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PubMed1989

Anaerobic induction of alanine aminotransferase in barley root tissue.

Alanine aminotransferase, otherwise called glutamate-pyruvate aminotransferase (GPT), activity increases up to fourfold during several days of anaerobic induction in barley (Hordeum vulgare L.) roots, reaching a maximum activity of 13 international units per gram fresh weight. This increase in activity paralleled the increase in alcohol dehydrogenase activity in the same root tissue. Upon return to aerobic conditions, the induced GPT activity declined with an apparent half-life of 2 days. The isozyme profile of GPT in barley root tissue comprised one band of activity; in maize there were three bands of activity, the bands with greater mobility had much lower activity. Native polyacrylamide gel electrophoresis indicated that the induction of GPT activity results from an increase in the level of activity of these bands; no other activities were detected. When root tissue was induced under different levels of hypoxia (0%, 2%, 5%, and 21% O(2)), changes in GPT activity were found to increase with lower levels of oxygen. Comparisons of GPT induction in barley, maize (Zea mays), rye, (Secale cereale) and wheat (Triticum aestivum) indicate that this enzyme is induced in the root tissue of all of these cereals; however, anaerobic root conditions do not result in the induction of GPT activity in leaf tissue. The dependence of GPT induction on high levels of nitrate in the media was tested by comparing activity levels in Hoagland solution and a nitrate-free nutrient solution. GPT activity was induced to similar levels under both conditions. These results indicate that alanine aminotransferase shows a very similar pattern of induction to alcohol dehydrogenase in barley root tissue and may be important in anaerobic glycolysis.

Journal Article↗

PubMed1992

Purification and Characterization of Glutamate 1-Semialdehyde Aminotransferase from Barley Expressed in Escherichia coli.

The immediate precursor in the synthesis of tetrapyrroles is Delta-aminolevulinate (ALA). ALA is synthesized from glutamate in higher plants, algae, and certain bacteria. Glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8) (GSA-AT), the third enzyme involved in this metabolic pathway, catalyzes the transamination of GSA to form ALA. The gene encoding this aminotransferase has previously been isolated from barley (Hordeum vulgare) and inserted into an Escherichia coli expression vector. We describe herein the purification of this recombinant barley GSA-AT expressed in Escherichia coli. Coexpression of GroEL and GroES is required for isolation of active aminotransferase from the soluble protein fraction of Escherichia coli. Purified GSA-AT exhibits absorption maxima characteristic of vitamin B(6)-containing enzymes. GSA-AT is primarily in the pyridoxamine form when isolated and can be interconverted between this and the pyridoxal form by addition of 4,5-dioxovalerate and 4,5-diaminovalerate. The conversion of GSA to ALA under steady-state conditions exhibited typical Michaelis-Menten kinetics. Values for K(m) (d,l-GSA) and k(cat) were determined to be 25 micromolar and 0.11 per second, respectively, by nonlinear regression analysis. Stimulation of ALA synthesis by increasing concentrations of d,l-GSA at various fixed concentrations of 4,5-diaminovalerate supports the hypothesis that 4,5-diaminovalerate is the intermediate in the synthesis of ALA.

Journal Article↗

PubMed2001

The structure of human mitochondrial branched-chain aminotransferase.

X-ray crystal structures of three forms of human mitochondrial branched-chain aminotransferase (BCAT) were solved by molecular-replacement methods, using Escherichia coli BCAT as the search model. The enzyme is a homodimer and the polypeptide chain of each monomer has two domains. The small domain is composed of residues 1--175 and the large domain is composed of residues 176--365. The active site is close to the dimer interface. The 4'-aldehyde of the PLP cofactor is covalently linked to the epsilon-amino group of the active-site lysine, Lys202, via a Schiff-base linkage in two of the structures. In the third structure, the enzyme is irreversibly inactivated by Tris. The overall fold of the dimer in human mitochondrial BCAT is similar to the structure of two bacterial enzymes, E. coli BCAT and D-amino acid aminotransferase (D-AAT). The residues lining the putative substrate-binding pocket of human BCAT and D-AAT are completely rearranged to allow catalysis with substrates of opposite stereochemistry. In the case of human mitochondrial branched-chain aminotransferase, a hydrogen-bond interaction between the guanidinium group of Arg143 in the first monomer with the side-chain hydroxyl of Tyr70 in the second monomer is important in the formation of the substrate-binding pocket.

