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 NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.
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PMID:Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. 132 Mar 78

Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.
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PMID:Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. 132 65

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.
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PMID:Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity. 132 33

Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, profoundly influenced the activity of the NADPH oxidase of human neutrophils. It strongly inhibited stimulation of superoxide generation by phorbol 12-myristate 13-acetate (PMA) and impaired translocation of protein kinase activity and of the two cytosolic components p47-phox and p67-phox to the plasma membrane. The increase in the phosphorylation of the cytochrome b-245 subunits p22-phox and gp91-phox after stimulation was also blocked. Inhibition of activity was associated with a decrease in cytosolic free Ca2+ and was reversed by the Ca2+ ionophore A23187, which also restored protein translocation and phosphorylation of the cytochrome. This effect of A23187 was itself blocked by preincubation with cyclosporin A, suggesting that calcineurin might be involved in the re-activation process. In contrast with PMA, the response to the bacterial peptide fMet-Leu-Phe was greatly prolonged after an initial decrease in the rate of onset of NADPH oxidase activity.
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PMID:Okadaic acid produces changes in phosphorylation and translocation of proteins and in intracellular calcium in human neutrophils. Relationship with the activation of the NADPH oxidase by different stimuli. 141 26

Evidence for the existence of an NADPH oxidase-like enzyme in rainbow trout macrophages is given. Reduced-minus-oxidised difference spectroscopy revealed the presence of a cytochrome b with three absorbance peaks, at 430, 533, and 558 nm. The low midpoint potential of the latter peak suggests this cytochrome is the same as the terminal component of NADPH oxidase (i.e., cytochrome b-245). Subcellular fractionation of macrophages revealed two peaks of cytochrome b activity, in accord with the concept of a plasma membrane localisation of cytochrome b activity in addition to a mitochondrial localisation. Finally, that the rainbow trout oxidase is a multicomponent enzyme was suggested by inhibitor studies, where specific inhibitors of the flavin and cytochrome b-245 components of NADPH oxidase induced significant reduction in superoxide anion production.
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PMID:NADPH oxidase-like activity in rainbow trout Oncorhynchus mykiss (Walbaum) macrophages. 142 92

Chronic Granulomatous Disease (CGD) manifests as a predisposition to infection as a result of defective function of the NADPH oxidase of phagocytic cells. Proteins identified as part of this system include two subunits of a cytochrome b (cytochrome b-245) and two cytosolic factors. The affected oxidase component was determined in 63 CGD patients from 57 families, by Western blotting of extracts of their neutrophils with antibodies to those proteins. 38 (67%) of the families were X-linked with a defect of the beta subunit of the cytochrome. 13 (23%) lacked p47-phox, 3 (5%) p67-phox, and 3 (5%) the alpha subunit of the cytochrome.
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PMID:Identification of the defective NADPH-oxidase component in chronic granulomatous disease: a study of 57 European families. 163 35

Professional phagocytes (neutrophils, eosinophils, monocytes and macrophages) possess an enzymatic complex, the NADPH oxidase, which is able to catalyze the one-electron reduction of molecular oxygen to superoxide, O2-. The NADPH oxidase is dormant in non-activated phagocytes. It is suddenly activated upon exposure of phagocytes to the appropriate stimuli and thereby contributes to the microbicidal activity of these cells. Oxidase activation in phagocytes involves the assembly, in the plasma membrane, of membrane-bound and cytosolic components of the oxidase complex, which were diassembled in the resting state. One of the membrane-bound components in resting phagocytes has been identified as a low-potential b-type cytochrome, a heterodimer composed of two subunits of 22-kDa and 91-kDa. The link between NADPH and cytochrome b is probably a flavoprotein whose subcellular localization in resting phagocytes remains to be determined. Genetic defects in the cytochrome b subunits and in the cytosolic factors have been shown to be the molecular basis of chronic granulomatous disease, a group of inherited disorders in the host defense, characterized by severe, recurrent bacterial and fungal infections in which phagocytic cells fail to generate O2- upon stimulation. The present review is focused on recent data concerning the signaling pathway which leads to oxidase activation, including specific receptors, the production of second messengers, the organization of the oxidase complex and the molecular defects responsible for granulomatous disease.
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PMID:The superoxide-generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects. 165 1

