Oncology 35.- 73-75 (1978)
Expression of Hormonally Induced Tyrosine Aminotransferase in Host Liver and Morris Hepatoma No. 7777 During Cofactor Depletion J. K. Shuler and G. P. T ryfiates Department of Biochemistry, West Virginia University, Morgantown, West Virginia Supported by G eneral Research Support grants from the West Virginia University Medical Corp., the local chapter of the Am. Cancer Socity and the School of Dentistry, and in part by USPHS grant CA 13759 from the National Cancer Institute. J. K. Shuler supported by graduate training award IN-76K from the American Cancer Society.
Abstract. Cytoplasmic tyrosine aminotransferase (L-tyrosine: 2oxoglutarate aminotransferase, EC2.6.1.5) was partially purified from host liver and Morris hepatoma No. 7777 grown in pyridoxine depleted rats. The animals were sacrificed six hours following the intrapcritoneal administration of hydrocortisone hemisuccinate. Enzyme pre parations were subsequently resolved by electrophoresis on poly acrylamide gels. Enzyme activity was detected histochemically in situ on the gels. Six and at least three enzymatically active protein peaks were detected in host liver and the hepatoma, respectively, by this method. Key Words. Aminotransferase - Expression - Acrylamide - Hepatoma - Pyridoxine - Hydrocortisone - Depletion
Introduction It is well known that cytoplasmic hepatic tyrosine am inotrans ferase (TA T) responds to the intraperitoneal adm inistration of steroids and/or other agents. The response of the enzyme to hydrocortisone varies in different Morris hepatom a lines from com plete unresponsiveness to significant increases [1, 2], The pattern of its horm onal induction is drastically altered in host liver and this hepatom a [3], In animals without tum ors, cofac tor depletion, i. e. pyridoxine depletion, alters the am inotrans ferase horm onal induction pattern and effects further resolu tion of the enzyme on electrophoresis in polyacrylamide gel [4, 5, 6], Further, lack of dietary pyridoxine significantly inhibits the growth of several M orris hepatom a lines, including that of hepatom a No. 7777 [3, 7-10], R at liver cytoplasmic tyrosine am inotransferase consists of multiple forms which may be affected by horm onal adm inistration [11-19]. W hether cofactor depletion affects the expression of the enzyme in hepatom a-bearing animals has not been investigated. The ex pression of tyrosine am inotransferase in host liver and hepa tom a No. 7777 during pyridoxine depletion was investigated in this study using polyacrylamide gel electrophoresis for protein resolution and histochemical staining for in situ detection of enzymatic activity.
placed in individual cages in the animal quarters and fed ad libitum a commercially prepared (N utr. Biochemicals Corp., Cleveland. O H ) diet lacking pyridoxine [2, 8, 10]. A fter 21 days, cells from Morris hepatom a No. 7777 (gen. 101) were inoculated into the hinds legs of the animals [2, 8, 10]. The animals were maintained on a strict lighting schedule. Lights w ere on for 12 hours daily starting at 7:00 a. m. Animals were injected (7 m g /100 g body wt) intraperitoneally with hydrocortisone hemisuccinate (Schwartz/M ann) in 0.14 M N aC l six hours before sacrifice. They w ere killed before noon by decapitation and after 24 days of tum or growth. The tissues were excised and washed in ice cold 0.14 M KC1 con taining 10~3 M ED TA . The pooled livers and tum ors were homogenised in three volumes of 0.05 M potassium phosphate buffer, pH 7.6, containing 10~3 M E D T A , 10~3 M dithiothreitol and 0.2 mM pyridoxal phosphate. The enzyme was purified from the high speed supernatant. Procedures for enzyme purification, gel electrophoresis, histochemical staining of enzymatic activity in situ (on the gel), enzyme assay, etc., have been reported in detail [5, 20],
Results G el scans following electrophoresis and histochemical staining of partially purified am inotransferase preparations from host liver and hepatom a No. 7777 are shown in figures l and 2, respectively. O nly representative results are presented. Each sample was derived from five pooled livers or ten pooled hepatom as grown in pyridoxine depleted animals (samples from single livers and tumors were also tested). The pattern of expression of tyrosine am inotransferase activity in the hepatom a is definitely different from that of the respective host liver. The enzyme from the host liver was resolved into six enzymatically active peaks (figure 1) while the tum or enzyme was resolved in at least three active peaks (figure 2).
