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PubMed1976

[Histochemical evidence of the aminotransferases. V. Quantitative histochemical investigations of the aminotransferases in organs of the rat (author's transl)].

The quantitative histochemical method was employed the activity of some aminotransferases in the rat organs. Determination was based on extraction of the enzymatic reaction product formazan with alkaline dimethylsulfoxid. Evaluation of the enzymatic activity was given by the formazan quantity related to 1 mug tissue protein. The following transaminases have been studied: aspartate-, alanine-, tyrosine-, phenylalanine-, tryptophane-, glycine-, and serine aminotransferases, as the leucine aminotransferase with substrate leucine, isoleucine and valine. Difference was observed as with respect to one and the same aminotransferase in the examined organs, as well, with respect to the different aminotransferases in one and the same organ.

Animals↗

PubMed1983

Late phase of liver restoration following partial hepatectomy in phenobarbital-treated rats. II. Effect of phenobarbital on aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and UDP-glucuronyltransferase activity.

Rats were treated with 50 mg phenobarbital (PB) per kg body weight for 7 days prior to or after partial hepatectomy. The activities of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase were measured in the regenerating liver 1 week following liver amputation. UDP-glucuronyl transferase activity was determined at the time of surgery as well as 7 days later at the time of death. Alanine aminotransferase was induced by PB in rats only treated in the postoperative period, while aspartate aminotransferase and glutamate dehydrogenase were not. The activity of UDP-glucuronyl transferase was increased more than twofold by repeated PB treatment in both normal and regenerating liver. After cessation of therapy, however, the enhanced activities returned to a normal level. It is concluded that UDP-glucuronyl transferase activity in regenerating liver is as inducible as in normal rat liver by repeated PB treatment despite incomplete hepatic regeneration. Preoperative PB treatment alone is not sufficient to stimulate the glucuronylating pathway in the late phase of liver regeneration.

Alanine Transaminase↗

PubMed1990

Incidence and properties of aspartate aminotransferase-immunoglobulin complexes in patients with a high serum aspartate to alanine aminotransferase ratio.

Sera of 260 patients with high serum aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1; AST)/alanine aminotransferase (L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2; ALT) ratio (greater than 2.0) and high serum AST (greater than 45 IU/1) were selected and tested for the presence of immunoglobulin complexed-AST, by using immunoprecipitation reaction and counterimmunoelectrophoresis. The macromolecular AST was confirmed by size-exclusion high-performance liquid chromatography (HPLC). 34 patients out of 260 were found to have AST-immunoglobulin complexes (13.1%). The classes of AST-linked immunoglobulins were identified to be alpha in 28 cases (82.4%, P less than 0.01), mixed type of alpha and gamma in 5 cases (14.7%) gamma in one case (2.9%). Positive frequency was the highest in liver malignancies, either primary (9/26, 34.6%) or metastatic (7/17, 42.2%), followed by other malignancies (6/55, 10.9%) and chronic liver diseases (4/22, 18.2%). Thus, it can be strongly suggested that the immunoglobulin A complexed-AST is frequently found in association with liver malignancies.

Adolescent↗

PubMed2001

Influence of convulsants on rat brain activities of alanine aminotransferase and aspartate aminotransferase.

There exist differences between 12-day-old and adult rats in the onset of seizures induced by some inhibitors of glutamate decarboxylase (GAD). The aim of study was to investigate if there are differences between both groups in activities of rat brain alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the enzymes involved in glutamate metabolism, after the administration of 3-mercaptopropionic acid as specific GAD inhibitor or isoniazid as less specific general inhibitor of pyridoxal enzymes. Activities of both aminotransferases in a supernatant 20,000 g of the whole brain (containing predominantly cytosolic isoforms of enzymes) were increased at the beginning of 3-mercaptopropionic acid-induced generalized tonic-clonic seizures. At isoniazid-induced generalized tonic-clonic seizures, a significant increase in both enzyme activities was observed in adult rat brain. In the 12-day-old rat brain, ALT and AST activities reached about 40% and about 50-60% of adult control levels, respectively. In in vitro experiments, no influence of 3-mercaptopropionic acid on transaminase activities was found and an inhibitory effect of isoniazid on the enzymes was confirmed. Increased aminotransferase activities might participate in the enhanced synthesis of excitatory amino acid neurotransmitters in the nervous system, which may take a part in the initiation of epileptic seizures. Alternatively, the increased AST activity may be connected with an increased transport of NADH from the cytosol to mitochondria, while the increased ALT activity would represent the transformation of pyruvate to alanine as a consequence of increased glycolysis.

3-Mercaptopropionic Acid↗

PubMed2005

Stereochemistry of reactions of the inhibitor/substrates L- and D-beta-chloroalanine with beta-mercaptoethanol catalysed by L-aspartate aminotransferase and D-amino acid aminotransferase respectively.