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the participation of both membrane and cytosolic components. We reported that ammonium sulfate fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143, 4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of cytosol in two fractions (sigma 1 and sigma 2) that support O2- production by solubilized membrane synergistically. We now describe the purification of sigma 1 to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction sigma 2. Sigma 1 was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether sigma 1 was added to cell-free systems composed of: (a) solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b) purified cytochrome b559, incorporated in liposomes, and purified sigma 2. Sigma 1 was purified by a sequence comprising ammonium sulfate fractionation, hydrophobic chromatography on phenyl-Superose, absorption with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sigma 1 of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the presence of both 24- and 22-kDa bands. We suggest that, in its native form, sigma 1 might represent a complex of the 24- and 22-kDa proteins. The specific roles of each molecule in NADPH oxidase function remain to be determined.
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PMID:Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages. 166 Aug 77

The superoxide (O2-) forming NADPH oxidase complex of resting phagocytes can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). For O2- production to occur, the participation of both membrane-associated and cytosol-derived components is required. The purpose of this investigation was to isolate and characterize the membrane component of NADPH oxidase. For this purpose, guinea pig macrophage membranes were extracted with 1 M NaCl, solubilized by 40 mM octyl glucoside, and subjected to a purification sequence consisting of absorption with DEAE-Sepharose, affinity chromatography on heparin-agarose, and chromatography on hydroxylapatite. At each purification step, fractions were assayed for their ability to support SDS-elicited, cytosol-dependent O2- production, following incorporation in liposomes of phosphatidylcholine. We found that membrane oxidase activity copurified strictly with cytochrome b559. Peak hydroxylapatite fractions exhibited specific O2(-)-forming activity in the range of 81-115 mumol of O2-/mg protein/min and a specific cytochrome b559 content of 7-14 nmol of cytochrome b559/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the peak oxidase activity fractions, derived by hydroxylapatite chromatography, revealed essentially two bands that were identified as the beta (54-60 kDa) and alpha (21/22 kDa) subunits of guinea pig cytochrome b559. The relation of the two polypeptides to cytochrome b559 was established by correlation with a spectral signal characteristic of cytochrome b559, immunoblotting with antibodies against defined human cytochrome b559 beta and alpha chain peptides, cross-linking studies, and deglycosylation experiments. Hydroxylapatite-purified membrane oxidase preparations did not contain FAD and were free of cytochrome c reductase activity. Purified membrane oxidase preparations were also capable of cooperating with purified cytosolic components in SDS-elicited cell-free O2- production. We conclude that the membrane-associated component of the O2- generating NADPH oxidase is identical to cytochrome b559.
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PMID:The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559. 184 35

The capacity to generate superoxide anion (O2-) can be induced in U937 cells by various agents known to cause myeloid cell differentiation. Other reported differentiation events include diminished cell proliferation and the induction by gamma-interferon (IFN gamma) of Fc receptors for immunoglobulin G1 (Fc gamma RI). In this study, we differentiated U937 cells and high Fc gamma RI-expression mutants of U937 cells by treating them with IFN gamma. We compared the time courses over which surface Fc gamma RI became maximal, NADPH oxidase activity was induced, and the antiproliferative effect of IFN gamma was detected. Oxidase activity was measured by stimulating cells with PMA or by activating surface Fc gamma RI using aggregated human IgG1 or second antibody crosslinking of mAb 32/Fc gamma RI complexes. We found that IFN gamma in the absence of additional lymphokines induced high levels of oxidase activity in maximally differentiated U937 cells with even higher levels in the fully differentiated high-Fc gamma RI expression mutants (greater than 8 nmoles/10(6) cells/min for A12.13 cells). Over the course of differentiation, maximal induced levels of Fc gamma RI were reached after 1 to 2 days of IFN gamma treatment, prior to the antiproliferative effect of the lymphokine. In contrast, oxidase activity was induced after a lag of approximately 2 days, becoming maximal only after 4 to 6 days of IFN gamma treatment. This comparison of the induction of Fc gamma RI with that of oxidase activity triggered through Fc gamma RI indicated that the rapid increase of surface receptor was not accompanied by a completion of the pathway of Fc gamma RI-mediated oxidase activity. However, the time courses of induction detected by PMA and Fc gamma RI-agonists were coincident suggesting that the development of oxidative capacity could be due to the induction of components required by both the PMA- and surface receptor-mediated pathways. There are several oxidase components that are known to be IFN gamma-inducible, such as the oxidase flavoprotein, a b558 cytochrome peptide, and oxidase-requiring cytosolic components, and it is possible that one or a set of these components could be the limiting factor(s) for IFN gamma-induced oxidase activity.
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PMID:Functional comparison of the inductions of NADPH oxidase activity and Fc gamma RI in IFN gamma-treated U937 cells. 216 Jun 4


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