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)

Recombinant interferon-gamma (rIFN-gamma) therapy has become an effective form of prophylaxis for patients with chronic granulomatous disease (CGD). Preliminary studies with CGD suggested that rIFN-gamma treatment enhanced phagocyte oxidase activity and increased superoxide (O2-) production. We evaluated several aspects of neutrophil NADPH oxidase activity in 19 CGD patients (representing all four known types of CGD) receiving prolonged rIFN-gamma therapy (6 to 27 months). In contrast to earlier studies, we failed to detect any improvement in neutrophil NADPH oxidase activity in 18 of the 19 CGD patients as determined by (1) intact cell O2- production (continuous assay), (2) nitroblue tetrazolium (NBT) staining, (3) cytochrome b558 spectroscopy, and (4) activity levels of cytosol and membrane oxidase components using a cell-free activation system. One patient with a variant form of X-linked CGD had a transient increase in neutrophil O2- production following 3 months of rIFN-gamma therapy. However, this was not sustained, and was not associated with any change in cytochrome b levels. In some patients, rIFN-gamma therapy was associated with the appearance of a small subset of circulating monocytes (1% to 20%) that were NBT-positive. Although the functional significance of this monocyte subpopulation needs to be determined, these results suggest that one possible mechanism by which rIFN-gamma may benefit CGD patients is by partially correcting the respiratory burst defect in a subset of monocytes. We conclude that the clinical benefit of prolonged rIFN-gamma therapy in the vast majority of CGD patients is not due to enhanced neutrophil NADPH oxidase activity. The mechanism of action of rIFN-gamma in most CGD patients remains unknown.
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PMID:Prolonged recombinant interferon-gamma therapy in chronic granulomatous disease: evidence against enhanced neutrophil oxidase activity. 131 72

Human B lymphocytes express components of the superoxide generating system of phagocytes, NADPH oxidase. We studied regulation of this 'B-cell oxidase' during in vitro blast transformation, using Lucigenin-amplified chemiluminescence (CL) to detect superoxide release. While freshly isolated tonsil B lymphocytes showed no CL responses, culture with phorbol myristate acetate (PMA) and ionomycin induced susceptibility to CL triggering by anti-IgM and anti-HLA-DR. Maximal effects were observed after 3 days of culture with 0.4 ng/ml PMA + 1 microgram/ml ionomycin. Cells from such B lymphoblast cultures showed no CL responses to opsonized zymosan. In contrast, peripheral blood mononuclear cells, where monocytes are the predominant oxidant source, showed CL responses to opsonized zymosan but not to anti-IgM and anti-HLA-DR, either before or after culture with PMA + ionomycin. Culture of B cells with the surface immunoglobulin cross-linking agent staphylococcus aureus Cowan I also led to emergence of a CL, response to anti-IgM, which was enhanced by interferon-gamma. Interestingly, markedly fewer B blasts than freshly isolated B lymphocytes expressed cytochrome b-558 surface antigen. Thus, the B-cell oxidase is up-regulated during blast transformation and can be triggered via surface IgM and HLA-DR; however, this appears to be restricted to a subset of B lymphoblasts.
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PMID:B lymphoblasts show oxidase activity in response to cross-linking of surface IgM and HLA-DR. 131 85

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

Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytes prepared from pig blood was carried out with concentrated cellular and subcellular fractions to characterize EPR signals of cytochrome b-558 (cyt b-558). A thick cell suspension (approximately 2 x 10(9) cells/ml), containing mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not only these signals but also low spin heme signals at g values of 2.86, 2.13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130-132). MPO and eosinophil peroxidase (EPO) were released from the membrane fractions with 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and then were highly concentrated, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O2(-)-forming NADPH oxidase associated in the membranes was solubilized with heptyl-thio-glucoside at 0 degree C and concentrated up to 45 microM cyt b-558 with no modification of the heme moiety confirmed by its O2(-)-generating activity and lack of carbon monoxide-binding capacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 +/- 0.05, which was cyanide-insensitive and reducible with reductants. The signal intensity was concentration dependent, suggesting that the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.
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PMID:Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pig blood granulocytes. 132 37

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

We have compared assays for products of the neutrophil respiratory burst in normal EBV-transformed B cell lines stimulated with agonists of protein kinase C. Those measuring O2- directly or its immediate product, H2O2, were successful. Of these, the most sensitive were the lucigenin- and luminol-based chemiluminescence assays for O2- and H2O2 respectively. Cell lines from CGD patients, with X-linked or autosomal recessive genetic defects in the neutrophil NADPH oxidase, did not respond in these assays, indicative of their inability to produce O2-. The defects in the lines studied encompass both proteins forming the cytochrome b-245 membrane component, and the 47 kDa cytosolic component of the NADPH oxidase. The possession of the disease associated phenotype by these cell lines provides evidence that in the normal situation both neutrophils and B cells produce O2- via the same system.
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PMID:Superoxide production by normal and chronic granulomatous disease (CGD) patient-derived EBV-transformed B cell lines measured by chemiluminescence-based assays. 133 Dec 41

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

Activation of the NADPH oxidase of phagocytes in the cell-free system requires the association of several cytosolic components with membrane-bound cytochrome b. In this study we were able to fully reconstitute NADPH oxidase activity in the cell-free system with three recombinant proteins: p67-phox, p47-phox, p21rac1, and pure cytochrome b-245. Activity was dependent upon the concentration of the proteins, with maximal activity observed with roughly equimolar ratios of the cytochrome b and p67-phox (133 and 163 mol/s/mol, respectively) and concentrations of the other two proteins approximately 1 order of magnitude greater. No activity was observed in the absence of any one of these components. In addition, activation was dependent upon p21rac1 being preloaded with GTP, the cytochrome b being reconstituted with lipid, and the presence of FAD during activation. Half-maximal activity was observed at a concentration of NADPH of approximately 50 microM. These findings confirm our recent description of the membrane-bound cytochrome b as a FAD-containing flavocytochrome b containing the NADPH binding site, and implicate the three cytosolic proteins in its activation.
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PMID:Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245. 151 17

The NADPH oxidase of phagocytic cells is an electron transport chain in the wall of the endocytic vacuole that is activated upon engulfment of the microbe, and is important for its efficient killing and digestion. The oxidase consists of a heterodimeric cytochrome b in the membrane, which is the site of the haem and FAD groups, and two cystolic factors p47-phox and p67-phox that appear to activate the system. Absence of this oxidase as a result of defects in each of these specialized proteins causes the syndrome of chronic granulomatous disease (CGD), that is characterized by a profound predisposition to pyogenic infection.
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PMID:Biochemistry and molecular biology of chronic granulomatous disease. 152 26


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