Discussion
Buffalo strain female weanling rats were purchased from Simonsen Labs, Inc., Gilroy, CA. Upon arrival, they were
H epatic tyrosine am inotransferase consists of multiple forms which may be differentially affected by different environm ent al stimuli such as hormones, am ino acids, cyclic nucleotides.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:53:13 AM
Materials and Methods
Shuler, Tryfiates: Enzyme Expression in Host Liver Hepatoma
74
Figure 1. Polyacrylamide gel electrophoresis of partially purified host liver tyrosine aminotransferase from pyridoxine depleted, hydrocorti sone induced, hepatoma-bearing animals. After electrophoresis enzyme activity was located histochemically and gel scans were taken. This gel scan, as well as that of figure 2, shows representative results. In all cases, the results were repeated at different times using different preparations. Preparations were also made six months apart and electrophoresed, etc, at that time. The specific activity prior to purification (105,000 x g; 1 hr; 4° c) was 114.6 units/mg protein. The yield was 100 %. The animals were fed the pyridoxine depleted diet for 45 days. The average weight of five livers in grams was 2.5 ± 0.3 (Standard Deviation). Approximately 10 ug of protein (5.73 enzyme units) were electrophoresed. Animal growth curves were as reported elsewhere [6], Other information in the Materials and Methods, and as previously described [5]. The cathode (-) is at the left of the figures. The specific activity of the purified preparation (prior to electro phoresis) was 575 units/mg protein.
etc. [14, 15, 21]. The response of the enzyme to hydrocorti sone adm inistration in animals bearing M orris hepatom as varies significantly [1, 2, 7], The horm onal enzyme induction pattern in the presence of the growing tum or is completely altered [3]. Results presented here show the multiple form nature of the horm onally induced enzyme as well as its varied pattern of expression in the host liver and this hepatom a. While six enzymatically active peaks are expressed in host liver only three (possibly four) are seen in the tum or itself. The opposite was observed in animals bearing this hepatom a and fed the pyridoxine supplem ented diet [20], Enzyme expression was highly limited (repressed) in the most liver-like (highly differentiated) hepatom a No. 7794A but was most fully ex pressed in hepatom a No. 7777 (i. e. the least liver-like tum or). Both the host liver and hepatom a No. 7794A showed one enzymatically active peak by the histochemical m ethod [20]. T hat cofactor lack results in TAT derepression was also shown in the liver of animals without hepatom as [5]. M oreover, TA T derepression was further verified by the im m unoprécipitation of the resolved gel proteins with antibody prepared against highly purified (> 3 ,0 0 0 fold) hepatic tyrosine am inotrans ferase [22, 23], Since lack of cofactor results in inhibition of tum or growth [3, 7 -1 0 ] and also enzyme derepression [5, 20, 22, 23] it is suggested herewith that the vitamin exercises a type of regulation over enzyme expression which could be essential for norm al tum or growth. It has been reported that the proportion of each T A T form could be varied by varying the pH of the homogenising medium [24]. Furtherm ore, a particulate liver fraction com po nent has been described which interconverts T A T multiple forms and whose catalytic action is pH dependent [25, 26]. It is possible that horm onal effects on T A T expression could reflect effects on the liver factor [25, 26], As suggested previously [23], this could also be the case with T A T derepression seen during cofactor depletion, i. e. TA T derepression may reflect a cofactor influence on the liver factor [25. 26]. D uring the course of purification the samples w ere handled identically and all protective factors were present [19]. The possibility of dénaturation taking place seems, therefore, unlikely. No attem pt was made to achieve com plete separation of the enzyme forms and/or characterise these further. A dditional tests are planned in this regard.
Acknowledgment Thanks are due to Dr. H. P. M o rris for giving us gratis the hepatom a donor animal and his continued interest and encouragem ent.
References 1
Potter , V. R.; W atanabe, M.; P itot, H. C., and Morris , H.
