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)

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.
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PMID:Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes. 894 64

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.
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PMID:Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox. 906 49

The leukocyte iodonitrotetrazolium violet (INT) reductase activity of disrupted bovine polymorphonuclear neutrophils is closely associated with the activation of the O2(-)-generating NADPH oxidase in a cell-free system. It is dependent upon NADPH, cytosolic factors, and amphiphiles (such as arachidonate), the same factors required for O2- generation. Both O2- generation and INT reductase activity are inhibited by phenylarsine oxide, an inhibitor of the activation of the NADPH oxidase [Li, J., & Guillory, R. J. (1997) J. Biochem. Mol. Biol. Biophys. (in press)]. In this report, the INT diaphorase activity of disrupted bovine polymorphonuclear neutrophils is shown to be resolved by DEAE-Sepharose chromatography into two fractions: an NADPH-cytochrome c reductase-containing fraction and a cytochrome b558-associated fraction. The diaphorase activity in the NADPH-cytochrome c reductase-containing portion is not dependent upon the presence of an amphiphile or phospholipid and is not associated with O2- generation. Upon incorporation into liposomes, the cytochrome b558-containing fraction demonstrates high O2- and INT reductase activities in the presence of cytosolic factors. Both O2- generation and INT reductase activities are SDS and FAD dependent and further stimulated by GTPgammaS. Phenylarsine oxide inhibits both O2- generation and INT reductase activities when added prior to activation by SDS. With the cytochrome b-containing liposomes, the Km values (O2- formation) for NADPH and NADH are 27.2 microM and 810 microM, and for INT reductase the Km values are 27.5 microM and 1017 microM, respectively. Under anaerobic conditions and thus in the absence of O2- formation, the NADPH-dependent INT reductase activity does not change, indicating that the dye reduction is not due to its direct reduction by O2 anion but is an intrinsic property of the superoxide-generating NADPH oxidase. Cytochrome b558 is the essential component of the NADPH oxidase and contains all the redox centers necessary for electron flow between NADPH and oxygen. The correlation of the activation and inhibition patterns for O2- generation and INT reduction by cytochrome b558 incorporated into artificial liposomes strongly indicates that the two activities are associated with the same membrane protein, cytochrome b558.
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PMID:Purified leukocyte cytochrome b558 incorporated into liposomes catalyzes a cytosolic factor dependent diaphorase activity. 915 36

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47(phox), p67(phox), and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47(phox) undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47(phox) using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47(phox) with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47(phox), whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47(phox) fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47(phox). Protein kinase C phosphorylation of p47(phox) in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47(phox) was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47(phox). Thus, our studies provide direct evidence linking conformational changes in p47(phox) to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47(phox) that are similar to those mediated by phosphorylation in vivo.
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PMID:Analysis of activation-induced conformational changes in p47phox using tryptophan fluorescence spectroscopy. 936 11

We examined the ability of C-terminal deletion mutants of p47PHOX, a cytosolic subunit of the leukocyte NADPH oxidase, to support the activity of the oxidase in two different cell-free systems, one using protein kinase C and the other an anionic amphiphile (SDS or arachidonic acid) as the oxidase-activating agent. Two deletion mutants were studied: p47PHOXdelta330 and p47PHOXdelta348, each named according to the first residue of the deleted polypeptide. Wild-type (WT) p47PHOX and both mutants were phosphorylated by protein kinase C, but the WT protein was the most heavily phosphorylated, containing 6.0 +/- 0.5 mol phosphate/mol protein. Of the two deletion mutants, only p47PHOXdelta348 could support oxidase activity, and then only in the amphiphile-activated system; neither of the mutants supported oxidase activity in the system activated by protein kinase C. Translocation correlated with activity: WT p47PHOX translocated to the membrane in response to both protein kinase C and amphiphile, but p47PHOXdelta348 translocated only in the amphiphile-activated system. Comparison of these findings with the results of earlier studies suggests that the phosphorylation of p47PHOX is an important component of oxidase activation. The findings provide no information, however, about whether amphiphiles participate in the activation process in intact cells. Consequently, a mechanism of in vivo oxidase activation involving both phosphorylation and the generation of an amphiphile remains a distinct possibility.
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PMID:Activation of the leukocyte NADPH oxidase in a cell-free system: phosphorylation vs. amphiphiles. 943 May 12

Plasma membranes of neutrophil cells contain the redox component of the O2(-)-generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an alpha subunit of 22 kDa and a beta subunit of 85-105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane-bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell-free system. To determine the PAO-binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4-[N-(4-azido-2-nitrophenyl)amino-[3H]acetamido]phenylarsine oxide, ([3H]azido-PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the beta subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85-105 kDa was shifted to 50-60 kDa as was the immunodetected beta subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the beta subunit of the membrane-bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell-free system. Incorporation of [3H]azido-PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the beta subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.
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PMID:Phenylarsine oxide as an inhibitor of the activation of the neutrophil NADPH oxidase--identification of the beta subunit of the flavocytochrome b component of the NADPH oxidase as a target site for phenylarsine oxide by photoaffinity labeling and photoinactivation. 949 37

The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN.
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PMID:Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica. 949 88

We have examined the kinetics of NADPH oxidase activation induced by arachidonic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b-245. Activation of oxidase activity required the simultaneous presence of p47(phox), flavocytochrome b-245, and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this difference represents an intermediate activation state of NADPH oxidase in which electron flow can proceed from NADPH to enzyme flavin (and hence to iodonitrotetrazolium violet) but not from flavin to heme (or not between the hemes). A model for NADPH oxidase activation is presented that is consistent with these observations.
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PMID:Simultaneous presence of p47(phox) and flavocytochrome b-245 are required for the activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation. 1033 45

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.
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PMID:Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system. 1033 47

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).
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PMID:Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox. 1125 27


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