Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes.

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.     

Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes.

The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.     

Purification and characterization of rat liver mitochondrial phenylalanine pyruvate aminotransferase.

Phenylalanine pyruvate aminotransferase in rat liver was found in both the mitochondrial and supernatant fractions. Phenylalanine pyruvate aminotransferase was purified from rat liver mitochondria. The purified enzyme was specific for pyruvate, exhibiting no activity with 2-oxoglutarate as aminoacceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order of activity: L-phenylalanine > L-tyrosine > L-histidine > 3,4-dihydroxy-DL-phenylalanine. Very little activity was observed with L-tryptophan and 5-hydroxy-L-tryptophan. The apparent Km values for L-phenylalanine and L-histidine were 2.6 mM and 2.7 mM, respectively. The Km values for pyruvate were 5.0 mM and 1.5 mM with phenylalanine and histidine as amino donors, respectively. The pH optimum was near 9.0. Sucrose density gradient centrifugation gave a molecular weight of approximately 68,000. On the basis of subcellular distributions, substrate specificities, substrate inhibition, pH optima, polyacrylamide gel electrophoresis and some other properties, it was suggested that mitochondrial phenylalanine pyruvate aminotransferase was identical with mitochondrial histidine pyruvate aminotransferase.     

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.     

Studies on the kynurenine adminotransferase activity in rat liver and kidney.

The kynurenine aminotransferase activity of supernatant and mitochondrial fractions obtained from rat liver and kidney was studied with L-kynurenine and L-3-hydroxykynurenine as substrates. A substrate inhibition with L-kynurenine at concentrations higher than 6-7mM was observed with all four enzyme preparations. This did not happen with L-3-hydroxykynurenine as a substrate. Moreover, the liver mitochondrial enzyme shows a Km for pyridoxal phosphate 2-4 times smaller than the other preparations when assayed with L-3-hydroxykynurenine as a substrate. Therefore, the accumulation of xanthurenic acid and not of kynurenic acid in B6 deficiency could be related both to this high activity of liver mitochondrial kynurenine aminotransferase with L-3-hydroxykynurenine, even at small concentrations of B6, and to substrate inhibition observed with L-kynurenine and not with L-3-hydroxykynurenine.     

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.     

Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.     

Purification,characterization and identification of tryptophan aminotransferase from rat brain

Tryptophan aminotransferase was purified from rat brain extracts. The purified enzyme had an isoelectric point at pH 6.2 and a pH optimum near 8.0. On electrophoresis the enzyme migrated to the anode. The enzyme was active with oxaloacetate or 2-oxoglutarate as amino acceptor but not with pyruvate, and utilized various L-amino acids as amino donors. With 2-oxoglutarate, the order of effectiveness of the L-amino acids was aspartate > 5-hydroxytryptophan > tryptophan > tyrosine > phenylalanine. Aminotransferase activity of the enzyme towards tryptophan was inhibited by L-glutamate. Sucrose density gradient centrifugation gave a molecular weight of approx. 55,000. The enzyme was present in both the cytosol and synaptosomal cytosol, but not in the mitochondria. The isoelectric focusing profile of tryptophan: oxaloacetate aminotransferase activity was identical with that of L-aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1) activity, with both subcellular fractions. On the basis of these data, it is suggested that the enzyme is identical with the cytosol aspartate: 2-oxoglutarate aminotransferase.     

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.     

CEREBRAL AROMATIC AMINOTRANSFERASE

—Aromatic: 2-oxoglutarate aminotransferase has been purified about 950-fold from rat brain mitochondria. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis and had a molecular weight of approx 63,000. On the basis of substrate specificity, substrate inhibition, purification ratio, yield, polyacrylamide gel electrophoresis and some other properties of the enzyme it has been suggested that brain mitochondrial tyrosine:2-oxoglutarate aminotransferase (l -tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) is identical with brain mitochondrial phenylalanine and kynurenine: 2-oxoglutarate aminotransferases (l -kynurenine: 2-oxoglutarate aminotransferase, EC 2.6.1.7), and also with aspartate: 2-oxoglutarate aminotransferase (l -aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).     

