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S Minakami

Publications and source records attributed to S Minakami.

At least 91 records · Page 5Linked to original sources
PubMed1985

Ubiquinone-5 is reduced by superoxide in the aerobic state by NADPH oxidase of guinea pig macrophages.

The reduction of ubiquinone-5 (Q1) by the phagocytosis-specific NADPH oxidase of guinea pig macrophages was not inhibited by superoxide dismutase (SOD) at concentrations usually used for O2- assay but was inhibited at about 100-times higher concentrations. Titration of the reaction with SOD and a comparison with that of xanthine oxidase showed that the inhibition was not due to the semiquinone oxidation accelerated by a removal of O2- but due to the accelerated dismutation of O2- which otherwise reduces the quinone. Molecular oxygens are therefore preferential electron acceptors in the NADPH oxidase even in the presence of Q1.

Aerobiosis↗
PubMed1985

The role of Ca2+ and Ca2+-activated phospholipid-dependent protein kinase in degranulation of human neutrophils.

The degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.

Bucladesine↗
PubMed1984

A cell-free preparation of human neutrophils catalyzing NADPH-dependent conversion of leukotriene B4.

The sonicate of human neutrophils converted leukotriene B4 to a polar product in aerobic condition in the presence of NADPH at a rate comparable to that of the intact cells. NADH could scarcely replace NADPH. The conversion was not observed in anaerobic conditions and was inhibited by carbon monoxide (CO/O2 = 4/1) or by 1 mM p-chlormercuribenzoate, while it was not affected by 1 mM KCN, 5 mM NaN3, 200 micrograms/ml catalase, 100 mM mannitol, and 10 micrograms/ml superoxide dismutase. These observations suggest that the myeloperoxidase-H2O2-halide system and active oxygen species are not involved in the reaction. The activity was observed in the 100,000xg supernatant from the homogenate, in which cytochrome P-450 was not detected.

Humans↗
PubMed1984

Protein phosphorylation in intact pig leukocytes.

The phosphorylation of proteins in intact pig polymorphonuclear leukocytes loaded with H3(32)PO4 was investigated by two-dimensional gel electrophoresis and subsequent autoradiography. The incorporation of 32P into at least 17 proteins began to increase and into one to decrease, relative to resting cells, upon exposure of the cells to phorbol 12-myristate 13-acetate. These changes in the autoradiographic patterns were accompanied by changes in the protein patterns obtained by staining with Coomassie brilliant blue, including the appearance, the acidic shift and the increase or decrease of the intensity of the spots. Among these proteins, Mr = 64 000, 31 000, 22 000, 21 000, 18 000 and 13 000 proteins were correlated well with the superoxide anion production of the cells in respect to the time-courses and the dose-responses. By taking the effects of EGTA into consideration, the phosphorylation of Mr 64 000 and 21 000 proteins, of which the latter was identified as the light chain of myosin, seemed to be involved in the signal-transmission mechanism of the induction of the NADPH oxidase responsible for the 'respiratory burst'. These two proteins were also phosphorylated in the cells stimulated by NaF or oil droplets opsonized with IgG.

Animals↗
PubMed1984

Superoxide release and intracellular free calcium of calcium-depleted human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine.

The superoxide release and the change in the intracellular free calcium ions on stimulation with N-formyl-methionyl-leucyl-phenylalanine were studied in human neutrophils deprived of divalent cations by treatment of the cells with ionophore A23187 in the presence of EGTA. The depleted cells showed no release of superoxide on stimulation with the chemotactic peptide when calcium ions were absent in the medium, but the activity was completely recovered when the cells were preincubated with calcium for at least 3 min before the stimulation. The cells pretreated with Cd2+ showed slight activity of the release, but no recovery was observed with other divalent cations such as Mg2+, Sr2+, Co2+, Ba2+ and Zn2+. The recovery with calcium ions was dependent on the time of the addition relative to the time of the stimulation with the chemotactic peptide: a simultaneous addition of both calcium and the peptide elicited about half of the full activity, while no release was observed when calcium was added later than 2 min after the stimulation with the peptide, though a marked elevation of the intracellular free calcium monitored by quin-2 fluorescence was found. Comparison of the time-courses of the superoxide release and the change in the fluorescence suggest that, besides the elevation of intracellular free calcium, a transient reaction which is also dependent on calcium is required for the full induction of the superoxide-producing activity.

