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The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Iα aspartate and aromatic aminotransferases is critical for T. cruzi’s TAT activity.

Complementary DNA clones encoding human tyrosine aminotransferase (TAT) were isolated by screening a normal adult woman liver lambda gt11 library with rat TAT cDNA. The largest isolated cDNA is 2051 bp long (EMBL accession number X55675). This cDNA was subcloned downstream of the cytomegalovirus promoter in the pCMV vector for transfection into human cervical carcinoma HeLa cells. Expression of the TAT cDNA resulted in the synthesis of a protein with a molecular mass of approximately 50 kDa, as assessed by Western analysis, a value which is in close agreement with the predicted molecular weight of 50,399, for a deduced sequence of 454 amino acids. The expressed protein catalyzed specifically the conversion of L-[14C]tyrosine into p-[14C]hydroxyphenylpyruvate. The availability of a functional TAT cDNA provides a useful tool for detailed study of the structure-function relationship of the enzyme and its mutated derivatives.

Carnitine-deficient jvs mice expressed reduced levels of a group of genes which are preferentially expressed in the liver, including urea cycle enzyme genes (Biochim. Biophys. Acta 1138, 167-171, 1992). The expression of alpha-fetoprotein and aldolase A was elevated, indicating that the liver of jvs mice is undifferentiated or dedifferentiated (FEBS Lett. 311, 63-66, 1992). Studies of the hormone signal transduction pathway showed that serum cortisol and plasma glucagon levels of jvs mice were 2 and 3 times higher, respectively, than those of normal mice, and that the hormone binding activity of glucocorticoid receptor (GR) in the cytosol of jvs liver was 50% of normal mice, which reflected the amount of receptor protein in the cytosol. On the other hand, GR protein accumulated in the nuclear fraction in jvs mice. Exogenously administrated dexamethasone induced carbamoyl phosphate synthetase (CPS) and tyrosine aminotransferase (TAT) mRNAs in jvs mice, indicating that CPS and TAT genes in jvs mice are responsive to induction by glucocorticoid and cAMP. Analysis of transacting factors by gel retardation assay revealed that HNF-1, COUP-TF and SP-1 were detected at almost the same level in the hepatic nuclear fraction of jvs mice as in normal littermates, and C/EBP and CREB were a little higher in jvs mice, suggesting that these factors are probably not targets of jvs mutation causing abnormal gene expression in the liver. On the other hand, AP-1 binding activity was much higher in jvs mice from an early age, preceding the abnormal expression of urea cycle enzyme, and carnitine administration normalized AP-1 binding activity. We suggest that elevated AP-1 binding induced by carnitine deficiency is closely connected with the abnormal gene expression in the liver.

The differential response of the tyrosine aminotransferase (TAT) gene to glucocorticoids and insulin in HTC cells and cell clones derived from Reuber H35 cells (FaO and Fu5.5) have been analyzed by nuclear run-on assay. It has been previously shown that clones of cells from HTC and Reuber H35 cell lines, exhibit different sensitivities for the induction of TAT mRNA and enzyme activity. The purpose of the present study was to determine whether this difference in TAT expression between hepatocytes and hepatoma cell lines occurs at the level of TAT gene transcription or mRNA stability. A study of the TAT mRNA accumulation in all cell types showed that TAT mRNA in the Reuber H35 cell clones and hepatocytes was synthesized at a higher rate than in HTC cells. However, dexamethasone induction of α 1 AGP mRNA and glutamine synthetase was comparable to glucocorticoid bound receptors. In addition, cycloheximide decreased the rate at which induced levels of TAT mRNA were degraded. We also show that a heterologous fusion gene constructed from 3.0 kilobases (kb) 5′ to the transcription initiation site of the rat TAT gene and the bacterial chloramphenicol acetyltransferase gene (CAT) responds similarly to dexamethasone in Fu5.5 and HTC cells as determined by transient transfection assay; and insulin inhibits dexamethasone mediated transcription in Reuber H35 cells and primary adult hepatocytes. These data indicate that DNA sequences involved in the differential response of the TAT gene to hormone treatments between HTC and Reuber H35 cell lines are not located in the first 3.0 kb fragment.

Adaptive increase of the tyrosine-α-ketoglutarate transaminase occurred in the livers of rats following injections of tyrosine or of hydrocortisone. The increases were as great as 10-fold and occurred in 5 hours. Analysis showed that hydrocortisone itself was a sufficient inducing stimulus. Tyrosine was an effective inducing stimulus only if the adrenal glands or hydrocortisone also were present.

