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Plants produce various l-tyrosine (Tyr)-derived compounds that are critical for plant adaptation and have pharmaceutical or nutritional importance for human health. Tyrosine aminotransferases (TATs) catalyze the reversible reaction between Tyr and 4-hydroxyphenylpyruvate (HPP), representing the entry point in plants for both biosynthesis of various natural products and Tyr degradation in the recycling of energy and nutrients. To better understand the roles of TATs and how Tyr is metabolized in planta, here we characterized single and double loss-of-function mutants of TAT1 (At5g53970) and TAT2 (At5g36160) in the model plant Arabidopsis thaliana. As reported previously, tat1 mutants exhibited elevated and decreased levels of Tyr and tocopherols, respectively. The tat2 mutation alone had no impact on Tyr and tocopherol levels, but a tat1 tat2 double mutant had increased Tyr accumulation and decreased tocopherol levels under high-light stress compared with the tat1 mutant. Relative to WT and the tat2 mutant, the tat1 mutant displayed increased vulnerability to continuous dark treatment, associated with an early drop in respiratory activity and sucrose depletion. During isotope-labeled Tyr feeding in the dark, we observed that the tat1 mutant exhibits much slower (13)C incorporation into tocopherols, fumarate, and other tricarboxylic acid (TCA) cycle intermediates than WT and the tat2 mutant. These results indicate that TAT1 and TAT2 function together in tocopherol biosynthesis, with TAT2 having a lesser role, and that TAT1 plays the major role in Tyr degradation in planta. Our study also highlights the importance of Tyr degradation under carbon starvation conditions during dark-induced senescence in plants.

Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [(3)H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GR(dim)) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.

Macromolecular translocation inhibitor II (MTI-II), which was first identified as an in vitro inhibitor of binding between the highly purified glucocorticoid receptor (GR) and isolated nuclei, is an 11.5-kDa Zn(2+)-binding protein that is also known as ZnBP or parathymosin. MTI-II is a small nuclear acidic protein that is highly conserved in rats, cows, and humans and widely distributed in mammalian tissues, yet its physiological function is unknown. To elucidate its in vivo function in relation to GR, we transiently transfected mammalian cells with an expression plasmid encoding MTI-II. Unexpectedly, we found that the expression of MTI-II enhances the transcriptional activity of GR. The magnitude of the transcriptional enhancement induced by MTI-II is comparable with that induced by the steroid receptor coactivator SRC-1. In contrast, MTI-II had little effect on the transcriptional activity of estrogen receptor. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, GR coprecipitates with MTI-II, and, vice versa, MTI-II coprecipitates with GR. The expression of various deletion mutants of MTI-II revealed that the central acidic domain is essential for the enhancement of GR-dependent transcription. Microscopic analysis of MTI-II fused to green fluorescent protein and GR fused to red fluorescent protein in living HeLa cells showed that MTI-II colocalizes with GR in discrete subnuclear domains in a hormone-dependent manner. Coexpression of MTI-II with the coactivator SRC-1 or p300 further enhances GR-dependent transcription. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, p300 and CREB-binding protein are coprecipitated with MTI-II. Furthermore, the knockdown of endogenous MTI-II by RNAi reduces the transcriptional activity of GR in cells. Moreover, expression of MTI-II enhances the glucocorticoid-dependent transcription of the endogenous glucocorticoid-inducible enzyme in cells. Taken together, these results indicate that MTI-II enhances GR-dependent transcription via a direct interaction with GR in vivo. Thus, MTI-II is a new member of the GR-coactivator complex.

The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood.

Glucocorticoid induction of the tyrosine aminotransferase gene deviates from that of many glucocorticoid-responsive genes by having a lower EC50 and displaying more agonist activity with a given antiglucocorticoid. A cis-acting element, located 3646 base pairs upstream of the start of tyrosine aminotransferase gene transcription, has been found to be sufficient to reproduce these variations with heterologous genes and promoters (Oshima, H., and Simons, S.S., Jr. (1992) Mol. Endocrinol. 6, 416-428). This element has been called a glucocorticoid modulatory element, or GME. Others have called this sequence a cyclic AMP-responsive element (CRE) due to the binding of the cyclic AMP response element binding protein (CREB). We now report the partial purification and characterization of two new proteins (GMEB1 and -2) of 88 and 67 kDa that bind to the GME/CRE as a heteromeric complex. This purification was followed by the formation of a previously characterized, biologically relevant band in gel shift assays. By several biochemical criteria, the GMEBs differed from many of the previously described CREB/CREM/ATF family members. Partial peptide sequencing revealed that the sequences of these two proteins have not yet been described. Size exclusion chromatography and molecular weight measurements of the gel-shifted band demonstrated that the GMEBs bound to the GME as a macromolecular complex of about 550 kDa that could be dissociated by deoxycholate. Similar experiments showed that CREB bound to the GME as heteromeric complexes of about 310 and 360 kDa. As determined from gel shift assays, GMEB1 and -2 are not restricted to rat liver cells but appear to be ubiquitous. Thus, these novel GMEBs may participate in a similar modulation of other glucocorticoid-inducible genes in a variety of cells.

