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PubMed1988

Identification of mammalian aminotransferases utilizing glyoxylate or pyruvate as amino acceptor. Peroxisomal and mitochondrial asparagine aminotransferase.

The subcellular distribution of asparagine:oxo-acid aminotransferase (EC 2.6.1.14) in rat liver was examined by centrifugation in a sucrose density gradient. About 30% of the homogenate activity after the removal of the nuclear fraction was recovered in the peroxisomes, about 56% in the mitochondria, and the remainder in the soluble fraction from broken peroxisomes. The mitochondrial asparagine aminotransferase had identical immunological properties with the peroxisomal one. Glucagon injection to rats resulted in the increase of its activity in the mitochondria but not in the peroxisomes. Immunological evidence was obtained that the enzyme was identical with alanine:glyoxylate aminotransferase 1 (EC 2.6.1.44) which had been reported to be identical with serine:pyruvate aminotransferase (EC 2.6.1.51) (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The same results as described above were obtained with mouse liver. All of alanine:glyoxylate aminotransferase 1 in livers of mammals other than rodents, which cross-react with the antibody against rat liver alanine:glyoxylate aminotransferase 1, had no asparagine aminotransferase activity.

Animals↗
PubMed1978

Enzyme-activated irreversible inhibitors of L-ornithine:2-oxoacid aminotransferase. Demonstration of mechanistic features of the inhibition of ornithine aminotransferase by 4-aminohex-5-ynoic acid and gabaculine and correlation with in vivo activity.

L-Ornithine:2-oxoacid aminotransferase is a specific enzyme with respect to the amino group donor. Nevertheless it was found that this enzyme is inhibited by some 4-aminobutyrate analogs, 4-aminohex-5-ynoic acid and 5-amino-1,3-cyclohexadienyl-carboxylic acid (gabaculine), which are currently considered to be enzyme-activated irreversible inhibitors of 4-aminobutyrate:2-oxoglutarate aminotransferase. The inhibitory mechanisms for the two omega-aminotransferases are identical. A close structural analog of these inhibitors, 4-aminohex-5-enoic acid, is not inhibitory for ornithine aminotransferase, whereas it effectively inhibits 4-aminobutyrate aminotransferase. The reasons for this difference are discussed. The in vitro findings are entirely transferable to the in vivo situation: 4-aminohex-5-ynoic acid and gabaculine cause a long-lasting inhibition of ornithine aminotransferase in brain and liver, and reduce significantly in vivo ornithine degradation, whereas 4-aminohex-5-enoic acid is inactive both in vivo and in vitro toward this enzyme. The enzyme-activated irreversible inhibitors allow one for the first time to study the physiological consequences of irreversible ornithine aminotransferase inhibition.

Aminocaproates↗
PubMed1985

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase.

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.

Alanine↗
PubMed1993

Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for symbiotic nitrogen fixation is atypical.

In Rhizobium meliloti, an aspartate aminotransferase (AspAT) encoded within a 7.3-kb HindIII fragment was previously shown to be required for symbiotic nitrogen fixation and aspartate catabolism (V. K. Rastogi and R.J. Watson, J. Bacteriol. 173:2879-2887, 1991). A gene coding for an aromatic aminotransferase located within an 11-kb HindIII fragment was found to complement the AspAT deficiency when overexpressed. The genes encoding these two aminotransferases, designated aatA and tatA, respectively, have been localized by subcloning and transposon Tn5 mutagenesis. Sequencing of the tatA gene revealed that it encodes a protein homologous to an Escherichia coli aromatic aminotransferase and most of the known AspAT enzymes. However, sequencing of the aatA gene region revealed two overlapping open reading frames, neither of which encoded an enzyme with homology to the typical AspATs. Polymerase chain reaction was used to selectively generate one of the candidate sequences for subcloning. The cloned fragment complemented the original nitrogen fixation and aspartate catabolism defects and was shown to encode an AspAT with the expected properties. Sequence analysis showed that the aatA protein has homology to AspATs from two thermophilic bacteria and the eukaryotic tyrosine aminotransferases. These aminotransferases form a distinct class in which only 13 amino acids are conserved in comparison with the well-known AspAT family. DNA homologous to the aatA gene was found to be present in Agrobacterium tumefaciens and other rhizobia but not in Klebsiella pneumoniae or E. coli.