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.

1. 1. The effect of a portacaval anastomosis on the activities of hepatic enzymes related to cholesterol metabolism was investigated in rats. 2. 2. Portacaval anastomosis led to a fall in body weight and liver weight/body weight ratio, and to a rise in the activities of hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase per g of liver. The net effect was to maintain a normal activity of both enzymes per 100 g of rat. Diurnal rhythm in the activities of both enzymes was maintained after portacaval anastomosis. 3. 3. The rate of excretion of total bile acids, per 100 g of rat, in bile fistula rats was not significantly decreased by portacaval anastomosis.

Incubation of rat hepatocytes for 3 h in a medium containing amino acids, salts and albumin resulted in a 2-fold increase in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Inclusion of 10% human plasma, rat serum or dialysed rat serum in the medium resulted in an approximate 7-fold increase in reductase activity. These increases were specific since there was little change in the rate of fatty acid synthesis or in the activity of tyrosine aminotransferase. Reductase levels were increased above control values when high density lipoproteins or lecithin dispersions were added to the cells. Lecithin dispersions were also shown to increase the rate of efflux of cell cholesterol to the medium. In contrast, reductase levels were reduced when cells were incubated with low density lipoproteins or cholesterol added as an equimolar cholesterollecithin dispersion. This inhibition of the reductase by cholesterol dispersions was dependent on the continued de novo protein synthesis. Our data indicate that in normal rat hepatocytes the relative rates of efflux and influx of cholesterol may be critical to the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity and cholesterogenesis.

Tyrosine aminotransferase induction has been studied in hepatocytes from untreated, partially and fully glucocorticoid-induced rats: enzyme activities were initially 12.9 +/- 1.7 (n = 16), 41.4 +/- 3.2 (n = 6) and 117.9 +/- 10.5 (n = 7) munits/mg protein, respectively. Untreated or fully induced hepatocytes maintain initial levels, whereas partially induced hepatocytes increase their tyrosine aminotransferase activity even in the presence of actinomycin D. Fully induced hepatocytes possess a normal protein synthetizing machinery and the mechanisms to degrade selectively tyrosine aminotransferase. The effect of progesterone treatment is consistent with these cells retaining a high dexamethasone level. Glucagon induces tyrosine aminotransferase via its second messenger, cyclic AMP. This induction decreases dramatically with in vivo glucocorticoid treatment. Time courses and effects of inhibitors are consistent with these in vivo and in vitro treatments being alternative methods of inducing tyrosine aminotransferase by the same basic pretranslational step.

In primary rat hepatocyte cultures, growth hormone was shown to depress tyrosine aminotransferase levels induced with hydrocortisone. Both induction by glucocorticoid and repression by growth hormone could be demonstrated in cultures several days old.

The effects of dibutyryl cyclic AMP, dexamethasone, and asparagine as inducers of ornithine decarboxylase (EC 4.1.1.17) and tyrosine aminotransferase (EC 2.6.1.5) in H-35 rat hepatoma cells were studied; a comparison was made between cells incubated in Eagle's minimal essential medium and cells incubated in Earle's balanced salt solution (salts/glucose solution). The addition of either 1 mM dibutyryl cyclic AMP or 1 μM dexamethasone increased both ornithine decarboxylase and tyrosine aminotransferase activities of H-35 hepatoma cells maintained in Eagle's minimal essential medium, whereas 10 mM asparagine increased the activity of ornithine decarboxylase and had no effect on tyrosine aminotransferase activity. The actions of dibutyryl cyclic AMP and dexamethasone but not asparagine, as inducers of ornithine decarboxylase activity, appeared to be dependent on the incubation medium used; dibutyryl cyclic AMP and dexamethasone were ineffective in increasing ornithine decarboxylase activity of H-35 hepatoma cells maintained in the salts/glucose solution, while asparagine was fully effective. Parallel studies on tyrosine aminotransferase demonstrated that both dibutyryl cyclic AMP and dexamethasone elicited maximal increases of tyrosine aminotransferase activity of H-35 hepatoma cells maintained in the salts/glucose solution. Experiments on the activation of cyclic AMP-dependent protein kinase and the uptake/binding of [3H]dexamethasone to whole cells revealed no significant difference between H-35 hepatoma cells maintained in Eagle's minimal essential medium and cells maintained in the salts/glucose solution. The half-lives of ornithine decarboxylase of cells maintained in minimal essential medium and salts/glucose solution were 45 and 38 min, respectively; the corresponding values for tyrosine aminotransferase were 2.5 and 1.5 h, respectively. It is proposed that the actions of dibutyryl cyclic AMP and dexamethasone as inducers of ornithine decarboxylase may involve the participation of component(s), possibly amino acids, present in the Eagle's minimal essential medium but not in the salts/glucose solution. These components may either mediate or modulate the actions of dibutyryl cyclic AMP and dexamethasone in the induction of ornithine decarboxylase activity.

