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)

NADPH-dependent superoxide generation was activated by anionic amphiphiles plus GTP gamma S in a cell-free system consisting of plasma membranes plus recombinant p47-phox, p67-phox, and the small GTP-binding protein Rac1. Rac1 was expressed in Escherichia coli both as the native form and as a mutant form (Rac1(C189S)) lacking the prenylation site. When preloaded with GTP gamma S, both Rac proteins supported activity to a level comparable to that seen using cytosol. A peptide corresponding to the carboxyl-terminal region of Rac1 was used to investigate oxidase assembly and activation. Rac1(178-188), but not several control peptides, inhibited activity. The peptide inhibited competitively (Ki = 15 microM) with respect to Rac1(C189S), while inhibition was noncompetitive or mixed with respect to p47-phox and p67-phox. This indicated specific inhibition of the interaction of the Rac protein with its target, possibly cytochrome b558. The peptide was effective only when added prior to activation with arachidonic acid, suggesting that it affects assembly rather than activity. Consistent with this possibility, the peptide prevented translocation of p47-phox and p67-phox to the plasma membrane. Thus, Rac plays a central role in the assembly of the neutrophil NADPH oxidase.
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PMID:Participation of the small molecular weight GTP-binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase. Inhibition by a carboxyl-terminal Rac peptide. 830 77

To further define the role played by protein kinase C (PKC) in the activation of the neutrophil NADPH oxidase, we have utilized a pseudosubstrate of PKC which was myristoylated at the N terminus. In electropermeabilized neutrophils, the myristoylated pseudosubstrate Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-psi PKC) inhibited PMA-induced protein phosphorylations and activation of the NADPH oxidase, induced either by PMA or by the receptor agonist formyl-methionyl-leucyl-phenylalanine. Both the pseudosubstrate lacking the N-terminal myristate (psi PKC) and a myristoylated control peptide (Phe-Ala-Glu-Asp-Gly-Ala-Leu-Glu-Gln, myr-CP) were without effect on these responses. The myristoylated pseudosubstrate was also tested in a cell-free system, in which NADPH oxidase activation can be achieved by addition of SDS and guanosine 5'-3-O-(thio)triphosphate in a staurosporine-insensitive manner. Myr-psi PKC, but not psi PKC or myr-CP, proved to be a potent inhibitor of NADPH oxidase activity in the cell-free system, indicating that the inhibition observed in permeabilized neutrophils may have been caused by an effect other than PKC inhibition. In the presence of myr-psi PKC, translocation in the cell-free system of the cytosolic oxidase components p47-phox and p67-phox to the plasma membrane was inhibited. From these results we conclude that myristoylation profoundly increases the ability of pseudosubstrates of PKC to inhibit not only PKC-mediated phosphorylations, but also NADPH oxidase activation. The latter effect, however, is most probably not related to PKC inhibition but may indicate a critical role of the membrane surface charge in the translocation of the cytosolic oxidase components p47-phox and p67-phox.
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PMID:Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C. 836 Jan 54

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 results presented in this paper demonstrate that the potentiation of phagocytosis of erythrocyte (E) IgG by TNF-alpha or PMA is not due to an oxygen-dependent mechanism. In fact, the potentiation of phagocytosis occurs normally in human neutrophils 1) when the respiratory burst is inhibited by diphenyleneiodonium, 2) in conditions where the reactive oxygen metabolites produced by the activation of NADPH oxidase, that accompanies the phagocytosis, were removed by catalase or superoxide dismutase, 3) of a patient lacking NADPH oxidase activity due to a genetic defect of p67-phox, 4) treated with staurosporine which allowed PMA to potentiate the ingestion of E-IgG at concentrations which inhibited the activation of the respiratory burst. Evidence is also presented that staurosporine not only did not inhibit, but amplified the potentiation of phagocytosis by PMA and TNF-alpha. This last finding suggests that the activation of protein kinase C plays a modulatory rather than a positive role in the mechanism of potentiation of phagocytosis.
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PMID:The potentiation by TNF-alpha and PMA of Fc receptor-mediated phagocytosis in neutrophils is independent of reactive oxygen metabolites produced by NADPH oxidase and of protein kinase C. 839 10