P.: Systematic oscillations in metabolic activity in rat liver and hepatomas. Survey of normal diploid and other hepatoma lines. Cancer Res. 29:55-78 (1969).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:53:13 AM
Figure 2. Polyacrylamide gel electrophoresis of partially purified tyro sine aminotransferase from Morris hepatoma No. 7777. Hepatomas were grown in pyridoxine depleted rats for 24 days. The animals were fed the pyridoxine depleted diet for a period of 45 days. The average weight of ten hepatomas in grams was 5.7 ± 0.5 (Standard Deviation). Animals were sacrificed following induction with hydrocortisone. The specific activity prior to purification (105,000 x g; 1 hr; 4° C) was 120.2 units/mg protein. After the heat step the specific activity was 610 units/mg protein. The yield was 100 %. Approximately 5 ug of protein (3.05 units) were electrophoresed. Other details as in figure 1.
Shuler, Tryfiates: Enzyme Expression in Host Liver Hepatoma
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T ryfiates, G. P.; Shuler , J. K., and Morris , H. P.: Enzyme
activities in vitamin B6 deficient, normal and tumor-bearing animals. Effect of hydrocortisone. Proc. Soc. exp. Biol. Med. 145:1363-1367(1974). T ryfiates, G. P.; Saus, F. L., and Morris , H. P.: Hormonal induction of tyrosine transaminase in host liver and hepatoma No. 7777 of normal and cofactor depleted animals. J. natn. Cancer Inst. 55:839-841 (1975). Puskar, T. and T ryfiates, G. P.: Induction of tyrosine transa minase by hydrocortisone in vitamin B6 deficient rats. J. Nutr. 104:1407-1415(1974). Shuler , J. K. and T ryfiates, G. P.: Effect of cofactor depletion on liver tyrosine aminotransferase expression. Enzyme 22: 262-265(1977). T ryfiates, G. P. and Saus, F. L.: Hormonal induction patterns of tyrosine aminotransferase in normal and cofactor depleted animals. Cancer Biochem. Biophys. 1:63-68 (1975). T ryfiates, G. P.; Shuler , J. K.; H efner , M. J., and Morris , H. P.: Effect of Bs deficiency on hepatoma 7794A growth rate: activities of tyrosine transaminase and serine dehydratase before and after induction by hydrocortisone. Eur. J. Cancer 10: 147-154(1974). T ryfiates, G. P. and Morris, H. P.: Effect of pyridoxine defi ciency on tyrosine transaminase activity and growth of four Morris hepatomas. J. natn. Cancer Inst. 52:1259-1262 (1974). T ryfiates, G. P. and Morris, H. P.: Growth of Morris hepatoma No. 7794A with and without vitamin Bé: effect of inoculation time. Eur. J. Cancer 12:9-12 (1976). T ryfiates, G. P.: Effect of pyridoxine on the growth of Morris hepatoma No. 7288Ctc and enzyme activity. Oncology 33: 209-211 (1976). A uricchio , F.; Valeriote , F. A.; T omkins, G. M., and R iley, W. D.: Studies on the structure of tyrosine transaminase. Biochim. biophys. Acta 221:307-313 (1970). G elehrter , T. D.; E mannuel, J. R.. and Spencer, C. H.: Induction of tyrosine aminotransferase by dexamethasone, in sulin and serum. Characterization of the induced enzyme. J. biol. Chem. 247:6197-6203 (1972). Holt . P. G. and O liver , I. T.: Multiple forms of tyrosine amino transferase in rat liver and their hormonal induction in the ceonate. FEBS Lett. 5: 89-91 (1969). Iwasaki, Y.; L amar, C.; D anenberg , K., and P itot , H. C.: Studies on the induction and expression of enzymes in rat liver. Characterization and metabolic regulation of multiple forms of tyrosine aminotransferase. Eur. J. Biochem. 34: 347-357 (1973).