Cyclic AMP derivatives increase the rate of synthesis of tyrosine aminotransferase in Reuber H35 hepatoma cells. Various studies lend support to the hypothesis that cyclic AMP increases the synthesis of tyrosine aminotransferase by acting at a posttranscriptional site. The presence of a limited non-translatable pool of tyrosine aminotransferase mRNA prior to the formation of the translatable tyrosine aminotransferase mRNA implicates a possible site of action of cyclic AMP. We compared the capacity of N 6 ,O 2 ′- dibutyryl cyclic AMP to induce tyrosine aminotransferase synthesis when untranslatable tyrosine aminotransferase mRNA sequences are present or absent. The transition of a condition in which non-translatable tyrosine aminotransferase mRNA sequences were present to a condition in which they were absent was established by preinduction of Reuber H35 cells with dexamethasone, followed by addition of actinomycin D. In the time period thereafter, the amount of non-translatable mRNA decreased and 1.5–2 h after addition of actinomycin D, only translatable tyrosine aminotransferase mRNA was present. It can be seen that the induction of tyrosine aminotransferase synthesis by dibutyryl cyclic AMP follows the normal decrease of tyrosine aminotransferase mRNA. We present evidence that dibutyryl cyclic AMP in Reuber H35 hepatoma cells regulates tyrosine aminotransferase synthesis at a posttranscriptional site independent of the pool of nontranslatable tyrosine aminotransferase mRNA sequences, but influencing the efficiency of translation of active tyrosine aminotransferase mRNA.

Of the proteinase inhibitors derived from Streptomyces spp., chymostatin is the most effective inhibitor of non-lysosomal proteolysis. As part of a systematic study of the structural features of the chymostatin molecule that are responsible for this inhibitory activity, a series of fifteen di- and tripeptide analogues of chymostatin were tested for their ability to suppress protein degradation in isolated primary hepatocytes. Protein degradation was assessed in two ways: by the release of radiolabel from proteins prelabelled in vivo (to which both lysosomal and non-lysosomal processes contribute) and by the rate of inactivation of tyrosine aminotransferase, a process that is exclusively non-lysosomal. All inhibitors were relatively non-toxic and did not affect the intracellular ATP levels, although some suppression of gluconeogenesis was observed in the presence of leupeptin, chymostatin or the analogues. Tripeptide phenylalanine aldehydes or semicarbazones were at least as effective as chymostatin in reducing protein degradation, whereas peptide alcohols were relatively ineffective. Replacement of the basic capreomycidine moiety in chymostatin with an arginine residue improved the inhibitory activity but equally, substitution of the arginine residue with an uncharged norleucine residue was without significant effect. The structural features that are optimal for inhibition of chymotrypsin or other serine proteinases (previously defined) are not as critical for inhibition of protein degradation in vivo.

Insulin stimulates a 2-fold increase in the amount of tyrosine aminotransferase and a 5-10-fold increase in the rate of amino acid transport in dexamethasone-treated rat hepatoma cells. In order to determine whether these effects are mediated by insulin receptors or receptors for insulin-like growth factors, we have examined the binding of 125I-labeled insulin and 125I-labeled multiplication-stimulating activity, a prototype insulin-like growth factor, and compared the biological effects of these polypeptides. Insulin and multiplication-stimulating activity cause an identical increase in transaminase activity and transport velocity; half-maximal biological effects were observed at 35 ng/mg (5.5 nM) insulin and 140 ng/ml multiplication-stimulating activity. The hepatoma cells display typical insulin receptors of appropriate specificity; half-maximal displacement of tracer insulin binding occurred at 33 ng/ml unlabeled insulin, but only at 2500 ng/ml unlabeled multiplication-stimulating activity. Specific multiplication-stimulating activity receptors also were demonstrated with which insulin did not interact even at 10 micrograms/ml. Half-maximal displacement of tracer multiplication-stimulating activity occurred at 200 ng/ml unlabeled multiplication-stimulating activity. We conclude that insulin cannot act via the multiplication-stimulating activity receptor and presumably acts via typical insulin receptors. The effects of multiplication-stimulating activity on enzyme induction and amino acid transport are probably mediated primarily via the multiplication-stimulating activity receptor.

