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)

The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membrane and cytosol by anionic amphiphiles such as sodium dodecyl sulfate and arachidonate (McPhail, L. C., Shirley, P. S., Clayton, C. C., and Snyderman, R. (1985) J. Clin. Invest. 75, 1735-1739; Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743; Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221). Herein, the activity thus obtained is shown to be very labile at 37 degrees C. The rate of inactivation varied inversely with cytosol concentration. The stabilizing factor(s) was destroyed by heat and trypsin, indicating that it is protein in nature. Whereas cytosol from normal cells and from a chronic granulomatous disease patient lacking p67phox stabilized the oxidase activity, that from a chronic granulomatous disease patient lacking p47phox did not. Also, dialdehyde NADPH-treated cytosol showed no stabilizing effect, indicating that p47phox and a putative NADPH-binding component both participate in stabilization. The mechanism of inactivation was further explored by examining the stabilizing effect of agents that can act as chemical cross-linkers. Of several tested, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was the most effective, but others that utilize different chemical mechanisms were also partially effective. EDC extended the half-life at 37 degrees C from 2 to 120 min, protected against the inactivating effects of Triton X-100 and high salt, and did not affect the Km for NADPH. Stabilization required prior activation in the presence of both cytosol and membrane; and EDC treatment of cytosol, membrane, or a mixture of the two prior to the addition of sodium dodecyl sulfate failed to induce stabilization. EDC eliminated the requirement for the continuous presence of cytosol and activator. Dialysis did not cause a loss in activity, whereas control activity was diminished with dialysis and was largely restored with added sodium dodecyl sulfate. In the absence of EDC, the separation of cytosol from the membrane fraction resulted in a significant loss of activity, which was largely restored by the addition of cytosol. However, EDC treatment allowed the isolation of a nearly fully active oxidase in the membrane fraction, the activity of which was not influenced by added cytosol. These results support a model in which the active NADPH oxidase consists of a dissociable complex among membrane and cytosolic components and indicate that the longevity of the activated state requires continuous association of these components.
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PMID:Stabilization of human neutrophil NADPH oxidase activated in a cell-free system by cytosolic proteins and by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. 131 6

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.
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PMID:Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins. 131 93

During the past 5 years, the discovery of cell-free superoxide generation system (Bromberg Y, Pick E: Cell Immunol 88:213-221, 1984) has been revolutionized our understanding of phagocyte superoxide generation. Using cell-free system, it was clarified that NADPH oxidase for superoxide generation was comprised of components present in both the plasma membrane as well as in the cytosol. This oxidase could be kept inactive by keeping its components separated from each other within the cell and then quickly bringing them together in the plasma membrane upon activation. We analyzed cytosol components with column method, and clarified that 3 neutrophil cytosol factors (NCF-1/-2/-3) was necessary for reconstitute the cytosol activity which was missing in an autosomal recessive type of Chronic Granulomatous Disease (CGD) patients (Nunoi H, et al:Science 242:1298-1301, 1988). NCF-1/-2 were analyzed with B1 antibody and found their molecular weight as 47 and 67 kilodalton respectively. One of autosomal CGD patients was missing NCF-67k and the others were missing NCF-47k. NCF-47k and -67k were cloned with this antibody and sequenced and expressed as recombinant NCF-47k/-67k using baculovirus/insect cell system. Using these recombinants, we are trying to purify NCF-3 which is reported as small G protein in these days. Using monoclonal antibodies against these recombinants, we analyzed tissues with immunohistochemical methods and are trying to classify the type of CGD patients in Japan. In summary, at least five oxidase components now have been identified (alpha and beta chain of cytochrome b558 (gp91-phox, p22-phox) and NCF-47k/-67k/-3 (p47-phox/p67-phox/delta-1 or SOCI)).
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PMID:[Molecular bases of chronic granulomatous disease--analysis of the involvement of cytosol factors for NADPH oxidase]. 131 71

The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.
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PMID:Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. 131 79

We tried to purify a new protein component required for the activation of NADPH oxidase from the guinea pig neutrophil cytosolic fraction which did not contain p47phox and p67phox, using HAC-5CP, IEC-QA and Superose 12HR columns. The NADPH oxidase-activating activity was separated into three fractions on IEC-QA anion-exchange HPLC. However, when each of the fractions was purified by Superose 12HR gel filtration, the active fraction eluted at the same position, and was found to contain a common protein with a molecular weight of 28.5 kDa on SDS-PAGE. These results suggest that the 28.5 kDa protein is a novel NADPH oxidase activating protein.
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PMID:Purification of the 28.5 kDa cytosolic protein involved in the activation of NADPH oxidase from guinea pig neutrophils. 158 57

