Biomedical subjects

H Oya

Publications and source records attributed to H Oya.

At least 73 records · Page 4Linked to original sources

PubMed1990

Structural studies on three flavin-interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria.

The flavoprotein (Fp) subunit of mitochondrial complex II contains covalently bound FAD as a prosthetic group. In this study, the primary structure of the flavin-bound tryptic peptide from the Fp subunit of Ascaris complex II was determined and found to be highly similar to those of the corresponding flavin-binding regions of bovine heart and bacterial Fp subunits. Furthermore, the Ascaris Fp subunit was shown to contain two regions exhibiting striking sequence similarity to the segments that have been predicted to interact noncovalently with the AMP moiety of FAD in bacterial Fp subunits. The conservation of these two regions also in the mitochondrial Fp subunit suggests their functional importance.

Amino Acid Sequence↗

PubMed1990

Human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of iron sulfur (Ip) subunit of liver mitochondria.

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex of both the tricarboxylic acid cycle and of the aerobic respiratory chains of mitochondria in eukaryotic cell and prokaryotic organisms. In this study, the amino acid sequence of iron sulfur-subunit in human liver mitochondria was deduced from cDNA which was isolated by immunoscreening a human liver lambda gtll cDNA library. An isolated clone contains an open reading frame of 786 nucleotides and encodes a mature protein of 252 amino acids with a molecular weight of 28,804. The amino acid sequence was highly homologous with that of bovine heart (94.1%) which has been determined from the purified peptide and that of Escherichia coli sdh B product (50.8%). Striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme. This is the first report on the cDNA sequence of mitochondrial complex II.

Amino Acid Sequence↗

PubMed1990

Oxidation-reduction potentials of cytochromes in Ascaris muscle mitochondria: high-redox-potential cytochrome b558 in complex II (succinate-ubiquinone reductase).

Oxidation-reduction midpoint potentials (Ems) were determined at pH 7.0 for cytochromes in the anaerobic respiratory chain of Ascaris mitochondria by redox titration techniques. Cytochrome b558, which is associated with complex II that functions as fumarate reductase in the terminal step of the respiratory chain, was shown to have an Em of -34 mV in the isolated complex II and -54 mV in mitochondria. These values are much higher than the value of Ascaris cytochrome b558. In contrast, Ems of cytochromes C + C1 and cytochrome b559.5 were determined in situ to be 235 mV and 78 mV, respectively, which are comparable to those of their mammalian counterparts.

Animals↗

PubMed1989

Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease.

Immunoblotting studies on mitochondria prepared from the striata of patients who died of Parkinson's disease were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with Parkinson's disease, the 30-, 25- and 24-kDa subunits of Complex I were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and Parkinson's disease. Deficiencies in Complex I subunits seem to be one of the most important clues to elucidate pathogenesis of Parkinson's disease.

Corpus Striatum↗

PubMed1989

Inhibitory effects of tetragalloylglucose on the complex II of mitochondrial respiratory chain of Ascaris muscle.

The effects of tetragalloylglucose (1,2,3,6-tetra-O-galloyl-beta-D-glucose) on purified complex II (succinate-ubiquinone oxidoreductase) of the mitochondrial electron transport system of Ascaris muscle were studied. Both succinate-ubiquinone-1 (Q1) oxidoreductase, and succinate dehydrogenase measured with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) in the presence of phenazine methosulfate (PMS) were inhibited by tetragalloylglucose. The inhibitions of both reductase activities of complex II were of competitive type, and the inhibitor constant (Ki) for Ascaris complex II (148 nM) was lower than that for rat liver complex II (1.5 microM). Thus, Ascaris complex II is much more sensitive to this inhibitor than the mammalian counterpart.

Animals↗

PubMed1988

ESR studies on iron-sulfur clusters of complex II in Ascaris suum mitochondria which exhibits strong fumarate reductase activity.

Complex II of Ascaris suum mitochondria, which functions as fumarate reductase in physiological conditions, contains three types of iron-sulfur clusters. These correspond to clusters S-1, S-2 and S-3 and are distinguishable by low-temperature ESR studies. Cluster S-1 is reduced by succinate, giving ESR signals with gz, gy and gx values at 2.033, 1.939 and 1.920. The existence of cluster S-2 is suggested by an enhancement of the S-1 spin relaxation induced upon reduction of S-2 by dithionite. Cluster S-3 is ESR detectable under air-oxidized conditions and gives a strong signal at g = 2.025. Cluster S-3 was only partially reduced even with an excess amount of sodium succinate, which is a common characteristic of fumarate reductase but this is not seen in the mitochondrial complex II.

