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)

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O-2 from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. In whole cells and under certain circumstances in the cell-free system the phosphorylation of p47phox mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47phox may be an important part of the activation mechanism. We show here that the total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47phox substantially decreased when p47phox was treated with anionic amphiphiles. A similar decrease in fluorescence was also observed when p47phox was phosphorylated with protein kinase C. Furthermore, a red shift of emission maximum and an increase of quenching by ionic quenchers and acrylamide were observed in the presence of activators. These results indicate the occurrence of a conformational change in the protein structure of p47phox. We propose that this alteration in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.
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PMID:Conformational changes of the leukocyte NADPH oxidase subunit p47(phox) during activation studied through its intrinsic fluorescence. 974 57

This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2- was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2- in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.
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PMID:Nicotinamide-adenine dinucleotide phosphate oxidase assembly and activation in EBV-transformed B lymphoblastoid cell lines of normal and chronic granulomatous disease patients. 979 33

Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.
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PMID:The Ku70 autoantigen interacts with p40phox in B lymphocytes. 991 62

The role of magnesium ions in the activation of NADPH oxidase has been investigated using flavocytochrome b-245 and either neutrophil cytosol or mixtures of recombinant p40phox, p47phox, p67phox and Rac2. Purified flavocytochrome b-245 is highly active (turnover number 120-150 mol of O2(-)/s per mol of cytochrome haem) in the absence of Mg2+, in marked contrast to neutrophil membranes or detergent-solubilized membranes, which have an absolute requirement for Mg2+ for NADPH oxidase activity. It was also found that Mg2+ affected the anionic amphiphile requirement for oxidase activation, and this was dependent on whether neutrophil cytosol or mixtures of recombinant cytosolic proteins were used in the assay. Unexpectedly we found that, using purified flavocytochrome b-245 and recombinant cytosolic proteins, NADPH oxidase undergoes spontaneous activation in the absence of anionic amphiphiles under Mg2+-free conditions. The results suggest that Mg2+ ions play an important role in NADPH oxidase function, perhaps stabilizing the 260 kDa complex of cytosolic phox proteins or the regulation of a guanine nucleotide-binding protein. We provide evidence that if the latter explanation is correct, the identity of the guanine nucleotide-binding protein is unlikely to be Rap1a.
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PMID:Spontaneous activation of NADPH oxidase in a cell-free system: unexpected multiple effects of magnesium ion concentrations. 993 20

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (approximately 5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.
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PMID:Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease. 1023 5

In addition to the extracellular production of O2- by NADPH oxidase in neutrophils stimulated by soluble stimuli, the intracellular formation of oxygen reactive species has been described. Cytochrome b559, the redox component of the NADPH oxidase complex, is mainly associated with specific granule membrane in resting neutrophils. We examined whether these granules could be a site for intracellular production of O2-. Phorbol myristate acetate (PMA)-stimulated neutrophils were fractionated by differential centrifugation, and generation of O2- was detected in both the granule and the plasma membrane-enriched fractions, but more in the granules. Translocation of p47phox and p67phox, two cytosolic components of the NADPH oxidase, was also quantitatively more important in the granules than in the plasma membrane fraction. After separation of the specific from the azurophil granules, p47phox and p67phox were found to be present only in the specific granules of PMA-activated cells. As a control, the production of O2- was studied in retinoic acid-differentiated NB4 cells that lack specific granules. During stimulation of NB4 cells with PMA, only the plasma membrane-enriched fraction was the site of O2- production. Together, these results indicate that NADPH oxidase can be functionally assembled in specific granules.
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PMID:NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils. 1033 91