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P itot, H. C. et al: Regulation of the levels of multiple forms of
serine dehydratase and tyrosine aminotransferase in rat tissues. Gann 13: 191-204(1972). Sadlf.ir , J. W.; Holt , P. G., and O liver, 1. T.: Fractionation of rat liver tyrosine aminotransferase during the course of purifica tion. Further evidence for multiple forms of the enzyme. FEBS Lett. 6:46-48(1970). Spencer , C. J. and G elehrter , T. D.: Pseudoisozymes of hepatic tyrosine aminotransferase. J. biol. Chem. 249: 577-583 (1974). T ryfiates, G. P.: On the synthesis of tyrosine transaminase in vitro. Biochim. biophys. Acta 169:779-781 (1969). Vai.f.riote, F. A.; A uricchio , F.; Tomkins, G. M., and R iley, W. D.: Purification and properties of rat liver tyrosine amino transferase. J. biol. Chem. 244:3618-3624 (1969). Shuler , J. K. and T ryfiates, G. P.: Expression of hormonally induced tyrosine transaminase in normal, host liver and three Morris hepatomas. Oncology 33:212-214 (1976). T ryfiates, G. P. and L itwack, G.: Appearance of an increment of tyrosine aminotransferase in a cell-free system. Biochemistry N.Y. 3: 1483-1487(1964). T ryfiates, G. P. and Saus, F. L.: Hepatic and tumor proteins reactive with tyrosine transaminase-specific rabbit antiserum. Eur. J. Cancer 12: 833-837(1976). T ryfiates, G. P.: Vitamin B6 effects on the growth of Morris hepatomas and the development of enzymatic activity. In Morris and C riss, Morris hepatomas. Mechanisms of regulation, pp. 607-642 (Plenum Press. New York 1977). Johnson, R. W. and G rossman, A.: Problems in interpreting the significance of multiple form patterns of rat liver tyrosine aminotransferase. Biochem. biophys. Res. Commun. 59: 520-526(1974). Smith, G. J.; Pearce , P. H., and O liver , I. T.: A liver factor that interconverts multiple forms of tyrosine aminotransferase. LifeSci. 16:437-450 (1975). R odriguez , J. H. and P itot, H. C.: The interconversion of multiple forms of tyrosine aminotransferase. Biochem. biophys. Res. Commun. 65:510-518 (1975).
Request reprints from: Dr. G. P. T ryfiates, Department of Bio chemistry, West Virginia University, School of Medicine, Morgan town, WV 26506 (USA).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:53:13 AM
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Expression of Hormonally Induced Tyrosine Aminotransferase in Host Liver and Morris Hepatoma No. 7777 During Cofactor Depletion J. K. Shuler and G. P. T ryfiates Department of Biochemistry, West Virginia University, Morgantown, West Virginia Supported by G eneral Research Support grants from the West Virginia University Medical Corp., the local chapter of the Am. Cancer Socity and the School of Dentistry, and in part by USPHS grant CA 13759 from the National Cancer Institute. J. K. Shuler supported by graduate training award IN-76K from the American Cancer Society.
Abstract. Cytoplasmic tyrosine aminotransferase (L-tyrosine: 2oxoglutarate aminotransferase, EC2.6.1.5) was partially purified from host liver and Morris hepatoma No. 7777 grown in pyridoxine depleted rats. The animals were sacrificed six hours following the intrapcritoneal administration of hydrocortisone hemisuccinate. Enzyme pre parations were subsequently resolved by electrophoresis on poly acrylamide gels. Enzyme activity was detected histochemically in situ on the gels. Six and at least three enzymatically active protein peaks were detected in host liver and the hepatoma, respectively, by this method. Key Words. Aminotransferase - Expression - Acrylamide - Hepatoma - Pyridoxine - Hydrocortisone - Depletion
Introduction It is well known that cytoplasmic hepatic tyrosine am inotrans ferase (TA T) responds to the intraperitoneal adm inistration of steroids and/or other agents. The response of the enzyme to hydrocortisone varies in different Morris hepatom a lines from com plete unresponsiveness to significant increases [1, 2], The pattern of its horm onal induction is drastically altered in host liver and this hepatom a [3], In animals without tum ors, cofac tor depletion, i. e. pyridoxine depletion, alters the am inotrans ferase horm onal induction pattern and effects further resolu tion of the enzyme on electrophoresis in polyacrylamide gel [4, 5, 6], Further, lack of dietary pyridoxine significantly inhibits the growth of several M orris hepatom a lines, including that of hepatom a No. 7777 [3, 7-10], R at liver cytoplasmic tyrosine am inotransferase consists of multiple forms which may be affected by horm onal adm inistration [11-19]. W hether cofactor depletion affects the expression of the enzyme in hepatom a-bearing animals has not been investigated. The ex pression of tyrosine am inotransferase in host liver and hepa tom a No. 7777 during pyridoxine depletion was investigated in this study using polyacrylamide gel electrophoresis for protein resolution and histochemical staining for in situ detection of enzymatic activity.