Two members of the alpha-family of PLP-dependent enzymes, L-aspartate aminotransferase and D-amino acid aminotransferase, have been shown to catalyse beta-substitution of L- and D-beta-chloroalanine respectively with beta-mercaptoethanol, reactions typical of the beta-family of PLP-dependent enzymes. The reaction catalysed by L-aspartate aminotransferase has been shown to occur with retention of stereochemistry, a typical outcome for reactions catalysed by beta-family enzymes. There are also indications that the reaction catalysed by D-amino acid aminotransferase may involve retention of stereochemistry. Both enzymes have been shown to catalyse exchange at C-3 when the appropriate enantiomer of beta-chloroalanine is the substrate.

Aspartate Aminotransferases↗

PubMed1990

Evolutionary and biosynthetic aspects of aspartate aminotransferase isoenzymes and other aminotransferases.

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase are homologous proteins. Both are encoded by nuclear DNA and synthesized on free polysomes. The organization of their genes is very similar, five out of a total of eight introns are located at the same nucleotide position. A variant consensus sequence was observed at the 3' splice site of introns of genes of imported mitochondrial proteins which may reflect the existence of splicing factors specific for the genes of this particular group of nuclear-encoded proteins. To date the amino acid sequences of 22 aminotransferases are known. A rigorous analysis yielded clear evidence that aspartate, tyrosine, and histidinol-phosphate aminotransferases are homologous proteins despite their low degree of sequence identity. The evolutionary relationship among the vitamin B6-dependent enzymes in general appears less clear. Conceivably, their common structural and mechanistic features are dictated by the chemical properties of pyridoxal 5'-phosphate rather than being due to a common ancestor of their protein moieties. In agreement with this notion, the ubiquitous active-site lysine residue that forms a Schiff base with the coenzyme can be replaced in the case of aspartate aminotransferase by a histidine residue without complete loss of catalytic competence.

Animals↗

PubMed1976

Influence of auxiliary enzymes on the spectrophotometric measurement of alanine aminotransferase and aspartate aminotransferase activities.

We investigated the enzyme activity of the blank in the spectrophotometric determination of the aminotransferase activities and aspartate aminotransferase activity. 6 lactate dehydrogenase and 3 malate dehydrogenase preparations from different manufactures and from different organs showed additional and contaminating activity. The additional activity depends upon the 2-oxoglutarate concentration. The contaminating activity is caused by alanine aminotransferase and aspartate aminotransferase in the auxiliary enzymes. We propose that exact definitions must be given for the auxiliary enzymes in the recommendations of standard determinations for enzyme activities.

Alanine Transaminase↗

PubMed1982

Biosynthesis of aspartate aminotransferases. Both the higher molecular weight precursor of mitochondrial aspartate aminotransferase and the cytosolic isoenzyme are synthesized on free polysomes.

The site of synthesis of the higher molecular weight precursor of mitochondrial aspartate aminotransferase (Sonderegger, P., Jaussi, R., and Christen, P. (1980) Biochem. Biophys. Res. Commun. 94, 1256-1260) has been determined by separation of free and membrane-bound polysomes under ionic conditions imitating the intracellular milieu and in vitro read-out translation of the two polysome fractions in a rabbit reticulocyte lysate. The amounts of the precursor of mitochondrial aspartate aminotransferase synthesized by free and membrane-bound polysomes were compared with the relative extent of the synthesized of cytosolic aspartate aminotransferase in the same fractions. Only a small (less than 10% of total) and for both isoenzymes quantitatively equivalent fraction was found to be produced by the membrane-bound polysome fraction; very likely, it has to be attributed to contaminating free polysomes. Apparently, the import of mitochondrial aspartate aminotransferase into the mitochondria does not involve an association of polysomes with intracellular membranes.

Animals↗

PubMed1977

Alpha-Aminoadipate aminotransferase and kynurenine aminotransferase. Purification, characterization, and further evidence for identity.

Alpha-Aminoadipate aminotransferase and kynurenine aminotransferase activities were co-purified from the rat kidney supernatant fraction. The resulting preparation was determined to be nearly homogenous by analytical disc gel electrophoresis at pH 8.9 and 7.5 isoelectric focusing on polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of approximately 85,000 was determined on Sephadex G-200 chromatography and sucrose density gradient analysis. The enzyme was determined to be comprised of two subunits of approximately the same molecular weight (45,500 +/- 850) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An isoelectric pH of 6.56 +/- 0.06 was determined by focusing on polyacrylamide gels. Further evidence is provided to support the idea that the alpha-aminoadipate aminotransferase and kynureine aminotransferase activities are properties of a single protein: (a) co-purification of the two activities from the rat kidney supernatant fraction with the ratio of their specific activities remaining constant, (b) similar chromatographic behavior, (c) a similarity in their dependence on added pyridoxal-P for activity, and (d) a similar pattern of heat inactivation.

2-Aminoadipic Acid↗

PubMed1988

Effects of inhibition of ornithine aminotransferase or of general aminotransferases on urea and citrulline synthesis and on the levels of acetylglutamate in isolated rat hepatocytes.

