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M Ishimoto

Publications and source records attributed to M Ishimoto.

At least 37 records · Page 2Linked to original sources
PubMed1987

Properties of glutamate dehydrogenase purified from Bacteroides fragilis.

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.

Bacteroides fragilis↗
PubMed1987

Regulation of synthesis and reversible inactivation in vivo of dual coenzyme-specific glutamate dehydrogenase in Bacteroides fragilis.

Regulation of the dual coenzyme-specific glutamate dehydrogenase (GDH; EC 1.4.1.3) was studied in the anaerobic bacterium Bacteroides fragilis. Cells grown at a low concentration of ammonia had a specific activity for the enzyme 10-fold higher than that for cells grown with excess ammonia. Immunochemical determination with a GDH-specific antiserum showed that the content of immuno-precipitated protein was about 8% of the total protein in the former cells and was 4% in the latter cells. When cells grown on 50 mM-NH4Cl were transferred to a fresh medium containing 0.5 mM-NH4Cl, an increase in the molecular activity of the enzyme occurred, and synthesis of immuno-reactive protein started. Rapid inactivation of the GDH occurred when cells grown on 1 mM-NH4Cl were exposed to 50 mM-NH4Cl. However, the amount of immuno-precipitated protein was not decreased. The inactivation was specifically induced by ammonia and was reversed by transferring the cells to an ammonia-limited medium even in the presence of chloramphenicol. These findings suggest that the synthesis of the GDH is stimulated under low ammonia conditions and that the enzyme activity is controlled by means of a reversible activation/inactivation mechanism which is regulated by ammonia. However, no phosphorylation of GDH was observed before and after exposure of cells to high concentrations of ammonia.

Ammonium Chloride↗
PubMed1986

Purification and properties of nitrate reductase from Mitsuokella multiacidus.

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).

Bacteroides↗
PubMed1986

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein.

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.

Catalysis↗
PubMed1985

Proton translocation coupled to nitrite reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to the reduction of nitrite was studied in anaerobically grown Escherichia coli. Extrusion of protons occurred by adding nitrite to an anaerobic suspension of wild-type cells. This extrusion was sensitive to a proton conductor, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Dicyclohexylcarbodiimide, an inhibitor of H+-ATPase, prevented the proton extrusion linked to nitrite reduction, whereas this reagent had no effect on respiratory nitrate reduction to nitrite. Proton extrusion was undetectable when nitrite was added to a suspension of mutant cells defective in H+-ATPase. These results indicate that the proton extrusion associated with nitrite reduction to ammonia is not by redox pumps but by H+-ATPase. From the results obtained by the measurement of proton extrusion in nitrite reductase-deficient mutants, NADH-nitrite reductase system is suggested to involve the proton extrusion in whole cells of E. coli.

Ammonia↗
PubMed1985

Purification and properties of thiosulfate reductase from Desulfovibrio vulgaris, Miyazaki F.

Thiosulfate reductase was purified to an almost homogeneous state from Desulfovibrio vulgaris, strain Miyazaki F, by ammonium sulfate precipitation, chromatography on DEAE-Toyopearl, Ultrogel AcA 34, and hydroxylapatite, and disc electrophoresis. The specific activity was increased 580-fold over the crude extract. The molecular weight was determined by gel filtration to be 85,000-89,000, differing from those reported for thiosulfate reductases from other Desulfovibrio strains. The enzyme had no subunit structure. When coupled with hydrogenase and methyl viologen, it stoichiometrically reduced thiosulfate to sulfite and sulfide with consumption of hydrogen. It did not reduce sulfite or trithionate. Cytochrome c3 was active as an electron donor. More than 0.75 mM thiosulfate inhibited the enzyme activity. o-Phenanthroline and 2,2'-bipyridine inhibited the enzyme and ferrous ion stimulated the reaction.

Cytochrome c Group↗
PubMed1985

Characterization of a dissimilatory-type sulfite reductase, desulfoviridin, from Desulfovibrio africanus Benghazi.