The degradation of rat liver tyrosine aminotransferase has been studied after transfection of suitable expression vectors into mammalian cells in culture. A normal rapid rate of degradation (half-life about 6 h) was observed in cells under stable transfection conditions. However, the higher enzyme levels produced during transient transfections or after amplification with methotrexate caused the apparent half-life of degradation to increase substantially. Analysis of expression in Chinese hamster ovary (CHO)-DG44 cells from vectors with deletions near either end of the tyrosine aminotransferase coding sequence showed that approximately the first 40 and the last 12 amino acid residues are not required to obtain normal catalytic function. When catalytically active deletion mutants were examined for effects on tyrosine aminotransferase degradation in stably transfected CHO-DG44 cell populations, short sequences near each end of the protein were found to be necessary for rapid degradation. The required sequence near the amino terminus is located between amino acids 30 and 40 and includes the highly basic region RKKGRKAR, a potential ubiquitin attachment site. The other essential sequence (EECDK) is located at the very COOH terminus of the 454-amino acid chain and is part of an acidic domain rich in cysteines and having PEST characteristics (rich in Pro, Glu, and Thr). Ser448, a potential casein kinase II phosphorylation site, is not required for activity or rapid degradation of tyrosine aminotransferase. No correlation was observed between the intracellular degradation rates of the various mutant proteins and their heat stabilities in vitro.

The mechanism of glucocorticoid resistance was studied in a rat hepatoma cell variant (6.10.2) which contains low levels of glucocorticoid receptor. These cells seem to have lost glucocorticoid-induced transcriptional responses as measured by the induction of expression of stably integrated mouse mammary tumor virus gene and the endogenous tyrosine aminotransferase gene, as well as the transcriptional suppression of glucocorticoid receptor gene expression. However, characterization of the glucocorticoid resistance in 6.10.2 cells revealed that the receptor is indistinguishable from the wild-type receptor with respect to hormone binding and affinity for both nonspecific and specific DNA sequences. The levels of the receptor mRNA and the total immunoreactive protein found in 6.10.2 cells were about 20% of those found in wild-type cells. Further analysis of 6.10.2 cells demonstrated that the receptor was indeed biologically functional. First, treatment of 6.10.2 cells with 8-bromo-cAMP elevated the endogenous glucocorticoid receptor levels 2-fold and restored responsiveness to glucocorticoids. Second, pretreatment of the cells with cycloheximide also led to acquisition of cellular responsiveness to glucocorticoids. We propose that there exists a "threshold" level of glucocorticoid receptor which is required for responsiveness and that under normal culture conditions, the level of glucocorticoid receptor in 6.10.2 cells is below this threshold. However, glucocorticoid responsiveness can be restored by raising the glucocorticoid receptor level above the threshold with 8-bromo-cAMP or, alternatively, by removing the threshold barrier with cycloheximide.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.

The native and one of the modified forms of tyrosine aminotransferase were purified from rat liver and characterized. Several hydrodynamic properties of the native enzyme are: Stokes radius, 46 A; subunit isoelectric point, 5.6; sedimentation coefficient, 5.6 S, frictional ratio, 1.44; diffusion coefficient, 4.65 X 10(-7) cm2 s-1; extinction coefficient of a 1% solution (w:v) at 280 nm, 10.5 cm-1. The molecular weight of the dimeric protein is 110,500 as calculated from the Stokes radius and sedimentation coefficient. The subunit of the modified form is of lower molecular weight than the subunit of the native enzyme and has a pI of about 5.9. During isoelectric focusing, both forms of the enzyme separate into two components. The more acidic component that is resolved from the native enzyme is phosphorylated, but the other component is not. The amino acid composition of native tyrosine aminotransferase differs from values reported for mixtures of the three forms of this enzyme. Neither the native nor the modified forms of the enzyme possess a free alpha-amino group as judged by dansylation, nor can they be digested with leucine aminopeptidase, implying that the NH2-terminus is blocked. The possibility that tyrosine aminotransferase is acetylated was examined by translating poly(A)+RNA from hepatoma cells in a cell-free translational system in the presence and absence of inhibitors of protein acetylation. [35S]Tyrosine aminotransferase synthesized in the presence of the inhibitors has a more basic isoelectric point than the native enzyme as determined by isoelectric focusing, suggesting that the enzyme is acetylated either at the NH2-terminal or the epsilon-amino group of an internal lysine. When digested by either of two lysosomal proteases, tyrosine aminotransferase is cleaved to a smaller size. These data show that tyrosine aminotransferase is susceptible to several post-translational modifications.

