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T Osumi

Publications and source records attributed to T Osumi.

At least 127 records · Page 7Linked to original sources
PubMed1984

Molecular cloning of cDNA for rat acyl-CoA oxidase.

Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes. This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA. Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776. By differential colony hybridization using [32P]cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA. On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats. The cleavage maps of the cDNA inserts showed they overlap with each other. We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.

Acyl-CoA Oxidase↗
PubMed1984

Radioimmunoassay of diethylstilbestrol in plasma of patients with prostatic carcinoma.

A method for the radioimmunoassay of diethylstilbestrol (DES) was established and it was used to measure the plasma concentration of DES in patients with prostatic carcinoma receiving intravenous drip infusion of 500 mg of diethylstilbestrol diphosphate (DES-DP). The sensitivity of this assay system was 40 pg per tube, with a recovery of 98%. The cross reactivity of the anti-DES antibody with estradiol, testosterone, and ethynylestradiol was less than 0.01% of DES, and that with DES-DP was 0.7%. In patients with prostatic carcinoma, plasma DES concentration at 6 and 24 hr after was declined to 10.2% and 4.4% of the level at the termination of intravenous administration of DES-DP, respectively; however, the concentration in patients with renal dysfunction was higher than that in ones with normal renal function.

Diethylstilbestrol↗
PubMed1984

Physico-chemical properties and stabilities of a new positive inotropic agent, 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl] -2(1H)-quinolinone (OPC-8212).

3,4-Dihydro-6-[4-(3,4- dimethoxybenzoyl )-1-piperazinyl]-2(1H)- qu inolinone ( OPC -8212), a new positive inotropic agent, was examined to clarify its physico-chemical properties, i.e. elemental analysis, melting point, spectra (UV, IR, NMR, MS), X-ray diffraction pattern, thermal analysis, solubilities, pKa, partition coefficient and chromatography (HPLC, TLC). Some degradation products of OPC -8212 were identified in acidic and basis solutions. OPC -8212 was determined by nonaqueous titration.

Cardiotonic Agents↗
PubMed1983

Purification and properties of carnitine acetyltransferase from rat liver.

Carnitine acetyltransferase was purified from rat liver after induction of the enzyme by feeding with di(2-ethylhexyl)phthalate. Two enzyme sources were used: the mitochondrial fraction and the homogenate of the liver. The purification procedure was essentially the same for the two enzyme sources. The enzyme purified from the mitochondrial fraction consisted of two different polypeptides with molecular weights of 36,500 and 27,000, whereas that from the homogenate consisted of one polypeptide with a molecular weight of 67,500. Amino acid compositions and peptide maps of the limited proteolytic products of the two enzyme preparations were nearly the same. Their antibodies were cross-reactive. Catalytic properties of the two preparations were nearly the same: the specific enzyme activities, double reciprocal plots of initial velocity study, substrate specificities for acylcarnitines having various carbon chain lengths, apparent Michaelis constants for substrates. On electrophoresis of the immunoprecipitate obtained after incubation of the mitochondrial extract, the two immunoreactive polypeptides with molecular weights of 36,500 and 27,000 were found. But only one polypeptide, with molecular weight of 67,500, was detected when the protease inhibitors were added to the mitochondrial extract. It was concluded that the enzyme in the mitochondrial fraction was a monomeric form but was converted into a dimeric form by proteolytic modification after the disruption of mitochondria. The preparation from the post-mitochondrial fraction, which had a lower specific activity, contained two polypeptides whose molecular weights were 69,000 and 67,500. They could not be separated from each other throughout the purification. The peptide maps of the products of the limited proteolysis were very similar.

Acetyltransferases↗
PubMed1983

Biosynthesis and turnover of carnitine acetyltransferase in rat liver.

Male Wistar rats were fed on a diet with and without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine acetyltransferase in the liver was increased about 100-fold by administration of DEHP. The results of in vivo experiments showed that the incorporation of L-[4,5-3H]leucine into the enzyme was 12-fold higher and the half-life of the labeled enzyme was elongated by a factor 4.6. The results of in vitro translation experiments with total hepatic RNA in a rabbit reticulocytelysate system and the results concerning the synthesis of the enzyme in isolated hepatocytes indicate that the translatable mRNA for the enzyme was increased upon administration of DEHP and that the enzyme is synthesized as a precursor (Mw = 69,000) larger than the mature enzyme (Mw = 67,500). RNA in the free polysomes directed the synthesis of the enzyme precursor five times more actively than RNA in membrane-bound polysomes.

Acetyltransferases↗
PubMed1983

Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver.

The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.

Acyltransferases↗
PubMed1983

Biosynthesis of carnitine octanoyltransferase and carnitine palmitoyltransferase.

