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)

Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.
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PMID:Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system. 165 5

A major action of the microbicidal system of human neutrophils is the formation of superoxide anion (O2-) by a multicomponent oxidase that transfers electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen. The mechanism of assembly and activation of the oxidase from its cytosolic and membrane-bound components is unknown, but may require the activity of a guanosine 5'-triphosphate (GTP)-binding component. A cytosolic GTP-binding protein (Gox) that regulates the NADPH oxidase of neutrophils was identified. Gox was purified and shown to augment the rate of O2- production in a cell-free oxidase activation system. Sequence analysis of peptide fragments from Gox identified it as Rac 2, a member of the Ras superfamily of GTP-binding proteins. Antibody to a peptide derived from the COOH-terminus of Rac 2 inhibited O2- generation in a concentration-dependent manner. These results suggest that Rac 2 is a regulatory component of the human neutrophil NADPH oxidase, and provide new insights into the mechanism by which this oxygen radical-generating system is regulated.
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PMID:Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. 166 Jan 88

The effects of anti-allergic drugs with or without H1-receptor antagonism on the NADPH oxidase from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Three anti-allergic drugs with H1-receptor antagonism, azelastine, ketotifen and oxatomide, were found to inhibit the superoxide generation of human neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of three drugs required for 50% inhibition of the oxidase (IC50) were as follows: azelastin--0.7 microM in the whole-cell system and 0.5 microM in the cell-free system, ketotifen--60 microM in the whole-cell system and 6.8 microM in the cell-free system, and oxatomide--25 microM in the whole-cell system and 9.7 microM in the cell-free system. In addition, in the cell-free system, these drugs did not change the Km values for the NADPH of the oxidase. However, micromoles of tranilast, an anti-allergic drug without H1-receptor antagonism, did not inhibit neutrophil NADPH oxidase in the whole-cell and cell-free systems. The IC50 of hydrocortisone in the cell-free system was 60 microM. These results suggest that anti-allergic drugs with H1-receptor antagonism inhibit reconstitution of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in cell-free systems and that they have a strong anti-inflammatory action. Anti-allergic drugs are not the drugs of first choice for asthma, but these drugs, especially basic anti-allergic drugs, should be used more frequently for the treatment of chronic asthma, infectious-typed asthma and mixed-typed asthma closely associated with acute and chronic inflammation of the airways as well as atopic asthma.
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PMID:[Biochemical study on anti-inflammatory action of anti-allergic drugs--with regard to NADPH oxidase]. 168 45

Neutrophils possess a plasma-membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which catalyzes superoxide (O2-) formation and is activated by a variety of stimuli. Recently, neutrophils of patients with essential hypertension (EHT) have been reported to generate O2- at rates up to fourfold higher than those of normotensive (NT) subjects upon exposure to the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). We studied regulation of O2- formation in neutrophils of 25 EHT subjects and 25 age- and sex-matched NT subjects. The intercellular signal molecules fMet-Leu-Phe, platelet-activating factor and leukotriene B4 activated O2- formation in neutrophils, but the latter two receptor agonists were less effective than the former. fMet-Leu-Phe activated O2- formation with a 50% effective concentration (EC50) of about 30 nmol/l, the effect of the chemotactic peptide being maximal at 0.1-1 mumol/l. fMet-Leu-Phe-induced O2- formation was potentiated by platelet-activating factor, adenosine 5'-[gamma-thio]triphosphate and cytochalasin B and was inhibited by the activators of adenylyl cyclase, isoproterenol, prostaglandin E1 and histamine. 4 beta-Phorbol 12-myristate 13-acetate, 1,2-dioctanoyl-sn-glycerol, gamma-hexachlorocyclohexane and arachidonic acid, which circumvent receptor stimulation, also activated O2- formation. Significant differences between NT and EHT subjects were not evident in respect of any of the parameters studied. Our data suggest that regulation of the neutrophil NADPH oxidase is not disturbed in EHT and that altered O2- formation does not represent a genetic marker for abnormalities in plasma-membrane signal transduction in EHT.
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PMID:Regulation of the superoxide-forming NADPH oxidase of human neutrophils is not altered in essential hypertension. 184 30

NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocytes or in a reconstituted system consisting of membranes, cytosolic components and arachidonate or SDS. To delineate mechanism of oxidase activation in the cell-free system, hydrolysis of phosphoinositides in the combined membrane-cytosol oxidase mixture was investigated. Arachidonate promoted hydrolysis of membrane-[3H]-phosphatidylinositol by cytosolic phospholipase C. PI hydrolysis was similarly supported by other unsaturated fatty acids and by SDS. Unlike activation of the NADPH oxidase, PI hydrolysis required the presence of calcium ions. Implications of these findings to the mechanism of NADPH oxidase activation are discussed.
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PMID:Arachidonate supports hydrolysis of phosphatidylinositol by neutrophil cytosolic phospholipase C: relation to NADPH oxidase. 190 42