Aminoquinolines↗
PubMed1984

Diacylglycerol, 1-oleoyl-2-acetyl-glycerol, stimulates superoxide-generation from human neutrophils.

1-Oleoyl-2-acetyl-glycerol which activates Ca2+-activated phospholipid-dependent protein kinase, induced the superoxide-production of human neutrophils, while other diacylglycerols did not. The induction was independent of extracellular calcium and did not accompany the increase of the intracellular free calcium. The superoxide-release by the diacylglycerol was inhibited by retinal, the inhibitor of the protein kinase. The diacylglycerol stimulated the phosphorylation of at least 4 proteins in intact neutrophils, the phosphorylation of which was stimulated by phorbol 12-myristate 13-acetate, the activator of the protein kinase. These observations indicate the possible involvement of the kinase in the induction process.

Diglycerides↗
PubMed1984

Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B4.

Leukotriene B4 stimulated a transient production of superoxide anions (O2-) by human polymorphonuclear leukocytes which continued for only about 1 min. The production was dependent on Ca2+ in the suspending medium and no production was observed without the addition of calcium. The concentrations of leukotriene B4 and calcium for the half-maximal production were about 1 microM and 200 microM, respectively. 8-(N,N,-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist, did not inhibit the O2- production stimulated by leukotriene B4 in the presence of calcium, while N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, did. When leukotriene B4 was added to the cells treated with cytochalasin B, the production of O2- was biphasic: an initial rapid phase, followed by a slow one. The slow phase was also dependent on Ca2+ concentrations but it could be induced even without the addition of Ca2+ to the medium. The cells treated with both cytochalasin B and TMB-8 in Ca2+-free medium showed a negligible production of superoxide on addition of leukotriene B4, but the production appeared upon addition of CaCl2. These findings suggest that the superoxide production stimulated by leukotriene B4 is associated with the influx of Ca2+.

Animals↗
PubMed1984

NADPH-dependent reduction of ubiquinone-1 associated with the superoxide-forming oxidase of pig polymorphonuclear leucocytes.

NADPH-dependent ubiquinone-1 reductase activity was present in the phagocytic vesicles of pig polymorphonuclear leucocytes. The apparent Km-value of the reductase for NADPH was 29 microM which is similar to that of the NADPH-dependent superoxide formation. Increase of the quinone-reductase activity by increasing the concentrations of ubiquinone-1 was associated with the decrease of the superoxide forming activity, the rate of the NADPH oxidation being constant independent of the quinone concentration. p-Chloromercuribenzoate inhibited both superoxide formation and reduction of the quinone, whereas low concentrations of cetyltrimethylammonium bromide which inhibit the superoxide formation did not inhibit the reduction of the quinone. The reduction of 2,6-dichlorophenolindophenol which has been shown not to be inhibited by both inhibitors. The quinone-reductase activity could be extracted with a mixture of deoxycholate and Tween 20 which extracts the superoxide forming activity. The observations indicate that a region of the superoxide-forming NADPH oxidase between a mercurial-sensitive site and a site sensitive to the cationic detergent is responsible for the reduction of ubiquinone.

Animals↗
PubMed1984

Phosphorylation of myosin light chain in intact pig leukocytes stimulated by phorbol 12-myristate 13-acetate.

Phosphorylation of myosin light chain was investigated in intact pig polymorphonuclear leukocytes. The labeling of the myosin light chain (Mr = 21000) was increased by exposure of the cells to phorbol 12-myristate 13-acetate. Two-dimensional gel electrophoresis revealed three forms of Mr = 21000 light chain, of which two were phosphorylated. The phosphorylation of the myosin light chain was inhibited by neither N-(6-aminohexyl)-5-chloro-1-naphthalene sufonamide nor trifluoperazine, suggesting that calmodulin is not involved in the phosphorylation.

