimulates the activity of n T A T in adult rats. The activities of m T A T of immature, and of c- and m T A T of adult rats are inhibited by adrenalectomy. Hydrocortisone induces and actinomycin D inhibits the induction of the isoenzymes in adrenalectomized rats of both ages. However, the induction and inhibition are more pronounced in the adult than in the immature.
INTRODUCTION We have initiated studies on the alterations in the levels 1 and induction2, a of certain isoenzymes of the rat in an attempt to understand the mechanism of ageing at the genetic level. Tyrosine aminotransferase (TAT; EC 2.6.1.5) is a suitable enzyme for such studies as it is rapidly induced by gluco-corticoids4, 5. It has three isoenzymes, cytoplasmic (c-), mitochondrial (m-) and nuclear T A T (nTAT)5, 6. m T A T has a broader tissue distribution v. In the pathological condition of tyrosinemia, the liver has only m T A T and no cTAT. Though there is clear demonstration that cTAT is induced by hydrocortisone, the induction of the two other isoenzymes by various steroid hormones remains disputed s. We report here that hydrocortisone induces all the three isoenzymes of TAT, and that there are age-related differences in their induction. MATERIALS AND METHODS Immature (6-) and adult (35-week) male albino rats of Wistar strain, maintained in standard laboratory conditions, were used. Rats of each age were divided into four groups, each having 4-5 rats. G r o u p I consisted of normal rats which were administered 1.0 ml of 0.9 ~ NaCl intraperitoneally and served as control. Groups II, I I I and IV rats were bilaterally adrenalectomized and were given 0.9 ~ NaC1 ad lib
188 instead of tap water for 10 days after operation. On the 1 lth day, G r o u p II rats were given 1.0 ml of 0.9 ~o NaCI, Group III rats were administered 5 mg hydrocortisone/100 g body wt., and Group IV rats were administered actinomycin D (0.1 mg/100 g body wt.) one hour prior to hydrocortisone administration. All the injections were given intraperitoneally in 1.0 ml of 0.9 ~ NaC1. Group II rats served as control for the induction of TAT. The hormone was administered at 3.00 p.m. and the rats were killed by dislocation of the neck five hours after. The liver was removed immediately, washed in 0 . 9 ~ NaCI and blotted dry. A 10 percent, homogenate of the liver was prepared in 0.25 M sucrose containing 10-aM EDTA. It was then centrifuged in an International refrigerated centrifuge. The 700 × g pellet of the crude homogenate, and the 14 000 x g pellet of the supernatant constituted the n- and m T A T respectively. The supernatant obtained after the separation of mitochondria constituted the cTAT. The nuclear and mitochondrial pellets were suspended in 0.25 M sucrose and recentrifuged at 700 x g and 14 000 x g before use. The particulate fractions were washed once with 0.44 M sucrose and suspended in 0.05 M potassium phosphate buffer, pH 7.2 containing 0.01 mmole pyridoxal phosphate and 0.1 mmole a-ketoglutarate, and kept overnight before assaying the enzyme spectrophotometrically 9,1°. The protein content was estimated H. One unit of the enzyme was taken as that amount which catalysed the formation of one #mole of p-hydroxyphenyl pyruvate per min at 37°C. RESULTS AND DISCUSSION Table I shows that the specific activity of nTAT is higher than those of c- and m T A T of both the immature and the adult. Also, the activities of m- and nTAT are significantly lower in the adult. There is a significant decrease in m T A T after adrenalectomy in both the ages. n T A T of the immature, and cTAT of the adult also decrease after adrenalectomy, whereas cTAT of the immature and nTAT of the adult do not show any alteration. Administration of hydrocortisone to adrenalectomized rats causes very significant induction of all the three isoenzymes in both the ages, but that of the adult is far greater. It is of interest that actinomycin D also suppresses the induction of all the three isoenzymes to a far greater extent in the adult. T A T is a tetramer 12. Four forms of T A T have been observed by hydroxylapatite chromatography 13. cTAT has three electrophoretically separable forms, cTAT is anionic and m T A T is cationic 1°. It has been reported that different forms of T A T appear in a sequential manner during embryonic development of the rat 12,t4. This indicates that different isoenzymes of T A T are under the control of different genes, and they may have different physiological roles. Our studies show a decrease in the activity of m- and nTAT in the adult. This may be either due to a decrease in transcription or translation of specific messenger RNAs. Thyroxine and growth hormone are known to have an inhibitory effect on the activity of TAT. The levels of these two hormones increase during the growth period which may in turn inhibit the synthesis of TAT. Also it is seen that when the thyroxine level increases at the time of metamorphosis, the level of T A T decreases 15. This may
189 TABLE 1 SPECIFIC ACTIVITY (UNITS PER MG PROTEIN × 103) OF THE ISOENZYMES OF HEPATIC TYROSINE AMINOTRANSFERASE OF IMMATURE AND ADULT MALE RATS Cytoplasmic Mean* S.D.