Amino Acid Sequence↗
PubMed2005

Lysine is catabolized to 2-aminoadipic acid in Penicillium chrysogenum by an omega-aminotransferase and to saccharopine by a lysine 2-ketoglutarate reductase. Characterization of the omega-aminotransferase.

The biosynthesis and catabolism of lysine in Penicillium chrysogenum is of great interest because these pathways provide 2-aminoadipic acid, a precursor of the tripeptide delta-L-2-aminoadipyl-L-cysteinyl-D-valine that is an intermediate in penicillin biosynthesis. In vivo conversion of labelled L-lysine into two different intermediates was demonstrated by HPLC analysis of the intracellular amino acid pool. L-lysine is catabolized to 2-aminoadipic acid by an omega-aminotransferase and to saccharopine by a lysine-2-ketoglutarate reductase. In lysine-containing medium both activities were expressed at high levels, but the omega-aminotransferase activity, in particular, decreased sharply when ammonium was used as the nitrogen source. The omega-aminotransferase was partially purified, and found to accept L-lysine, L-ornithine and, to a lesser extent, N-acetyl-L-lysine as amino-group donors. 2-Ketoglutarate, 2-ketoadipate and, to a lesser extent, pyruvate served as amino group acceptors. This pattern suggests that this enzyme, previously designated as a lysine-6-aminotransferase, is actually an omega-aminotransferase. When 2-ketoadipate is used as substrate, the reaction product is 2-aminoadipic acid, which contributes to the pool of this intermediate available for penicillin biosynthesis. The N-terminal end of the purified 45-kDa omega-aminotransferase was sequenced and was found to be similar to the corresponding segment of the OAT1 protein of Emericella (Aspergillus) nidulans. This information was used to clone the gene encoding this enzyme.

2-Aminoadipic Acid↗
PubMed1989

Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol.

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.

Alanine Transaminase↗
PubMed1987

Identity of beta-alanine-oxo-glutarate aminotransferase and L-beta-aminoisobutyrate aminotransferase in rat liver.

L-beta-Aminoisobutyrate served as an amino donor for purified beta-alanine-oxo-glutarate aminotransferase from rat liver when 2-oxoglutarate was employed as an amino acceptor, but the D-isomer did not. L-beta-Aminoisobutyrate acted as a competitive inhibitor with respect to beta-alanine and had a Ki of approximately 2.6 mM, which is the same value as the Km of 2.7 mM. When the crude extract was applied to a DEAE-Sepharose CL-6B column, L-beta-aminoisobutyrate aminotransferase and beta-alanine-oxo-glutarate aminotransferase activities were found in the same fractions with a single peak. Antiserum to rat liver beta-alanine-oxo-glutarate aminotransferase inhibited L-beta-aminoisobutyrate aminotransferase activity in rat liver in the same way as beta-alanine-oxo-glutarate aminotransferase activity.

Aminoisobutyric Acids↗
PubMed1981

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.

Alanine Transaminase↗
PubMed1976

A purine auxotroph deficient in phosphoribosylpyrophosphate amidotransferase and phosphoribosylpyrophosphate aminotransferase activities with normal activity of ribose-5-phosphate aminotransferase.

