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)

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91(phox) to function as an H(+) pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H(+) flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H(+) channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67(phox) and p47(phox), to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b(558) spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H(+) pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.
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PMID:Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites. 1151 29

The generation of superoxide by the NADPH oxidase is an electrogenic process resulting in a rapid depolarisation of the membrane potential of the cell. The efflux of H+ ions through an arachidonate-activatable, Zn(2+)-inhibitable H+ pathway accompanies the efflux of electrons and provides the necessary charge compensation. Inhibition of H+ flux leads to inhibition of superoxide generation. The protein gp91phox, a transmembrane component of the NADPH oxidase, was demonstrated to be capable of acting as the NADPH oxidase-associated H+ channel in a stable CHO cell line, CHO91. The N-terminal 230 amino acids contain all that is required for the protein to form an H+ channel and specifically histidine 115 is important to the ability of gp91phox to conduct H+ ions. The recording of outward currents from CHO91 cells, in the whole-cell configuration, demonstrated that gp91phox is also capable of functioning as a voltage-gated H+ conductance pathway. The similarity in properties between voltage-elicited outward currents, from both wild type and the mutations, and the arachidonate-activated H+ flux strongly suggests that these H+ pathways are one in the same. Among the recently identified homologues of gp91phox only NOH-1S has so far been demonstrated to also act as an H+ conductance pathway.
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PMID:NADPH oxidase subunit gp91phox: a proton pathway. 1173 36

The NADPH oxidase is a multicomponent enzyme that transfers electrons from NADPH to O2 to generate superoxide (O2*-), the precursor of microbicidal oxygen species that play an important role in host defense. Flavocytochrome b558, a heterodimeric oxidoreductase comprised of gp91(phox) and p22(phox) subunits, contains two nonidentical, bis-histidine-ligated heme groups imbedded within the membrane. Four histidine residues that appear to serve as noncovalent axial heme ligands reside within the hydrophobic N terminus of gp91(phox), but the role of p22(phox) in heme binding is unclear. We compared biochemical and functional features of wild type flavocytochrome b558 with those in cells co-expressing gp91(phox) with p22(phox) harboring amino acid substitutions at histidine 94, the only invariant histidine residue within the p22(phox) subunit. Substitution with leucine, tyrosine, or methionine did not affect heterodimer formation or flavocytochrome b558 function. The heme spectrum in purified preparations of flavocytochrome b558 containing the p22(phox) derivative was unaffected. In contrast, substitution of histidine 94 with arginine appeared to disrupt the intrinsic stability of p22(phox) and, secondarily, the stability of mature gp91(phox) and abrogated O2*- production. These findings demonstrate that His94 p22(phox) is not required for heme binding or function of flavocytochrome b558 in the NADPH oxidase.
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PMID:Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function. 1204 18

The NADPH oxidase of neutrophils is a transmembrane electron transfer complex, containing a flavin adenine dinucleotide and two hemes, all of which are suggested to be contained within gp91 (phox), one of four subunits of the enzyme. The transfer of electrons through the NADPH oxidase is associated with an efflux of protons. gp91 (phox) has previously been demonstrated to function as the proton conduction pathway. The mutation of histidines 111, 115, and 119 to leucines and of histidine 115 to leucine within the N-terminal 230-amino-acid fragment of gp91 (phox) has previously been demonstrated to result in the loss of proton conduction through this N-terminal fragment. In this paper we have investigated the role of these histidines in proton conduction by the full-length gp91 (phox). Stable CHO cell lines were established which expressed full-length gp91 (phox) in which histidines 111, 115, and 119 had been mutated to leucines (CHO91H111/115/119) and in which histidine 115 had been mutated to leucine (CHO91H115L). The expression of gp91 (phox) and its cellular localisation in these cell lines were comparable between wild-type and the mutant gp91 (phox). The mutation of histidines 111, 115, and 119 to leucines or just histidine 115 to leucine resulted in an almost total loss of both the arachidonate-activated influx and efflux of protons, in comparison with that observed for wild-type gp91 (phox). Therefore, histidine 115 is required for proton conduction by both full-length gp91 (phox) and the N-terminal 230-amino-acid fragment of gp91 (phox). Histidine 115 has recently been proposed to act as a coordinating ligand for the outer heme iron of the NADPH oxidase. On the basis of observations for cytochrome c oxidase, we propose a model for this dual role of histidine 115.
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PMID:Proton conduction through full-length gp91phox requires histidine 115. 1276 47