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.

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.

Hepatic tyrosine aminotransferase (EC 2.6.1.5) was induced in rats by intubation of amino acid mixtures (complete or tryptophan-free). Enzyme activity was increased 4-fold by the complete mixture and 8-fold by the tryptophan-free mixture. The enzyme was analyzed by chromatography on CM-Sephadex. Chromatographic patterns were characteristic of the type of inducer rather than of the chronology of the induction cycle: after induction by the complete amino acid mixture the three forms of the enzyme were equally increased whereas after induction by the tryptophan-free mixture Form I was preferentially increased.

The regulation of hepatic tyrosine aminotransferase ( l -tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) in the rat has been extensively studied but little is known about the enzyme from other sources. We have studied the regulation of this enzyme in the frog Rana temporaria and in this paper we report that: 1. 1. Cortisone acetate, adrenocorticotropic hormone and α -methyl- p -tyrosine, an agent known to induce hepatic tyrosine aminotransferase in the rat via activation of the pituitary-adrenal axis, have no effect on the activity of the enzyme in the frog. 2. 2. Dibutyryl-3′, 5′-cyclic AMP induces the enzyme to about 2-fold. 3. 3. Injection of tyrosine methyl ester and a protein-rich diet result in an increase in the enzyme activity. This increase is of the same order of magnitude as that caused by dibutyryl cyclic AMP. 4. 4. Glucose significantly reduces tyrosine aminotransferase activity in frog liver. These results suggest that cyclic AMP induces the enzyme via a mechanism independent of glucocorticoids. The frog offers a model for studies on the regulation of hepatic tyrosine aminotransferase in vivo without interference from secondary effects mediated by the adrenals.

The hormone-responsive enzymes tyrosine aminotransferase and glycerol-3-phosphate dehydrogenase were studied with respect to current models of the mechanism of glucocorticoid/cAMP interaction during the induction of enzyme activity in responsive cell hybrids between rat C6 glioma cells and rat FU5AH hepatoma cells. The results of experiments involving protein and mRNA synthesis inhibitors, sequential addition of inducers, and the assay of cyclic-AMP-dependent protein kinase could not be adequately explained by any one model of inducer interaction. Comparison of the hybrid clones revealed the presence of factors that may modify induction but that are not essential for synergistic induction.

The effect of protein feeding and the addition of amino acids on the activity of hepatic phospho enol pyruvate carboxykinase (GTP: oxalacetate carboxylyase (transphosphorylating), EC 4.1.1.32) was investigated in vivo and in the isolated perfused rat liver. Protein feeding resulted in a considerable increase in phospho enol pyruvate carboxykinase activity within 6 h. This rise was independent of the presence of glucocorticoids. In the isolated perfused liver system amino acids per se had a small effect on phospho enol pyruvate carboxykinase activity and led to an increase by 20% when glucocorticoids were present, but resulted in a rise by 100% when glucocorticoids plus dibutyryl cyclic AMP were added to the perfusion medium. The effect of amino acids in the presence of dibutyryl cyclic AMP could also be observed in the liver of glucocorticoid-deprived rats. Cycloheximide, a translational inhibitor, totally blocked all effects of amino acids on enzyme activity. These results indicate that the concentration of amino acids in the portal vein modify the regulation of phospho enol pyruvate carboxykinase by cyclic AMP.

The stabilities of nine rat liver cytosol enzymes were compared at a variety of pH values. The cytosol enzymes studied were (a) those with half-lives in vivo of 3 days or longer: lactate dehydrogenase, arginase, glyceraldehyde phosphate dehydrogenase and alanine aminotransferase, (b) those with half-lives in vivo shorter than 2 days; glucokinase, dihydroorotase, serine dehydratase and tyrosine aminotransferase and (c) catalase, which has an intermediate half-life of 2.5 days for the protein portion. All the enzymes were stable in vitro at neutral and alkaline pH values. However, at acidic pH values (pH 4): the long-lived enzymes (a) were stable; the short-lived enzymes (b) were completely inactivated with one exception; and catalase was partially inactivated. Tyrosine aminotransferase was the exception in that it is a short-lived enzyme in vivo but stable under all conditions tested in vitro. The finding that long-lived enzymes are stable in an acid milieu and short-lived enzymes are generally unstable was only observed if certain ligands (NAD+, pyridoxal 5′-phosphate, Mn2+, amino acids) were added to the iv vitro systems. Lysosomal extracts did not accelerate the rate of inactivation of any cytosol enzyme in acidic solutions. These results indicate that if degradation of intracellular enzymes occurs in lysosomes, acid inactivation and denaturation of enzymes may be the initial event in determining the functional half-lives of the enzymes in vivo.

Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translational system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified from the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.

Low doses of cycloheximide or emetine cause rat liver tyrosine aminotransferase activity to rise up to twice the control levels in 2 h. By contrast, in the same interval no changes, or only a slight decrease, are produced by either drug at high dosage. Adrenalectomised animals display the same pattern of response. High doses of either antibiotic virtually afford a complete inhibition of 14C-labelled amino acid incorporation into liver and plasma proteins, whereas no more than a 30% decrease is observed with low doses. When administered in the course of the induction by cortisol, high doses of inhibitor prevent any further change in tyrosine aminotransferase activity, stabilising it at the level already attained; low doses, while slightly affecting the synthetic phase evoked by cortisol, drastically interfere with the deinduction. Six hours after various doses of either inhibitor the tyrosine aminotransferase activity is markedly increased, this late effect being largely dependent on the presence of adrenals. The amino acid incorporating activity of the liver may exceed that of controls, as observed particularly after small doses of emetine.

The glycogenic action of cortisol in cultured fetal rat hepatocytes was completely abolished by the absence of NaHCO 3 from the medium, while its presence stimulated the action in relation to its concentration. The absence of NaHCO 3 slightly reduced glycogen storage by insulin but did not affect glucose-dependent glycogen deposition in the basal state. Also, the cortisol-induced increase in glycogen synthase a activity was reduced but that in total synthase activity was not affected. The absence of NaHCO 3 did not reduce the cortisol-induced increase in tyrosine aminotransferase activity and the incorporation of [ 3 H]dexamethasone into the nuclei. These results show that the absence of NaHCO 3 specifically inhibits the glycogenic action of glucocorticoids in cultured fetal rat hepatocytes and indicate the need for further investigation into the role of HCO − 3 in universally used bicarbonate-buffered media.

Pyridoxine-deficient animals have normal basal levels of hepatic tyrosine aminotransferase, tryptophan aminotransferase and tryptophan peroxidase, but decreased levels of aromatic amino acid decarboxylase. Response of the aminotransferases to inducers is essentially normal in pyridoxal-deficient animals as is the circadian rhythm of this enzyme. The results argue against the physiological importance of induction of these enzymes by cofactor in adult animals and against the view that the circadian rhythm of tyrosine aminotransferase results from competition between norepinephrine and apoenzyme for cofactor.

1. 1. A tyrosine-catabolizing system similar to that found in rat liver was found to be present in Tetrahymena pyriformis , strain W. The five constituent enzymes of the system, i.e. tyrosine aminotransferase, p -hydroxyphenylpyruvate oxidase, homogentisate oxidase, maleylacetoacetate isomerase and fumarylacetoacetate hydrolase were assayed in extracts of the cells. Fumarylacetoacetic acid was prepared enzymatically from homogentisic acid with a protein fraction of the cells. 2. 2. All the tyrosine-catabolizing enzymes and phosphoenolpyruvate (PEP) carboxykinase were repressed by glucose and by acetate. The first enzyme of the catabolic sequence, tyrosine aminotransferase, and PEP carboxylase were repressed by glucose in both logarithmic and stationary phases of growth while the four subsequenct catabolis enzymes were repressed essentially only in stationary phase. A close metabolis relationship was found to exist between tyrosine aminotransferase and PEP carboxykinase. 3. 3. The repression of enzymes by glucose and acetate was accompanied by a repression of carbon flow from tyrosine to glycogen. 4. 4. An inverse relationship exists between the activities of tyrosine aminotransferase and PEP carboxykinase on the one hand and isocitrate lyase on the other. This relationship and the repression of the catabolic enzymes by acetate, a glucogenic precursor in Tetrahymena , suggests that the regulation of the tyrosine-catabolizing enzymes and particularly of tyrosine aminotransferase is geared to the demand for gluconeogenesis from tyrosine in these cells.