Animals↗

PubMed1988

Electron-transfer complexes of Ascaris suum muscle mitochondria. III. Composition and fumarate reductase activity of complex II.

Complex II of the anaerobic respiratory chain in Ascaris muscle mitochondria showed a high fumarate reductase activity when reduced methyl viologen was used as the electron donor. The maximum activity was 49 mumol/min per mg protein, which is much higher than that of the mammalian counterpart. The mitochondria of Ascaris-fertilized eggs, which require oxygen for its development, also showed fumarate reductase activity with a specific activity intermediate between those of adult Ascaris and mammals. Antibody against the Ascaris flavoprotein subunit reacted with the mammalian counterparts, whereas those against the Ascaris iron-sulfur protein subunit did not crossreact, although the amino acid compositions of the subunits in Ascaris and bovine heart were quite similar. Cytochrome b-558 of Ascaris complex II was separated from flavoprotein and iron-sulphur protein subunits by high performance liquid chromatography with a gel permeation system in the presence of Sarkosyl. Isolated cytochrome b-558 is composed of two hydrophobic polypeptides with molecular masses of 17.2 and 12.5 kDa determined by gradient gel, which correspond to the two small subunits of complex II. Amino acid compositions of these small subunits showed little similarity with those of cytochrome b-560 of bovine heart complex II. NADH-fumarate reductase, which is the final enzyme complex in the anaerobic respiratory chain in Ascaris, was reconstituted with bovine heart complex I, Ascaris complex II and phospholipids. The maximum activity was 430 nmol/min per mg protein of complex II. Rhodoquinone was essential for this reconstitution, whereas ubiquinone showed no effect. The results clearly indicate the unique role of Ascaris complex II as fumarate reductase and the indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase system.

Amino Acid Sequence↗

PubMed1988

Acivicin inhibits Crithidia fasciculata growth in a serum-free medium and inactivates carbamoyl-phosphate synthetase II in vivo.

1. Crithidia fasciculata was grown in a serum-free medium. 2. Twenty-six hours after addition of 2, 5, 20, 50, and 150 microM acivicin to logarithmically growing organisms, cell counts were decreased to 46, 23, 14, 9.1, and 8.6% of the control, respectively. 3. Guanosine plus cytidine (0.1 mM each) provided complete protection against growth inhibition by 5 microM acivicin. 4. Cells exposed to 10 microM acivicin showed a time-dependent, irreversible inactivation of L-glutamine-dependent carbamoyl-phosphate synthetase II activity; ammonia-dependent synthetase II activity was increased up to 34% of the control. 5. Glutamine (20 mM) protected the enzyme from inactivation in vivo. 6. These results indicate that acivicin acts as an affinity analog of L-glutamine in vivo as it does in vitro.

Animals↗

PubMed1987

Inactivation of Crithidia fasciculata carbamoyl phosphate synthetase II by the antitumor drug acivicin.

In Crithidia fasciculata, carbamoyl phosphate synthetase II, which catalyses the first step of de novo pyrimidine biosynthesis, was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. The antitumor drug acivicin competitively inhibited the synthetase II activity with respect to L-glutamine, yielding an apparent Ki of 2 microM. In the absence of L-glutamine, acivicin resulted in a selective, time-dependent inactivation of L-glutamine-dependent activity of the enzyme, with an inactivation constant (Kinact) of 100 microM and a minimum inactivation half-time (T) of 0.2 min. L-Glutamine protected the enzyme from inactivation. These results are consistent with a postulate that acivicin is an active site-directed affinity analogue of L-glutamine, achieving irreversible inactivation. The inactivated enzyme retained ammonia-dependent activity. Acivicin stimulated the ammonia-dependent activity by increasing the Vmax value of the enzyme; apparent Km values for ammonia and MgATP were not affected. Differential action of acivicin on the Crithidia and mammalian synthetase II is discussed.

Ammonia↗

PubMed1987

Regulatory properties of carbamoyl-phosphate synthetase II from the parasitic protozoan Crithidia fasciculata.

1. At the lowered concentrations of 0.5 mM ATP and 1.5 mM MgCl2, 2.0 mM UTP, UDP and UMP inhibited the activity of Crithidia fasciculata carbamoyl-phosphate synthetase II by about 65, 80 and 40% respectively. 2. The result suggests that feedback inhibition of the activity by uridine nucleotides is a mechanism of regulation of the de novo pyrimidine biosynthetic pathway in C. fasciculata. 3. ADP, AMP and CDP inhibited the activity (about 70, 40 and 40%). 4. Excess Mg2+ at around 1 mM, relative to the ATP concentration, was required for the maximum activity. 5. 5-Phosphoribosyl 1-pyrophosphate had no significant effect on the activity under various conditions examined.

