Ontogeny of tyrosine aminotransferase in Xenopus laevis.

The development of tyrosine aminotransferase (TAT) activity in Xenopus laevis embryos was studied. Undivided eggs can transaminate tyrosine to some extent. The enzyme activity increases after hatching on the third day of development. In the early stages of development, the transamination of tyrosine is due to aspartate aminotransferase (ASAT, EC 2.6.1.1), both isoenzymes of which are present in the undivided egg. No specific TAT (EC 2.6.1.5) can be detected until the age of about 1 day, at which time neurulation is complete and the rapid development of the foregut and visceral pouches and arches has begun. The appearance of the enzyme is immediately preceded by a steep increase in the concentration of free tyrosine. Tyrosine aminotransferase is known to be induced by its substrate in the adult liver, and a similar effect may operate in the embryo.     

Endogenous factor in rat liver that converts tyrosine aminotransferase form III to form I: a rapid assay of this converting factor.

After induction by cortisol, tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) present in rat liver homogenates can be resolved into three peaks of activity by CM-Sephadex chromatography. Based on differential elution of these forms by a linear KCl gradient, a three-tube assay was developed that quantitates the amount of form III relative to total enzyme. The assay was used to determine the presence of a factor in the liver that converts tyrosine aminotransferase form III to form I. Definitive evidence for the liberation of such a factor is presented.     

Assignment of the human tyrosine aminotransferase gene to chromosome 16

Summary The liver enzyme tyrosine aminotransferase (TAT; EC 2.6.1.5) catalyzes the rate-limiting step in the catabolic pathway of tyrosine. Deficiency in TAT enzyme activity underlies the autosomally inherited disorder tyrosinemia II (Richner-Hanhart syndrome). Using a human TAT cDNA clone as hybridization probe, we have determined the chromosomal location of the TAT structural gene by Southern blot analysis of DNAs from a series of human x rodent somatic cell hybrids. The results assign the TAT gene to human chromosome 16.     

Characterization of differentiated and dedifferentiated clones from a rat hepatoma

An analysis of clonal variability of derivatives of the rat hepatoma line H4IIEC3 has shown that the overwhelming majority of clones express in a stable fashion a number of liver specific functions, including secretion of serum albumin, activity of the liver specific isozymes of alcohol dehydrogenase (EC1.1.1.1) and aldolase (EC4.1.2.13), and high basal activity and hormone inducibility of tyrosine aminotransferase (EC2.6.1.5) and alanine aminotransferase (EC2.6.1.2). The differences in level of expression of these functions cover a range of five to ten-fold, and the variations do not appear coordinated within or between clones.Seven clones, which differ from the above ones both in morphology and in the expression of liver specific functions, have been isolated. In five of them, no expression of any of the functions is detectable, while two of them show diminished but significant expression of two or three of the functions. In addition, an unexplained negative correlation between activity of glucose-6-phosphate dehydrogenase (EC1.1.1.49) and the expression of liver specific functions is described.     

Control of ketogenesis from amino acids. III. In vitro and in vivo studies on ketone body formation lipogenesis and oxidation of tyrosine by rats.

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferse (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rat limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate. For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1- 14C]tyrosine. Then their respiratory 14CO2 and the incorporation of 14C into total lipids of liver were measured. The amounts of radioactivity in CO2 and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U- 14C]acetate the radioactivities in CO2 and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studied gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

On the induction of tyrosine aminotransferase in rat liver by α-methyl-p-tyrosine

Hepatic tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) is known to be induced by α-methyl-p-tyrosine, a well-known catecholamine depletor, in both intact and adrenalectomized rats. The authors have studied this subject further and their results show that α-methyl-p-tyrosine does not influence the activity of tyrosine aminotransferase in the isolated, perfused liver and that hypophysectomy totally abolishes the induction of the enzyme by this agent. The involvement of hypophyseal hormones is discussed.     