Messenger RNA specific for tyrosine aminotransferase was quantitated by microinjection into oocytes of Xenopus laevis. The heterologously translated enzyme was identified by specific immunoprecipitation and found to be identical with authentic aminotransferase by several criteria. The level of functional message present in rat liver increases during hydrocortisone induction, and this increase is directly proportional to the increased rate of synthesis of the enzyme. Kinetic analysis of the changes in tyrosine aminotransferase mRNA levels during induction and withdrawal indicates that the steroid does not affect the stability of the message, which has a half-life of approximately 1.2 h. Hydrocortisone, therefore, acts to increase the rate of synthesis of the specific messenger by stimulating either its transcription or processing to functional mRNA.

We have investigated the p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase activity in cultured hepatoma cells. The similarity of the effect of p-chlorophenylalanine on phenylalanine hydroxylase in the hepatoma cells and that reported from studies in vivo indicates that the loss of phenylalanine hydroxylase activity is due to a direct interaction of the amino acid analogue with the liver. We can find no evidence that the loss of phenylalanine hydroxylase activity is due to: a direct inactivation of the hydroxylase by p-chlorophenylalanine or an inhibitor produced by p-chlorophenylalanine treatment; an effect similar to that of p-fluorophenylalanine; or leakage of enzyme from the cells during p-chlorophenylalanine treatment. The data presented indicate: (a) the p-chlorophenylalanine effect is rather specific for phenylalanine hydroxylase; (b) following p-chlorophenylalanine removal, new protein synthesis is necessary for restoration of the hydroxylase activity; (c) the rate of loss of phenylalanine hydroxylase activity after the addition of p-chlorophenylalanine is much faster than the rate of restoration of the hydroxylase activity after removal of p-chlorophenylalanine; (d) even in the presence of p-chlorophenylalanine, hydrocortisone greatly stimulates the hydroxylase activity; (e) the cell density-dependent increase of phenylalanine hydroxylase activity is blocked by p-chlorophenylalanine. A discussion of the possible mechanisms of p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low leanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low levels of phenylalanine hydroxylase activity, a new procedure, based on isotope dilution, was developed for isolating the tyrosine formed during the enzymatic reaction.

Reuber H35 hepatoma cells growing in monolayer cultures have been found to contain the soluble enzyme phosphoenolpyruvate carboxykinase. The activity of this enzyme can be increased 2- to 3-fold by the addition of either N6,O2'-dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP) or the synthetic glucocorticoid dexamethasone (Δ1,2,9α-fluoro,16α-methyl cortisol). Tyrosine transaminase responds similarly to DBcAMP but its activity was elevated 5- to 8-fold by dexamethasone. The effects of each inducer on both enzymes were similar with respect to kinetics, dose-response relationships, analogues, and cycloheximide inhibition. The response of both enzymes to DBcAMP, however, was more rapid than to dexamethasone. The rate of transaminase synthesis was increased 2- to 3-fold by DBcAMP but no effect of the cyclic nucleotide could be detected on the rate of transaminase degradation. Elevation of carboxykinase activity appears to be due to an increase in the rate of synthesis of this enzyme based on immunological evidence and cycloheximide inhibition of the response to both dexamethasone and DBcAMP. Insulin completely suppressed the effects of both DBcAMP and dexamethasone on the carboxykinase at the same time that it produced additive increases in transaminase activity with these agents. On the other hand, progesterone completely prevented the effects of dexamethasone on both enzymes but did not inhibit the effects of DBcAMP on either enzyme.

Although the hydrocortisone-mediated induction of hepatic tyrosine transaminase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) is not inhibited by the administration of 8-azaguanine with the hormone, the later phases of the induction cycle are drastically modified. Within 6 hours after administering hydrocortisone to adrenalectomized rats (controls), the enzyme activity begins to return from the induced to the basal level. In contrast, the activity rises to approximately 30% above the induced level of the controls (i.e. is superinduced) in animals treated simultaneously with hydrocortisone and 8-azaguanine. The activity is maintained at this superinduced level for approximately 3 hours before falling to the induced level of the controls. There follows another phase in animals receiving both hydrocortisone and 8-azaguanine during which the activity is maintained at or near the induced level until approximately 18 hours after hormone administration (i.e. 12 hours later than in the controls). The delay in the fall of enzyme activity, but not the superinduction phenomenon, is observed when the analogue is administered 4 hours after hydrocortisone. Results of experiments in which 8-azaguanine was given 4 or 5.5 hours after the hormone indicate that there is a lag of approximately 2 hours before the analogue becomes effective in this system. Other bases having primary amino groups (guanine, cytosine, or adenine), when given in conjunction with hydrocortisone, elicit the superinduction but not the maintenance of enzyme activity. Bases without primary amino groups (uracil or 8-azaxanthine) do not alter the induction cycle. Preliminary treatment of rats receiving 8-azaguanine and hydrocortisone, with 4-amino-5-imidazolecarboxamide, abolishes the superinduction phase but not the prolonged maintenance of enzyme activity at or near the induced level. The data indicate that the superinduction and the maintenance phenomena can be dissociated. It appears that the superinduction is due to an increase in free amino acids in liver following ammonia production by deamination of the bases; on the other hand, the delay in the fall of tyrosine transaminase activity, which is restricted to 8-azaguanine-treated rats, appears to be a consequence of the incorporation of the analogue into RNA.