placed in individual cages in the animal quarters and fed ad libitum a commercially prepared (N utr. Biochemicals Corp., Cleveland. O H ) diet lacking pyridoxine [2, 8, 10]. A fter 21 days, cells from Morris hepatom a No. 7777 (gen. 101) were inoculated into the hinds legs of the animals [2, 8, 10]. The animals were maintained on a strict lighting schedule. Lights w ere on for 12 hours daily starting at 7:00 a. m. Animals were injected (7 m g /100 g body wt) intraperitoneally with hydrocortisone hemisuccinate (Schwartz/M ann) in 0.14 M N aC l six hours before sacrifice. They w ere killed before noon by decapitation and after 24 days of tum or growth. The tissues were excised and washed in ice cold 0.14 M KC1 con taining 10~3 M ED TA . The pooled livers and tum ors were homogenised in three volumes of 0.05 M potassium phosphate buffer, pH 7.6, containing 10~3 M E D T A , 10~3 M dithiothreitol and 0.2 mM pyridoxal phosphate. The enzyme was purified from the high speed supernatant. Procedures for enzyme purification, gel electrophoresis, histochemical staining of enzymatic activity in situ (on the gel), enzyme assay, etc., have been reported in detail [5, 20],
Results G el scans following electrophoresis and histochemical staining of partially purified am inotransferase preparations from host liver and hepatom a No. 7777 are shown in figures l and 2, respectively. O nly representative results are presented. Each sample was derived from five pooled livers or ten pooled hepatom as grown in pyridoxine depleted animals (samples from single livers and tumors were also tested). The pattern of expression of tyrosine am inotransferase activity in the hepatom a is definitely different from that of the respective host liver. The enzyme from the host liver was resolved into six enzymatically active peaks (figure 1) while the tum or enzyme was resolved in at least three active peaks (figure 2).
Discussion
Buffalo strain female weanling rats were purchased from Simonsen Labs, Inc., Gilroy, CA. Upon arrival, they were
H epatic tyrosine am inotransferase consists of multiple forms which may be differentially affected by different environm ent al stimuli such as hormones, am ino acids, cyclic nucleotides.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:53:13 AM
Materials and Methods
Shuler, Tryfiates: Enzyme Expression in Host Liver Hepatoma
74
Figure 1. Polyacrylamide gel electrophoresis of partially purified host liver tyrosine aminotransferase from pyridoxine depleted, hydrocorti sone induced, hepatoma-bearing animals. After electrophoresis enzyme activity was located histochemically and gel scans were taken. This gel scan, as well as that of figure 2, shows representative results. In all cases, the results were repeated at different times using different preparations. Preparations were also made six months apart and electrophoresed, etc, at that time. The specific activity prior to purification (105,000 x g; 1 hr; 4° c) was 114.6 units/mg protein. The yield was 100 %. The animals were fed the pyridoxine depleted diet for 45 days. The average weight of five livers in grams was 2.5 ± 0.3 (Standard Deviation). Approximately 10 ug of protein (5.73 enzyme units) were electrophoresed. Animal growth curves were as reported elsewhere [6], Other information in the Materials and Methods, and as previously described [5]. The cathode (-) is at the left of the figures. The specific activity of the purified preparation (prior to electro phoresis) was 575 units/mg protein.
