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S Minakami

Publications and source records attributed to S Minakami.

At least 109 records · Page 6Linked to original sources
PubMed1982

Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte.

A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. The activity of the homogenate of the phagocytosing cells was 40 times that of the resting cells, and 70% of the activity in the homogenate was recovered in the phagocytic vesicle fraction. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mumol/min per mg of protein with NADPH, and one-fifth of this with NADH, Km values being similar to those of the vesicle fraction (40 microM for NADPH and 400 microM for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH +2O2 leads to NADP+ + 2O2 -. + H+. The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The Km value for FAD was 0.077 microM. The activities in both vesicle fraction and extract were labile even on refrigeration, but could be kept for several months at -70 degrees C.

Animals↗
PubMed1982

Hexose-6-phosphate dehydrogenase of rat liver microsomes. Isolation by affinity chromatography and properties.

Hexose-6-phosphate dehydrogenase was purified from rat liver microsomal fraction more than 500-fold with a 45% recovery using DEAE-cellulose and 2',5'-ADP-Sepharose 4B columns. The purified enzyme appeared to be immunologically and electrophoretically homogeneous and had broad substrate and cofactor specificities. The enzyme activity was not inhibited by p-chloromercuribenzoate. The purified enzyme was a glycoprotein in nature, having a Stokes radius of about 55 A, a sedimentation coefficient of about 8.2 s, and an isoelectric point of about 6.4. Minimum molecular weight of the enzyme was about 108,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the product cross-linked with glutaraldehyde or dimethyl suberimidate had Mr approximately equal to 220,000, suggesting that the active enzyme existed as a dimer of identical subunits. Antiserum raised against the purified enzyme inhibited the activity of the solubilized enzyme but did not inhibit the cytosol glucose-6-phosphate dehydrogenase activity. The antigenic sites of the enzyme were latent in intact microsomes. Comparison was also made between the enzymes isolated from untreated and phenobarbital-pretreated animals.

Animals↗
PubMed1982

Alteration of inner-membrane components and damage to electron-transfer activities of bovine heart submitochondrial particles induced by NADPH-dependent lipid peroxidation.

We investigated the changes of the inner-membrane components and the electron-transfer activities of bovine heart submitochondrial particles induced by the lipid peroxidation supported by NADPH in the presence of ADP-Fe3+. Most of the polyunsaturated fatty acids were lost as a result of the peroxidation, and phospholipids were changed to polar species. Ubiquinone was also modified to polar substances as the peroxidation proceeded. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the disappearance of 27000-Mr and 30000-Mr proteins and the appearance of highly polymerized substances. Flavins and cytochromes were not diminished, but the respiratory activity was lost. The reactions of NADH oxidase and NADH-cytochrome c reductase were most sensitive to the peroxidation, followed by those of succinate oxidase and succinate-cytochrome c reductase. Succinate dehydrogenase and duroquinol-cytochrome c reductase were inactivated by more extensive peroxidation, but cytochrome c oxidase was only partially inactivated. NADH-ferricyanide reductase was not inactivated. The pattern of the inactivation indicated that the lipid peroxidation affected the electron transport intensively between NADH dehydrogenase and ubiquinone, and moderately at the succinate dehydrogenase step and between ubiquinone and cytochrome c.

Animals↗
PubMed1982

Release of the membrane-calcium and its relation to the superoxide formation by polymorphonuclear leukocytes.

The relationship between the intracellular translocation of calcium from the storage pool and the oxidative metabolism was studied. An intracellular calcium-antagonist, TMB-8, inhibited the release of superoxide induced by a calcium ionophore A23187 and the inhibition was relieved by the addition of calcium ions. The release induced by cytochalasin D or by the ingestion of bacteria was similarly inhibited by TMB-8. The mobilization of intracellular divalent cations of leukocytes was monitored by a fluorescent probe, CTC. When the CTC-loaded cells were stimulated with cytochalasin D or E. coli, a fluorescence change ascribable to the release of calcium from the intracellular hydrophobic environment was observed. The dose-response curve of the fluorescence change and that of the superoxide release of th cells were very similar. TMB-8 inhibited both metabolic and fluorescence changes in parallel. The results support the hypothesis that an intracellular translocation of calcium is stimulated the oxidative metabolism of leukocytes.

Calcimycin↗
PubMed1982

Hexose-6-phosphate and 6-phosphogluconate dehydrogenases of rat liver microsomes. Involvement in NADPH and carbon dioxide generation in the luminal space of microsomal vesicles.

Rat liver microsomal fraction generates 14CO2 from [1(-14)C]glucose 6-phosphate in the presence of NADP+ and a detergent. The activity is mediated through an enzyme system consisting of hexose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase inherent to the microsomes, with the latter enzyme reaction being a rate-determining step. Both enzymes of the system in microsomes are extremely resistant to trypsin digestion, thereby distinguishing them from the corresponding cytosol enzymes. A stoichiometric relationship was obtained between the generations of NADPH and 14CO2 (2: 1 on a molar basis), indicating that the observed generation of NADPH in microsomes could entirely be accounted for by the action of the enzyme system. A method was devised to measure NADP(H) inside or outside the microsomal vesicles, and it was found that a considerable amount of the cofactor was present within the vesicles. Subfractionation of various intracellular fractions on sucrose density gradients confirmed the close association of NADP(H) with liver microsomes. It is suggested that both enzymes of the system function to generate the reduced form of NADP+ in the luminal space of the endoplasmic reticulum, where NADP(H) and glucose 6-phosphate are available.