Animals↗

PubMed1987

Kinetic properties of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) in the parasitic protozoan Crithidia fasciculata and separation of the enzyme from aspartate carbamoyltransferase.

A high specific activity of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing; EC 6.3.5.5) was demonstrated in extract of the cultured Crithidia fasciculata. The enzyme was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. Apparent Km for the synthetase for L-glutamine, NH4+, MgATP or bicarbonate was 0.27, 26, 1.7 or 1.7 mM at 2.0% dimethyl sulfoxide plus 0.3% glycerol. 8.6% dimethyl sulfoxide plus 1.4% glycerol decreased Km for L-glutamine to 0.10 mM, while Km for MgATP was unaffected. The higher solvent concentrations made Vmax markedly reduced, yielding the inhibition of the activity. These properties are unique to the Crithidia synthetase, compared with the mammalian enzyme.

Ammonium Sulfate↗

PubMed1986

Electron-transfer complexes of Ascaris suum muscle mitochondria. II. Succinate-coenzyme Q reductase (complex II) associated with substrate-reducible cytochrome b-558.

A succinate-coenzyme Q reductase (complex II) was isolated in highly purified form from Ascaris muscle mitochondria by detergent solubilization, ammonium sulfate fractionation and gel filtration on a Sephadex G-200 column. The enzyme preparation catalyzes electron transfer from succinate to coenzyme Q1 with a specific activity of 1.2 mumol coenzyme Q1 reduced per min per mg protein at 25 degrees C. The isolated complex II is essentially free of NADH-ferricyanide reductase, reduced CoQ2-cytochrome c reductase and cytochrome c oxidase and consists of four major polypeptides with apparent molecular weights of 66 000, 27 000, 12 000 and 11 000 and two minor ones with Mr of 36 000 and 16 000. The complex II contained cytochrome b-558, a major constituent cytochrome of Ascaris mitochondria, at a concentration of 3.6 nmol per mg protein, but neither other cytochromes nor quinone. The cytochrome b-558 in the complex II was reduced with succinate. In the presence of Ascaris NADH-cytochrome c reductase (complex I-III) (Takamiya, S., Furushima, R. and Oya, H. (1984) Mol. Biochem. Parasitol. 13, 121-134), the cytochrome b-558 in complex II was also reduced with NADH and reoxidized with fumarate. These results suggest the cytochrome b-558 to function as an electron carrier between NADH dehydrogenase and succinate dehydrogenase in the Ascaris NADH-fumarate reductase system.

Animals↗

PubMed1985

Biosynthesis of lipids during embryogenesis of Ascaris lumbricoides eggs.

Alteration in lipid metabolism of Ascaris lumbricoides eggs during development in either air or nitrogen gas was studied using acetate-1-14C and labeled fatty acids. It is thought that formation of 14C-lipids and 14C-palmitoleate from acetate-1-14C and palmitate-1-14C in developing eggs may be influenced by the concentration of molecular oxygen in the medium. Acetate-1-14C was incorporated into palmitate, palmitoleate and unsaturated 18-carbon acid of fatty acids in the eggs, but the incorporation into saturated 18-carbon acid was slight. Radioactive fatty acid methyl esters were major lipid component of the radioactive lipid classes in the first and second stage larvae incubated with palmitate-1-14C. Isopentadecanoic acid, which is a small component of fatty acids in Ascaris eggs, contains a high percentage of the total radioactivity in the second stage larvae incubated with acetate-1-14C under both air and nitrogen. It appears that a conversion of the fatty acid biosynthesis system may occur during development in to the second stage larvae.

Acetates↗

PubMed1985

Quantitative determination of cytochromes in the aerobic respiratory chain of Escherichia coli by high-performance liquid chromatography and its application to analysis of mitochondrial cytochromes.

A method was established for quantitative determination of the composition and the amounts of cytochromes in the aerobic respiratory chain of Escherichia coli. Cytochromes were solubilized with 3%(W/V) Sarkosyl and the extract was analyzed by high-performance liquid chromatography on a gel permeation column of TSK gel-G3000SW. This analytical system required only about 0.5 mg of membrane protein and 20 min per assay. The effects of the gene dosage of F-prime factors and plasmids on cytochromes were analyzed with this system, and the system was applied to the analysis of mitochondrial cytochromes.

Aerobiosis↗

PubMed1984

Chromosomal location of the Escherichia coli cytochrome b556 gene, cybA.