A desulfoviridin-type sulfite reductase having the alpha band at 638 nm was purified from Desulfovibrio africanus Benghazi (NCIB 8401) by chromatography on DEAE-cellulose, Sephadex G-200, and DEAE-Sepharose columns and by disc gel electrophoresis. The content of desulfoviridin in the soluble protein was estimated to be about 6% from the purification indexes. Like the typical desulfoviridin from D. vulgaris Miyazaki K, it formed mainly trithionate besides thiosulfate and sulfide in sulfite reduction coupled to hydrogenase and methyl viologen. No significant differences in the amino acid compositions, CD patterns in the UV (205-250 nm) region, and subunit structures were found, except for a pI value about 1 unit larger (pI 5.3). The split Soret (410 +/- 2 nm, less intense peak at 391 +/- 2 nm with a shoulder around 380 nm) and beta (584 +/- 2 nm) band maxima of the enzyme as isolated, and the visible absorption and fluorescence spectra of the acidic acetone-extracted chromophore were almost identical to those ascribed to sirohydrochlorin in spite of the reported difference in the native enzyme (alpha band maxima at 638 nm as against 628 +/- 2 nm in a typical desulfoviridin). Iron was the only significant chelatable metal contained in the chromophore. Some differences between africanus and vulgaris desulfoviridins were observed in the CD patterns in the UV to near UV region (250-340 nm) and also in the visible absorption spectra in the presence of dithionite.

Amino Acids↗
PubMed1983

Characterization of periplasmic hydrogenase from Desulfovibrio vulgaris Miyazaki K.

Periplasmic hydrogenase [hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki K (MK) was purified to homogeneity. Its chemical and immunological properties were examined and compared with those of other Desulfovibrio hydrogenases. The pure enzyme showed a specific activity of 1,000 mumol H2 evolution min-1 (mg protein)-1. The enzyme had a molecular weight of 50,000 as estimated by gel filtration and consisted of a single polypeptide chain. The absorption spectrum of the enzyme was characteristic of an iron-sulfur protein and the extinction coefficients at 400 and 280 nm were 34 and 104 mM-1. cm-1, respectively. It contained 9.4 mol iron and 6.9 mol of acid-labile sulfide per mol. The amino acid composition of the preparation was very similar to the value reported for D. desulfuricans NRC 49001 hydrogenase. Rabbit antisera were prepared against the enzyme of D. vulgaris MK. Ouchterlony double diffusion and immunotitration tests of crude extracts from several strains of Desulfovibrio revealed that the enzyme from MK cells was immunologically identical with those from D. vulgaris Hildenborough and D. desulfuricans NRC 49001, but different from those from D. vulgaris Miyazaki F (MF) and Miyazaki Y, and D. desulfuricans Essex 6 strains. It is concluded that among Desulfovibrio hydrogenases, those from D. vulgaris MK, D. vulgaris Hildenborough and D. desulfuricans NRC 49001 form one group in terms of both subunit structure and antigenicity.

Amino Acids↗
PubMed1983

Purification and some properties of nitrite reductase from Clostridium perfringens.

Nitrite reductase from Clostridium perfringens was purified by chromatographies on DEAE-cellulose, DEAE-Sephadex, Sephadex G-150, and hydroxylapatite and by isoelectric focussing to a homogeneous state, showing essentially a single protein band in disc gel electrophoresis and a single immuno-precipitation line in double diffusion against antiserum obtained from immunized rabbits. The reductase was induced in the presence of nitrate. It had a molecular weight of 54,000 and showed no absorption peak in the visible region. The pH optimum was 6.2 and Km for nitrite was 5 mM. Ferredoxin, as well as viologen dyes, was found to be an electron donor. The product of nitrite reduction was hydroxylamine. This reductase was inhibited by o-phenanthroline and azide but not by cyanide or diethyldithiocarbamate.