Canaline and gabaculine, inhibitors of gamma-aminotransferases and thus of ornithine aminotransferase (E.C. 2.6.1.13), decreased the flow through ornithine carbamoyl transferase (E.C. 2.1.3.3) in isolated rat hepatocytes incubated with 10 mM NH4Cl and ornithine. The levels of acetylglutamate, an essential activator of carbamoyl phosphate synthetase (ammonia) (E.C. 6.3.4.16), were also decreased, suggesting that the inhibitors had also caused a decrease in the rate of carbamoyl phosphate synthesis. Under these conditions, ornithine appears to be a precursor of acetylglutamate, via ornithine aminotransferase, possibly as a consequence of glutamate synthesis. The influence of aminooxyacetate, an aminotransferase inhibitor, has also been examined.

Aminobutyrates↗

PubMed1976

Identity of isoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase.

After glucagon injection, rats showed virtually identical percentage increases in hepatic histidine-pyruvate aminotransferase and serine-pyruvate aminotransferase activities, both in the mitochondria and in the cytosol. Histidine-pyruvate aminotransferase isoenzyme 1, with pI8.0, was purified to homogeneity from the mitochondrial fraction of liver from glucagon-injected rats. The purified enzyme catalysed transamination between a number of amino acids and pyruvate or phenylpyruvate. For transamination with pyruvate, the activity with serine reached a constant ratio to that with histidine during purification, which was unchanged by a variety of treatments of the purified enzyme. Serine was found to act as a competitive inhibitor of histidine transamination, and histidine of serine transamination. These results suggest that histidine-pyruvate amino-transferase isoenzymes 1 is identical with serine-pyruvate aminotransferase. The enzyme is probably composed of two identical subunits with mol. wt. approx. 38000. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents strongly indicate the presence of pyridoxal phosphate.

Animals↗

PubMed1999

Alanine aminotransferase and glycine aminotransferase from maize (Zea mays L.) leaves.

Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids prompted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio.

Alanine Transaminase↗

PubMed1990

Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2.

Dimethylarginine:pyruvate aminotransferase, which plays a role in the metabolism of dimethylarginines, has been purified to homogeneity from rat kidney. The enzyme has a molecular weight of approximately 200,000 and an isoelectric point at about pH 6.3. The enzyme consists of four similar subunits having a molecular weight of about 50,000. The enzyme catalyzes the effective transaminations of guanidino-N methylated L-arginines (e.g. NG,NG-dimethyl-L-arginine, NG,N'G-dimethyl-L-arginine and NG-monomethyl-L-arginine) and the alpha-amino group of L-ornithine to pyruvate or glyoxylate. The enzyme was always accompanied by the known alanine:glyoxylate amino-transferase activity with the ratios of their specific activities remaining constant during the purification steps. The physicochemical and immunological properties of the purified enzyme were shown to be identical with those of the isozyme of alanine:glyoxylate aminotransferase (EC 2.6.1.44), designated as alanine:glyoxylate aminotransferase 2 (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The distribution profiles in tissues and the negative response to glucagon treatment further supported the identity of the two enzymes. The present data show that alanine:glyoxilate aminotransferase 2 functions in dimethylarginine metabolism in vivo in rats.

Alanine Transaminase↗

PubMed1989

The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases.

The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli. The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226. Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E. coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size. Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar. The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene.

Amino Acid Sequence↗

PubMed1995

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Aminotransferases↗

PubMed1978

The apoenzyme of aspartate aminotransferase and alanine aminotransferase in the serum of healthy persons and patients suffering from liver diseases.

In activity determination with addition of pyridoxal 5'-phosphate (P-5-P), aspartate aminotransferase (AST) activity increases by 6.5 U/l and that of alanine aminotransferase (ALT) by 2.5 U/l in the serum of healthy persons. This corresponds to a relative stimulation of initial activity by 37% and 15.2%, respectively. ApoAST activity in patients with chronic liver diseases is not changed as compared with that of healthy persons, the relative stimulation rate, however, is significantly smaller. ApoALT activity and corresponding relative stimulation is significantly greater as compared with healthy persons. In the case of acute viral hepatitis, a decrease of AST and ALT activity is followed by a decrease of apoenzyme activity in the course of disease. Diagnostic evidence of determinations of aminotransferase activities could not be improved by addition of P-5-P.

Alanine Transaminase↗

PubMed2003

How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase.

Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100-270-fold increases in k(cat)/K(m)(Phe) and had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001 or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection TAT) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT-TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15 A of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.

Amino Acid Sequence↗

PubMed1987

Aminotransferases in elderly people: influence of pyridoxal 5'-phosphate addition on the determination of serum aminotransferases in healthy elderly subjects.

Aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activity was measured in a group of 81 healthy subjects over 70 years of age and another of 180 younger persons, in order to investigate histograms and means of aminotransferase values in both groups and the variation in stimulation of these enzyme activities by pyridoxal 5'-phosphate (P5P) added in vitro. It was found that ASAT and ALAT mean values were significantly higher in both groups in the presence of P5P, than in the absence of P5P. ASAT and ALAT mean values obtained with and without P5P in old people were similar to those found in young subjects. In addition, mean stimulation percentages by P5P were identical in both groups.

Adolescent↗