Three enzyme reactions have been reported to catalyze the synthesis of phosphoribosylamine in eukaryotic cells. These activities are glutamine phosphoribosylpyrophosphate (P-Rib-P-P) amidotransferase [amidophosphoribosyl-transferase; 5-phosphoribosylamine: pyrophosphate phosphoribosyltransferase (glutamate-amidating) EC 2.4.2.14], ammonia P-Rib-P-P aminotransferase, and ammonia ribose-5-phosphate aminotransferase. A purine auxotroph derived from a cell line of Chinese hamster fibroblasts was shown to be deficient in catalytic activities of glutamine P-Rib-P-P amidotransferase and ammonia P-Rib-P-P aminotransferase. Extracts from this cell line had normal ammonia ribose-5-phosphate aminotransferase activity. The defect in purine biosynthesis in the mutant cell line was localized to the synthesis of phosphoribosylamine. These results indicate that glutamine P-Rib-P-P amidotransferase or ammonia P-Rib-P-P aminotransferase or both are important for phosphoribosylamine synthesis, but that ammonia ribose-5-phosphate aminotransferase activity probably does not play a significant role in this eukaryotic cell line. The simultaneous disappearance of both P-Rib-P-P-dependent activities suggests these two enzyme activities are closely related structurally or genetically.

Amidophosphoribosyltransferase↗
PubMed1995

Enzymatic synthesis of aminocyclitol moieties of aminoglycoside antibiotics from inositol by Streptomyces spp.: detection of glutamine-aminocyclitol aminotransferase and diaminocyclitol aminotransferase activities in a spectinomycin producer.

Extracts of stationary-phase mycelia of the spectinomycin producer Streptomyces flavopersicus ATCC 19756 catalyzed inositol dehydrogenase, L-glutamine:inosose aminotransferase, 2-epi-streptamine:inosose aminotransferase, streptamine:inosose aminotransferase, N3-methyl-2-deoxystreptamine:inosose aminotransferase, and aminodeoxy-scyllo-inositol:inosose aminotransferase reactions, as detected with a new rapid assay procedure. These results suggest that one or both amino groups of the N1,N3-dimethyl-2-epi-streptamine moiety of spectinomycin are derived by transamination from the alpha-amino group of L-glutamine. An enzymatic procedure for distinguishing among N1- and N3-monomethyl diaminocyclitol derivatives and their diaminocyclitol biosynthetic precursors is described. A scheme showing key roles of glutamine-aminocyclitol aminotransferases in biosynthesis of major aminoglycoside antibiotics is presented.

Glutamine↗
PubMed1977

Glutamate-glyoxylate aminotransferase in rat liver cytosol. Purification, properties and identity with alanine-2-oxoglutarate aminotransferase.

After cortisone injection, virtually identical increases in rat liver cytosol alanine-2-oxoglutarate aminotransferase and glutamate-glyoxylate aminotransferase activities were observed. The two activities were co-purified to homogeneity from rat liver cytosol. The purified enzyme was specific for L-alanine with 2-oxoglutarate as amino acceptor. With glyoxylate, however, the enzyme utilized various L-amino acids as amino donors in the following order of activity: glutamate greater than alanine greater than glutamine greater than methionine. The ratio of alanine-2-oxoglutarate aminotransferase activity to glutamate-glyoxylate aminotransferase activity remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. These results suggest that glutamate-glyoxylate aminotransferase is identical with alanine-2-oxoglutarate aminotransferase. Evidence was obtained that the two enzyme activities in the cytosol of dog, cat and human liver are also properties of the same protein.

Alanine Transaminase↗
PubMed1984

Measurement of aminotransferases: Part 1. Aspartate aminotransferase.

Aminotransferases are ubiquitous enzymes of mammalian cells and several are of important diagnostic use. The application of aspartate aminotransferase activity measurements in serum from individuals suffering from myocardial infarction brought about a new dimension in clinical laboratory testing in the 1950s. This review focuses on measurement techniques for aspartate aminotransferase and their application (a subsequent article will review other aminotransferases). Assay techniques measuring enzyme activity are direct spectrophotometric measurements, manometric techniques, assays using dye substances, coupled enzyme techniques, and radiometric procedures. Of these procedures, the one employing malate dehydrogenase and NADH is the most important and is covered in particular detail. The estimation of the mitochondrial isoenzyme of aspartate aminotransferase is also of clinical interest, in particular for estimating severity of disease or in specific applications (e.g., chronic alcoholism). Methods reviewed for estimation of this enzyme are electrophoresis, chromatography, differential kinetic behavior, and immunochemical separation. Determination of the enzyme protein by techniques independent of its catalytic activity are also reviewed.