The alteration in free amino acid patterns in various responsive tissues and in plasma has been investigated 4 h after the administration of a relatively low dose of cortisone acetate. In liver, in which cortisone evokes elevated tyrosine transaminase ( L -tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) levels, a marked depression in the level of tyrosine and an elevation in concentrations of glutamate, aspartate, and alanine are evident. In the liver, in which protein synthesis is augmented under corticoid influences, the concentrations of numerous other amino acids were depressed following cortisone administration. In tissues in which cortisone produces a decrease in protein content, namely, thymus and muscle, the steroid elicited smaller elevations in the concentrations of many amino acids in addition to marked increments in the amounts of glutamate, aspartate, and alanine. Thus the alterations in amino acid patterns in tissues responsive to glucocorticoid seem to be consequent to the hormonally induced elevation of tyrosine α-ketoglutarate transaminase in the liver and to protein loss from lymphoid tissues and muscle.

Tryptophan pyrrolase ( L -tryptophan: H 2 O 2 oxidoreductase, EC 1.11.1.4) and tyrosine-α-ketoglutarate transaminase ( L -tyrosine: 2-oxoglutarate amino transferase, EC 2.6.1.5) activities of rat liver homogenate were increased about 2–2.5 fold in 4–6 h after laparotomy, and 4–6 fold in 8–10 h after partial hepatectomy, without loading of gluccorticoid or substrate. The rise of tryptophan pyrrolase was inhibited by actinomycin D administered immediately after operation. In adrenalectomized rats, the induced tryptophan pyrrolase activity was inhibited. The increased activity in tryptophan pyrrolase after partial hepatectomy has been attributed to change in supernatant fraction, and was not affected by administration of cortisone.

The effect of l -canaline, a structural analogue of l -ornithine, was studied on several mammalian enzymes in vitro. The results obtained with three ornithine metabolizing enzymes indicated that canaline is not an effective competitor of ornithine in these reactions. However, canaline strongly inhibited the activity of all seven pyridoxal-dependent enzymes studied, including amino acid decarboxylases [ornithine decarboxylase (EC 4.1.1.17), 5-hydroxytryptophan decarboxylase (EC 4.1.1.28)], aminotransferases [ornithine-ketoacid aminotransferase (EC 2.6.1.13), tyrosine aminotransferase (EC 2.6.1.5)], ornithine transcarbamylase (EC 2.1.3.3) and plasma diamino-oxidase (EC 4.1.3.6). The reversibility of this inhibition by excess pyridoxal phosphate, as well as a strong interaction between canaline and pyridoxal phosphate in aqueous solution, support the view that canaline inhibition is due to complex formation between canaline and the pyridoxal coenzyme. l -canaline is one of the most potent inhibitors of pyridoxal enzymes. Ornithine-ketoacid amino-transferase, for example, was inhibited by 50% in the presence of 3 ° 10 −6 M l -canaline.

Intraperitoneal injection of histones and other polypeptides into adrenalectomized mice increases hepatic tyrosine-alpha-ketaglutarate transaminase and tryptophan pyrolase activity

An oral load of glucose in fasted rats led to a marked and sustained decrease in the activity of liver tyrosine transaminase. Intraperitoneal administration of actinomycin D, 1 h prior to the glucose feeding, inhibited this “glucose affect”. Further study using 14 C-labelled glucose showed that 3 h after incubation, 68% of t he glucose was retained in the stomach and intestine of rats pretreated with the drug as compared to 28% for the controls. Concurrently, less glucose appeared in bollod, muscle and liver glycogen in the actinomycin D-treated rats. Thus, the action of actinomycin D on the effect upon liver tyrosine transminase is most likely to be secondary to inhibition of glucose absorption.

1. 1.This paper describes the purification and characteristics of tyrosine-α-ketoglutarate transaminase ( l -tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) from bovine thyroid tissue. The most active preparations appeared to be more than 80% homogeneous on the basis of ultracentrifugal and immunoelectrophoretic analysis. 2. 2.Bovine thyroid tyrosine-α-ketoglutarate transaminase was compared with the same enzyme isolated from bovine liver and found to be similar with respect to heat tolerance, pH optima, substrate affinity, and response to most inhibitors. Immunologically the proteins appeared to be identical. 3. 3.The only difference between the two enzymes was the effect of diiodotyrosine. Diiodotyrosine did not inhibit thyroid transaminase and would partially protect it against inhibition by the sulfhydryl inhibitor, p -chloromercuriphenylsulfonic acid. On the other hand, diiodotyrosine inhibited liver transaminase 50% at 2.5 mM concentration and did not protect it from the inhibitory effect of p -chloromercuriphenylsulfonic acid. 4. 4.Both thyroid and liver transaminase utilized tyrosine and monoiodotyrosine as substrates but used diiodotyrosine poorly.