Alanine Transaminase↗

PubMed2003

Involvement of conserved asparagine and arginine residues from the N-terminal region in the catalytic mechanism of rat liver and Trypanosoma cruzi tyrosine aminotransferases.

Rat liver and Trypanosoma cruzi tyrosine aminotransferases (TATs) share over 40% sequence identity, but differ in their substrate specificities. To explore the molecular features related to these differences, comparative mutagenesis studies were conducted on full length T. cruzi TAT and N-terminally truncated rat TAT recombinant enzymes. The functionality of Arg315 and Arg417 in rat TAT was investigated for comparison with the conserved Arg292 and Arg386 in aspartate and bacterial aromatic aminotransferases (ASATs and ARATs). The rat TAT Arg315Lys variant remained fully active indicating that, as in T. cruzi TAT and contrary to subfamily Ialpha aminotransferases, this residue is not critical for activity. In contrast, the Arg417Gln variant was inactive. The catalytic relevance of the putative rat TAT active site residues Asn54 and Arg57, which are strictly conserved in TATs (Asn17 and Arg20 in T. cruzi TAT) but differ in ASATs and ARATs, was also explored. The substitutions Arg57Ala and Arg57Gln abolished enzymatic activity of these mutants. In both variants, spectral studies demonstrated that aromatic but not dicarboxylic substrates could efficiently bind in the active site. Thus, Arg57 appears to be functionally equivalent to Arg292 of ASATs and ARATs. Asn54 also appears to be involved in the catalytic mechanism of rat TAT since its exchange for Ser lowered the k(cat)/K(m) ratios towards its substrates. Mutation of the analogous residues in T. cruzi TAT also lowered the catalytic efficiencies (k(cat)/K(m)) of the variants substantially. The results imply that the mamalian TAT is more closely related to the T. cruzi TAT than to ASATs and ARATs.

Amino Acid Sequence↗

PubMed1981

Selective inhibition by sodium butyrate of glucocorticoid-induced tyrosine aminotransferase synthesis in hepatoma tissue-cultured cells.

Sodium butyrate in a 5 mM concentration prevents the induction of tyrosine aminotransferase in hepatoma culture cells, without affecting the basal level of the enzyme. This effect is reversible immediately after the removal of butyrate, or after a lag, if butyrate was present for more than 2 h. Neither the amount of cellular RNA nor the rate of total RNA synthesis were affected by sodium butyrate. Furthermore, butyrate does not inhibit protein synthesis: [35S]methionine incorporation into proteins, measured in a reticulocyte lysate system, shows no significant difference between the translation capacity of the RNAs from butyrate-treated cells and from dexamethasone-induced or uninduced cells. Nevertheless, when tyrosine aminotransferase was isolated from the translation products by its specific antiserum and analyzed by gel electrophoresis, we observed that the amount of the enzyme synthetized in the presence of RNAs from dexamethasone/butyrate-treated cells was strongly diminished relative to that synthesized in the presence of RNA from dexamethasone-induced cells. These experiments indicate that the treatment of the cells with butyrate decreases the activity of the specific messenger RNA for tyrosine aminotransferase to a level close to the basal level.

Animals↗

PubMed1986

Biosynthesis of 4-aminobutyrate aminotransferase.

Mitochondrial 4-aminobutyrate aminotransferase was synthesized in a cell-free reticulocyte lysate using polysomal RNA isolated from pig brain. Its primary translation product has a higher molecular mass than the mature enzyme. The difference in relative molecular mass is approximately 2000 as revealed by SDS/polyacrylamide gel electrophoresis. The precursor of 4-aminobutyrate aminotransferase recognizes polyvalent antibodies raised against the mature enzyme. The precursor of 4-aminobutyrate aminotransferase binds pyridoxal-5-P and displays catalytic activity. Enzymatic activity was detected using a sensitive fluorimetric method, which is based on the formation of condensation products between succinic semialdehyde and cyclohexane-1,3-dione. It is concluded that removal of an extra peptide from the precursor is not an obligatory first step in the production of biological active species.