Mitochondrial P
Mean* S.D.
Nuclear P
Mean* S.D.
P
(a) 6 week (immature)
Normal
9.125 ~ 0.867
Ad + Saline Ad + Hc
8.675 ~ 1.17 (--5.0%) 37.65 ~ 3.83 (+334%) Ad + 26.00 i 1.63 Act.D + (--30.0%) Hc
> 0.5 (NS) < 0.001 < 0.005
22.55 ± 3.74 16.00 ± 1.73 (--29.0%) 78.85 ~ 3.59 (+393.0%) 46,50 ± 6.75 (~41.0%)
< 0.02 < 0.001 < 0.001
42.70 ± 8.08 36.00 ± 7.3 (--15.3%) 81.88 ± 6.3 (+127.3%) 53.20 ~ 5.55 (--35.0%)
> 0.25 (NS) < 0.001 < 0.005
(b) 35 week (adult)
Normal Ad + Saline Ad + Hc
10.37 ~ 0.896
6.10 ± 0.525 (~1.2%) 78.3 ~ 10.27 (+1183.5%) Ad + 4.8 + 1.2 Act.D. + (--94.0%) Hc
< 0.005 < 0.00l < 0.001
16.72 ± 3.12 10.5 ± 1.98 (--37.2%) 100.34 ± 9.175 (+855.6%) 10.30± 1.88 (--89.8 %)
< 0.005 < 0.00l < 0.001
22.60 £
4.41
2 2 . 1 5 i 3.4 (--2.0%) 260.0 ± 45.8 (+1073.8%) 23.5 ± 2.06 (--91.0%)
> 0.8 (NS)
< 0.001 < 0.001
* The mean values of the data from 4-5 rats are given. Ad, adrenalectomized; Act.D, actinomycin D; Hc, hydrocortisone.
be because the availability o f tyrosine, a substrate for thyroxine, decreases d u r i n g this period. It has been previously r e p o r t e d t h a t in the early n e o n a t a l life, t o t a l T A T is inducible by glucagon, b u t n o t by h y d r o c o r t i s o n e 16. As g r o w t h proceeds, however, the responsiveness to h y d r o c o r t i s o n e increases. Thus the responsiveness o f T A T a p p e a r s to be d e p e n d e n t on a d r e n a l h o r m o n e s . O u r finding t h a t the three isoenzymes o f T A T have different responsiveness to h y d r o c o r t i s o n e in the i m m a t u r e a n d the a d u l t m a y be related to the a m o u n t o f the h o r m o n e p r o d u c e d by the a d r e n a l cortex d u r i n g the g r o w t h period. This is in c o n f o r m i t y with the previous studies on the responsiveness o f T A T to the h o r m o n e in n e o n a t a l life. It is k n o w n t h a t the i n d u c t i o n o f T A T by h y d r o c o r t i s o n e is b r o u g h t a b o u t by s t i m u l a t i o n o f specific T A T - m e s s e n g e r R N A synthesis 17. The s u p p r e s s i o n o f the i n d u c t i o n o f the three isoenzymes b y actinomycin D shows t h a t there are three distinct genes which are responsible for their synthesis. W h e t h e r o r n o t the i n d u c t i o n o f each isoenzyme by h y d r o c o r t i s o n e is m e d i a t e d t h r o u g h separate c y t o p l a s m i c receptors is n o t clear. O u r studies indicate t h a t the i n d u c t i o n o f the isoenzymes o f T A T by steroid h o r m o n e s occurs in a sequential m a n n e r till a d u l t h o o d . This m a y be o f p h y s i o l o g i c a l significance. This is consistent
190
with the gene regulation theory of aging is according to which in each organism programmed changes in the activities of genes occur that govern the levels of various enzymes and hence the activity of the individual at various phases of its life span. ACKNOWLEDGEMENTS
This research was supported by grants from the Nuffield Foundation, London, and PL-480 (In-ARS-24) made to M.S.K.