Animals↗
PubMed1984

Oxygen metabolism of human colostral macrophages: comparison with monocytes and polymorphonuclear leukocytes.

Human colostral macrophages stimulated by opsonized zymosan or phorbol myristate acetate (PMA) released superoxide anions (O2-) and hydrogen peroxide (H2O2) with activities comparable to those of monocytes and about one-fourth of those of polymorphonuclear leukocytes (PMNL) of blood. The O2- -forming oxidase in the macrophages stimulated by PMA was dependent on NADPH as an electron donor with an apparent Km value for NADPH of 27.6 +/- 4.0 microM, which is comparable to those obtained for the stimulated monocytes and PMNL of blood. The Vmax was 1.86 +/- 0.33 nmol O2/min/10(6) cells, which is essentially the same as that of monocytes and about half of that of PMNL. p-Chloromercuribenzoate or cetyltrimethylammonium bromide completely inhibited oxidases of all three types of phagocytes. A b-type cytochrome was identified in the macrophages but the concentrations in the macrophages and monocytes were less than half of that in PMNL. These results suggest that the differences in the O2- -forming activities of the three types of phagocytes are quantitative rather than qualitative. The macrophages and monocytes showed very low activities of myeloperoxidase [EC 1.11.1.7] in contrast to PMNL. The activity of beta-glucuronidase [EC 3.2.1.31] in the macrophages was much higher than those of the monocytes and PMNL, but little difference was observed in the activities of lysozyme [EC 3.2.1.17], catalase [EC 1.11.1.6] and superoxide dismutase [EC 1.15.1.1] among the three types of phagocytes examined. Electron micrographs of the macrophages showed little increase of vacuoles upon exposure to PMA, in contrast to the cases of monocytes and PMNL.

Catalase↗
PubMed1983

Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats.

Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.

Animals↗
PubMed1983

Affinity labeling of erythrocyte band 3 protein with pyridoxal 5-phosphate. Involvement of the 35,000-dalton fragment in anion transport.

Transport of pyridoxal 5-phosphate (PLP) into erythrocytes was inhibited by inhibitors of anion transport including stilbene disulfonate compounds, indicating that it is mediated by Band 3 protein. When erythrocytes were treated with PLP and large amounts of free lysine and NaBH4, two membrane-spanning fragments of Band 3 (Mr = 17,000 and 35,000) were specifically labeled. When the cells were pretreated with 4,4'-dinitrostilbene 2,2'-disulfonate, the labeling in the 35,000-dalton fragment was inhibited. Erythrocytes labeled by PLP in both the 17,000- and 35,000-dalton fragments transported PLP at a decreased rate, whereas the cells labeled in only the 17,000-dalton fragment had essentially the same transport activity as the control when 4,4'-dinitrostilbene 2,2'-disulfonate was removed. The extent of inhibition of transport of inorganic phosphate in the labeled cells was similar to that of PLP. The results indicate that the 35,000-dalton fragment participates in the anion transport of the cell membrane.

Affinity Labels↗
PubMed1983

NADPH-dependent reduction of 2,6-dichlorophenol-indophenol by the phagocytic vesicles of pig polymorphonuclear leucocytes.

NADPH-dependent 2,6-dichlorophenol-indophenol (DCIP) reductase activity in the homogenate of phagocytosing pig polymorphonuclear leucocytes was twice that of the resting cells and the activity in the phagocytic vesicles corresponded to the activity increment due to phagocytosis. The apparent Km value of the reductase activity in the vesicles for NADPH was 30 microM, which is similar to that of the NADPH-dependent superoxide (O2-) formation. Increasing the DCIP reductase activity by increasing the DCIP concentration caused a decrease in the O2- -forming activity, the NADPH oxidation rate being constant and independent of the dye concentration. p-Chloromercuribenzoate and cetyltrimethylammonium bromide at low concentrations inhibited the O2- -forming activity of the vesicles without inhibiting the DCIP reductase. Quinacrine inhibited both O2- formation and DCIP reduction. The DCIP reductase activity could be extracted with a mixture of deoxycholate and Tween-20, which extracts the O2- -forming activity. The reductase activity in the extract was enhanced 2-fold by the addition of FAD, and its apparent Km was 0.085 microM. These results indicate that the NADPH-dependent DCIP reductase activity of the phagocytic vesicles is catalysed by a flavin-containing component of the O2- -forming system.