The level of tyrosine transaminase (l-tyrosine:2-oxoglutarate transaminase, EC 2.6.1.5) in cultured hepatoma cells was markedly elevated by increasing the concentration of amino acids in the medium. The active component of the amino acid mixture was identified as l-leucine. d-Leucine, dl-trifluoroleucine, and the deaminated product of leucine, α-ketoisocaproic acid, were inactive. At the optimum concentration (5 mm) l-leucine elicited an 8- to 10-fold increase in the transaminase level; the response was maximum at 12 to 14 hours and persisted for at least 24 hours. The half-life of the transaminase, estimated both indirectly from kinetics of change in enzyme activity after addition or withdrawal of the amino acid and directly by isotopic-immunochemical methods, was 6 hours in the presence of 5 mm l-leucine and 2 hours after return of the cells to the basal medium (0.2 mm l-leucine). Pulse labeling experiments indicated that l-leucine also increased the rate of tyrosine transaminase synthesis by a factor of 3 to 4. Thus l-leucine increased the level of tyrosine transaminase by a dual effect on both synthesis and degradation of the enzyme. Combinations of l-leucine with the hormonal inducers hydrocortisone and insulin yielded additive effects on the transaminase levels. Hydrocortisone or insulin effectively induced the transaminase in leucine-free medium, and l-leucine further increased, in an additive manner, the enzyme activity of the cells which had been maximally previously induced by hydrocortisone or insulin. These results indicate that induction of the enzyme by either hydrocortisone or insulin is not mediated by l-leucine and imply that the mechanism of the induction by l-leucine is different from that effected by either of these hormones.

Halogenated tyrosine transaminase has been purified 30-fold from a sonic extract of rat kidney mitochondria. Purification involves ammonium sulfate fractionation and chromatography on diethylaminoethyl cellulose and hydroxylapatite. The purified enzyme is highly specific for tyrosine analogues with halogen substituents in positions 3 or 3 and 5, for thyroid hormones, and, to a lesser extent, for naturally occurring aromatic amino acids such as tyrosine, tryptophan, and phenylalanine. The identity of the enzyme and thyroid hormone transaminase has been confirmed. Halogenated tyrosine transaminase requires pyridoxal 5'-phosphate as a coenzyme and α-ketoglutarate as amino acceptor. Inhibition studies indicate that a sulfhydryl group is essential for enzyme activity. 3,5-Diiodo-4-hydroxyphenylacetate has been found to be a powerful inhibitor of the enzyme, functioning as a mixed type of inhibitor. The reaction catalyzed by this enzyme is: halogenated tyrosine + α-ketoglutarate → halogenated p-hydroxyphenylketo acid + glutamate.

A several fold increase in the activity of tyrosine-α-ketoglutarate transaminase occurs in intact or adrenalectomized rats following treatment with an enzymatic hydrolysate of casein, whereas tryptophan pyrrolase is unresponsive to this treatment. Doses of tyrosine, tryptophan, or the combination of these amino acids equivalent in amount to those contained in an effective dose of casein hydrolysate did not produce a rise in tyrosine transaminase activity. The effect of casein hydrolysate is inhibited by actinomycin D or cycloheximide, indicating that the synthesis of new RNA and protein is associated with the response of tyrosine transaminase to casein hydrolysate. Additive and, in some cases, synergistic effects on tyrosine transaminase are observed when casein hydrolysate and hydrocortisone are given together or sequentially. In contrast to the response of this enzyme, no additive effect on tryptophan pyrrolase or serine dehydrase occurs when 1 g of casein hydrolysate is given at the same time as a large dose of hydrocortisone. Treatment of adrenalectomized rats with casein hydrolysate, 4 or 8 hours after they were treated with hydrocortisone, stimulates a marked further increase in the activity of tyrosine transaminase. In intact rats, tyrosine transaminase shows a synergistic response to casein hydrolysate when it is given concomitantly with a small dose of hydrocortisone; under these conditions tryptophan pyrrolase is also responsive, but this effect appears to be due to tryptophan stabilization of the enzyme against degradation. When a low dosage of hydrocortisone is administered to intact rats, tryptophan pyrrolase, but not tyrosine transaminase, shows an increase in activity. On the other hand, of these two enzymes, only the transaminase responds to casein hydrolysate. Each of these treatments stimulates the incorporation of orotic acid-6-14C into liver RNA to about the same extent, indicating either that this is a selective effect or that factors in addition to new RNA synthesis are involved in the induction of these enzymes.