4-Aminobutyrate Transaminase↗

PubMed1987

Transcription activation of the tyrosine aminotransferase gene by glucocorticoids and cAMP in primary hepatocytes.

The expression of the tyrosine aminotransferase (TAT) gene of the rat was analyzed in primary hepatocytes. The TAT gene remains active in primary cultured cells at a level similar to that in liver cells. Expression can be induced by glucocorticoids and cAMP, glucocorticoids lead to a 8-10-fold increase in TAT mRNA level, cAMP to a 20-30-fold increase. The elevation of the TAT mRNA is preceeded by a rise in the relative rate of transcription of the gene. Surprisingly transcription of the albumin gene, which steadily declines with the age of the culture, can also strongly be stimulated by glucocorticoids in primary hepatocytes. cAMP antagonists, which act as competitive inhibitors of the cAMP-dependent protein kinase, prevent induction of transcription of the tyrosine aminotransferase gene by cAMP suggesting that the effect of cAMP on expression of the tyrosine aminotransferase gene is mediated by a cAMP-dependent protein kinase. The cAMP antagonist does not interfere with induction by glucocorticoids which suggests that phosphorylation of the glucocorticoid receptor by the cAMP-dependent protein kinase is not required for its function. We thus conclude that the two inducers affect transcription by independent mechanisms.

Animals↗

PubMed1990

Purification and characterization of serine-glyoxylate aminotransferase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

Serine--glyoxylate aminotransferase was purified to complete homogeneity from a serine-producing methylotrophic bacterium, Hyphomicrobium methylovorum GM2, which possesses the serine pathway. This is the first microbial serine--glyoxylate aminotransferase to be purified. The enzyme has a molecular mass of about 140 kDa and consists of four subunits of identical mass, i.e. 40 kDa. The holoenzyme exhibited absorption maxima at 282 nm and 408 nm, and a shoulder at about 315-345 nm in potassium phosphate pH 7.0; it contained 4 mol pyridoxal 5'-phosphate/mol enzyme. Isoelectric focusing showed that the enzyme had a pI value of 6.9. The Km values for glyoxylate and L-serine were 0.23 mM and 4.98 mM, respectively, and the enzyme showed high specificity for these substrates. The transamination between glyoxylate and L-serine seemed to be nearly irreversible. These data indicated that this serine--glyoxylate aminotransferase plays an essential role in methanol assimilation through the serine pathway in H. methylovorum GM2.

Bacteria↗

PubMed1994

The efficiency of nuclear processing of the tyrosine aminotransferase mRNA transcript increases after partial hepatectomy.

Following a two-thirds partial hepatectomy, an approximately fivefold increase in the levels of nuclear and total mRNA for tyrosine aminotransferase was observed at 1 h and 1.5 h, respectively, and a return to the levels of the quiescent state, i.e. the levels found in non-operated livers from adrenalectomized rats, was established 16 h post-hepatectomy. The increase in mRNA levels was not accounted for by a comparable change in the rate of transcription of the gene which, at 0.5 h post-hepatectomy, reached a maximum value that amounted to only 1.4-fold the value for quiescent liver. Subsequent changes in the transcription rate largely accounted for the changes in mRNA levels observed later on. Although tyrosine aminotransferase mRNA levels were equal in quiescent and 16-h-regenerating liver, the rate of transcription of the gene in quiescent liver was threefold higher than the rate in 16-h-regenerating liver. The maintenance of a higher rate of gene transcription in quiescent liver, as compared to regenerating liver, was shown to depend on ongoing protein synthesis. The possibility that the high rate of gene transcription was due to blockage or pausing during transcript elongation in quiescent liver was excluded. The inference is that the pronounced increase in tyrosine aminotransferase mRNA levels within 1 h of partial hepatectomy is largely due to a rapid increase in the efficiency of nuclear processing of the primary transcript.

Animals↗

PubMed1995

Reciprocal regulation of prothrombin secretion and tyrosine aminotransferase induction in hepatocytes.