Kynurenine pyruvate transaminase and its inhibitor in rat intestine

Kynurenine pyruvate transaminase was found to be present in rat small intestine, partially purified and characterized. The enzyme catalysed the conversion of L-kynurenine to kynurenic acid. Transamination rates of 3-hydroxy-DL-kynurenine and 5-hydroxy-DL-kynurenine by the enzyme were 1/2.9 and 1/2.6 that of L-kynurenine. The enzyme showed higher preference for pyruvate than 2-oxoglutarate as aminoacceptor. The pH optimum of the reaction was 8.0 to 8.5. Purification of the enzyme lowered markedly apparent Km for L-kynurenine but not for pyruvate. It was shown that the inhibitor of kynurenine pyruvate transaminase was present in the intestine, on the basis of the inhibition produced by heating a portion of each purification step enzyme preparation in 50% ethanol, centrifuging, concentrating it, and adding it to an incubate of the unheated preparation. The possible interrelationship of enzyme and inhibitor was discussed and comparisons with kynurenine transaminase in liver, kidney and brains were noted.     

Purification and characterization of yeast L-kynurenine aminotransferase with broad substrate specificity

L-Kynurenine aminotransferase [L-kynurenine:2-oxoglutarate aminotransferase (cyclizing), EC 2.6.1.7] has been purified to homogeneity and crystallized from cell-free extracts of a yeast, Hansenula schneggii, grown in a medium containing L-tryptophan as an inducer. The enzyme has a molecular weight of about 100,000 and consists of two subunits identical in molecular weight (52,000). The enzyme exhibits absorption maxima at 280, 335, and 430 nm, and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. The enzyme-bound pyridoxal 5'-phosphate shows negative circular dichroic extrema, in contrast with other pyridoxal 5'-phosphate acting on L-amino acids. In addition to L-kynurenine and alpha-ketoglutarate, which are the most preferred substrates, a large number of L-amino acids and alpha-keto acids can serve as substrates; the extremely broad substrate specificity is the most characteristic feature of this yeast enzyme. The enzyme activity is significantly affected by both carbonyl and sulfhydryl reagents. Certain dicarboxylic acids such as adipate and pimelate act as competitive inhibitors. Addition of various substrate amino acids to the culture medium results in the inductive formation of aminotransferases which are immunochemically indistinguishable from L-kynurenine aminotransferase.     

Subcellular distribution and properties of kynurenine pyruvate transaminase in rat kidney.

Kynurenine transaminase activity in rat kidney was found in both the mitochondrial and supernatant fractions. These fractions contained (a) kynurenine pyruvate transaminase, which showed a preference for pyruvate as amino acceptor, and had a pH optimum between 8.0 and 8.5, and (b) kynurenine 2-oxoglutarate transaminase, with a preference for 2-oxoglutarate and a pH optimum between 6.0 and 6.5. The apparent Km value of the former enzyme for L-kynurenine was much lower than that of the latter enzyme.     

Characteristics of hepatic alanine-glyoxylate aminotransferase in different mammalian species.

Mitochondrial extracts of dog, cat, rat and mouse liver contain two forms of alanine-glyoxylate aminotransferase (EC 2.6.1.44): one, designated isoenzyme 1, has mol.wt. approx. 80 000 and predominates in dog and cat liver; the other, designated isoenzyme 2, has mol.wt. approx. 175 000 and predominates in rat and mouse liver. In rat and mouse liver, isoenzyme 1 activity was increased by the injection in vivo of glucagon, but not isoenzyme 2 activity. Isoenzyme 1 was purified and characterized from liver mitochondrial extracts of the four species. Both rat and mouse enzyme preparations catalysed transamination between a number of L-amino acids and glyoxylate, and with L-alanine as amino donor the effective amino acceptors were glyoxylate, phenylpyruvate and hydroxypyruvate. In contrast, both dog and cat enzyme preparations were specific for L-alanine and L-serine with glyoxylate, and used glyoxylate and hydroxypyruvate as effective amino acceptors with L-alanine. Evidence that isoenzyme 1 is identical with serine-pyruvate aminotransferase (EC 2.6.1.51) was obtained. Isoenzyme 2 was partially purified from mitochondrial extracts of rat and mouse liver. Both enzyme preparations were specific for L-alanine and glyoxylate. On the basis of physical properties and substrate specificity, it was concluded that isoenzyme 2 is a separate enzyme. Some other properties of isoenzymes 1 and 2 are described.     

