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)

In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp91-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H+ translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H+ extrusion. CGD cells expressing diminished amounts of cytochrome displayed H+ conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+. Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity.
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PMID:Activation of H+ conductance in neutrophils requires assembly of components of the respiratory burst oxidase but not its redox function. 816 76

Superoxide (O2-) production by neutrophils stimulated with chemotactic peptides [e.g., formylmethionyl-leucyl-phenylalanine (fMLP)] is transient but increases in rate and duration after pretreatment of the cells with dihydrocytochalasin B (dhCB), suggesting a possible role for the plasma membrane and membrane skeleton in the regulation of the O2- generating system. Analysis of plasma membrane isolated from these cells by isopycnic sucrose density gradient sedimentation showed that there were no significant variation in the distribution of plasma membrane markers between control and dhCB-treated cells, whereas a significant redistribution of plasma membrane markers was observed in dhCB + fMLP-treated cells. Instead of sedimenting to 31-35% sucrose, as in the former two groups, plasma membrane markers were broadly distributed over 25-50% sucrose in the dhCB + fMLP-treated cells. In addition, approximately 80% degranulation was achieved in these cells, whereas little granule release (< 5%) was observed in control and dhCB-treated cells. Analysis of the gradient fractions for membrane skeletal (actin and fodrin) and NADPH oxidase (cytochrome b and p47-phox) components in dhCB + fMLP-treated cells demonstrated that the distribution of fodrin, actin, cytochrome b, and p47-phox followed the broad distribution of plasma membrane markers, with an overall eightfold increase in membrane-associated actin. Despite the broad redistribution of plasma membrane markers, the distribution of O2- generating activity remained confined to a narrower peak at approximately 50% sucrose. These results demonstrate that a heterogeneous surface membrane of higher density with a differential distribution of proteins and O2- generating activity are created after dhCB + fMLP treatment; however, domain structure is conserved in the new membrane, with only a subfraction of the reorganized plasma membrane containing all of the components necessary for active O2- generation. Our results support a role for plasma membrane lateral organization and participation of the membrane skeleton in the regulation of the O2- generating system.
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PMID:Remodeling of the plasma membrane after stimulation of neutrophils with f-Met-Leu-Phe and dihydrocytochalasin B: identification of membrane subdomains containing NADPH oxidase activity. 819 93

Erythropoietin (Epo)-producing hepatoma cells (HepG2) reveal, in addition to the cytochromes of the respiratory chain, a photometrically measurable haem signal with absorbance maxima at 559 nm and 427 nm, suggesting the presence of a b-type cytochrome. This activity exhibited a low midpoint potential, CO-binding spectra and reduction which was insensitive to both cyanide and antimycin. This haem possessed a 22 kDa subunit and might be part of an electron transfer chain similar to the NADPH oxidase, since the NADPH oxidase cytosolic activating factor (p47) could be identified by Western blot analysis. H2O2, which was detected inside the cells by confocal microscopy, might therefore be produced by the suggested electron transfer chain. This cyanide- and antimycin-insensitive but hypoxia-sensitive cytochrome b would be an attractive candidate for controlled Epo production in response to pO2.
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PMID:Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2). 838 44

The O2- generating NADPH oxidase of human Epstein-Barr virus immortalized B lymphocytes (EBV-B lymphocytes) and the NADPH oxidase of human neutrophils were compared. The capacity of the oxidase of EBV-B lymphocytes to generate O2- is 100-fold less than that of neutrophils. Like the oxidase of neutrophils, the oxidase of EBV-B lymphocytes is decreased or abolished in chronic granulomatous disease (CGD). Activation of neutrophil oxidase in an heterologous cell-free system, using human neutrophil membranes and EBV-B lymphocyte cytosol from healthy and CGD patients, combined with immunoblotting investigations of the cytosolic activating factors p47 and p67 involved in O2- production, suggests that neutrophils and EBV-B lymphocytes possess similar complements of cytosolic factors p47 and p67. Cytochrome b -245, the major membrane redox component of the O2- generating oxidase, is only slightly expressed in the membrane of EBV-B lymphocytes. A sensitive and specific immunocytochemical method for detection of the two subunits of cytochrome b -245 is described; it shows that both subunits are virtually absent in EBV-B lymphocytes from CGD patients deficient in the large subunit.
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PMID:The O2- generating oxidase of B lymphocytes: Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulomatous disease. 839 41

Superoxide is produced by phagocytic cells at rates sufficient to have cytocidal effects. A wide variety of receptor-dependent and -independent agonists triggers this respiratory burst, including immunoglobin aggregates, complement fragments, and leukotriene B4. Lower rates of O2-. production are triggered by addition of specific cytokines into B-lymphocytes, endothelial cells, fibroblasts, and kidney mesangial cells; low concentration of radicals may act as signals for proliferation or other changes. The NADPH oxidase of phagocytes, characterized by the presence of FAD and a low potential cytochrome b, is organized to transfer electrons electrogenically across the plasma membrane from NADPH to O2. A proton channel permits movement of compensating H+.
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PMID:The mechanism of the production of superoxide by phagocytes. 839 50