In the isolated perfused rat liver, synthesis of the enzyme tyrosine transminase (EC 2.6.1.5) is stimulated by either insulin or glucocorticoid hormone. The antibiotic cycloheximide blocks both synthesis and degradation of tyrosine transminase in insulin-treated perfused livers, whereas it blocks only synthesis of the enzyme in glucocorticoid-treated or untreated livers. When insulin is given in additions to cycloheximide, degradation of the enzyme is blocked in glucocorticoid-treated livers as well. The inhibitory effects on degradation are enzyme-specific, since tryptophan oxygenase (EC 1.13.1.12), the synthesis of which is stimulated by glucocorticoid hormone but not by insulin, is degraded at the same rate under all conditions. It is suggested that both cycloheximide and insulin affect tyrosine transminase degradation indirectly, bvy augmenting the amounts of amino acids or other metabolites that inhibit degradation.

1. 1. A single oral glucose load in normal, fasted rats effected a very early, transient increase of the level of liver tyrosine transaminase activity. Afterwards the enzyme activity was strongly depressed for at least 16 h, thus corresponding to the well-known “glucose effect” in mammals. Glycerol and fructose had similar inhibitory effects; but mannose and galactose were ineffective; ribose and galactosamine enhanced the tyrosine transaminase level. The glucose load was no longer effective in streptozotocin-diabetic rats. 2. 2. Data presented favor an alteration in the enzyme protein synthesis as the mechanism for the “glucose effect” rather than a mere inhibition of its activity. 3. 3. When administered together with cortisone, glucose strongly altered tyrosine transaminase induction and most of the early parameters of the glucocorticoid action. 4. 4. Glucose effect was largely relieved by simultaneous administration of glucagon thus indicating that the liver adenyl cyclase system might be involved. 5. 5. It is suggested that the nutritional state of the organism could modulate, in an as yet unknown manner, the hepatic biochemical responses to cortisone administration.

It was shown that cortisol increases the level of tyrosine-α-ketoglutarate transaminase ( l -tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) of the isolated rat liver, when perfused with diluted rat blood containing an amino acid mixture; a more striking increase was obtained in the presence of a supplementary amount of tyrosine. This enhancement of activity was blocked by inhibitors both of protein synthesis (chloramphenicol, puromycin, ethionine) and of ribonucleic acid synthesis (actinomycin D and mitomycin C). Adding [6-14C]orotate to the perfusing blood, caused a rapid incorporation of radioactivity into nuclear ribonucleic acid to take place; cytoplasmic ribonucleic acid was poorly labelled after 30 min, but its radioactivity rose thereafter. Cortisol stimulated the labelling of both the nuclear and the cytoplasmic ribonucleic acid; moreover it induced the increase of tyrosine transaminase after a lag of about 30 min. In both cases, the effect of the hormone was prevented by actinomycin D, or by mitomycin C. It was finally found that livers perfused with cortisol have a higher nuclear ribonucleic acid polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) activity than control ones. The facts that the synthesis of nuclear ribonucleic acids precedes the increase of tyrosine transaminase and also that cortisol stimulates ribonucleic acid polymerase strongly suggest that the action of the hormone can be related to an increase in the synthesis of messenger ribonucleic acid molecules for the synthesis of the enzyme.

1. During the induction of tyrosine aminotransferase by insulin, histone phosphorylation and acetylation occurred prior to the stimulation of RNA synthesis. Increased methylation of histones ran parallel to the rise in RNA synthesis. 2. Hydrocortisone led only to a slight increase in acetylation prior to RNA synthesis and increased methylation at the height of enzyme activity.