The induction and subsequent inactivation of hepatic tyrosine transaminase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) have been studied in the isolated perfused rat liver. This study was undertaken to confirm that the inhibition in vivo by 8-azaguanine and cycloheximide of tyrosine transaminase inactivation results from a direct action of these inhibitors on the liver. When hydrocortisone was administered at the start of perfusion, the maximal activity of tyrosine transaminase attained was approximately 50% of that normally observed after induction in vivo. Furthermore, the return of the activity to the basal level occurred 3 to 4 hours sooner than it did in vivo. This limited response appears to be attributable to the lower protein-synthetic capacity of the perfused liver, as witnessed by a gradual conversion of a portion of the polyribosomes in the postmitochondrial supernatant of the liver to monomers and dimers during the course of perfusion. The effect of the inhibitors was studied with this system after the induction of tyrosine transaminase by hydrocortisone had been allowed to proceed for 4 hours in vivo. 8-Azaguanine or cycloheximide given at the start of perfusion prevented the normal fall in enzyme activity from the steroid-induced to the basal level. No such inhibition was observed when 8-azaxanthine, pyridoxal, l-tyrosine, l-tryptophan, α-ketoglutarate, glucagon, or insulin was added to the perfusate. The data show conclusively that the prevention of tyrosine transaminase inactivation results from a direct action of 8-azaguanine and cycloheximide on the liver, and confirm that this phenomenon depends upon an inhibition of protein synthesis.

Decapitation of fetuses in utero rendered rat liver tyrosine transaminase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) occasionally inducible by hydrocortisone. By placing fetal rat liver in organ culture, reproducible induction by the steroid could be obtained. Histological and biochemical studies supported the conclusion that the hepatocytes remained healthy over a 3-day period in culture. The response to hormone was very low in fresh explants for the first 12 hours, but thereafter a 4- to 8-fold elevation in enzyme activity was obtained within 4 to 6 hours. The induced level of the transaminase was maintained unless steroid was removed, and then the activity fell rapidly back toward the control level. The optimal effective concentration of hydrocortisone was 10-6 m, but levels as low as 10-8 m produced a limited response. Only the glucocorticoids and the protein hormones insulin and glucagon were active inducers among a number of hormones tested. Cycloheximide and actinomycin D prevented enzyme induction, but hydrocortisone did not affect either general RNA or protein synthesis. Lactic dehydrogenase activity in explants of fetal liver was unaffected by hydrocortisone. Titrations against antibody showed that the tyrosine transaminases in fetal and adult liver are immunologically identical and proved that the increase in enzyme activity in explants exposed to hydrocortisone was due to an increase in enzyme protein. Immunochemical labeling experiments confirmed that synthesis de novo of enzyme protein is stimulated by hydrocortisone. The results are consistent with the hypothesis that the synthesis of this enzyme is repressed during gestation.

Moderate doses of the hypophysial growth hormone can effect a selective repression of the synthesis of rat liver tyrosine transaminase (l-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) in hypophysectomized, as well as in adrenalectomized, rats. The response is erratic, which may reflect the action of competing hormones or the fact that the hepatic repression is secondary to another metabolic alteration brought about by growth hormone. When repression occurs, the hepatic level of the enzyme falls at approximately the rate at which "degradation" occurs in normal turnover. The rate of transaminase synthesis is reduced nearly to zero, as determined by immunochemical-isotopic analyses, while labeling of the bulk of the liver proteins is increased by the hormone. Repression is blocked when RNA synthesis is inhibited by actinomycin.

The effects of cortisone on the dietary induction and repression of serine dehydrase, ornithine transaminase, and tyrosine transaminase were studied. In rats pretreated with cortisone dietary induction was affected relatively little, but carbohydrate repression was largely eliminated. The effects of cortisone and phenobarbital on the dietary induction of serine dehydrase and ornithine transaminase were studied. Pretreatment with phenobarbital had little effect on the induction of serine dehydrase and ornithine transaminase, but pretreatment with cortisone and phenobarbital together caused an enhanced response of these enzymes to casein hydrolysate feeding, with respect to both total amount of enzyme produced and activity per g of liver protein. Neither cortisone nor phenobarbital acted as an inducing agent when administered alone or in combination; their effects were manifested only in the presence of dietary inducer.

The reaction catalyzed by purified rat liver cytosol tyrosine aminotransferase (l-tyrosine + 2 oxoglutarate = p-hydroxyphenylpyruvate + l-glutamate, EC 2.6.1.5) has been analyzed with regard to product inhibition and combination with each of its rate-limiting substrates by the Hill equation. Glutamate is a linear competitive inhibitor with respect to tyrosine, a linear noncompetitive inhibitor with respect to coenzyme or α-ketoglutarate. p-Hydroxyphenylpyruvate is a linear noncompetitive inhibitor with respect to tyrosine or pyridoxal-P but is a linear competitive inhibitor with respect to α-ketoglutarate. These data, considered with replots of slopes and intercepts, indicate a "ping-pong" mechanism random in the sense that either coenzyme or Schiff's base of coenzyme and amino acid can add to the apoenzyme. A study of the half-reaction with apoenzyme, pyridoxamine-P, and α-ketoglutarate showed that 90% or more of the complete chemical reaction rate was achieved when freshly isolated apoenzyme was used. The pH dependence of the half-reaction paralleled that of the complete chemical reaction and supports the conclusion that the same protein catalyzes the complete and half-reaction. Furthermore, the magnitude of the half-reaction compared to the complete transamination would dictate that the mechanism does not switch between ping-pong and ordered depending upon the level of substrates available. By use of the Hill equation, the reaction between enzyme and substrates is first order with respect to tyrosine, pyridoxal-P, and α-ketoglutarate. Reduction of the concentration of α-ketoglutarate to a rate-limiting value does not change the order with respect to tyrosine or to pyridoxal-P. Reduction of the concentration of pyridoxal-P, or of tyrosine, or of both of these reactants to rate-limiting levels when the concentration of α-ketoglutarate is varied does not affect the order of the reaction with respect to α-ketoglutarate. Thus, homotropic effects are not manifested by this system.