Superoxide anions (O2-) are supposedly involved in the pathogenesis of endothelial dysfunction. We investigated whether the enhanced formation of O2- is involved in the attenuation of endothelium-dependent relaxation induced by lipopolysaccharide (LPS). Rats were injected with LPS (10 mg/kg IP), the aorta was removed after 12 or 30 hours, and generation of O2-, H2O2, and ONOO- was measured using chemiluminescence assays. Protein tyrosine nitration and expression of xanthine oxidase (XO), NAD(P)H oxidase, and manganese superoxide dismutase were determined by Western or Northern blotting, and endothelium-dependent relaxation in aortic rings was studied. LPS treatment increased vascular O2- (from 35+/-2 cpm/ring at baseline to 166+/-21 cpm/ring at 12 hours and 225+/-16 cpm/ring at 30 hours) and H2O2 formation, which was partially sensitive to the NAD(P)H oxidase inhibitor diphenylene iodonium at both time points studied and to the XO inhibitor oxypurinol only 30 hours after LPS treatment. Expression of XO and NAD(P)H oxidase (p22phox, p67phox, and gp91phox) were increased by LPS in a time-dependent manner, as were protein tyrosine nitration and ONOO- formation. LPS also induced expression of the oxidative stress-sensitive protein manganese superoxide dismutase. Endothelium-dependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase. Inhibition of O2- with superoxide dismutase, oxypurinol, tiron, or the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride did not restore but further deteriorated the relaxation of LPS-treated rings. In summary, treatment of rats with LPS enhances vascular expression of XO and NAD(P)H oxidase and increases formation of O2- and ONOO-. Because removal of O2- compromised rather than restored endothelium-dependent relaxation, a direct role of O2- in the induction of endothelial dysfunction is unlikely. Other mechanisms, such as prolonged protein tyrosine nitration by peroxynitrite (which is formed from NO and O2-) or downregulation of the NO effector pathway, are more likely to be involved.
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PMID:Role of increased production of superoxide anions by NAD(P)H oxidase and xanthine oxidase in prolonged endotoxemia. 1033 19

p67phox and p47phox are phosphorylated in the course of stimulation of the NADPH oxidase in neutrophils. Isolated neutrophil cytosol can phosphorylate both of these proteins in vitro. Phosphoamino acid analysis showed that isolated membranes can tyrosine-phosphorylate p67phox in vitro. Further experiments with anti-phosphotyrosine antibodies did not support a role for tyrosine phosphorylation of p67phox in the cell. A phosphopeptide analysis showed that the phosphorylation of p67phox is unchanged in the absence of p47phox. These results further characterise the phosphorylation of p67phox and provide evidence that this is a cytosolic event independent of interaction with p47phox and the membrane.
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PMID:Phosphorylation of p67phox in the neutrophil occurs in the cytosol and is independent of p47phox. 1033 37

It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules.
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PMID:The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex. 1039 79

Phagocytic cells possess a tightly regulated multicomponent enzyme complex, the NADPH oxidase, which produces superoxide, a reactive oxygen molecule that is an essential component of host defense against infection. Upon stimulation, a functional NADPH oxidase is assembled when the cytosolic proteins, Rac, p67phox, p47phox, and possibly p40phox, associate with the gp91phox and p22phox transmembrane proteins. Rac is a GTPase that in the GTP-bound state binds p67phox to activate NADPH oxidase. The function of p40phox is not known; it is believed to have a regulatory function in sequestering p67phox and p47phox in a cytosolic complex. We investigated binding interactions between p40phox, p67phox, and Rac and found that Rac1-GTP displaced p67phox bound to p40phox. In contrast, Cdc42, a GTPase homologous to Rac, did not displace p67phox from p40phox. A synthetic peptide corresponding to p67phox amino acids 170-199, a region identified previously as a Rac binding domain, significantly reduced the ability of Rac1-GTP to disrupt p67phox/p40phox binding. We hypothesize that Rac-GTP binds the p67phox N-terminal domain encompassing amino acids 170-199 that transmits a conformational change which causes p40phox to dissociate from its binding site in the p67phox C-terminus.
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PMID:Rac1 disrupts p67phox/p40phox binding: a novel role for Rac in NADPH oxidase activation. 1048 63


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