etc. [14, 15, 21]. The response of the enzyme to hydrocorti sone adm inistration in animals bearing M orris hepatom as varies significantly [1, 2, 7], The horm onal enzyme induction pattern in the presence of the growing tum or is completely altered [3]. Results presented here show the multiple form nature of the horm onally induced enzyme as well as its varied pattern of expression in the host liver and this hepatom a. While six enzymatically active peaks are expressed in host liver only three (possibly four) are seen in the tum or itself. The opposite was observed in animals bearing this hepatom a and fed the pyridoxine supplem ented diet [20], Enzyme expression was highly limited (repressed) in the most liver-like (highly differentiated) hepatom a No. 7794A but was most fully ex pressed in hepatom a No. 7777 (i. e. the least liver-like tum or). Both the host liver and hepatom a No. 7794A showed one enzymatically active peak by the histochemical m ethod [20]. T hat cofactor lack results in TAT derepression was also shown in the liver of animals without hepatom as [5]. M oreover, TA T derepression was further verified by the im m unoprécipitation of the resolved gel proteins with antibody prepared against highly purified (> 3 ,0 0 0 fold) hepatic tyrosine am inotrans ferase [22, 23], Since lack of cofactor results in inhibition of tum or growth [3, 7 -1 0 ] and also enzyme derepression [5, 20, 22, 23] it is suggested herewith that the vitamin exercises a type of regulation over enzyme expression which could be essential for norm al tum or growth. It has been reported that the proportion of each T A T form could be varied by varying the pH of the homogenising medium [24]. Furtherm ore, a particulate liver fraction com po nent has been described which interconverts T A T multiple forms and whose catalytic action is pH dependent [25, 26]. It is possible that horm onal effects on T A T expression could reflect effects on the liver factor [25, 26], As suggested previously [23], this could also be the case with T A T derepression seen during cofactor depletion, i. e. TA T derepression may reflect a cofactor influence on the liver factor [25. 26]. D uring the course of purification the samples w ere handled identically and all protective factors were present [19]. The possibility of dénaturation taking place seems, therefore, unlikely. No attem pt was made to achieve com plete separation of the enzyme forms and/or characterise these further. A dditional tests are planned in this regard.
Acknowledgment Thanks are due to Dr. H. P. M o rris for giving us gratis the hepatom a donor animal and his continued interest and encouragem ent.
References 1
Potter , V. R.; W atanabe, M.; P itot, H. C., and Morris , H.
P.: Systematic oscillations in metabolic activity in rat liver and hepatomas. Survey of normal diploid and other hepatoma lines. Cancer Res. 29:55-78 (1969).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:53:13 AM
Figure 2. Polyacrylamide gel electrophoresis of partially purified tyro sine aminotransferase from Morris hepatoma No. 7777. Hepatomas were grown in pyridoxine depleted rats for 24 days. The animals were fed the pyridoxine depleted diet for a period of 45 days. The average weight of ten hepatomas in grams was 5.7 ± 0.5 (Standard Deviation). Animals were sacrificed following induction with hydrocortisone. The specific activity prior to purification (105,000 x g; 1 hr; 4° C) was 120.2 units/mg protein. After the heat step the specific activity was 610 units/mg protein. The yield was 100 %. Approximately 5 ug of protein (3.05 units) were electrophoresed. Other details as in figure 1.
Shuler, Tryfiates: Enzyme Expression in Host Liver Hepatoma
3
4
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7
8
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14
T ryfiates, G. P.; Shuler , J. K., and Morris , H. P.: Enzyme