The superoxide-generating NADPH oxidase system in phagocytes consists of membrane-associated cytochrome b558 and three cytosolic components named p67-phox, p47-phox, and rac p21s. In a cell-free system consisting of membrane and cytosol, the oxidase can be activated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and an unsaturated fatty acid such as arachidonic acid (AA). Incubation of cytosol and membrane with AA alone caused clear translocation of p47-phox and p67-phox to the membrane, but only slight translocation of rac p21s. GTP gamma S alone did not significantly induce the translocation of rac p21s. However, GTP gamma S in combination with AA markedly augmented rac p21s translocation to the membrane. The translocation of rac p21s is not induced by GDP or GDP beta S. These results indicate that the GTP-bound active form of rac p21s is the entity that is translocated to the membrane by the action of AA.
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PMID:Combination of arachidonic acid and guanosine 5'-O-(3-thiotriphosphate) induce translocation of rac p21s to membrane and activation of NADPH oxidase in a cell-free system. 839 27

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

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.
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PMID:Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca(2+)-depleted human neutrophils. 843 86

Chronic granulomatous disease is an uncommon inherited disorder of phagocytes in which the defective production of microbicidal oxidants leads to an enhanced susceptibility to bacterial and fungal infections. Despite the near uniform absence of the respiratory burst in CGD phagocytes, there is a striking clinical and genetic heterogeneity in this disorder. The recent elucidation of the molecular basis of CGD now provides an explanation for this heterogeneity. CGD is caused by a defect in any one of four components of NADPH oxidase, the enzyme responsible for the generation of the antimicrobial oxidants. X-linked inheritance is seen in approximately 65% of patients and results from mutations in the gene encoding the gp91-phox subunit of the cytochrome b558 component of the oxidase. The remaining 35% of patients inherit CGD in an autosomal recessive manner due to mutations in the genes encoding the remaining three oxidase components: p22-phox (chromosome 16), p47-phox (chromosome 7), and p67-phox (chromosome 1). Deletions, insertions, and point mutation leading to premature stop codons, amino acid substitutions, and splice site defects have all been identified. Most CGD patients have mutations unique to their families. The diversity of these mutations and the multiple genes affected provide an explanation for the clinical and genetic heterogeneity of CGD.
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PMID:Chronic granulomatous disease: the solving of a clinical riddle at the molecular level. 850 Feb 77

Generation of the microbicidal oxidative burst in human neutrophils requires participation of four proteins, a membrane bound flavocytochrome beta-558, two soluble proteins termed p47-phox and p67-phox, and the Ras-related GTPase Rac. Because plant cells exposed to pathogens produce a similar oxidative burst, we have looked for similarities between the oxidase complexes of the two systems. Antibodies against human neutrophil p47-phox and p67-phox were used to immunoblot cell extracts from several plant cell lines and were found to cross-react with proteins of the same molecular weight. Furthermore, plant cell lines not previously shown to produce an oxidative burst, yet found to express these immunoreactive proteins, rapidly generated hydrogen peroxide in response to elicitation. Finally, diphenylene iodonium (DPI) and alpha-naphthol, known specific inhibitors of the NADPH oxidase in neutrophils, also inhibited the oxidative burst in soybean cell suspensions with similar Ki values (about 15 microM and 30 microM respectively). These results provide evidence for involvement of proteins related to the neutrophil oxidase complex in the defense-related oxidative burst of plants.
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PMID:Plant and human neutrophil oxidative burst complexes contain immunologically related proteins. 860 Sep 79

The yeast two-hybrid system is finding increased use in the study of interactions between proteins. In this method, two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain (bd) and activating domain (ad), respectively. Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters. The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 (comprised of subunits p22-phox and gp91-phox) and three cytosol proteins (p47-phox, p67-phox, and p21rac) that translocate to membrane and bind to cytochrome b558. This is the first report to demonstrate that two of cytosolic components of cytochrome b558, p47-phox binding to p67-phox each other. We encountered several methodological problems in the two-hybrid system which are the focus of this report.
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PMID:Elimination of false negative results in the two-hybrid system in the phagocyte NADPH oxidase. 867 37


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