Regulation of ornithine decarboxylase activity by putrescine and spermidine in rat liver

The marked enhancement of the activity of ornithine decarboxylase (EC 4.1.1.17) in rat liver at 4 h following partial hepatectomy or the treatment with growth hormone could be almost completely prevented by intraperitoneal administration of putrescine. A single injection of putrescine to partially hepatectomized rats caused a remarkably rapid decline in the activity of liver ornithine decarboxylase with an apparent half-life of only 30 min, which is almost as rapid as the decay of the enzyme activity after the administration of inhibitors of protein synthesis. Under similar conditions putrescine did not have any inhibitory effect on the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) or tyrosine aminotransferase (EC 2.6.1.5). Spermidine given at the time of partial hepatectomy or 2 h later also markedly inhibited ornithine decarboxylase activity at 4 h after the operation and, in addition, also caused a slight inhibition of the activity of adenosylmethionine decarboxylase.     

Effects on induction of tyrosine aminotransferase in fetal mouse liver in vitro of prednisolone,insulin and thyroxine

The hormonal requirements for formation of tyrosine aminotransferase (EC 2.6.1.5) in fetal mouse liver were investigated in organ culture using chemically defined medium. The hormones tested were insulin, thyroxine and prednisolone. Prednisolone alone resulted in a two-fold increase in tyrosine amino-transferase activity in explanted liver in hormone-free medium on day 6, and its effect was dose dependent, but neither insulin nor thyroxine alone induced the enzyme. Addition of prednisolone plus thyroxine and prednisolone plus insulin increased the enzyme activity 1.4- and 1.3-fold, respectively, over that of explants with prednisolone alone. These three hormones together had the greatest effect, causing induction of 1.5-fold more activity than that with prednisolone plus insulin or plus thyroxine. The three hormones were not all needed continuously during the culture period: prednisolone and insulin were required during the early part of cultivation and thyroxine during the later part. The effects of these hormones were blocked by actinomycin D or puromycin, suggesting that these hormones increase de novo synthesis of tyrosine aminotransferase. Phase-contrast microscopy showed that prednisolone stimulated liver epithelial cell outgrowth, probably acting with insulin.     

Involvement of cyclic GMP in the initial stage of hepatocytes proliferation.

A transient rise in cyclic guanosine 3' : 5' monophosphate (c-GMP) in the liver was observed in rats in vivo 10--20 min after partial hepatectomy. A similar increase in c-GMP in the liver was also found in rats in vivo 15 min after infusion of TGH solution (a mixture of triiodothyronine, glucagon, and heparin). In both cases, inductions of ornithine decarboxylase [EC 4.1.1.17] and tyrosine aminotransferase [EC 2.6.1.5] were found 4 hr after the beginning of the experiments. Later, 22 hr after the surgical intervention or hormone infusion, thymidine kinase [EC 2.7.1.21] was activated and liver slices were able to incorporate [3H]thymidine into DNA. These biochemical phenomena were observed commonly in regenerating liver as well as in the liver of rats infused with TGH solution. c-GMP, but not c-AMP, could induce ornithine decarboxylase and tyrosine aminotransferase in isolated, perfused liver.     

Purification of the native form of tyrosine aminotransferase from rat liver

A procedure that provides a homogeneous, native form of tyrosine aminotransferase (l-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver in exceptionally high yield is described. This goal is accomplished by rapidly inactivating the lysosomal converting factor that generates two additional, lower-molecular-weight forms of tyrosine aminotransferase, and by separating the native enzyme from the altered forms by chromatography on carboxymethyl-Sephadex C-50 and an optional hydroxylapatite step. Homogeneity appears to be achieved after the carboxymethyl-Sephadex C-50 step, and the final yield of the enzyme exceeds 30%.     

DIFFERENCES IN THE REGULATION OF TYROSINE AMINOTRANSFERASE IN BRAIN AND LIVER

Abstract— l -Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) activity in rat brain is not regulated in the same way as in rat liver. No diurnal rhythm in the activity of the cerebral enzyme was found in rats fed ad lib. although there was a marked diurnal variation in the activity of the hepatic enzyme. In adrenalectomized rats, hydrocortisone and glucagon induced the enzyme in liver but had no effect on the enzyme in brain. In normal rats, treatment with reserpine or exposure to cold elevated the activity of the hepatic enzyme without affecting the enzyme in brain. Thus, the tyrosine aminotransferase of brain differed from the enzyme in liver since it did not exhibit diurnal variations of activity and was not affected by hormones, drugs, or stress.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells.