2,6-Dichloroindophenol↗
PubMed1983

Ribose. An oxidation product of glucose 6-phosphate in microsomal fraction.

An oxidative metabolism of glucose 6-phosphate was studied in rat liver microsomal fraction. Although radioactive 14CO2 was formed from [1-14C]glucose 6-phosphate in the microsomal fraction (Hino, Y., and Minakami, S. (1982) J. Biochem. (Tokyo) 92, 547-557), the formation was negligible when [2-14C]glucose 6-phosphate was used as a starting substrate. These results indicated an inability of the microsomal fraction to rearrange [2-14C]glucose 6-phosphate to form [1-14C] glucose 6-phosphate, and it was expected that a certain compound derived from glucose 6-phosphate accumulated as an end-product of the reaction. We, therefore, have tried to identify the product by high performance liquid chromatography, and found that ribose accumulated as the end-product. The formation of ribose was inhibited in the same manner as that of 14CO2 by antibodies against rat liver microsomal hexose-6-phosphate dehydrogenase, and the ratios of ribose to 14CO2 formed in the reaction were 0.5-0.8 on a molar basis. The finding of ribose formation further suggested the involvement of ribose phosphate isomerase and phosphatase activities in the reaction.

Animals↗
PubMed1983

Human NADH-cytochrome b5 reductases: comparison among those of erythrocyte membrane, erythrocyte cytosol, and liver microsomes.

NADH-cytochrome b5 reductases purified from human red cell membranes and cytosol were compared with those prepared from human liver microsomes. Minimal molecular weights of the membrane and the cytosol enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were 36,000 and 32,000 daltons, respectively, which are comparable to those of the detergent-solubilized reductase (dfp) and the protease-solubilized one (tfp) of liver microsomes, respectively. All the enzymes contained FAD and had essentially the same turnover numbers and apparent Km values for NADH and protease-solubilized cytochrome b5. The membrane enzyme and liver dfp reduced cytochrome c in the presence of detergent-solubilized cytochrome b5 70-80 times faster than in the presence of trypsin-solubilized cytochrome b5, whereas the cytosol enzyme and liver tfp showed essentially the same low activities with both preparations of cytochrome b5. SDS-PAGE mapping of the limited proteolytic products of the reductases obtained by digestion with staphylococcal protease or a-chymotrypsin showed essentially the same patterns of peptides between the red cell membrane enzyme and liver dfp and between the red cell cytosol enzyme and liver tfp. These results suggest that the NADH-cytochrome b5 reductase of human red cell membranes is identical with that of liver microsomes and that the enzyme of red cell cytosol is a proteolytic product of the membrane enzyme.

Cytochrome Reductases↗
PubMed1983

Heat production of pig platelets in relation with glycolysis and respiration.

Heat production of pig platelets was measured on a flow microcalorimeter with simultaneous measurements of the oxygen consumption and metabolite change of the suspension. The heat production associated with the glycolytic reaction was estimated from the decrease in the heat production caused by the addition of sodium fluoride. The glycolytic heat production was about -75 kJ per mol of lactate formed, which is the sum of the enthalpy values for the conversion of glucose to lactic acid and that for the neutralization of the acid. The heat production due to the respiration was estimated from the heat production of the cells at various pH. The respiratory heat production was about -475 kJ per mol oxygen consumed, which agrees with the enthalpy change for the non-phosphorylating respiration of mitochondria or for the complete oxidation of glucose or fatty acids. The heat production of the cells increased by the alkalinization of the medium, and the increase of the heat production was parallel with the increase in the lactate formation.

Animals↗