Changes in isoenzyme spectrum of tyrosine aminotransferase in rat liver have been investigated under cortisol induction and during liver regeneration after partial hepatectomy. Electrophoresis in agar gels reveals two groupos of isoenzymes of liver tyrosine aminotransferase. One of them, moving to the anode during electrophoresis in agar gel, exhibits a sharp rise of activity after cortisol administration. The second one, moving to the cathode, does not exhibit any change of activity. When the action of the hormone-inductor is stopped, the activity of inducible anode isoenzymes falls to its initial level, while the activity of cathode isoenzymes remains unchanged. During liver regeneration, the rise of activity only of anode isoenzymes of tyrosine aminotransferase is noted, while that of cathode ones remains unchanged. The increase of total activity of tyrosine aminotransferase after cortisol induction and partial hepatectomy is a result of an increase in the activity of anode isoenzymes only.

1. 1. The effects of electrical stimulation of the autonomic nerves on glycogen phosphorylase (α-1,4-glucan: orthophosphate glycosyltransferase, EC 2.4.1.1) and on glucose-6-phosphatase ( d -glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) of rabbit liver were investigated. The activities of these two enzymes increased markedly after stimulation of the splanchnic nerve (sympathetic), and the effect was not eliminated by adrenalectomy or pancreatectomy of the rabbits. 2. 2. The response of the enzymes reached a maximum within 30 sec after the onset of splanchnic-nerve stimulation, and the half-time of the increase was approximately 14 sec. 3. 3. The effect of splanchnic-nerve stimulation was completely counteracted by simultaneous stimulation of the vagus nerve (parasympathetic), although vagal stimulation alone had little effect on the enzymes. 4. 4. The dephosphophosphorylase and dephosphophosphorylase kinase (ATP: dephosphophosphorylase phosphotransferase, EC 2.7.1.38) from rabbit liver were partially purified and their properties were studied. 5. 5. Total phosphorylase of the liver was determined by assaying phosphorylase activity after conversion of dephosphophosphorylase to the active form. The total amount of phosphorylase was not altered significantly by splanchnic-nerve stimulation or vagal stimulation. In the resting state, about one fourth of the total phosphorylase in the liver was active. The amount increased to three fourths of the total after stimulation of the splanchnic nerve, but was unchanged during vagal stimulation. 6. 6. Previous treatment of the rabbits with actinomycin S failed to block the increase in phosphorylase activity after splanchnic-nerve stimulation. These results indicate that the increase in phosphorylase activity caused by splanchnic-nerve stimulation can be attributed to enzymic conversion of the dephosphophosphorylase to the active form during stimulation.

Prostaglandin E1 suppressed the induction of rat liver tyrosine aminotransferase by glucagon, l -tryptophan or insulin, but failed to affect the hydrocortisone-mediated induction of the enzyme. The role of prostaglandin E1 in enzyme induction was discussed in relation to cyclic AMP.

Structural studies were performed on hepatic tyrosine aminotransferase isolated from rats injected with triamcinolone. The enzyme, treated with guanidine · HCl, was partially dissociated into a species of mol. wt. 32 000. The number of peptides separated after tryptic digestion or reaction with cyanogen bromide suggests that the enzyme is composed of four similar or identical subunits. No free N-terminal amino acid could be detected after dinitrophenylation of the denatured enzyme.

The tyrosine metabolites, p- hydroxyphenylpyruvate , homogentisate, and fumarylacetoacetate were examined as inhibitors and inactivators of purified rat liver tyrosine α-ketoglutarate transaminase. Of these, homogentisate proved to be a very powerful inactivator when the enzyme was preincubated with it. This effect was blocked by prior addition of pyridoxal phosphate, α-ketoglutarate and dithiothreitol, but could not be reversed by these compounds. The homogentisate inactivated enzyme retained its ability to interact with the antibody to native tyrosine α-ketoglutarate transaminase.

1. 1. This study was designed to examine the influence of glucose consumption on induction of tyrosine aminotransferase (EC 2.6.1.5) and tryptophan oxygenase (EC 1.11.1.4) by glucocorticoids, trytophan and casein. 2. 2. Glucose feeding lowers basals levels of these enzymes and the magnitude of the rise in levels after inducer. The ratio between induced and basal enzymes and the magnitude was essentially unaffected. Glucose treatment did not simply delay induction of these enzymes by inducer but rather depressed enzyme levels throughout the period of enzyme induction. 3. 3. In adrenalectomized animals, glucose pretreatment lower the induction of these enzymes by casein but not by tryptopha. 4. 4. These and other observations lead us to suggest that tryptophan oxygenase functions to regulate intracellular tryptophan levels and that tryptophan levels play a key role in shifting metabolism between protein synthesis and gluconeogenesis.