Corticosteroid-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 X g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I leads to II leads to III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cell subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Control of ketogenesis from amino acids: III. In vitro and in vivo studies on ketone body formation, lipogenesis and oxidation of tyrosine by rats

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferase (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rate limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate.For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1-¹⁴ C]tyrosine. Then their respiratory ¹⁴CO₂ and the incorporation of ¹⁴C into total lipids of liver were measured. The amounts of radioactivity in CO₂ and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U-¹⁴C] acetate the radioactivities in CO₂ and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studies gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

Isolation of two different molecular weight polypeptides copurifying with rat liver tyrosine aminotransferase.

An affinity chromotography resin highly specific for rat liver tyrosine aminotransferase (EC 2.6.1.5) has been synthesized and used in the purification of this enzyme. The structure of the resin, N-(5′-phosphopyridoxyl)-l-tyrosyl-aminoocytl-Sepharose 4B, was designed to resemble the tyrosine-pyridoxal phosphate Schiff's base intermediate in the reaction pathway catalyzed by this enzyme. Use of this resin in combination with octyl-agarose chromatography on partially purified enzyme resulted in a tyrosine aminotransferase preparation with a specific activity of about 450 units/mg protein. When analyzed on one-dimensional polyacrylamide-sodium dodecyl sulfate slab gels, the highly purified enzyme was composed of two polypeptides with molecular weights of about 56,000 and 53,000. Radioiodinated tryptic peptides from each of these polypeptides were essentially identical following two-dimensional analysis. Although the two polypeptides could not be separated from each other in an active form, it was found that (i) both polypeptides have pyridoxal phosphate-binding sites, (ii) the coenzyme is probably bound to both polypeptides as a Schiff's base, (iii) both polypeptides have binding sites for l-tyrosine and l-glutamic acid, the two specific substrates for the enzyme, and (iv) both polypeptides can catalyze the formation of the initial amino acid-pyridoxal phosphate Schiff's base adduct in the overall reaction pathway. Since the ratios of these polypeptides differed from preparation to preparation of purified enzyme, the 53,000 M_r species probably arises by proteolysis of tyrosine aminotransferase in crude liver extracts. These results imply that if tyrosine aminotransferase isozymes exist, they are not the result of translation products produced by different structural genes.     

Role of coenzyme in aminotransferase turnover.

The role of coenzyme in determining intracellular contnet of pyridoxal enzymes was assessed by analyzing effects of pyridoxine deficiency on the rapidly degraded, readily dissociable tyrosine aminotransferase (EC 2.6.1.5) and the slowly degraded, nondissociable alanine aminotransferase (EC 2.6.1.2) of rat liver. Synthesis of the tyrosine enzyme was reduced, leading to a decreased amount of this enzyme, much of which was present as active apoenzyme. Synthesis of alanine aminotransferase was unchanged but much of this enzyme was present as an inactive apoenzyme which retained immunological reactivity. Degradation rates of both enzymes (t1/2 about 1.5 h, tyrosine aminotransferase; about 3 days, alanine aminotransferase) were not changed in pyridoxine deficiency. Hence, interaction with coenzyme is not a significant determinant in intracellular degradation of these aminotransferases. Coenzymes dissociation and intracellular stability probably reflect structural features of the proteins which determine both properties.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells

Corticosteroi-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 × g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I → II → III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cells subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Immunological study of carbon tetrachloride-mediated induction of tyrosine aminotransferase in rat liver

Administration of CCl₄ (1.0 ml/kg) to rats resulted in a rise of liver tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) activity to a maximum of about 3.6 times the normal level 6 hr later. An immunological titration study proved that the phenomenon was due to increased enzyme content. Using an isotopic-immunochemical procedure the half-life of liver tyrosine aminotransferase at 3.5 hr after CCl₄ administration was shown to be 11.9 hr in contrast to 2.1 hr in the normal liver. Immunochemical analysis revealed that enzyme synthesis was decreased by CCl₄. Thus, in the early stage of CCl₄ poisoning, enzyme synthesis proceeded at a moderate rate while degradation was markedly impaired, resulting in the rise of tyrosine aminotransferase in the liver tissue.Several hours after administration of hydrocortisone to adrenalectomized rats, induced tyrosine aminotransferase reached its peak activity and then subsided to the basal level. At any time following hydrocortisone administration, administration of CCl₄ consistently caused an elevation of the enzyme activity above the level in controls not treated with CCl₄. Actinomycin D (5 mg/kg) also increased the enzyme at an early period of induction cycle but failed to do so at a later period.The CCl₄-mediated “superinduction” of hormonally preinduced tyrosine aminotransferase, like the induction of this enzyme by CCl₄ at a basal level, was found to be caused by the differential inhibitory effect of CCl₄ on the synthesis and degradation of this enzyme.     