Two of the cytosolic NADPH oxidase components, p47-phox and p67-phox, translocate to the plasma membrane in normal neutrophils stimulated with phorbol myristate acetate (PMA). We have now studied the translocation process in neutrophils of patients with chronic granulomatous disease (CGD), an inherited syndrome in which the oxidase system fails to produce superoxide due to lesions affecting any one of its four known components: the gp91-phox and p22-phox subunits of cytochrome b558 (the membrane-bound terminal electron transporter of the oxidase), p47-phox, and p67-phox. In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b558. In one patient with a rare X-linked form of CGD caused by a Pro----His substitution in gp91-phox, but whose neutrophils have normal levels of this mutant cytochrome b558, translocation was normal. In two patients with p47-phox deficiency, p67-phox failed to translocate, whereas p47-phox was detected in the particulate fraction of PMA-stimulated neutrophils from two patients deficient in p67-phox. Our data suggest that cytochrome b558 or a closely linked factor provides an essential membrane docking site for the cytosolic oxidase components and that it is p47-phox that mediates the assembly of these components on the membrane.
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PMID:Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558. 198 7

The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole cell and fully soluble (cell-free) systems were investigated. Three NSAIDs, indomethacin, salicylic acid and acetylsalicylic acid (aspirin), were found to inhibit the superoxide generation by human neutrophils exposed to phorbol myristate acetate in a whole cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. Concentrations of these NSAIDs required for 50% inhibition of the oxidase (IC50) were: indomethacin (180 microM in both systems), salicylic acid (1.30 mM in the cell-free system, and more than 3.0 mM in the whole cell system) and acetylsalicylic acid (1.35 mM in the cell-free system, and more than 3.0 mM in the whole cell system). In addition, in the cell-free system, these NSAIDs did not change the Km values for NADPH of the oxidase. These results suggest that these NSAIDs, especially indomethacin, concentration-dependently inhibit the reconstitution of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in the cell-free system.
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PMID:Effects of non-steroidal anti-inflammatory drugs on human neutrophil NADPH oxidase in both whole cell and cell-free systems. 216 17

Activation of the phagocyte NADPH oxidase requires participation of membrane-bound cytochrome b558 and cytosol proteins of 47 kDa (p47) and 67 kDa (p67). We examined the sequence of participation of p47 and p67 in activation of the oxidase using an arachidonate-activated cell-free superoxidase (O2-) generating assay requiring phagocyte membrane and cytosol. Neutrophil cytosol from patients with certain forms of autosomal recessive chronic granulomatous disease (CGD) lack either p47 or p67. Initial incubation of membrane and arachidonate with CGD cytosol deficient in either p47 or p67 fails to generate superoxide in the cell-free assay until addition of complementary cytosol. CGD cytosol was incubated with arachidonate and membrane for 5-15 min and the lag time of O2- generation was measured after addition of complementary CGD cytosol. The lag time is shortened when p47, but not p67, is present in the initial incubation. We have previously shown that the peptide, RGVHFIF, corresponding to a cytoplasmic carboxyl-terminal domain of the large subunit of cytochrome b558, inhibits activation of NADPH oxidase in the cell-free assay, but does not affect the enzyme activity of fully assembled oxidase. Experiments with sequential addition of complementary CGD cytosols were performed as above, except that RGVHFIF was added after the initial incubation. The peptide failed to inhibit when added after initial incubation if p47 was present during that incubation. In contrast, the peptide markedly inhibited oxidase activity if p47 was absent during the initial incubation. These results suggest that p47, but not p67, is a participant with membrane and/or other cytosol components in early arachidonate-dependent reactions. In the absence of p67, these reactions culminate in the irreversible formation of a metastable activation intermediate that is insensitive to inhibition by RGVHFIF. After addition of p67, this activation intermediate subsequently reacts to form the active NADPH oxidase.
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PMID:The phagocyte 47-kilodalton cytosolic oxidase protein is an early reactant in activation of the respiratory burst. 216 17

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.
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PMID:Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1). 239 22

The plasma membrane-bound NADPH-cytochrome c reductase of guinea pig macrophages (M phi) was found to be phosphorylated when [32P]phosphate-labeled cells were stimulated with 12-phorbol 13-myristate acetate (PMA). The time course of phosphorylation was parallel to that of O2- -generating activity elicited. These results suggest that the reductase participates as a flavoprotein in activation of the respiratory burst NADPH oxidase, when phosphorylated.
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PMID:Phosphorylation of NADPH-cytochrome c reductase in guinea pig peritoneal macrophages stimulated with phorbol myristate acetate. 253 48


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