The amounts of cytochrome b556 in the cytoplasmic membranes of several Escherichia coli K12 strains having F-prime factors and a lambda transducing phage were determined. The amount was amplified about two-fold in strains having F100-12 and F152, but not in strains having F100-11, F8 and lambda psu+2glnS+. The strain TK3D11, which lacks the kdp-gltA region (deletion D-01) of the E. coli chromosome, did not synthesize cytochrome b556 at all. From these results, the gene cybA encoding cytochrome b556 was located in the kdp-gltA region. In the cytochrome b556-deficient mutant, a novel b type cytochrome, cytochrome b561 which is a product of the gene cybB, was identified. It seems to function as a physiological electron transferring cytochrome in place of cytochrome b556 in this mutant.

Bacteriophage lambda↗

PubMed1984

Electron transfer complexes of Ascaris suum muscle mitochondria: I. Characterization of NADH-cytochrome c reductase (complex I-III), with special reference to cytochrome localization.

An NADH-cytochrome c reductase (complex I-III) was isolated from Ascaris suum muscle mitochondria. The enzyme preparation catalyzed the reduction of 1.68 mumol cytochrome c min-1 mg-1 protein at 25 degrees C with NADH but not with NADPH, and retained its sensitivity to rotenone, piericidin A and 2-heptyl-4-hydroxyquinoline-N-oxide as with the submitochondrial particles. The isolated complex I-III, essentially free of succinate-cytochrome c reductase and cytochrome c oxidase, consisted of fourteen polypeptides with apparent molecular weights ranging from 76 000 to 12 000. The complex I-III contained three cytochromes, b-559.5, b-563 and c1-550.5 and Pigment-558 at concentrations of 1.28, 0.211, 1.23 and 0.321 nmol mg-1 protein, respectively. Cytochrome b-558, a major constituent cytochrome of Ascaris mitochondria and previously suggested to participate in the fumarate reductase system, was not fractionated in the complex I-III. Localization of the cytochromes in Ascaris electron transfer complexes is discussed.

Animals↗

PubMed1984

Ultrastructural evidence for eosinophil-mediated destruction of Angiostrongylus cantonensis transferred into the pulmonary artery of non-permissive hosts.

Ultrastructural and cytochemical analyses were carried out on cellular reactions to the young adult worms of Angiostrongylus cantonensis surgically transferred into the pulmonary arteries of permissive (rat) and non-permissive (rabbit and guinea-pig) hosts. In permissive hosts, no appreciable cellular reactions could be found around worms throughout the course of the observations. By contrast, the infiltration of neutrophils along with eosinophils was observed around worms in non-permissive hosts even at early stages (days 2 to 4). At day 7 and later, the prominent degranulation (solubilization of the whole granule or the matrix alone with preserved crystalloid, tubulovesicular structure formation, and vacuole formation containing lysosomal contents, etc.) of eosinophils, and subsequent release of the lysosomal contents on to the worm surface were noted. Discharge of large amounts of peroxidase on to the worm surface was also demonstrated. The worms were thus damaged and their cuticular fragments were frequently found removed. In addition to this, degenerative changes, such as lipid-droplet and vacuole formations, were detectable in the hypodermis, somatic musculature and intestine of the parasites transferred into the non-permissive hosts, as early as day 4 after transfer. These data suggest that eosinophils would serve as a potential effector cell for killing of pulmonary arterial A. cantonensis in non-permissive hosts.

Angiostrongylus↗

PubMed1983

Mechanism of inactivation of a Fasciola proteolytic enzyme by peptide aldehydes and alkylating agents.

A proteolytic enzyme of the liver fluke Fasciola sp. was purified as described previously by ammonium sulfate fractionation, gel filtration on Sephadex G-200 column and L-phenylalanine-agarose chromatography. Leupeptin, a peptide aldehyde of microbial origin, competitively inhibited the enzyme activity with respect to the substrate alpha-N-benzoyl-L-argininamide; the apparent Ki value for leupeptin is 45 000-fold less than the apparent Km for the substrate. Incubation of the enzyme with leupeptin resulted in time-dependent inactivation of the globinolytic activity, with an inactivation constant (Kinact) of 0.4 microM giving the half-maximum inactivation velocity, and with a minimum inactivation half-time (T) of 2.7 min at infinite concentration of this compound. The inactivated enzyme was not reactivated by extensive dialysis. These results imply that leupeptin yields an affinity labelling of an active site of the enzyme. The activity of the Fasciola proteolytic enzyme was also inactivated by other peptide aldehydes and alkylating agents and inactivation constants observed were 0.5 microM for chymostatin, 13 microM for antipain, 2 microM for p-toluenesulfonyl-L-lysine chloromethyl ketone, 140 microM for p-toluenesulfonyl-L-phenylalanine chloromethyl ketone and 40 microM for iodoacetate under the conditions used.

Aldehydes↗