Effect of cofactor depletion on liver tyrosine aminotransferase expression.

Hepatic tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) was partially purified from pyridoxine depleted and control rats and subsequently resolved by electrophoresis on polyacrylamide gels. Enzyme activity was detected histochemically in situ on the gel. Six enzymatically active forms were detected. Cofactor depletion effected further resolution of the enzyme into seven active forms as revealed by the bifurcation of the major active peak.     

Enhancement by streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine of the tyrosine aminotransferase activity in cultured rat liver cells: role of dexamethasone and NAD

The activity of tyrosine aminotransferase (TAT) (EC 2.6.1.5) was enhanced 3-fold after a 5-h exposure of cultured rat liver cells (RLC) to streptozotocin (SZ) at concentrations higher than 100 microgram/ml (0.38 mM) in the presence of 10 nM dexamethasone, a potent glucocorticoid inducer for the enzyme. The structurally related carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also enhanced the aminotransferase in the presence of the glucocorticoid, but its optimal concentration was at 100 ng/ml (0.68 microM). While the cellular NAD (NAD+ + NADH) concentration was reduced to 60% of the control levels, the rate of poly(ADP-ribose) formation in the isolated cell nuclei was unaffected by treating the cells with SZ. The enhancement of tyrosine aminotransferase by SZ and MNNG was effectively prevented by nicotinamide. Using nicotinamide and its derivatives such as 1-methyl-, N'-methyl- or 6-amino-derivatives it was found that the degree of enzyme induction is almost inversely proportional to the cellular NAD content, though the activity of nuclear poly(ADP-ribose)polymerase remains unchanged. The results indicate that SZ or MNNG, in combination with dexamethasone, stimulate the induction of tyrosine aminotransferase through their NAD lowering action.     

Effects of 6- and 8-substituted analogs of adenosine 3':5'-monophosphate on phosphoenolpyruvate carboxykinase and tyrosine aminotransferase in hepatoma cell cultures.

A variety of 6- and 8-substituted analogs of cAMP (cyclic adenosine 3:5-monophosphate) have been tested for their ability to increase activity of tyrosine aminotransferase (EC 2.6.1.5) in cultured Reuber H35 hepatoma cells. Some analogs, particularly the 8-thio-substituted ones, produced effects approximately equivalent to those generated by N-6, O2'-dibutyryl cAMP. In contrast, cAMP and its O-2-monobutyryl derivative were relatively ineffective even at very high concentrations, whereas three other analogs actually depressed the activity of the aminotransferase. Changes in enzyme activity generated by the various analogs were paralleled closely by changes in the relative rate of aminotransferase synthesis. An excellent correlation was found to exist between the ability of any given analog to influence the activity of tyrosine aminotransferase and that of phosphoenolpyruvate carboxykinase (EC 4.1.1.32). A similar correlation was found to exist between the ability of various analogs to evelate the activity of these enzymes and to inhibit reversibly the growth of H35 cells. Only one of five inhibitors of cAMP phosphodiesterase activity tested produce any increase in aminotransferase activity when added alone. All of the 6- and 8-substituted analogs tested, including noniducers, stimulated f1 histone phosphorylation in crude rat liver extracts with approximately equal potencies. On the other hand, dibutyryl cAMP was only a weak activator of protein kinase in vitro, even though it is a potent enzyme inducer. A possible resolution of this apparent discrepancy has been provided by preliminary analyses of site-specific f1 histone phosphorylation in whole cells. Only compounds active as aminotransferase inducers are capable of stimulating phosphorylation of the serine-37 residue of endogenous f1 histone (3- to 10-fold).