REFERENCES 1 S. N. Singh and M. S. Kanungo, Alterations in lactate dehydrogenase of the brain, heart, skeletal muscle and liver of rats of various ages, J. Biol. Chem., 243 (1968) 4526. 2 M. S. Kanungo and B. S. Gandhi, Induction of malate dehydrogenase isoenzymes in livers of young and old rats, Proc. Nat. Acad. Sci. U.S., 69 (1972) 2035. 3 S. K. Patnaik and M. S. Kanungo, Different patterns of induction of the two isoenzymes of alanine aminotransferase of liver of rat as a function of age, Biochem. Biophys. Res. Commun., 56 (1974) 845. 4 F. T. Kenney and R. M. Flora, Induction of tyrosine-a-ketoglutarate transaminase in rat liver. I. Hormonal nature, J. Biol. Chem., 236 (1961) 2699. 5 G. Litwack, M. L. Sears and T. I. Diamondstone, Intracellular distribution of tyrosine-a ketoglutarate transaminase and 4-14C-hydrocortisone activities during induction, J. Biol. Chem., 238 0963) 302. 6 J. E. Miller and G. Litwack, Subcellular distribution of tyrosine aminotransferase in rat brain, Arch. Biochem. Biophys., 134 (1969) 149. 7 J. Fellman, P. J. Vanbellinghen, R. T. Jones and R. D. Koler, Soluble and mitochondrial forms of tyrosine aminotransfcrase. Relationship to human tyrosinemia, Biochemistry, 8 0969) 615. 8 J. E. Miller and G. Litwack, Studies on soluble and mitochondrial tyrosine aminotransferase. Evidence for a physical change in the cytosol enzyme during induction, Biochem. Biophys. Res. Commun., 36 (1969) 35. 9 F. T. Kenney, D. L. Greenman, W. D. Wicks and W. L. Albritton, R N A synthesis and enzyme induction by hydrocortisone, Advan. Enzyme Regul., 3 (1965) 1. 10 J. E. Miller and G. Litwack, Purification, properties and identity of liver mitochondrial tyrosine aminotransferase, J. Biol. Chem., 246 (1971) 3234. 11 E. W. Sutherland, C. F. Cori, R. Haynes and N. S. Olsen, Purification of the hyperglycemicglycogenolytic factor from gastric mucosa, J. Biol. Chem., 180 (1949) 825. 12 F. Auriscchio, F. Valeriote, G. M. Tomkins and W. D. Riley, Studies on the structure of tyrosine aminotransferase, Biochim. Biophys. Acta, 221 (1970) 307. 13 I. Yoshifumi and H. C. Pitot, The regulation of four forms of tyrosine aminotransferase in adult and developing rat liver, Life Sci., 10 (1971) 1071. 14 P. G. Holt and I. T. Oliver, Multiple forms of soluble rat liver tyrosine aminotransferase. Premature induction and postnatal development, Int. J. Biochem., 2 (1971) 212. 15 C. Shung-Kai and P. P. Cohen, Activity of three transaminases during spontaneous and induced metamorphosis in Rana catesbianca, Arch. Biochem. Biophys., 104 0964) 325. 16 G. Olga and H. K. Dewey, Initiation by glucagon of the premature development of tyrosine aminotransferase, serine dehydratase and glucose-6-phosphatase in fetal rat liver, J. Biol. Chem., 242 (1967) 1986. 17 B. Peterkofsky and G. M. Tomkins, Evidence for the steroid-induced accumulation of tyrosine aminotransferase messenger R N A in the absence of protein synthesis, Proc. Nat. Acad. Sci. U.S., 60 (1968) 222. 18 M. S. Kanungo, Biochemistry of aging, Biochem. Rev. India, 41 (1970) 13.