Animals↗
PubMed1978

Optimization of methods for aspartate aminotransferase and alanine aminotransferase.

Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.

Alanine Transaminase↗
PubMed1975

[Histochemical evidence of aminotransferases. IV. Histochemical and electrophoretical investigation of aminotransferases in rat organs (author's transl)].

The histochemical localization and isoenzyme profil of some aminotransferases in different organs of the rat has been studied. A positive histochemical reaction with unequal intensity, depending on the aminotransferase investigated, was been observed. The topochemical and cytochemical localization of the aminotransferases was typical for everyone organ. The studied aminotransferases possessed isoenzyme fractions with characteristic number, electrophoretic mobility and intensity in the different organs. The results were discussed in relation with substrate specifity of the aminotransferases.

Alanine Transaminase↗
PubMed1983

Stability of aspartate aminotransferase and alanine aminotransferase activities.

Because there are conflicting data regarding the effect of different temperatures and durations of storage on the stability of the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), a new study has been conducted to re-examine this important issue. Blood obtained from patients with varying aminotransferase levels was centrifuged, the resultant serum was divided into aliquots, the samples were stored at room, refrigerator, and freezer temperatures, and the aminotransferases measured on days 0, 1, 2, 3, 4, and 30. In all but one circumstance, both AST and ALT activities declined markedly beginning within 24 hr of venipuncture; the temperature of storage did not significantly affect the rate or degree of loss of enzyme activity. The exception was the evidence that ALT activity in samples obtained from individuals with initially normal values showed a rise during the first 3 to 4 days, followed thereafter by a decline to below baseline values. Thus, to ensure accuracy of aminotransferase measurement, testing of samples should be conducted on the day of venipuncture.

Alanine Transaminase↗
PubMed1977

Identity of rat kidney histidine-pyruvate aminotransferase with glutamine-oxo acid aminotransferase.

Histidine-pyruvate aminotransferase and glutamine-phenylpyruvate aminotransferase were purified from rat kidney by the same procedure. The ratio of the two activities remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. Glutamine was found to act as a competitive inhibitor of histidine-pyruvate aminotransferase. These results suggest that rat kidney histidine-pyruvate aminotransferase is identical with glutamine-oxo acid aminotransferase. Identity of the two enzymes in other tissues of rats is discussed.

Animals↗
PubMed1976

Identity of kynurenine: pyruvate aminotransferase with histidine: pyruvate aminotransferase.

Kynurenine pyruvate aminotransferase was purified from rat kidney. The purified enzyme had an isoelectric point of pH 5.2 and a pH optimum of 9.3. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors. L-Amino acids were effective in the following order of activity: histidine greather than phenylalanine greater than kynurenine greater than tyrosine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values were about 0.63 mM, 1.4 mM and 0.09 mM for histidine, kynurenine and phenylalanine, respectively. Km values for pyruvate were 5.5 mM with histidine as amino donor, 1.3 mM with kynurenine and 8.5 mM with phenylalanine. Kynurenine pyruvate aminotransferase activity of the enzyme was inhibited by the addition of histidine or phenylalanine. The molecular weights determined by gel filtration and sucrose density gradient centrifugation were approximately 76000 and 79000, respectively. On the basis of purification ratio, substrate specificity, inhibition by common substrates, subcellular distribution, isoelectric focusing and polyacrylamide-gel electrophoresis, it is suggested that kynurenine pyruvate aminotransferase is identical with histidine pyruvate aminotransferase and also with phenylalanine pyruvate aminotransferase. The physiological significance of the enzyme is discussed.

Animals↗