Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Superoxide production by phagocytic blood cells involves assembly of an active NADPH oxidase complex from components found both in membrane and cytosolic locations in resting cells. We recently cloned cDNAs encoding two cytosolic components (p47-phox and p67-phox) of the oxidase that are deficient in distinct forms of autosomal recessive chronic granulomatous disease. The precise roles of p47-phox and p67-phox were explored further using purified factors produced in large quantities using recombinant baculoviruses to infect cultured Sf9 insect cells. Neither p47-phox nor p67-phox are thought to represent the flavoprotein components of the oxidase, since neither of the purified recombinant factors contained or bound FAD. Recombinant p47-phox and p67-phox are capable of restoring the deficient cytosol from chronic granulomatous disease patient neutrophils to nearly normal levels in a cell-free reconstitution system. Both p47-phox and p67-phox, used together in the absence of neutrophil cytosol, are incapable of supporting cell free production of superoxide, confirming the involvement of other soluble factor(s) in the assembly of an active oxidase in vitro.
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PMID:Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses. 191 85

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.
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PMID:Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid. 215 45

The rat carotid body tissue reveals a photometrically measurable haem signal with absorbance maxima at 560 nm, 518 nm and 425 nm, suggesting the presence of a b-type cytochrome; this was confirmed by pyridine haemochrome and CO spectra. The quantity of cytochrome b was estimated to be 310 pmol.mg of protein-1. This haem is capable of H2O2 formation, which can be inhibited by 10 microM-diphenyliodonium (DPI). The hypoxia-induced increase in nervous chemoreceptor discharge and the reduction of FAD and NAD(P)+ were also inhibited by DPI (10 microM). These results suggest that an oxidase such as the NAD(P)H oxidase of neutrophils may act as a pO2 sensor protein in the rat carotid body, probably inducing the pO2 chemoreceptor process by H2O2 formation.
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PMID:Involvement of an NAD(P)H oxidase as a pO2 sensor protein in the rat carotid body. 226 99

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH----FAD----cytochrome b-245----O2.
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PMID:Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase. 255 3

The NADPH-dependent superoxide production induced by sodium dodecyl sulfate (SDS) in the sonicates of unstimulated pig neutrophils required both membrane fraction and two components of cytosol fraction. The potency of the cytosol fraction in the activation of the superoxide production could be reconstituted dose dependently by mixing two protein components with relative molecular masses of 300 kDa and 50 kDa. Another low-molecular-mass component (1.3 kDa) could substitute the 50-kDa component. In the cell-free system consisting of the 300- and 50-kDa components and the membrane fraction, the superoxide production was markedly enhanced by FAD with a required concentration for half-maximal effect of 0.16 microM and inhibited by divalent cations such as Ca2+, Ba2+, Co2+, Zn2+ and Mn2+ and not Mg2+. ATP was not necessary for the activation, indicating that protein kinases such as protein kinase C are not involved in the SDS-dependent activation of NADPH oxidase. The NADPH oxidase activated by SDS in the cell-free system was recovered in the membrane fraction, and the superoxide formation by the SDS-activated membrane exhibited a Km value for NADPH of 46 microM and optimum pH at 7.0. The formation did not require the addition of SDS and FAD to the reaction mixture and was scarcely inhibited by the divalent cations.
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PMID:Characterization of the NADPH-dependent superoxide production activated by sodium dodecyl sulfate in a cell-free system of pig neutrophils. 282 May 10

The cytosolic component of macrophage-derived superoxide generating NADPH oxidase was partially purified by affinity chromatography on 2',5'-ADP-agarose. Elution was nonspecific by elevated phosphate molarity. A single step attains at least 40-fold enrichment of specific activity, the recovery being over 20%. Elution with various ligands in the concentration range 2-3.5 mM was also tested. The most effective ligands were: ATP, dATP, GTP, NADPH and 2',5'-ADP. Ineffective were AMP, 2'-AMP, FMN, FAD and NADH. ADP was of medium potency. On the basis of the above and other results, we infer that the molecule (or complex) purified by us may contain the enzymatic NADPH binding site. This component is fully retained by a 100 kDa cutoff membrane and is labile at room temperature, the lability being cancelled by 2-mercaptoethanol.
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PMID:Macrophage-derived superoxide-generating NADPH oxidase in an amphiphile-activated, cell-free system; partial purification of the cytosolic component and evidence that it may contain the NADPH binding site. 282 78

NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.
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PMID:The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils. 284 66

Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.
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PMID:Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme. 285 Feb 66

The chemical composition, properties and activation mechanism of the O2(-)-forming NADPH oxidase of phagocytes were investigated, using partially purified enzyme preparations. Highly active NADPH oxidase was extracted as an aggregate of high Mr from the membranes of neutrophils and macrophages. The enzyme complex contained phospholipids and cytochrome b-245, very little FAD and almost no quinones or NAD(P)H-dye reductase activity. The purification of a polypeptide with a relative molecular mass of 31 500 strictly paralleled the purification of NADPH oxidase, suggesting that this polypeptide is a component of the enzyme. This protein was identified as cytochrome b -245 after dissociation of the proteolipid complex and purification of the cytochrome moiety. The 31 500 Mr protein was phosphorylated in enzyme preparations from activated but not from resting cells. The results indicate that: cytochrome b-245 is a major component of NADPH oxidase; the involvement of NAD(P)H dye reductases in the O2(-)-forming activity is questionable; the cytochrome b-245: FAD ratio in the enzyme complex is much higher than that indicated in crude preparations; the Mr of pig neutrophil cytochrome b-245 is 31 500; the activation of the O-2-forming system involves a process of phosphorylation of cytochrome b-245.
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PMID:Respiratory response of phagocytes: terminal NADPH oxidase and the mechanisms of its activation. 301 13

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.
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PMID:NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system. 303 Oct 60


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