Clostridium perfringens↗
PubMed1983

Nutritional aspect of methionine isomers studied by pulmonary exhalation of dimethyl sulfide and urinary excretion of alpha-keto-gamma-methiolbutyrate in humans.

With the view of evaluating the relative utilization of methionine isomers, the pulmonary exhalation of dimethyl sulfide and methyl mercaptan and the urinary excretion of alpha-keto-gamma-methiolbutyrate were studied in normal healthy subjects following oral or intravenous administration of L- or D-isomer of methionine. Dimethyl sulfide concentration in the expired alveolar gas (mean +/- SD) before methionine loading was 2.1 +/- 1.4 ng/dl (N = 23). Maximum concentrations of dimethyl sulfide in oral methionine loading tests were: 56.0 +/- 24.9 ng/dl (D-2g) (n = 4), 66.0 +/- 42.1 (D-1g) (N = 6) and 4.6 +/- 2.6 (L-2g) (N = 4); and in intravenous loading tests: 60.0 +/- 19.9 (D-Ig) (N = 4) and 4.2 +/- 2.1 (L-2g) (N = 3), respectively. The changes in methyl mercaptan in the expired alveolar gas were small and were disproportional to the changes in dimethyl sulfide following administration of both isomers. Preloading concentration of alpha-keto-gamma-methiolbutyrate in urine was 0.15 +/- 0.10 microgram/mg Creatinine (mean +/- SD) (N = 5). Postloading values during the initial two hours were 578 and 156 micrograms/mg Creatinine following 3g of D- and 0.20 and 29.7 micrograms/mg Creatinine following 3g of L-methionine ingestion. In view of the results obtained, significant amounts of D-methionine seem to be metabolized through the transaminative pathway of methionine metabolism.

Adult↗
PubMed1981

Siroheme as an active catalyst in sulfite reduction.

Siroheme extracted by acetone/HCl treatment of sulfite reductase from yeast and purified by column chromatography catalyzed the reduction of sulfite to thiosulfate and sulfide when coupled with a hydrogen-hydrogenase-methyl viologen system. The activity increased with decrease in pH from 7 to 4, and an apparent Km value of 50 mM for sulfite was obtained. In contrast to sirohydrochlorin plus Fe2+, addition of inorganic iron or 2,2'-bipyridine prior to the reduction reaction had scarcely any effect on the sulfite-reducing activity of siroheme. Hydroxylamine was reduced by siroheme at a much faster rate than sulfite, and the rate increased with increase in pH from 6 to 9. Siroheme extracted from Chromatium vinosum strain D sulfite reductase also reduced sulfite to thiosulfate and sulfide.

Catalysis↗
PubMed1981

Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.

Anaerobiosis↗
PubMed1980

Nitrate respiration of Klebsiella pneumoniae on amino acids, especially on serine.

Anaerobic growth on an amino acids mixture in the presence of nitrate of a strain of K. pneumoniae isolated from soil depended on the presence of serine in the mixture. This organism grew on serine as sole carbon source under anaerobic conditions in the presence of nitrate but not in its absence. Glycylglycine could replace serine. Serine was degraded to acetate, formate, pyruvate, acetone and acetoin with formation of nitrite under these conditions. These products were practically not different from those formed from glucose. Resting cells decomposed serine also in the absence of nitrate forming butanediol and a large amount of formate in place of decreased amounts of acetate and pyruvate. These cells also anaerobically decomposed alanine to products similar to those from serine and phenylalanine to phenylacetate and formate only in the presence of nitrate. Valine was not catabolized under the same conditions. Serine was decomposed to pyruvate and ammonia in the crude extract in the presence and absence of nitrate, indicating participation of serine dehydratase in its breakdown. Phenylalanine appears to be decomposed to phenylpyruvate by transamination coupled to glutamate oxidation and then to phenylacetate and formate. Little growth on serine under anaerobic conditions in the absence of nitrate may be accounted for by the small gain of ATP in the anaerobic breakdown of serine by this organism.

Amino Acids↗