Aspartate aminotransferase of Pediococcus cerevisiae.

A five-step procedure is described for preparing highly purified aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC.2.6.1.1) from cell-freee enzyme extracts of Pediococcus cerevisiae. An overall purification of 130-fold was achieved. Some of P. cerevisiae aspartate aminotransferase properties were studied, i.s. pH optimum (7.8--8.0), optimum of temperature (37 degrees), Michaelis constans for 4 enzyme substrates and substrate specificity of enzyme. The enzyme is very thermolabile. During purification the enzyme was stabilizated by 2-oxoglutarate. The highly purified preparation was stored in the solution containing ammonium sulphate. The obtained aspartate aminotransferase preparation was free of alanine and aromatic amino acids aminotransferase activites and did not reveal malate dehydrogenase activity.     

Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.     

Co-purification of alanine-glyoxylate aminotransferase with 2-aminobutyrate aminotransferase in rat kidney

Alanine-glyoxylate aminotransferase and 2-aminobutyrate aminotransferase were co-purified from rat kidney to a single protein (about 500-fold purified from the homogenate). The activity ratios of alanine-glyoxylate aminotransferase to 2-aminobutyrate aminotransferase were constant during co-purification steps suggesting the 2-aminobutyrate aminotransferase activity was catalysed by only alanine-glyoxylate aminotransferase. The molecular weight of the enzyme was estimated to be approx. 213 000, 220 000 and 236 000 by analytical ultracentrifugation, Sephadex G-150 gel filtration and sucrose density gradient centrifugation, respectively. From the polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the enzyme consisted of four apparently similar subunits having a molecular weight of approx. 56 000. The enzyme was almost specific to L-alanine and L-2-aminobutyrate as amino donor and to glyoxylate, pyruvate and 2-oxobutyrate as amino acceptor. The enzyme was identified with rat liver alanine-glyoxylate aminotransferase isoenzyme 2 but not with rat liver alanine-glyoxylate aminotransferase isoenzyme 1 from Ouchterlony double diffusion analysis. Absorption spectra and some kinetic properties of the enzyme were clarified.     

GABA-T in bovine medulla cells: kinetic properties and comparison with GABA-T from other tissues

4-aminobutyrate-2-oxoglutarate aminotransferase (GABA-T) has been found in adrenal medulla. The enzyme from this tissue is very similar to those found in other tissues in respect to their mitochondrial localization, optima pH and responses to cofactor. The enzyme from medulla has substrate km values similar to those for the brain enzyme, while it differs from those found for other tissues such as kidney, liver and platelets.     

A kinetic method for quantification of aspartate aminotransferase isoenzymes

A spectrophotometric assay is proposed to determine the levels of aspartate aminotransferase (AAT) isoenzymes from chicken liver by a steady-state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of substrate 2-oxoglutarate at pH 6.2. The use of a standard curve permits the determination of the percentage of chicken liver c-AAT and m-AAT isoenzymes. This method yields results in good correlation with those achieved by different extent adipate inhibition and by differential centrifugation.     

Purification and characterization of aromatic-amino-acid-glyoxylate aminotransferase from monkey and rat liver.

Aromatic-amino-acid-glyoxylate aminotransferase was highly purified from the mitochondrial fraction of livers from monkey and glucagon-injected rats. The two enzyme preparations showed physical and enzymic properties different from a kynurenine aminotransferase previously described. The two enzymes had nearly identical molecular weights (approximate 80 000), isoelectric points (pH 8.0) and pH optima (pH 8.0 - 8.5). However, a difference in substrate specificity was observed between the two enzymes. Both enzymes utilized glyoxylate, pyruvate, hydroxypyruvate and 2-oxo-4-methyl-thiobutyrate as effective amino acceptors. 2-Oxoglutarate was active for rat enzyme but not for monkey enzyme. With glyoxylate, amino donors were effective in the following order of activity; phenylalanine greater than histidine greater than tyrosine greater than tryptophan greater than 5-hydroxytrypotphan greater than kynurenine for the rat enzyme, and phenylalanine greater than kynurenine greater than histidine greater than tryptophan greater than 5-hydroxy-tryptophan for the monkey enzyme.