Multicellular spheroids of hepatocytes are known to maintain liver functions for a long period. Rat hepatocytes were isolated to form spheroids by rotation culture and immobilized within calcium alginate. Immobilized spheroids had a much higher extent of tyrosine aminotransferase induction, which is one of the liver-specific differentiated functions, than immobilized non-aggregated cells, while the spheroids secreted significantly less prothrombin than non-aggregated cells. Co-culture of hepatocytes and non-parenchymal liver cells in a monolayer enhanced tyrosine aminotransferase induction and suppressed prothrombin secretion, while conditioned medium prepared from non-parenchymal cells greatly stimulated tyrosine aminotransferase induction and suppressed the prothrombin secretion and DNA synthesis in monolayer-cultured hepatocytes. Prothrombin secretion in hepatocytes was subjected to cell-density-dependent regulation. In a similar manner to other growth-related functions, prothrombin secretion was stimulated at low cell density. It has been reported that thrombin activates the zymogen of hepatocyte growth factor activator [Shimomura, T., Kondo, J., Ochiai, M., Naka, D., Miyazawa, K., Morimoto, Y. & Kitamura, N. (1993) J. Biol. Chem. 268, 22,927-22,932]. Therefore, prothrombin secretion could be one of the growth-related functions and involved in wound healing and liver regeneration.

Animals↗

PubMed1995

Primary structure and tissue distribution of human 4-aminobutyrate aminotransferase.

cDNA encoding human 4-aminobutyrate aminotransferase (aminobutyrate:2-oxoglutarate aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA-deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina-Kauwe, L. K., Tillakaratne, N. J. K., Wu, J.-Y. & Tobin, A. J. (1994) J. Neurochem. 62, 1267-1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity of the two sequences is established if it is assumed that a frame-shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4-aminobutyrate aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme.

4-Aminobutyrate Transaminase↗

PubMed1994

Effect of protein and fat content in feed on plasma alanine-aminotransferase and hepatic fatty infiltration in mink.

The effect of the content of protein and fat in the feed on the development of fatty infiltration of the liver in the period from weaning until pelting was measured in two groups of male scanblack mink (Mustela vision) fed 20% and 45%, respectively, of metabolizable energy (ME) from protein. Furthermore, plasma activity of alanine-aminotransferase and the content of specifically chosen clinical-chemical variables in the blood were measured. At pelting time in December, the liver weights were absolutely and relatively heavier to body weight and had a considerably higher fat content at 20% of ME from protein than at 45% of ME from protein. From August to pelting time, the activity of alanine-aminotransferase in plasma was higher at a low protein level than at a higher protein level in the feed. It is concluded that the content of protein and fat in the feed affects the incidence of hepatic fatty infiltration in mink. In the growth period, it is possible, based on plasma activity of alanine-aminotransferase, to select animals with histological fatty infiltration of the liver.

Alanine Transaminase↗

PubMed1981

Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase: relationship to catecholamine metabolism.

Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of alpha-methyl-p-tyrosine (AMT) methyl ester HCl (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.

Animals↗

PubMed1996

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase, key enzymes for the assimilation of one-carbon compounds in methylotrophs, was performed using antibodies raised against these enzymes purified from Hyphomicrobium methylovorum GM2. Immunodiffusion studies indicated that serine-glyoxylate aminotransferase and hydroxypyruvate reductase of all seven Hyphomicrobium strains tested were immunochemically similar. In immunotitration experiments and Western blot analyses of both enzymes in the genera Hyphomicrobium and Methylobacterium, the serine-glyoxylate aminotransferase of the genus Methylobacterium exhibited low similarity to that of the genus Hyphomicrobium. For hydroxypyruvate reductase, no immunological relationship was observed between the genera Hyphomicrobium and Methylobacterium, which was in agreement with the differences in primary structure and enzymological properties.

Alcohol Oxidoreductases↗

PubMed1998

Alanine aminotransferase apoenzyme in dogs.

