Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
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 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.
 ...
PMID:p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. 828 52

Phagocytic white blood cells contain a multicomponent oxidase that generates microbicidal products by catalyzing electron transfer from NADPH to molecular oxygen. Activation of this oxidase requires interactions of a unique membrane flavocytochrome with the cytosolic proteins p47phox, p67phox, and p21Rac. This flavocytochrome, designated cytochrome b558, is a heteromer comprising a 22-kDa alpha-subunit (p22phox) and a glycosylated approximately 91-kDa beta-subunit (gp91phox). Cytochrome b558 was expressed in Sf9 insect cells coinfected with recombinant baculoviruses carrying cDNAs for p22phox and gp91phox. Membranes of these cells contained a b-type cytochrome with a dithionite-reduced minus oxidized difference spectrum similar to that of neutrophil cytochrome b558. The recombinant cytochrome b558 beta-subunit was heterogeneously N-glycosylated as demonstrated by its susceptibility to cleavage with endoglycosidases F and H. In contrast to the neutrophil cytochrome b558, a portion of the N-linked oligosaccharide was of the high mannose type. Recombinant cytochrome b558 supported superoxide production in a cell-free assay containing recombinant p47phox, p67phox, and p21Rac. The enzymatic turnover of the partially purified recombinant cytochrome b558 and neutrophil cytochrome b558 were similar (approximately 100-160 mol of superoxide generated/s/mol of cytochrome heme, range of two experiments) and the native and recombinant cytochromes showed similar requirements for NADPH and exogenous FAD. These studies represent the first reconstitution of the NADPH oxidase solely from recombinant proteins and define a model system to explore the structure and function of cytochrome b558.
 ...
PMID:Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins. 831 88

The effect of sulfite on the oxidative metabolism of human neutrophils was studied in vitro. Superoxide anion production of PMN was determined using superoxide dismutase-inhibitable lucigenin-dependent CL. The addition of sulfite in concentrations of 0.01 mM-1 mM results in an up to 6-fold increase in CL of nonstimulated neutrophils at 37 degrees C and pH 7. Neutrophils stimulated with zymosan or PMA have an additional 2-fold stimulation when sulfite is added. Higher sulfite concentrations (2 mM-10 mM) decrease the CL of both nonstimulated and stimulated cells. The activity of NADPH oxidase, responsible for O2.- production, is significantly increased in neutrophils incubated with 1 mM sulfite. Neutrophils from patients with chronic granulomatous disease, which are cytochrome b558 negative or have p47phox deficiency, exhibit no significant NADPH oxidase activity and show no increase in CL by sulfite. Inhibitors of protein kinase C, H7, and calphostin C, as well as inhibitors of Ca(2+)- and calmodulin-dependent processes, W7, and R 24 571, completely inhibited the increased CL of sulfite-treated neutrophils. These findings indicate that sulfite in low concentrations stimulates neutrophils to produce superoxide anions by activation of NADPH oxidase through a signal transduction pathway involving protein kinase C and Ca2+/calmodulin.
 ...
PMID:Sulfite stimulates NADPH oxidase of human neutrophils to produce active oxygen radicals via protein kinase C and Ca2+/calmodulin pathways. 839 22

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.
 ...
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

The NADPH oxidase is an electron transport chain found in lymphocytes and in the wall of the endocytic vacuole of 'professional' phagocytic cells. It is so called because NADPH is used as an electron donor to reduce oxygen to superoxide and hydrogen peroxide. The redox components are provided by a very unusual flavocytochrome b from the membrane, which is dependent upon cytosolic factors (including two specialized proteins, p47phox and p67phox) for activation. The small GTP-binding protein, p21rac, is also implicated in this system, possibly as the switch that triggers electron transport. This system provides a key to our understanding of the way in which these GTP-binding proteins function.
 ...
PMID:The biochemical basis of the NADPH oxidase of phagocytes. 848 57

Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).
 ...
PMID:Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins. 850 89

NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b558 of cytosolic components p47phox, p67phox, and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67phox; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67phox. Finally, we observe that p67phox interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67phox and that Rac2 is the main p67phox-interacting GTPase in human cells.
 ...
PMID:The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1. 855 Jun 29

In vivo loading of a synthetic peptide (peptide 4) corresponding to residues 314-331 (RSRKRLSQDAYRRNSVRF) consistently diminished the oxidative burst in response to either phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine and cytochalasin B (fMLP/CB) compared to other synthetic peptides derived from the p47phox sequence. The effects of peptide 4 were concentration dependent with respect to both PMA and fMLP/CB. In contrast, peptide 4 enhanced the oxidative burst in response to fMLP alone. Peptide 4 inhibited the PMA and fMLP-mediated phosphorylation of endogenous neutrophil cytosolic proteins including p47phox. The PMA-induced translocation of p47phox to the plasma membrane was diminished in neutrophils loaded with peptide 4. These data represent the first report of a synthetic peptide derived from p47phox that inhibits the NADPH oxidase in intact neutrophils and inhibits the protein kinase C-mediated phosphorylation of endogenous p47phox.
 ...
PMID:A synthetic peptide containing a predominant protein kinase C site within p47phox inhibits the NADPH oxidase in intact neutrophils. 855 59

The effect of the S-nitrosothiol (RSNO) on the activation of NADPH oxidase in human neutrophils was studied using an in vitro translocation system in which an anionic amphiphil, such as sodium dodecyl sulfate or arachidonate, plays a role as an activator. When membranes pretreated with RSNO and a cytosol fraction from resting neutrophils were combined to reconstitute the NADPH oxidase, both translocation of the cytosolic NADPH oxidase components such as p47phox and p67phox to the plasma membrane fraction and subsequent superoxide generation was inhibited. However, RSNO had no effect on O2- production when added after enzyme activation. A similar inhibition of translocation of recombinant p47phox was observed with RSNO-treated membrane. When the RSNO-treated membrane fraction was exposed to 2-mercaptoethanol the inhibition was reversed. The data suggest that RSNO inhibits translocation of p47phox or p47phox containing cytosolic complex via a direct effect on the membrane component of the NADPH oxidase.
 ...
PMID:Attenuation of p47phox and p67phox membrane translocation as the inhibitory mechanism of S-nitrosothiol on the respiratory burst oxidase in human neutrophils. 860 52

Src homology 3 (SH3) domains mediate specific protein-protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3-mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151-284), spanning both SH3 domains, was required for flavocytochrome-dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N-terminal domain (residues 1-246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain-mediated interaction involving the C-termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.
 ...
PMID:Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells. 863 53


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>