The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membranes, cytosol, and an anionic amphiphile such as sodium dodecyl sulfate (SDS) or arachidonate, and guanosine 5'-(3-O-thio)triphosphate (GTP(gamma)S) augments activation. We report herein that short-chain diacylglycerols (e.g. dioctanoylglycerol (diC8)) synergize with SDS in the activation of superoxide generation in a dose- and time-dependent manner, resulting in rates up to 1400 nmol/min/mg plasma membrane protein, or 250-700% higher than the rate seen with SDS alone. diC8 did not affect significantly the dose response for either cytosol or SDS, indicating that the activation was not due to increased sensitivity of the oxidase toward either of these components. At optimal concentrations of SDS and diC8, additional activation was observed in the presence of GTP(gamma)S, indicating that diC8 and GTP activate by separate mechanisms. In contrast to diC8, other known activators of protein kinase C (phorbol myristate acetate and mezerein) augmented SDS activation only minimally (typically 20-30%), and neither diacylglycerols nor tumor promoters activated in the absence of SDS. Activation by diC8 was calcium and phosphatidylserine independent, and the specificity for neutral lipids was atypical for protein kinase C. Inhibitors of protein kinase C (staurosporine and a peptide substrate analog) also failed to inhibit the response. Nevertheless, phosphorylation of several neutrophil proteins including p47phox was seen with both SDS and diC8, and synergistic phosphorylation of p47phox was seen when both activating factors were present. Thus, diacylglycerol synergizes with SDS in activating both superoxide generation and p47phox phosphorylation in the cell-free activation system, but the activation is atypical of a protein kinase C mechanism.
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PMID:Diradylglycerol synergizes with an anionic amphiphile to activate superoxide generation and phosphorylation of p47phox in a cell-free system from human neutrophils. 217 Mar 84

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.
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PMID:Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1). 239 22

For somatic gene therapy to become a realistic therapeutic strategy for chronic granulomatous disease (CGD), we have to be able to assign the molecular lesion to a specific component of the NADPH oxidase and to confirm that transfer of a functional copy of the corresponding defective gene will result in correction of the cellular defect. We used an adenovirus vector expressing p47phox to transduce monocytes from patients with CGD. We showed by nitroblue-tetrazolium staining that NADPH-oxidase activity was restored to these cells. This technique offers a rapid means for molecular diagnosis. In the short term, this approach may have therapeutic potential.
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PMID:Gene transfer to primary chronic granulomatous disease monocytes. 754 96

Cytosolic components of the phagocyte NADPH oxidase (p47phox, p67phox, and Rac2) translocate to the plasma membrane on cell activation where they interact with a membrane-bound cytochrome b to generate superoxide anion. Phosphorylation reactions are known to be important for activity of NADPH oxidase. Translocation of Rac2, p47phox, and p67phox were all enhanced in formyl-Met-Leu-Phe-stimulated neutrophils treated with 50 nM of the protein phosphatase 1/2A inhibitor calyculin A. Rac translocation was blocked by the tyrosine kinase inhibitors genistein (50 microM) and herbimycin (17 microM), whereas movement of p47phox and p67phox were not inhibited. Cell-free analysis of Rac translocation also demonstrated that translocation of p47phox and p67phox were not linked to the movement or availability of Rac2. Thus, Rac2 does not appear to regulate NADPH oxidase by controlling movements of the cytosolic components to the membrane-associated enzyme but may exert its effect at the level of the assembled complex. Tyrosine kinase activity is required for translocation of Rac in the chemoattractant-stimulated human neutrophil.
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PMID:Dissociation of Rac translocation from p47phox/p67phox movements in human neutrophils by tyrosine kinase inhibitors. 761 2

The Rac guanosine 5'-triphosphate (GTP)-binding proteins regulate oxidant production by phagocytic leukocytes. Two Ste20-related p21-activated kinases (PAKs) were identified as targets of Rac in human neutrophils. Activity of the approximately 65- and approximately 68-kilodalton PAKs was rapidly stimulated by chemoattractants acting through pertussis toxin-sensitive heterotrimeric GTP-binding proteins (G proteins). Native and recombinant PAKs phosphorylated the p47phox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component in a Rac-GTP-dependent manner. The action of PAKs during phagocyte activation by G protein-coupled pathways may contribute to regulation of NADPH oxidase activity.
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PMID:Regulation of human leukocyte p21-activated kinases through G protein--coupled receptors. 761 83


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