Orally intubated 3H-labeled tryptophan was retained significantly in the stomach and intestine of rats treated with actinomycin D (43%) as compared to controls (13%) 4 hours after intubation. Likewise, smaller amounts of the isotope subsequently appeared in the liver and serum of the drug-treated animals. In contrast, actinomycin had no effect on movement of intraperitoneally injected 3H-tryptophan out of the peritoneal cavity or its subsequent accumulation in the liver. Induction of tryptophan pyrrolase by intraperitoneally or intravenously administered tryptophan was unaffected by concomitant actinomycin D treatment. On the other hand, similar induction of tyrosine transaminase was completely blocked by the drug. When l-tryptophan was administered orally over a range of 5 to 20 mg/100 g, the maximum induction of tyrosine transaminase was obtained at the 5-mg dose with no further enhancement at higher levels. However, with tryptophan pyrrolase, there was a gradual response: no induction after 5 mg, a 3-fold increase at 10 mg, and a 6-fold enhancement at 20 mg. These experiments show that in the rat actinomycin D affects the transport and absorption of dietary metabolites from the gastrointestinal tract. Therefore it is important to consider any conclusion drawn from orally administered substrate induction experiments in the light of this action of actinomycin D.

l-Tryptophan, l-5-hydroxytryptophan, serotonin, and other indole amines increase tyrosine aminotransferase activity in mice liver. l-5-Hydroxytryptophan was as effective as serotonin, whereas l-tryptophan was far less effective at low doses. If inhibitors of aromatic l-amino acid decarboxylase were injected prior to the administration of indole compounds, the increase of tyrosine aminotransferase activity by 5-hydroxytryptophan or tryptophan was completely blocked, whereas the increase by serotonin was not reduced. On the other hand, a monoamine oxidase inhibitor enhanced the increase of tyrosine aminotransferase activity by serotonin or 5-hydroxytryptophan. These results indicate that indole amines are the active agents in the increase of enzyme level. Among various indole amines, serotonin, 5-methoxytryptamine, and tryptamine were effective in increasing the enzyme activity. Although the increase in the enzyme activity was partially mediated by adrenal hormones, these indole amines could increase tyrosine aminotransferase activity in adrenalectomized mice. Cycloheximide blocked the increase of tyrosine aminotransferase activity by serotonin, indicating that the increase is due to induction of new enzyme. The results suggest a possible role of indole amines in the regulation of metabolism.

The role of the pancreatic hormones, insulin and glucagon, in the regulation of hepatic tyrosine transaminase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) was studied in adrenalectomized rats. Each of these hormones initiated a rapid but limited increase in the enzyme level, which reached a maximum after 2.5 to 3 hours. The optimal doses of insulin and glucagon per 100 g of body weight were 0.75 unit (33 µg) and 150 µg, respectively. Alloxan-induced diabetes in adrenalectomized rats caused a significant increase in the transaminase level after 5 days. Immunochemical-isotopic analyses showed that the pancreatic hormones cause an increase in the rate of transaminase synthesis which is comparable to that caused by hydrocortisone. Effects of the pancreatic hormones on the rate of enzyme synthesis are not additive with one another but are additive with the effect of hydrocortisone. Induction by either insulin or glucagon was inhibited by actinomycin D. A discrete mechanism for induction of this enzyme, which is steroid-independent and which can be initiated in vivo by either insulin or glucagon, is indicated.

Orally administered ferric citrate exerts a pronounced synergistic effect on the induction of hepatic δ-aminolevulinic acid synthetase produced by two compounds. The effect appears to be specific for iron citrate, as opposed to other metals in complex with citrate, and specific also in affecting the level of δ-aminolevulinic acid synthetase as compared with another induced enzyme, tyrosine transaminase. Inorganic iron salts did not produce augmentation of the induction of hepatic δ-aminolevulinic acid synthetase. The data suggest that iron augments the rate of synthesis of the enzyme during induction rather than activating or stabilizing the enzyme.