activities in vitamin B6 deficient, normal and tumor-bearing animals. Effect of hydrocortisone. Proc. Soc. exp. Biol. Med. 145:1363-1367(1974). T ryfiates, G. P.; Saus, F. L., and Morris , H. P.: Hormonal induction of tyrosine transaminase in host liver and hepatoma No. 7777 of normal and cofactor depleted animals. J. natn. Cancer Inst. 55:839-841 (1975). Puskar, T. and T ryfiates, G. P.: Induction of tyrosine transa minase by hydrocortisone in vitamin B6 deficient rats. J. Nutr. 104:1407-1415(1974). Shuler , J. K. and T ryfiates, G. P.: Effect of cofactor depletion on liver tyrosine aminotransferase expression. Enzyme 22: 262-265(1977). T ryfiates, G. P. and Saus, F. L.: Hormonal induction patterns of tyrosine aminotransferase in normal and cofactor depleted animals. Cancer Biochem. Biophys. 1:63-68 (1975). T ryfiates, G. P.; Shuler , J. K.; H efner , M. J., and Morris , H. P.: Effect of Bs deficiency on hepatoma 7794A growth rate: activities of tyrosine transaminase and serine dehydratase before and after induction by hydrocortisone. Eur. J. Cancer 10: 147-154(1974). T ryfiates, G. P. and Morris, H. P.: Effect of pyridoxine defi ciency on tyrosine transaminase activity and growth of four Morris hepatomas. J. natn. Cancer Inst. 52:1259-1262 (1974). T ryfiates, G. P. and Morris, H. P.: Growth of Morris hepatoma No. 7794A with and without vitamin Bé: effect of inoculation time. Eur. J. Cancer 12:9-12 (1976). T ryfiates, G. P.: Effect of pyridoxine on the growth of Morris hepatoma No. 7288Ctc and enzyme activity. Oncology 33: 209-211 (1976). A uricchio , F.; Valeriote , F. A.; T omkins, G. M., and R iley, W. D.: Studies on the structure of tyrosine transaminase. Biochim. biophys. Acta 221:307-313 (1970). G elehrter , T. D.; E mannuel, J. R.. and Spencer, C. H.: Induction of tyrosine aminotransferase by dexamethasone, in sulin and serum. Characterization of the induced enzyme. J. biol. Chem. 247:6197-6203 (1972). Holt . P. G. and O liver , I. T.: Multiple forms of tyrosine amino transferase in rat liver and their hormonal induction in the ceonate. FEBS Lett. 5: 89-91 (1969). Iwasaki, Y.; L amar, C.; D anenberg , K., and P itot , H. C.: Studies on the induction and expression of enzymes in rat liver. Characterization and metabolic regulation of multiple forms of tyrosine aminotransferase. Eur. J. Biochem. 34: 347-357 (1973).
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18 19
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P itot, H. C. et al: Regulation of the levels of multiple forms of
serine dehydratase and tyrosine aminotransferase in rat tissues. Gann 13: 191-204(1972). Sadlf.ir , J. W.; Holt , P. G., and O liver, 1. T.: Fractionation of rat liver tyrosine aminotransferase during the course of purifica tion. Further evidence for multiple forms of the enzyme. FEBS Lett. 6:46-48(1970). Spencer , C. J. and G elehrter , T. D.: Pseudoisozymes of hepatic tyrosine aminotransferase. J. biol. Chem. 249: 577-583 (1974). T ryfiates, G. P.: On the synthesis of tyrosine transaminase in vitro. Biochim. biophys. Acta 169:779-781 (1969). Vai.f.riote, F. A.; A uricchio , F.; Tomkins, G. M., and R iley, W. D.: Purification and properties of rat liver tyrosine amino transferase. J. biol. Chem. 244:3618-3624 (1969). Shuler , J. K. and T ryfiates, G. P.: Expression of hormonally induced tyrosine transaminase in normal, host liver and three Morris hepatomas. Oncology 33:212-214 (1976). T ryfiates, G. P. and L itwack, G.: Appearance of an increment of tyrosine aminotransferase in a cell-free system. Biochemistry N.Y. 3: 1483-1487(1964). T ryfiates, G. P. and Saus, F. L.: Hepatic and tumor proteins reactive with tyrosine transaminase-specific rabbit antiserum. Eur. J. Cancer 12: 833-837(1976). T ryfiates, G. P.: Vitamin B6 effects on the growth of Morris hepatomas and the development of enzymatic activity. In Morris and C riss, Morris hepatomas. Mechanisms of regulation, pp. 607-642 (Plenum Press. New York 1977). Johnson, R. W. and G rossman, A.: Problems in interpreting the significance of multiple form patterns of rat liver tyrosine aminotransferase. Biochem. biophys. Res. Commun. 59: 520-526(1974). Smith, G. J.; Pearce , P. H., and O liver , I. T.: A liver factor that interconverts multiple forms of tyrosine aminotransferase. LifeSci. 16:437-450 (1975). R odriguez , J. H. and P itot, H. C.: The interconversion of multiple forms of tyrosine aminotransferase. Biochem. biophys. Res. Commun. 65:510-518 (1975).
Request reprints from: Dr. G. P. T ryfiates, Department of Bio chemistry, West Virginia University, School of Medicine, Morgan town, WV 26506 (USA).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:53:13 AM
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