Unusually low serum activity of alanine aminotransferase (ALT) was detected in a Rottweiler dog with gastric dilatation-volvulus. Activity of ALT in the same sample was found to be much higher (estimated increase of 14,225%) when measured by methods adding the cofactor pyridoxal-5'-phosphate (P5P), indicating that nearly all serum ALT was in the apoenzyme form. An investigation was undertaken to determine the frequency of high serum aminotransferase apoenzyme levels in dogs. Eighty canine serum samples submitted to the Clinical Pathology Laboratory at Cornell University were assayed for ALT and aspartate aminotransferase (AST) in the presence or absence of exogenous P5P. In 79 dogs, inclusion of P5P in the LAT assay resulted in a median decrease in AST activity of -6.3% (range -33.3% to 25.0%) in all 80 dogs. One dog had an increase of 336% in ALT activity after inclusion of P5P in the assay, but lacked a similar increase in AST activity. The reason for the high levels of ALT apoenzyme in the 2 dogs was not determined.

Journal Article↗

PubMed1968

Tyrosine aminotransferase: enzyme induction independent of adenosine 3', 5'-monophosphate.

The importance of adenyl cyclase and adenosine 3',5'-monophosphate in the induction of tyrosine aminotransferase by adrenocorticosteroids has been tested in HTC cells derived from a rat hepatoma and grown in tissue culture. Adrenocorticosteroids cause a 10-to 15-fold increase in the rate of synthesis of tyrosine aminotransferase in these cells. Under various experimental conditions, with or without glucocorticoids, neither adenyl cyclase nor cyclic adenosine mono-phosphate could be detected in HTC cells. In addition, neither the cyclic nucleotide nor N(6), O(2')-dibutyryl cyclic adenosine monophosphate caused increased activity of the transaminase in HTC cells. We conclude that induction of tyrosine aminotransferase by glucocorticoids is not mediated by the adenyl cyclase-cyclic adenosine monophosphate system.

Adenine Nucleotides↗

PubMed1981

Lectins mimic insulin in the induction of tyrosine aminotransferase.

Various lectins were found to induce tyrosine aminotransferase in H-35 rat hepatoma cells grown in monolayer culture. Wheat germ agglutinin gave a maximal induction of tyrosine aminotransferase 6 hours after its addition. The induction time course was similar to that elicited by insulin. Fourteen micrograms of wheat germ agglutinin per milliliter gave half-maximal enzyme induction and 50 micrograms per milliliter gave the maximal response. The induction of tyrosine aminotransferase by wheat germ agglutinin was additive with the induction by either dexamethasone or dibutyryl adenosine 3',5'-monophosphate, but was not additive with the tyrosine amino transferase induction by insulin. Wheat germ agglutinin also mimicked insulin in the inhibition of cellular protein degradation in the absence of serum. The insulin-like effects of lectins should be considered in lectin-mediated manipulations such as agglutination.

Animals↗

PubMed1997

Taurine-sulfur assimilation and taurine-pyruvate aminotransferase activity in anaerobic bacteria.

We demonstrated the ability of strictly fermentative, as well as facultatively fermentative, bacteria to assimilate sulfonate sulfur for growth. Taurine (2-aminoethanesulfonate) can be utilized by Clostridium pasteurianum C1 but does not support fermentative growth of two Klebsiella spp. and two different Clostridium spp. However, the latter are able to assimilate the sulfur of a variety of other sulfonates (e.g., cysteate, 3-sulfopyruvate, and 3-sulfolactate) anaerobically. A novel taurine-pyruvate aminotransferase activity was detected in cell extracts of C. pasteurianum C1 grown with taurine as the sole sulfur source. This activity was not detected in extracts of other bacteria examined, in C. pasteurianum C1 grown with sulfate or sulfite as the sulfur source, or in a Klebsiella isolate assimilating taurine-sulfur by aerobic respiration. More common aminotransferase activities (e.g., with aspartate or glutamate as the amino donor and pyruvate, oxalacetate, or (alpha)-ketoglutarate as the amino acceptor) were present, no matter what sulfur source was used for growth. Partial characterization of the taurine-pyruvate aminotransferase revealed an optimal temperature of 37(deg)C and a broad optimal pH range of 7.5 to 9.5.

Journal Article↗