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X-linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.
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PMID:X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox. 840 Feb 70

Activation of the superoxide-generating NADPH oxidase system of human neutrophils involves the assembly of several neutrophil components, some located on the plasma membrane and others in the cytosol. It has recently been established that one of the required components for NADPH oxidase activity is the GTP-binding protein Rac. To further investigate the role of Rac in the NADPH oxidase system, studies were carried out to determine its subcellular distribution in resting and activated human neutrophils. In resting cells, Rac and an associated guanine nucleotide regulatory factor, GDP dissociation inhibitor (GDI), were located only in the cytosol, along with other known oxidase factors, p47-phox and p67-phox. After activation of neutrophils with phorbol 12-myristate 13-acetate or formyl-methionyl-leucyl-phenylalanine, Rac was translocated from the cytosol to the plasma membrane, and this translocation corresponded temporally with the translocation of p47-phox and p67-phox and with the generation of superoxide. GDI remained localized to the cytosol, suggesting activation of the oxidase involved dissociation of the Rac-GDI complex prior to Rac translocation. Determination of the quantities of cytosolic factors associated with the plasma membrane indicated that Rac, p47-phox, and p67-phox are translocated to the plasma membrane simultaneously in equimolar amounts, but that the membrane-associated cytochrome b was present at 3-4-fold molar excess. These findings suggest that Rac may play a role in assembly of the active NADPH oxidase complex.
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PMID:Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. 840 34

The availability of sufficient quantities of highly purified phagocyte cytochrome b-558 has been necessary for many of the biochemical and immunological analyses of this important NADPH oxidase component, and it was only through the analysis of highly purified cytochrome b that the subunit composition was elucidated and the small subunit (p22-phox) was cloned and sequenced. In addition, the association of the small GTP-binding protein Rap1A with cytochrome b-558 was discovered through the analysis of purified cytochrome b. The procedures described here provide an easy, efficient, and highly reproducible method for the purification of cytochrome b as well as cytochrome b-Rap1A complexes. The ability to purify cytochrome b and cytochrome b-Rap1A complexes will also allow further analysis of the structure of this novel plasma membrane redox protein and the role of its association with low molecular weight GTP-binding proteins in the structure and regulation of the phagocyte NADPH oxidase.
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PMID:Purification of human neutrophil NADPH oxidase cytochrome b-558 and association with Rap 1A. 852 35

The immunochemical characterization of NADPH oxidase activity of cytochrome b558 purified from human neutrophils was determined after reconstitution in a cell-free assay using the native hemoprotein and recombinant purified cytosolic activating factors. The oxidase activity showed a strict dependence on the heme content at each step of the hemoprotein purification process. The immunochemical properties of the reconstituted oxidase made use of monoclonal antibodies raised against membrane-bound and octyl-glucoside-extracted cytochrome b. From nine specific monoclonal antibodies reacting with gp91-phox cytochrome b558, two were selected, both of which were found to bind to the beta subunit of cytochrome b558 and to inhibit superoxide formation in the oxidase reconstituted cell-free assay. The extent of inhibition was dependent on the phospholipid environment. Neutrophil membrane extracts from X-linked chronic granulomatous disease patients did not produce O2- in the reconstituted system and did not bind to the antibodies.
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PMID:Characterization of neutrophil NADPH oxidase activity reconstituted in a cell-free assay using specific monoclonal antibodies raised against cytochrome b558. 852 42

The latent NADPH oxidase activity of purified cytochrome b(558) from rabbit peritoneal neutrophils was expressed in a cell-free system consisting of either gel-filtrated cytosol from resting neutrophils, or a mixture of the three cytosolic activation factors, namely p47, p67 and the G protein Rac1. The cell-free system was supplemented with arachidonic acid and GTPgammaS. With gel-filtrated cytosol, the oxidase activity was relatively high (22 moles O(2)(-)/s/mole heme b in the absence of added FAD), and enhanced by less than one fourth upon addition of FAD. In contrast, with the purified cytosolic activation factors the rate of O(2)(-) production was low (8 moles O(2)(-)/s/mole heme b), and enhanced more than two-fold by a saturating concentration of FAD. The specificity of FAD was demonstrated by the lack of effect of FMN. FAD was determined together with heme b and the oxidase activity in eluates from a Sepharcryl column at the last step of the purification of cytochrome b(558). In the eluted fraction that contained both the maximal inducible oxidase activity and the highest amount of heme b, the molar amount of FAD was 20 times less than that of heme b. It is concluded that cytochrome b(558) is an NADPH-dependent flavocytochrome oxido-reductase (NADPH oxidase) in which one part of FAD is firmly bound and another, loosely attached. On the other hand, there may exist a parallel pathway of electron transfer from NADPH via distinct FAD dehydrogenase(s) to the heme b component of the NADPH oxidase.
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PMID:Assessment of the flavoprotein nature of the redox core of neutrophil NADPH oxidase. 864 81


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