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine octanoyltransferase (COT) in the liver was increased 23.5-fold in rats given DEHP. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rate of synthesis of COT was 14.1-fold higher and that of its degradation was 1.5-fold lower in the DEHP group. COT was translated much more effectively in free polysomes than in membrane-bound polysomes. The molecular size of the in vitro product was the same as that of the mature enzyme. The translation activity of mRNA coding for COT measured with total hepatic RNA was 16.6-fold higher in the DEHP group. Carnitine palmitoyltransferase (CPT) was increased 5.9-fold after administration of DEHP. The rate of synthesis of CPT measured in the in vivo experiment was 5.0-fold higher in the DEHP group. The rate of its degradation was the same in the two groups. CPT was also translated much more effectively in free polysomes. The size of the preenzyme was larger than that of the subunit of the mature enzyme by about 2,400 daltons. In contrast to COT, the increase in the translation activity of mRNA for CPT by administration of DEHP was markedly higher than the increase in the rate of its synthesis measured in the in vivo experiment.

Acyltransferases↗
PubMed1983

Cooperative binding of the sugar substrates and allosteric regulatory protein (enzyme IIIGlc of the phosphotransferase system) to the lactose and melibiose permeases in Escherichia coli and Salmonella typhimurium.

An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-beta-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-alpha-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-beta-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-alpha-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme III(Glc) to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme III(Glc)) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme III(Glc) to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme III(Glc), the postulated regulatory protein of the PTS.

Adenylyl Cyclase Inhibitors↗
PubMed1982

Regulation of lactose permease activity by the phosphoenolpyruvate:sugar phosphotransferase system: evidence for direct binding of the glucose-specific enzyme III to the lactose permease.

Interaction between the glucose-specific enzyme III (enzyme IIIglc) of the phosphoenolpyruvate:sugar phosphotransferase system and the lactose permease was studied with membrane fragments from an Escherichia coli strain that overproduces the lactose permease. Substrates of the permease markedly and specifically stimulated binding of enzyme IIIglc to the membranes. The sugar-stimulated binding of enzyme IIIglc was concluded to the specific to the lactose permease because it (i) was dependent on the amount of the permease, (ii) was promoted only by sugar substrates of the permease, and (iii) was completely eliminated by treatment of the membranes with N-ethylmaleimide in the absence (but not the presence) of thio-beta-D-digalactoside. The pH dependence of binding was similar to that reported for the binding of thio-beta-D-digalactoside to the permease. Phosphoenolpyruvate prevented the binding of enzyme IIIglc to the lactose permease in the presence (but not the absence) of the other phosphate transfer components of the phosphotransferase system. These results support the hypothesis that enzyme IIIglc, in its dephosphorylated form, modulates the activity of the lactose permease by a direct protein-protein interaction.

Allosteric Regulation↗
PubMed1981

Properties of peroxisomal 3-ketoacyl-coA thiolase from rat liver.

Peroxisomal 3-ketoacyl-CoA thiolase has a molecular weight of 89,000 and consists of 2 polypeptide chains of identical size. The enzyme has no interchain disulfide bonds and is reversibly dissociated to an inactive monomer in the cold. Mitochondrial 3-ketoacyl-CoA thiolase and acetoacetyl-CoA specific thiolase have molecular weights of 154,000 and 149,000, respectively. They each consist of 4 polypeptide chains of identical size. Peroxisomal thiolase and mitochondrial 3-ketoacyl-CoA thiolase operate by a ping-pong mechanism. The catalytic properties, including substrate specificity, of the peroxisomal enzyme were compared to those of mitochondrial 3-ketoacyl-CoA thiolase.

Acetyl-CoA C-Acetyltransferase↗
PubMed1980

Turnover of enzymes of peroxisomal beta-oxidation in rat liver.

Male Wistar rats were given a diet containing 2% (w/w) di-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal beta-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal beta-oxidation was 5--7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the beta-oxidation system and of catalase decreased to the control levels with a half-life of 2--3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.

Animals↗
PubMed1980

Purification and properties of acyl-CoA oxidase from rat liver.

Acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was purified from rat liver. The final preparation was judged to be nearly homogeneous from the results of sedimentation analysis. Ultrogel AcA-34 column chromatography, and phosphocellulose column chromatography. The molecular weight of the enzyme was determined to be 139,000 by the sedimentation equilibrium method and Ultrogel AcA-34 column chromatography. The S020,W of the enzyme was 7.85S. Three protein components, A, B, and C, were found in the enzyme preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights, 75,500, 50,100, and 19,000) and high-pressure liquid chromatrography in the presence of 6 M guanidine . HCl (molecular weights, 71,900, 51,700, and 20,500). It was concluded that components B and C were formed from component A, probably by proteolytic cleavage, based on the result of amino acid analysis of each component. The pI of the enzyme was 9.2. The enzyme contained FAD as a prosthetic group, and exhibited absorption maxima at 278, 378, and 450 nm. The FAD content was 1.22 mol/mol of enzyme. When palmitoyl-CoA was added to the enzyme solution under anaerobic conditions, the bound FAD was reduced. The Km values were lower for C14 to C18 acyl-CoA's than for others tested, whereas Vmax values were roughly the same for C8 to C18 acyl-CoA's. The Km value for O2 was 5 microM. The optimal pH was 8. 3-Ketohexadecanoyl-CoA inhibited the enzyme (Ki=0.47 microM), forming a charge-transfer complex with the enzyme.

Acyl Coenzyme A↗
PubMed1980

Properties of mitochondria and peroxisomal enoyl-CoA hydratases from rat liver.

Mitochondrial and peroxisomal enoyl-CoA hydratases were purified from rat liver. The mitochondrial enzyme, with a molecular weight of 161,000, was composed of 6 identical subunits. The molecular structure of the rat liver enzyme was very similar to that of the bovine liver enzyme. Acetoacetyl-CoA was a competitive inhibitor of the mitochondrial enzymes. The results of titration of the rat liver enzyme with acetoacetyl-CoA suggest that 3 subunits of the enzyme exhibit catalytic activity. The catalytic properties of the enzyme were studied. The peroxisomal enzyme was composed of one polypeptide with a molecular weight of 70,000-81,000. Some of the enzyme molecules were shown to be cleaved to two polypeptides in the cell by the following methods: amino acid analysis, peptide mapping and immunoprecipitin reaction. The catalytic properties of the peroxisomal enzyme were different from those of the mitochondrial enzyme. The peroxisomal enzyme is a bifunctional enzyme exhibiting 3-hydroxyacyl-CoA dehydrogenase activity. Studies on the titration with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.

Amino Acids↗
PubMed1980

Stereochemistry of dehydrogenation catalyzed by Acyl-CoA oxidase.

The stereochemical course of dehydrogenation catalyzed by acyl-CoA oxidase was investigated using the enzymes from rat liver peroxisomes and Candida lipolytica. Stearoyl-CoA and nonanoyl-CoA, stereospecifically labeled with deuterium at either C-2 or C-3, were incubated with the enzyme, the products were converted to methyl esters and their deuterium contents were measured by gas chromatography-mass spectrometry. The results suggested that acyl-CoA oxidase-catalyzed dehydrogenation occurred by anti-elimination of the pro 2R and pro-3R hydrogens of acyl-CoA.

Acyl Coenzyme A↗
PubMed1980

The presence of a new 3-oxoacyl-CoA thiolase in rat liver peroxisomes.

A novel 3-oxacyl-CoA thiolase was found in rat liver. This thiolase, mitochondrial general 3-oxacyl-CoA thiolase and acetoacetyl-CoA thiolase were purified from the rat liver after the induction of these activities by the administration to rats of di(2-ethylhexyl)phthalate, which enhanced the peroxisomal beta-oxidation activity. The new 3-oxoacyl-CoA thiolase was distinguished from mitochondrial and cytoplasmic thiolases by the following: DEAE-cellulose chromatography, phosphocellulose chromatography, immunochemical titration, and substrate specificity. Subcellular fractionation of liver and sucrose-density-gradient centrifugation of the light mitochondrial fraction revealed that the new thiolase was mainly in peroxisomes.

Acetyl-CoA C-Acyltransferase↗
PubMed1980

Effect of Bordetella pertussis vaccine on glucagon secretion in normal and alloxan dogs.

The effect of Bordetella pertussis vaccine on plasma glucose, insulin and glucagon secretion was investigated in normal and alloxan dogs. On the 8th day after the vaccine injection, in normal and alloxan dogs during the infusion of arginine and glucose, the plasma glucose level was lower and the IRI level was higher than in the saline controls. On the other hand, the plasma IRG level showed no significant alloxan dogs this vaccine made the plasma IRG level lower during arginine infusion than in the saline controls and suppressed it significantly during glucose infusion. These effects of the vaccine disappeared on the 30th day after its injection into normal and alloxan dogs. It is suggested that in normal dogs Bordetella pertussis vaccine decreased plasma glucose through the promotion of insulin secretion without any effect on glucagon secretion, while in alloxan dogs this vaccine might alleviate hyperglycemia through the enhancement of insulin and the inhibition of glucagon secretion.

Animals↗