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S Horie

Publications and source records attributed to S Horie.

At least 235 records · Page 13Linked to original sources
PubMed1980

Studies on the enzymatic reduction of C-nitroso compounds. III. The kinetic analysis of C-nitrosoreductase reaction catalyzed by the cytoplasmic enzyme from porcine liver.

The reaction kinetics of the major cytoplasmic NADH-nitrosoreductase, which is the identical enzyme to alcohol dehydrogenase [EC 1.1.1.1], was studied with a partially purified preparation from porcine liver. On the basis of the data obtained, the following scheme is proposed as the mechanism of this enzyme reaction: [Formula: see text], where E and E' are the enzyme unit (one subunit of alcohol dehydrogenase) and an intermediate form of the enzyme unit-substrate compound which appears by two-electron reduction of the enzyme unit-substrate compound, respectively, S is p-nitrosophenol (p-NSP), N is NADH, and P1 and P2 are NAD+ and p-aminophenol (p-AmP), respectively. In this case, it is assumed that k1 less than K2. Para-aminophenol, the reaction product, showed an inhibition competitive to p-NSP at fixed concentrations of NADH and also showed a mixed-type inhibition to NADH at fixed concentrations of p-NSP. NAD+ inhibited the reaction in a competitive manner to NADH at fixed concentrations of p-NSP and in a non-competitive manner to p-NSP at fixed concentrations of NADH. These results can also be accounted for by the proposed mechanism.

Alcohol Oxidoreductases↗
PubMed1980

Studies on the enzymatic reduction of C-nitroso compounds. IV. Partial purification and kinetic properties of porcine heart C-nitrosoreductase.

An NAD(P)H-dependent C-nitrosoreductase was purified 90 fold from porcine heart cytosol fraction by ammonium sulfate fractionation, gel filtration with Sephadex G-100, ion-exchange chromatography on DEAE-Sephadex A-50, and affinity chromatography on 5'-AMP-Sepharose. The enzyme had no aldehyde reductase activity and showed a pH optimum of 5.5. Its molecular weight estimated by gel filtration was about 65,000 and 70,000 daltons. In the reaction catalyzed by this enzyme, 2 mol of NADH were consumed per mol p-nitrosophenol (p-NSP) reduced to p-aminophenol (p-AmP). Nitrosobenzene and other aryl nitroso compounds were also reduced but neither phenylhydroxylamine nor hydroxylamine could serve as the electron acceptor. Kinetic measurements were also carried out and, based on the data obtained, the following scheme is proposed for the mechanism of the reaction: [Formula: see text], where E, E', and E" represent the active enzyme unit, the enzyme unit after two-electron reduction, and the enzyme unit after four-electron reduction, respectively, N in NADH, S is p-NSP, and P1 and P2 are NAD+ and p-AmP, respectively. Para-aminophenol showed an inhibition noncompetitive wih NADH and also one apparently noncompetitive with p-NSP. NAD+ showed an inhibition competitive with NADH and one uncompetitive with p-NSP. These results can also be accounted for by the proposed mechanism.

Alcohol Oxidoreductases↗
PubMed1979

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition.

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same cross-point, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, D-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2 : 1. The theoretical value of cross-point of the core calculated from from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.

Animals↗
PubMed1978

Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae cavara. II. Purification and properties of a beta-glucosidase.

Three components (GA, GB-1, and GB-2) of beta-glucosidase were detected in the culture filtrate of Pyricularia oryzae grown in a cellulose or cellulose derivative medium. Among them, GB-1 was induced most strongly. Purified GB-1 was homogeneous on polyacrylamide gel electrophoresis and showed an approximately 1,400-fold increase of specific activity over the starting material. The molecular weight was determined to be 240,000 by sodium dodecyl sulfate-gel electrophoresis. A similar value was also obtained by sucrose density gradient centrifugation. The enzyme contained a high proportion of acidic amino acids and mannose, and the isoelectric point of the enzyme was pH 4.15. The enzyme had a pH optimum of 5.5 and a temperature optimum at 55 degrees C. beta-Glucosidase activity was inhibited by Mn2+, Cu2+, Hg2+, p-chloromercuribenzoate, and glucono-delta-lactone. The enzyme split off glucose units one by one from the nonreducing ends of not only beta-glucooligosaccharides but also some beta-glucans, such as carboxymethylcellulose, laminaran, pustulan, and zeagallan. The affinity for cello- and laminari-oligosaccharides tended to increase in parallel with the chain length.