187
© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
A G E - D E P E N D E N T I N D U C T I O N OF I S O E N Z Y M E S OF T Y R O S I N E A M I N O T R A N S F E R A S E OF T H E L I V E R OF T H E R A T
B. K. RATHA and M. S. KANUNGO Biochemistry Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)
(Received May 7, 1974)
SUMMARY The effects of adrenalectomy, hydrocortisone and actinomycin D on the cytoplasmic (c-), mitochondrial (m-) and nuclear (n-) tyrosine aminotransferase (TAT) isoenzymes of the liver of immature (6-) and adult (35-week) male albino rats were studied. Adrenalectomy stimulates the activity of n T A T in adult rats. The activities of m T A T of immature, and of c- and m T A T of adult rats are inhibited by adrenalectomy. Hydrocortisone induces and actinomycin D inhibits the induction of the isoenzymes in adrenalectomized rats of both ages. However, the induction and inhibition are more pronounced in the adult than in the immature.
INTRODUCTION We have initiated studies on the alterations in the levels 1 and induction2, a of certain isoenzymes of the rat in an attempt to understand the mechanism of ageing at the genetic level. Tyrosine aminotransferase (TAT; EC 2.6.1.5) is a suitable enzyme for such studies as it is rapidly induced by gluco-corticoids4, 5. It has three isoenzymes, cytoplasmic (c-), mitochondrial (m-) and nuclear T A T (nTAT)5, 6. m T A T has a broader tissue distribution v. In the pathological condition of tyrosinemia, the liver has only m T A T and no cTAT. Though there is clear demonstration that cTAT is induced by hydrocortisone, the induction of the two other isoenzymes by various steroid hormones remains disputed s. We report here that hydrocortisone induces all the three isoenzymes of TAT, and that there are age-related differences in their induction. MATERIALS AND METHODS Immature (6-) and adult (35-week) male albino rats of Wistar strain, maintained in standard laboratory conditions, were used. Rats of each age were divided into four groups, each having 4-5 rats. G r o u p I consisted of normal rats which were administered 1.0 ml of 0.9 ~ NaCl intraperitoneally and served as control. Groups II, I I I and IV rats were bilaterally adrenalectomized and were given 0.9 ~ NaC1 ad lib
188 instead of tap water for 10 days after operation. On the 1 lth day, G r o u p II rats were given 1.0 ml of 0.9 ~o NaCI, Group III rats were administered 5 mg hydrocortisone/100 g body wt., and Group IV rats were administered actinomycin D (0.1 mg/100 g body wt.) one hour prior to hydrocortisone administration. All the injections were given intraperitoneally in 1.0 ml of 0.9 ~ NaC1. Group II rats served as control for the induction of TAT. The hormone was administered at 3.00 p.m. and the rats were killed by dislocation of the neck five hours after. The liver was removed immediately, washed in 0 . 9 ~ NaCI and blotted dry. A 10 percent, homogenate of the liver was prepared in 0.25 M sucrose containing 10-aM EDTA. It was then centrifuged in an International refrigerated centrifuge. The 700 × g pellet of the crude homogenate, and the 14 000 x g pellet of the supernatant constituted the n- and m T A T respectively. The supernatant obtained after the separation of mitochondria constituted the cTAT. The nuclear and mitochondrial pellets were suspended in 0.25 M sucrose and recentrifuged at 700 x g and 14 000 x g before use. The particulate fractions were washed once with 0.44 M sucrose and suspended in 0.05 M potassium phosphate buffer, pH 7.2 containing 0.01 mmole pyridoxal phosphate and 0.1 mmole a-ketoglutarate, and kept overnight before assaying the enzyme spectrophotometrically 9,1°. The protein content was estimated H. One unit of the enzyme was taken as that amount which catalysed the formation of one #mole of p-hydroxyphenyl pyruvate per min at 37°C. RESULTS AND DISCUSSION Table I shows that the specific activity of nTAT is higher than those of c- and m T A T of both the immature and the adult. Also, the activities of m- and nTAT are significantly lower in the adult. There is a significant decrease in m T A T after adrenalectomy in both the ages. n T A T of the immature, and cTAT of the adult also decrease after adrenalectomy, whereas cTAT of the immature and nTAT of the adult do not show any alteration. Administration of hydrocortisone to adrenalectomized rats causes very significant induction of all the three isoenzymes in both the ages, but that of the adult is far greater. It is of interest that actinomycin D also suppresses the induction of all the three isoenzymes to a far greater extent in the adult. T A T is a tetramer 12. Four forms of T A T have been observed by hydroxylapatite chromatography 13. cTAT has three electrophoretically separable forms, cTAT is anionic and m T A T is cationic 1°. It has been reported that different forms of T A T appear in a sequential manner during embryonic development of the rat 12,t4. This indicates that different isoenzymes of T A T are under the control of different genes, and they may have different physiological roles. Our studies show a decrease in the activity of m- and nTAT in the adult. This may be either due to a decrease in transcription or translation of specific messenger RNAs. Thyroxine and growth hormone are known to have an inhibitory effect on the activity of TAT. The levels of these two hormones increase during the growth period which may in turn inhibit the synthesis of TAT. Also it is seen that when the thyroxine level increases at the time of metamorphosis, the level of T A T decreases 15. This may
189 TABLE 1 SPECIFIC ACTIVITY (UNITS PER MG PROTEIN × 103) OF THE ISOENZYMES OF HEPATIC TYROSINE AMINOTRANSFERASE OF IMMATURE AND ADULT MALE RATS Cytoplasmic Mean* S.D.