p47(phox) is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for the production of the superoxide which kills invasive microorgamisms. A recombinant form of a histidine-tagged tandem SH3 domain of the p47(phox)-containing polybasic autoinhibited region was expressed in Escherichia coli and purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 6000 as a precipitant. Diffraction data were collected to 2.15 A resolution at 100 K using synchrotron radiation. The crystal belongs to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 100.02, c = 44.94 A. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.6 A(3) Da(-1) and a solvent content of 52% by volume.
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PMID:Crystallization and preliminary crystallographic analysis of the autoinhibited form of the tandem SH3 domain of p47(phox). 1287 58

Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G-->A mutation, the A- variant common in African ethnic groups. The proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-->A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease.
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PMID:Association of glucose-6-phosphate dehydrogenase deficiency and X-linked chronic granulomatous disease in a child with anemia and recurrent infections. 1497 96

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.
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PMID:Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. 1532 91

The hypersensitive reaction is a type of programmed cell death in plants. Cryptogein is a proteinaceous elicitor secreted from Phythophthora cryptogea. In one current model, active oxygen species (AOS) trigger programmed cell death in plants. In this study, we examined a variety of AOS scavengers to elucidate the function of AOS in the death program. Most of these AOS scavengers, including tiron, a scavenger for superoxide radical, catalase for hydrogen peroxide, and hydroquinone, sodium ascorbate and propyl gallate for free radicals, almost completely removed extracellular AOS. However, none of the reagents completely blocked the cell death process. Other reagents, such as histidine and dimethylfuran, scavengers for singlet oxygen, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, showed significant toxicity in BY-2 cells. These results indicate that AOS produced in the extracellular space do not play a role in hypersensitive cell death.
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PMID:Effects of scavengers for active oxygen species on cell death by cryptogein. 1569 53

Voltage-gated proton channels are highly proton selective ion channels that are present in many cells. Although their unitary conductance is 1000 times smaller than that of most ion channels, detection of single-channel currents supports their identification as channels rather than carriers. Proton channels are gated by membrane depolarization, but their absolute voltage dependence is also strongly regulated by the pH gradient, DeltapH (pHo-pHi). A model of this behavior postulates regulatory protonation sites that are alternately accessible to external or internal solutions. Consequently, proton channels open only when the electrochemical gradient is outward, and serve to extrude acid from cells. No "classical" blockers of proton channels that bind to and physically occlude the channel have been identified. A number of weak bases that inhibit proton currents probably act indirectly, perhaps by changing local pH. The best known and most potent inhibitors are polyvalent cations, especially Zn(2+) and Cd(2+). These cations are coordinated at two or more external protonation sites, most likely His residues where they compete with protons and interfere with gating. In phagocytes, proton channels are required to compensate for the electrogenic action of NADPH oxidase. During the "respiratory burst," i.e., when NADPH oxidase is active, proton channels in these cells adopt an "activated" gating mode. Recently, two labs identified a gene that codes for either the proton channel itself or a protein that is essential for proton channel activity. Expression of this protein results in currents with many similarities to the native channel.
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PMID:Pharmacology of voltage-gated proton channels. 1769 9

Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91(phox) and p22(phox), which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47(phox) and p67(phox). A fifth subunit, p40(phox), plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40(phox), in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40(phox)R105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40(phox)-deficient granulocytes, with premature loss of p40(phox)R105Q from phagosomes. Thus, p40(phox) binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.
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PMID:A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. 1969 3


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