Animals↗
PubMed1981

Rejuvenation of aged erythrocytes by incorporating phosphoenolpyruvate into the cells.

An acid-citrate dextrose solution which contains phosphoenolpyruvate and sucrose was used as a preservative and medium for rejuvenating depleted erythrocytes. 2,3-bisphosphoglycerate and ATP in washed erythrocytes, red cell concentrate or whole blood were increased effectively by incubating the cells with the solution at 37 degrees C for 30--60 min. The transport of phosphoenolpyruvate through the erythrocyte membrane was essential to the increase of 2,3-bisphosphoglycerate and ATP. During storage of the cells at 4 degrees C in the presence of phosphoenolpyruvate, no increase of 2,3-bisphosphoglycerate and ATP was observed because no transport of phosphoenolpyruvate into the cells occurred. By an incubation at 37 degrees C for 30 min at the end of storage periods, however, levels of ATP and 2,3-bisphosphoglycerate in the cells were raised.

2,3-Diphosphoglycerate↗
PubMed1980

Reduction of ferricytochrome c by human red cells.

Human red cells reduced extracellular ferricytochrome c to ferrocytochrome c under various conditions, suggesting that ferricytochrome c reducing systems are present at the outer surface of the red cell membrane.

Cytochrome c Group↗
PubMed1980

NADH- and NADPH-dependent lipid peroxidation in bovine heart submitochondrial particles. Dependence on the rate of electron flow in the respiratory chain and an antioxidant role of ubiquinol.

Malondialdehyde formations by bovine heart submitochondrial particles supported by NADH or NADPH in the presence of ADP and FeCl3 was studied. The NADH-dependent reaction was maximal at very low rate of electron input from NADH to the respiratory chain and it decreased when the rate became high. The reaction was stimulated by rotenone and inhibited by antimycin A when the input was fast, whereas it was not affected by the inhibitors when the input was slow. The input rate of the electrons from NADPH was also so low that the reaction supported by NADPH was not affected by the inhibitors. Most of the endogenous ubiquinone in the particles treated with antimycin A was reduced by NADH even in the presence of ADP-Fe3+ chelate, but uniquinone was not reduced by NADPH when ADP-Fe3+ was present. Succinate strongly inhibited both NADH- and NADPH-dependent lipid peroxidation. The inhibition was abolished when uniquinone was removed from the particles, and it appeared again when uniquinone was reincorporated into the particles. Reduced uniquinone-2 also inhibited the peroxidation, but duroquinol, which reduces cytochrome b without reducing endogenous uniquinone, did not. Thus the malondialdehyde formation appeared to be inversely related to the extent of the reduction of endogenous uniquinone. These observations suggest that both NADH- and NADPH-dependent liquid-peroxidation reactions are closely related to the respiratory chain and that the peroxidation is controlled by the concentration of reduced ubiquinone.

Animals↗
PubMed1980

Lipid peroxidation and the reduction of ADP-Fe3+ chelate by NADH-ubiquinone reductase preparation from bovine heart mitochondria.

The NADH-ubiquinone reductase preparation (Complex I) of bovine hart mitochondria catalysed in the presence of reduced coenzymes and ADP-Fe3+ the lipid peroxidation of liposomes prepared from mitochondrial lipids. The apparent Km values for the coenzymes and the optimal pH of the reactions agreed well with those of the lipid peroxidation of the submitochondrial particles treated with rotenone. On assay of the reduction of ADP-Fe3+ chelate by the reduction of cytochrome c in the presence of superoxide dismutase and antimycin A or by the oxidation of reduced coenzymes, the reactions were not affected by rotenone but were inhibited by thiol-group inhibitors. The properties of the ADP-Fe3+ reductase activity were highly consistent with those of the lipid-peroxidation reaction. These observations suggest that electrons from reduced coenzymes are transferred to ADP-Fe3+ chelate from a component between a mercurial-sensitive site and the rotenone-sensitive one of the NADH dehydrogenase and that the reduction of ADP-Fe3+ chelate by the NADH dehydrogenase is an essential step in the lipid peroxidation.

Adenosine Diphosphate↗
PubMed1980

Superoxide anion-generating activities of macrophages as studied by using cytochalasin E and lectins as synergistic stimulants for superoxide release.

Treatment of macrophages with cytochalasin E in combination with a lectin was found to stimulate the generation of superoxide anions (O2-) very efficiently. The macrophages stimulated with concanavalin A, phytohemagglutinin or wheat germ agglutinin released superoxide, but cells pretreated with cytochalasin E released much greater amounts of superoxide, without notable lag time, upon stimulation with the lectin. Wheat germ agglutinin was found to be the most efficient stimulant among the lectins tested. Superoxide generation in guinea pig macrophages was shown to be dependent largely on cytoplasmic glucose metabolism and to some extent on mitochondrial respiration, since the superoxide release was largely but not totally inhibited by 2-deoxyglucose and to a lesser extent by antimycin A or KCN. The method presented is sensitive and allows rapid assay of the superoxide-generating activity with only 1--5 X 10(5) macrophages for a single determination. In application of this technique, elevation of the superoxide-generating activity was shown with macrophages elicited by chemical inflammation or those obtained from mice after treatment with tubercle bacilli.

Animals↗