Mitochondrial P
Mean* S.D.
Nuclear P
Mean* S.D.
P
(a) 6 week (immature)
Normal
9.125 ~ 0.867
Ad + Saline Ad + Hc
8.675 ~ 1.17 (--5.0%) 37.65 ~ 3.83 (+334%) Ad + 26.00 i 1.63 Act.D + (--30.0%) Hc
> 0.5 (NS) < 0.001 < 0.005
22.55 ± 3.74 16.00 ± 1.73 (--29.0%) 78.85 ~ 3.59 (+393.0%) 46,50 ± 6.75 (~41.0%)
< 0.02 < 0.001 < 0.001
42.70 ± 8.08 36.00 ± 7.3 (--15.3%) 81.88 ± 6.3 (+127.3%) 53.20 ~ 5.55 (--35.0%)
> 0.25 (NS) < 0.001 < 0.005
(b) 35 week (adult)
Normal Ad + Saline Ad + Hc
10.37 ~ 0.896
6.10 ± 0.525 (~1.2%) 78.3 ~ 10.27 (+1183.5%) Ad + 4.8 + 1.2 Act.D. + (--94.0%) Hc
< 0.005 < 0.00l < 0.001
16.72 ± 3.12 10.5 ± 1.98 (--37.2%) 100.34 ± 9.175 (+855.6%) 10.30± 1.88 (--89.8 %)
< 0.005 < 0.00l < 0.001
22.60 £
4.41
2 2 . 1 5 i 3.4 (--2.0%) 260.0 ± 45.8 (+1073.8%) 23.5 ± 2.06 (--91.0%)
> 0.8 (NS)
< 0.001 < 0.001
* The mean values of the data from 4-5 rats are given. Ad, adrenalectomized; Act.D, actinomycin D; Hc, hydrocortisone.
be because the availability o f tyrosine, a substrate for thyroxine, decreases d u r i n g this period. It has been previously r e p o r t e d t h a t in the early n e o n a t a l life, t o t a l T A T is inducible by glucagon, b u t n o t by h y d r o c o r t i s o n e 16. As g r o w t h proceeds, however, the responsiveness to h y d r o c o r t i s o n e increases. Thus the responsiveness o f T A T a p p e a r s to be d e p e n d e n t on a d r e n a l h o r m o n e s . O u r finding t h a t the three isoenzymes o f T A T have different responsiveness to h y d r o c o r t i s o n e in the i m m a t u r e a n d the a d u l t m a y be related to the a m o u n t o f the h o r m o n e p r o d u c e d by the a d r e n a l cortex d u r i n g the g r o w t h period. This is in c o n f o r m i t y with the previous studies on the responsiveness o f T A T to the h o r m o n e in n e o n a t a l life. It is k n o w n t h a t the i n d u c t i o n o f T A T by h y d r o c o r t i s o n e is b r o u g h t a b o u t by s t i m u l a t i o n o f specific T A T - m e s s e n g e r R N A synthesis 17. The s u p p r e s s i o n o f the i n d u c t i o n o f the three isoenzymes b y actinomycin D shows t h a t there are three distinct genes which are responsible for their synthesis. W h e t h e r o r n o t the i n d u c t i o n o f each isoenzyme by h y d r o c o r t i s o n e is m e d i a t e d t h r o u g h separate c y t o p l a s m i c receptors is n o t clear. O u r studies indicate t h a t the i n d u c t i o n o f the isoenzymes o f T A T by steroid h o r m o n e s occurs in a sequential m a n n e r till a d u l t h o o d . This m a y be o f p h y s i o l o g i c a l significance. This is consistent
190
with the gene regulation theory of aging is according to which in each organism programmed changes in the activities of genes occur that govern the levels of various enzymes and hence the activity of the individual at various phases of its life span. ACKNOWLEDGEMENTS
This research was supported by grants from the Nuffield Foundation, London, and PL-480 (In-ARS-24) made to M.S.K.