Amino Acids↗
PubMed1976

Studies on P-450. X. On the coordination structure of hemoprotein P-450.

The spectral properties of a P-450 preparation from adrenocortical mitochondria were compared with those of both other hemoproteins and model compounds to examine the possibility suggested by previous authors that the mercaptide anion (thiolate anion, RS-) of a cysteine residue might be one of the axial ligands of P-45O. 1. The following features were found in addition to the well known anomalous spectral characteristics of P-450. The Soret band of the ferrous form was at anomalously short wavelengths compared with those of other ferrous high-spin hemoproteins. The regularity in the shifts of the Soret maximum observed with various forms of usual hemoproteins was disrupted in the case of P-450 by the maximum of the ferrous form. 2. The complex of methemoglobin with 2-mercaptoethanol, which was the model for P-450 used by previous authors for spectral and ESR studies, was studied more precisely. The model showed an absorption spectrum very similar to that of the imidazole complex of ferric P-450, whereas the complex of methemoglobin with imidazole showed both Soret and visible absorption maxima at shorter wavelengths. The rapid rate of reduction of methemoglobin by dithionite decreased on binding to mercaptoethanol and the slow rate of reduction of P-450 increased on binding to imidazole. Thus the half-reduction times for both complexes were of the same order of magnitude. 3. An absorption spectrum which was very similar to the spectrum of ferric high-spin P-45O could be obtained by quick scanning immediately after ferric protoheme had been dissolved in a 33% solution of mercaptoethanol in water containing Tween 20 and NaOH. 4. Protoheme dissolved in an aqueous solution of Nikkol BL9EX (a non-ionic detergent) containing NaOH, dithionite, and 1 mM mercaptoethanol formed a transient intermediate complex having a Soret maximum at 470 nm. Treatment of this complex with carbon monoxide resulted in the formation of a CO adduct having a Soret maximum at 450 nm and a single band at about 555 nm in the visible region. These maxima were similar to those of the carbon monoxide complex of P-450. Many of the results could be explained readily by assuming that a mercaptide anion was one of the axial ligands, and also that heme was held in a hydrophobic crevice in the P-450 molecule. The coordination structure of P-450 seems to be more flexible than those of usual hemoproteins.

Adrenal Cortex↗
PubMed1976

Isolation, properties, and crystallization of an iron-chlorin protein from Aspergillus niger.

A green iron-chlorin protein was purified 160-fold from a lyophilized extract of Aspergillus niger by ion-exchange chromatography and gel filtration with a yield of 25%. The purified preparation appeared nearly homogeneous on sedimentation analysis and the sedimentation coefficient of the protein at infinite dilution was 13.4S. Its molecular weight was calculated to be 2.8--3.2 X 10(5) from sedimentation and gel filtration data. The ferric form of the protein had absorption maxima at 587.5 and 708 nm in the visible region and a Soret band at 404 nm. High-spin ESR signals of a rhombically distorted ferric iron-chlorin complex were observed at g = 6.5 and 5.3 together with unidentified, weaker signals at g = 4.3 and 2.0. The ferric form reacted readily with cyanide to give a complex showing absorption peaks at 422 and 632 nm and a shoulder at about 595 nm. When the protein combined with cyanide its high-spin ESR signals disappeared and low-spin signals at g = 1.88, 2.29, anous form having absorption maxima at 622 and about 410 nm. The rate of reduction by dithionite was slightly reduced by the presence of either nitrite or sulfite, and greatly accelerated by the presence of hydroxylamine. The reduced spectrum obtained in the presence of hydroxylamine had maxima at 620 and about 420 nm. The ferric cyanide complex did not show any spectral change on addition of dithionite. The green prosthetic group could be extracted with acidified acetone and the absorption maxima of the pyridine ferrihemochrome were at 413 and 599 nm. On removal of metal from the prosthetic group the characteristic spectrum of a chlorin was obtained. The absorption maxima of a solution of the chlorin in benzene were at 403, 501, 537, 576, 595, and 655 nm, the 655 nm band being strongest of those in the visible region. No significant amount of flavin was detected in the purified preparation. The iron-chlorin protein catalyzed methyl viologen-linked reduction of hydroxylamine and also that of nitrite at a slower rate under the same conditions, but not evidence that it reduced sulfite was obtained in the present study. The purified preparation also had high catalase [ec 1.11.1.6] activity. Crystalline material was obtained by gradual concentration of the purified preparation at about pH 6.

Aspergillus↗