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)

Phagocytes produce superoxide by the assembly of a multicomponent complex that utilizes NADPH for the reduction of molecular oxygen (NADPH oxidase). The components participating in the assembly are a membrane-bound flavocytochrome and three cytosolic proteins, one of which was shown to be a dimer of the small GTP-binding protein (G protein) Rac1 p21 or Rac2 p21 with GDP dissociation inhibitor for Rho (Rho GDI). We determined the identity and quantity of the nucleotide bound to Rac1 p21 by high performance anion exchange chromatography of extracts prepared from highly purified Rac1 p21-Rho GDI, isolated from guinea pig macrophage cytosol. Rac1 p21 contained only GDP at a ratio of close to 1 mol of GDP per mol of G protein. The GDP-bound form of Rac1 p21 complexed to Rho GDI functioned as a potent activator of NADPH oxidase in a cell-free system that contained no free GTP or ATP. We propose that the GDP-bound form of Rac1 p21 might be the physiological activator of NADPH oxidase in macrophages, following its dissociation from Rho GDI, and that nucleotide exchange or conversion to GTP is not necessarily involved.
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PMID:The GDP-bound form of the small G protein Rac1 p21 is a potent activator of the superoxide-forming NADPH oxidase of macrophages. 812 10

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.
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PMID:A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox. 818 43

Neutrophils possess a multicomponent NADPH oxidase system capable of producing large quantities of superoxide in a process known as the respiratory burst (1). Upon stimulation of a phagocytic cell, two cytosolic components of the oxidase, p67phox and p47phox, associate with a membrane-bound flavocytochrome b and a small GTP-binding protein to form a functional enzyme complex. Each of the Phox proteins contains two src homology 3 (SH3) domains, which are of unknown function but are potential mediators of protein-protein interactions between components of the activated oxidase. We have isolated a 47-kDa protein from lysates of differentiated HL60 cells that specifically bound to the carboxyl-terminal SH3 domain of p67phox and not to any other SH3 domain tested. This protein was identified as p47phox, and the putative SH3 domain binding site was located to a carboxyl-terminal proline-rich region. Proline-rich synthetic peptides based on this carboxyl-terminal region specifically inhibited the binding of p47phox to the carboxyl-terminal SH3 domain of p67phox, and sequential truncation defined a unique minimal sequence, which, although similar, does not match the consensus sequence defined for other SH3-binding proteins.
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PMID:An SH3 domain and proline-rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex. 818 50

The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.
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PMID:p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. 828 52

Protein kinase C (PKC) inhibitors, staurosporine or 1,5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited NADPH oxidase activity and phosphorylation of 47 kDa protein (p47) in PMA-stimulated neutrophils in a dose-dependent manner. These PKC inhibitors, at the same doses, did not affect oxidase activity and caused only partial inhibition of p47 phosphorylation in OZ-stimulated neutrophils. There was residual (20%) phosphorylated p47 in the membranes of OZ-stimulated cells in the presence of PKC inhibitors, at concentrations which caused total inhibition of oxidase activity and p47 phosphorylation in PMA-stimulated neutrophils. In the presence of ionomycin, which increased intracellular calcium ion concentrations, staurosporine was less effective in inhibiting both superoxide generation and p47 phosphorylation stimulated by PMA, similar to its effect in OZ-stimulated cells. The results indicate that some phosphorylation of p47 always accompanied oxidase activation induced by PMA or OZ, though the degree of phosphorylation of membrane-bound p47 does not directly correlate with rates of superoxide production.
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PMID:The requirement of p47 phosphorylation for activation of NADPH oxidase by opsonized zymosan in human neutrophils. 830 97

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.
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PMID:Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase. 838 55

The human organism survives the constant attack by bacteria and other pathogens thanks to the surveillance function of the neutrophil leukocytes. At sites of infection, several messenger molecules are generated that attract neutrophils from the blood and direct their migration toward the microbes, a process termed chemotaxis. Neutrophils sense chemotactic agonists through a group of closely related, GTP-binding protein-coupled receptors. Several of these have been recently cloned and shown to belong to the superfamily of rhodopsin-like, seven-transmembrane-domain receptors. At the site of infection, the neutrophils engulf and kill the invading microbes. This critical function depends on the production of superoxide and related radicals by a tightly regulated, membrane-bound NADPH oxidase that is activated by chemotactic agonists and other inflammatory stimuli. The characteristics of the receptors as well as new insights into the mechanism of activation of the superoxide-forming oxidase as presented at a recent FASEB meeting symposium are reviewed.
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PMID:Activation of neutrophil leukocytes: chemoattractant receptors and respiratory burst. 839 40

The GTPase activity of membranes isolated from differentiated HL-60 cells was investigated to obtain information about the possible involvement of membrane-bound GTP-binding proteins in the regulation of the NADPH oxidase. A more than tenfold increase in the rate of hydrolysis of membrane-bound GTP was observed when cytosol and arachidonic acid were added simultaneously, i.e. under the same conditions where NADPH oxidase becomes activated. There were parallel changes in GTPase and NADPH oxidase activities when the concentration of arachidonic acid or the species of the fatty acid was varied or different detergents were applied. Separation of the GTP-binding proteins of the solubilized membrane by sucrose density gradient centrifugation, allowed us to ascribe the observed effect to the stimulation of the GTPase activity of small GTP-binding proteins by cytosolic component(s). Indirect evidence suggests that, in contrast to the effect upon recombinant ras and ras-GTPase-activating protein, in intact HL-60 membranes the interaction of rap1A with rap-GTPase-activating protein, is strongly enhanced by arachidonic acid.
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PMID:GTPase activity of small GTP-binding proteins in HL-60 membranes is stimulated by arachidonic acid. 840

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

A large number of publications recently have drawn strong analogies between the production of active oxygen species in plant cells and the "oxidative burst" of the phagocyte, even to the point of constructing elaborate models involving receptor mediated G-protein activation of a plasmalemma NADPH oxidase in plant cells. However there are potentially other active oxygen species generating systems at the plant cell surface. The present work examines these alternatives with particular emphasis on the rapid production of active oxygen species, in common with a number of other systems, by suspension-cultured cells of French bean on exposure to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. The cells show a rapid increase in oxygen uptake which is followed shortly afterwards by the appearance of a burst of these active oxygen species, as measured by a luminescence assay, which is probably all accounted for by hydrogen peroxide. An essential factor in this production of H2O2 appears to be transient alkalinization of the apoplast where the pH rises to 7.0-7.2. Dissipation of this pH change with a number of treatments, including ionophores and strong buffers, substantially inhibits the oxidative burst. Little evidence was found for enhanced activation of a membrane-bound NADPH oxidase. However the production of H2O2 under alkaline conditions can be modelled in vitro with a number of peroxidases, one of which, an M(r) 46,000 wall-bound cationic peroxidase, is able to sustain H2O2 production at neutral pH unlike the other peroxidases which only show low levels of this reaction under such conditions and have pH optima at values greater than 8.0. On the basis of such comparative pH profiles between the cells and the purified peroxidase and further inhibition studies a direct production of H2O2 from the wall peroxidase in French bean cells is proposed. These experiments may mimic some of the responses to plant pathogens, particularly the hypersensitive response, which is an important feature of resistance. A cell wall peroxidase-origin for the oxidative burst is clearly different from a model consisting of receptor activation of a plasmalemma-localised NADPH oxidase generating superoxide. An alternative simple and rapid mechanism thus exists for the generation of H2O2 which does not require such multiple proteinaceous components.
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PMID:The origin of the oxidative burst in plants. 857 46


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