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Biomedical subjects

E Okuno

Publications and source records attributed to E Okuno.

At least 73 records · Page 4Linked to original sources
PubMed1983

A new biosynthetic route of porphyrin precursors in common between animals and plants.

An additional enzyme, 4-oxo-5-hydroxyvalerate (OHV) dehydrogenase was identified and characterized. This enzyme catalyzes the conversion of OHV to 4,5-dioxovalerate, a direct precursor of 5-aminolevulinate. The enzyme was partially purified from rat liver supernatant as two isoenzyme (ca. 40,000 and 70,000 dalton). 5-Aminolevulinate was formed from OHV via 4,5-dioxovalerate by this dehydrogenase and alanine-4,5-dioxovalerate aminotransferase (EC 2.6.1.43). This dehydrogenase required NADP or NAD as a hydrogen acceptor. The enzyme was heat sensitive and catalyzed the reaction reversibly. The dehydrogenase was present in the high speed supernatants of liver and kidney of rat, rabbit and human, and that of spinach leaf.

Alcohol Oxidoreductases↗
PubMed1983

Purification and characterization of amino-acid N-choloyltransferase from human liver.

Amino-acid N-choloyltransferase was purified from human liver. The procedure resulted in about 100-fold enriched activity and glycine- and taurine-dependent activities co-purified and did not separate to any extent in any of the steps. The final enzyme preparation had an apparent molecular mass of 100 kDa by gel filtration, 118 kDa by sucrose density gradient centrifugation and was composed of two identical subunits which had a molecular mass of 52 kDa, as judged by dodecyl sulfate polyacrylamide disc gel electrophoresis. The glycine- and taurine-dependent activities showed optima at pH 7.2 and pH 6.5, respectively. Apparent Km values of purified enzyme were 3.2 and 0.6mM for glycine and taurine, respectively. The Km value for choloyl-CoA was 50 microM for the glycine-dependent activity and 87 microM for the taurine-dependent activity. Bile acid derivatives and cholesterol had an inhibitory effect on both glycine- and taurine-dependent activity in vitro; on the other hand, the reaction was stimulated by the addition of glutathione, EDTA and L-cysteine. Amino acid substrate specificity was restricted to glycine, taurine, beta-alanine and D-alpha-alanine. As well as choloyl-CoA, its deoxy derivatives were also good substrates for the enzyme.

Acyltransferases↗
PubMed1983

Effect of chronic administration of riboflavin 2',3',4',5'-tetrabutyrate on the hepatic enzymes of fatty acid oxidation in the rat.

Effects of long-term administration of riboflavin, sodium butyrate or riboflavin 2',3',4',5'- tetrabutyrate ( RTB ) on the activities of renal and hepatic enzymes that catalyze the beta-oxidation of fatty acid were determined in the rat. Feeding of riboflavin or sodium butyrate for 5 weeks had no effect on all the enzymes examined. By contrast, feeding of RTB resulted in an increase in the hepatic activity of 3-ketoacyl-CoA thiolase [EC 2.3.1.16] by 50% of the control level, while the activities of renal 3-ketoacyl-CoA thiolase and of hepatic and renal acyl-CoA synthetase [EC 6.2.1.3] and acyl-CoA dehydrogenase [EC 1.3.99.3] remained unaffected. The increase in hepatic 3-ketoacyl-CoA thiolase activity suggests that prolonged RTB administration results in an increased beta-oxidation of fatty acid in the liver, which may explain the reported reduction in the concentration of tryglyceride in plasma during RTB treatment.

Acetyl-CoA C-Acyltransferase↗
PubMed1982

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.

4-Aminobutyrate Transaminase↗
PubMed1980

Crystallization and characterization of human liver kynurenine--glyoxylate aminotransferase. Identity with alanine--glyoxylate aminotransferase and serine--pyruvate aminotransferase.

Kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s(20,w) value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine-glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine-glyoxylate aminotransferase, alanine-glyoxylate aminotransferase, serine-pyruvate aminotransferase and alanine-hydroxypyruvate aminotransferase reactions of the enzyme are presented.

Aged↗
PubMed1978

Subcellular distribution of pyruvate (glyoxylate) aminotransferases in rat liver.

The distribution of pyruvate (glyoxylate) aminotransferases in the particulate fraction of rat liver homogenates was examined by centrifugation in a sucrose density graident. Aminotransferase activities towards serine, phenylalanine and histidine with pyruvate and those towards phenylalanine and histidine with glyoxylate were nearly identically distributed. Some 50-55% of the particulate activity was localized in the peroxisomes and the remainder in the mitochondria. Most of alanine-glyoxylate aminotransferase activity was localized in the mitochondria, with some activity in the peroxisomes. Glucagon injection resulted in increases of these enzyme activities in the mitochondria, but not in the peroxisomes.

Animals↗
PubMed1978

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.

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 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↗
PubMed1976

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.

Animals↗
PubMed1976

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.

Animals↗
PubMed1975

Purification and characterization of kynurenine--2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats.

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.

Animals↗