REFERENCES 1 S. N. Singh and M. S. Kanungo, Alterations in lactate dehydrogenase of the brain, heart, skeletal muscle and liver of rats of various ages, J. Biol. Chem., 243 (1968) 4526. 2 M. S. Kanungo and B. S. Gandhi, Induction of malate dehydrogenase isoenzymes in livers of young and old rats, Proc. Nat. Acad. Sci. U.S., 69 (1972) 2035. 3 S. K. Patnaik and M. S. Kanungo, Different patterns of induction of the two isoenzymes of alanine aminotransferase of liver of rat as a function of age, Biochem. Biophys. Res. Commun., 56 (1974) 845. 4 F. T. Kenney and R. M. Flora, Induction of tyrosine-a-ketoglutarate transaminase in rat liver. I. Hormonal nature, J. Biol. Chem., 236 (1961) 2699. 5 G. Litwack, M. L. Sears and T. I. Diamondstone, Intracellular distribution of tyrosine-a ketoglutarate transaminase and 4-14C-hydrocortisone activities during induction, J. Biol. Chem., 238 0963) 302. 6 J. E. Miller and G. Litwack, Subcellular distribution of tyrosine aminotransferase in rat brain, Arch. Biochem. Biophys., 134 (1969) 149. 7 J. Fellman, P. J. Vanbellinghen, R. T. Jones and R. D. Koler, Soluble and mitochondrial forms of tyrosine aminotransfcrase. Relationship to human tyrosinemia, Biochemistry, 8 0969) 615. 8 J. E. Miller and G. Litwack, Studies on soluble and mitochondrial tyrosine aminotransferase. Evidence for a physical change in the cytosol enzyme during induction, Biochem. Biophys. Res. Commun., 36 (1969) 35. 9 F. T. Kenney, D. L. Greenman, W. D. Wicks and W. L. Albritton, R N A synthesis and enzyme induction by hydrocortisone, Advan. Enzyme Regul., 3 (1965) 1. 10 J. E. Miller and G. Litwack, Purification, properties and identity of liver mitochondrial tyrosine aminotransferase, J. Biol. Chem., 246 (1971) 3234. 11 E. W. Sutherland, C. F. Cori, R. Haynes and N. S. Olsen, Purification of the hyperglycemicglycogenolytic factor from gastric mucosa, J. Biol. Chem., 180 (1949) 825. 12 F. Auriscchio, F. Valeriote, G. M. Tomkins and W. D. Riley, Studies on the structure of tyrosine aminotransferase, Biochim. Biophys. Acta, 221 (1970) 307. 13 I. Yoshifumi and H. C. Pitot, The regulation of four forms of tyrosine aminotransferase in adult and developing rat liver, Life Sci., 10 (1971) 1071. 14 P. G. Holt and I. T. Oliver, Multiple forms of soluble rat liver tyrosine aminotransferase. Premature induction and postnatal development, Int. J. Biochem., 2 (1971) 212. 15 C. Shung-Kai and P. P. Cohen, Activity of three transaminases during spontaneous and induced metamorphosis in Rana catesbianca, Arch. Biochem. Biophys., 104 0964) 325. 16 G. Olga and H. K. Dewey, Initiation by glucagon of the premature development of tyrosine aminotransferase, serine dehydratase and glucose-6-phosphatase in fetal rat liver, J. Biol. Chem., 242 (1967) 1986. 17 B. Peterkofsky and G. M. Tomkins, Evidence for the steroid-induced accumulation of tyrosine aminotransferase messenger R N A in the absence of protein synthesis, Proc. Nat. Acad. Sci. U.S., 60 (1968) 222. 18 M. S. Kanungo, Biochemistry of aging, Biochem. Rev. India, 41 (1970) 13.