Synthesis of the messenger RNA that codes for tyrosine α-ketoglutarate transaminase (l-tyrosine:2-oxo-glutarate aminotransferase, EC 2.6.1.5) was blocked by actinomycin and its stability was determined in cultured hepatoma cells by measuring rates of transaminase synthesis. By this criterion the transaminase messenger RNA is degraded with a half-life of 2 to 3 hours in both H-35 and HTC cell lines; this rate is not changed by hydrocortisone. Transaminase synthesis is decelerated by high and low concentrations of actinomycin; high concentrations of this antibiotic block transaminase degradation as well. At low concentrations of actinomycin, transaminase induction by hydrocortisone is blocked but induction by insulin is unaffected for a period of time consistent with messenger stability. Preliminary treatment with hydrocortisone primes the cells to respond to insulin with a rapid increase in enzyme, but preliminary treatment with insulin does not alter the response to hydrocortisone. With the use of experimentally derived rates of degradation of both the transaminase and its messenger RNA, a mathematical model was developed which satisfactorily predicts kinetic behavior of the enzyme level upon addition and withdrawal of inducing hormones. The data are consistent with the conclusion that hydrocortisone acts transcriptionally while insulin acts on some posttranscriptional or translational step in enzyme synthesis. We find no indication of posttranscriptional mechanisms in the steroid-mediated induction. A discussion of the mathematical model used is appended.

When development is initiated in Dictyostelium discoideum by the removal of exogenous energy sources, there is an increase in the specific activity of tyrosine transaminase (l-tyrosine:2-oxoglutarate aminotransferase EC 2.6.1.5). The enzyme was partially purified from crude extracts of developed cells and shown to be specific for l-tyrosine among aromatic amino acids. The increase in specific activity which occurs 8 hours after the initiation of development appears to result from de novo synthesis of the enzyme rather than from activation of latent activity. Experiments with actinomycin D suggest that synthesis of tyrosine transaminase depends on prior RNA synthesis. Analysis of the kinetics of accumulation of tyrosine transaminase in a series of morphologically and temporally deranged mutant strains indicates that both the synthesis and subsequent partial inactivation of this enzyme are developmentally regulated in D. discoideum.

The possible functional significance of induction of the hepatic enzymes, tryptophan pyrrolase and tyrosine transaminase, has been studied in intact normal and adrenalectomized adult male rats and in the isolated perfused rat liver. In intact, normal rats, several-fold increases in tryptophan pyrrolase activity induced by treatment with hydrocortisone or tryptophan, or both, were associated with at most a 20% increase in the percentage dose of dl-tryptophan-3-14C converted to 14CO2. However, under conditions in which the activity of tryptophan pyrrolase did not increase further, for example, with increasing the tryptophan load from 17.5 to 35 mg, the conversion of labeled tryptophan to 14CO2 was substantially increased. In the adrenalectomized rat, hydrocortisone elicited a 6-fold increase in tryptophan pyrrolase activity without changing oxidation of a tracer dose of dl-tryptophan-3-14C to 14CO2. Although hydrocortisone added to the labeled tryptophan load led to a further 4-fold increase in tryptophan pyrrolase activity, no change in oxidation of tryptophan to 14CO2 was seen. In the adrenalectomized rat the percentage dose l-tyrosine-1-14C converted to 14CO2 increased substantially with a 10-mg or 30-mg load of l-tyrosine in spite of the fact that the level of hepatic tyrosine transaminase remains unchanged; in contrast, treatment with hydrocortisone leads to a 7- to 14-fold increase of enzyme activity, yet the increase in oxidation of l-tyrosine-1-14C to 14CO2 is trivial at several load levels. In the isolated perfused rat liver induction of tryptophan pyrrolase with hydrocortisone did not alter the rate of clearance of a graded load of l-tryptophan from the perfusate, did not enhance accumulation of kynurenine in the perfusate, and did not increase the rate of conversion of dl-tryptophan-3-14C to 14CO2. Thus, manifold increases in enzyme activity inducible in the intact or adrenalectomized rat and in the isolated perfused liver are not associated with parallel increases in oxidation of 14C-labeled amino acids to 14CO2. However, increasing the substrate load per se was associated with quantitatively large increases in oxidation to 14CO2 under conditions not associated with altered levels of enzyme activity.

The N6,O2'-dibutyryl analogue of cyclic adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) elevates the level of hepatic tyrosine-α-ketoglutarate transaminase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) some 3- to 6-fold in intact and adrenalectomized rats. The response of this enzyme is more rapid to dibutyryl cyclic AMP than to any other inducer of the transaminase. Hourly injection of dibutyryl cyclic AMP can maintain the elevated transaminase level for at least 5 hours. The rate of synthesis of tyrosine transaminase, as measured by an isotopic-immunochemical procedure, is enhanced by treatment with dibutyryl cyclic AMP. There is no detectable effect of dibutyryl cyclic AMP on the synthesis of total soluble proteins, however. Combinations of dibutyryl cyclic AMP and hydrocortisone produce additive or, in some cases, synergistic increases in transaminase activity, but the responses to combinations of dibutyryl cyclic AMP and either glucagon or insulin are not additive. Two other soluble liver enzymes, P-enolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase transphosphorylating, EC 4.1.1.32) and serine dehydrase (l-serine hydro-lyase, EC 4.2.1.13) are also elevated by treatment of intact rats with either dibutyryl cyclic AMP or glucagon, but another, tryptophan pyrrolase (l-tryptophan:oxygen oxidoreductase, EC 1.13.1.12, tryptophan oxygenase), is not affected by either agent. Hydrocortisone induces both the transaminase and pyrrolase 3- to 5-fold but the steroid has no effect on either the carboxykinase or the dehydrase. The results support the view that cyclic AMP is the intracellular mediator of the action of glucagon on hepatic enzyme synthesis.

1. An injection of d-galactosamine at the same time as an injection of dexamethasone inhibits the increase of rat liver tyrosine aminotransferase activity normally observed following injection of dexamethasone alone. The galactosamine-mediated inhibition is dose dependent: a 50% inhibition of the increase of enzyme activity is observed at 10 mg of galactosamine·HCl per 100 g of body weight, and a total inhibition is observed at 15 mg per 100 g of body weight. Following a single injection of 37.5 mg of galactosamine·HCl per 100 g of body weight, the basal level of tyrosine aminotransferase activity does not change for at least 16 hours. 2. Induction of the enzyme by glucagon or dibutyryl cyclic AMP is only partially inhibited by a dose of 37.5 mg of galactosamine·HCl per 100 g of body weight. 3. The total inhibition of the dexamethasone-induced enzyme increase is specific for galactosamine. A high dose of d-glucose or d-galactose has no inhibitory effect, and a high dose of d-glucosamine or 2-deoxy-d-galactose has only a partially inhibitory effect. 4. The inhibition by galactosamine of the dexamethasone-induced enzyme increase can be prevented by simultaneous injection of uridine. Determination of the uridine phosphate levels following injection of different doses of galactosamine reveals a partial inhibition of the enzyme induction below 100 nmoles of UTP+UDP per g of liver and a total inhibition of enzyme induction below 40 to 50 nmoles of UTP+UDP per g of liver. 5. There is little or no relationship between levels of galactosamine metabolites in the liver and inhibition of enzyme induction. 6. Unlike actinomycin D, injection of galactosamine at various times after injection of dexamethasone does not result in a superinduction of tyrosine aminotransferase.

The role of pyridoxine and its coenzymically active derivatives in determining the stability of rat liver tyrosine α-ketoglutarate transaminase in vitro and in vivo was studied. Incubation of crude or purified holoenzyme preparations with cysteine and other amino acids causes removal of the coenzyme and subsequent inactivation of the inherently unstable apoenzyme; under these conditions the relatively stable glutamic-pyruvic transaminase retains its coenzyme and remains active. Inactivation of tyrosine transaminase is prevented by small amounts of 5'-phosphate derivatives of pyridoxine; the enzyme is also stabilized in solutions of high ionic strength. Evidence is presented that protection by coenzyme involves its reaction with enzymically active sites. The induction of tyrosine transaminase by pyridoxine administration does not reflect stabilization in vivo. Induction involves an increased rate of transaminase synthesis, determined immunochemically, and is inhibited by actinomycin D, and is thus similar to the inductions of this enzyme brought about by hydrocortisone and other hormones.

Hormonal regulation of the synthesis of hepatic tyrosine transaminase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) was studied in the isolated, perfused rat liver. Hydrocortisone, insulin, and glucagon, each of which increases the rate of synthesis of tyrosine transaminase when administered in vivo, were all found to do so by direct action on the liver. Induction by continuous infusion of hydrocortisone continued throughout the time course of the experiments (9 to 11 hours). Infusion of either insulin or glucagon initiated a rapid but limited increase in tyrosine transaminase synthesis, the maximum activity being attained 2½ to 3 hours after the initial addition of these hormones. The hormonal inductions of this enzyme were all sensitive to actinomycin D. Growth hormone, which mediates a selective repression of tyrosine transaminase synthesis when administered to adrenalectomized rats in vivo, did not alter the level of this enzyme in the isolated, perfused rat liver, suggesting that this hormone acts indirectly.

The processes involved in the induction of the liver enzyme tyrosine transaminase by either hydrocortisone or adenosine 3',5'-monophosphate (cyclic AMP) in organ culture have been shown to exhibit different characteristics. Removal of cyclic AMP, as the N6,O2'-dibutyryl analogue (dibutyryl cyclic AMP) during the early phase of induction of either tyrosine transaminase or phosphoenolpyruvate carboxykinase, or after attainment of a new elevated steady state level, prompted a virtually immediate cessation of the induction process. In contrast, removal of hydrocortisone at either stage did not stop elevation of transaminase activity for 1½ to 2 hours even in the presence of actinomycin D. The sequential addition of one inducer after maximal induction by the other revealed that hydrocortisone markedly amplified the subsequent effect of dibutyryl cyclic AMP on the transaminase. Prior exposure to dibutyryl cyclic AMP did not lead to any amplification of the subsequent enzyme response to hydrocortisone. The results are interpreted to indicate that hydrocortisone and cyclic AMP act at sequential sites n the synthesis of tyrosine transaminase. The suggestion s made that cyclic AMP may act at a post-transcriptional evel in the case of both the transaminase and carboxykinase.