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)

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.
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PMID:Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD. 0 Mar 95

The phagocyte NADPH oxidase is a multicomponent membrane-bound electron transport chain that catalyzes the reduction of O2 to superoxide. Cytochrome b558, the terminal electron donor to O2, is an integral membrane heterodimer containing 91- and 22-kDa subunits (gp91-phox and p22-phox, respectively). Synthetic peptides, whose amino acid sequences correspond to a gp91-phox carboxyl-terminal domain, inhibit superoxide production by blocking assembly of the oxidase from membrane and cytosol components. In this study, we examined the amino acid sequence requirements of a series of synthetic truncated gp91-phox peptides for inhibition of human neutrophil NADPH oxidase activation. RGVHFIF, corresponding to gp91-phox residues 559-565, was the minimum sequence capable of inhibiting superoxide generation. Contributions of individual amino acids to overall RGVHFIF inhibitory activity were determined by comparing the abilities of alanine-substituted RGVHFIF peptides to inhibit superoxide production. Substitution of alanine for arginine, valine, isoleucine, or either of the phenylalanines (but not glycine or histidine) within RGVHFIF resulted in loss of inhibitory activity. Synthetic gp91-phox carboxyl-terminal peptides are likely to be competitive inhibitors of the corresponding carboxyl-terminal domain of native gp91-phox by virtue of amino acid identity. We conclude that properties of arginine valine, isoleucine, and phenylalanine side chains within an RGVHFIF-containing domain of gp91-phox contribute significantly to cytochrome b558-mediated activation of the oxidase.
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PMID:Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity. 131 44

Intraperitoneal administration of tuftsin-M [Thr-Lys-Pro-Arg-NH-(CH2)2-NH-CO-C15H31] to Balb/C mice has been shown to induce a respiratory burst in the peritoneal exudate cells. The macrophages exhibited enhanced levels of O2-, H2O2, NADPH oxidase and myeloperoxidase, but the activities of superoxide dismutase, catalase and glutathione peroxidase remained virtually unchanged. The magnitude of the oxidative burst depended directly on the dose of tuftsin-M; higher activity was observed at higher doses of the peptide. Tuftsin-M enhanced the generation of both O2- and H2O2 under in vitro conditions, as did phorbol myristate acetate. These results suggest that tuftsin-M could enhance non-specific defence against infections by activating the macrophages.
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PMID:Respiratory burst in peritoneal exudate cells in response to a modified tuftsin. 133 Jun 71

Brain nitric oxide synthase is a Ca2+/calmodulin-regulated enzyme which converts L-arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme-bound H4biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non-heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin-dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin.
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PMID:Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase. 171 90

Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2.
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PMID:Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells. 171 62

Human blood leukocytes generated large amounts of superoxide (O2-) following stimulation by certain "cocktails" of soluble agents consisting of poly-L-arginine (PARG), phytohemagglutinin, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O2- generation by the soluble cocktails, markedly depressed luminol-dependent chemiluminescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O2- by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O2- was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin (LL) and which lost their viability, and their O2- and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O2- generation. Since PARG acted both as a cytolytic agent and as a inducer of O2- generation, we postulate that lytic agents might also act as "primers" of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in "multiple hits," PARG could be totally replaced either by LL or by digitonin in the generation of O2- provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble "cocktails" may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes.
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PMID:NADPH and "cocktails" containing polyarginine reactivate superoxide generation in leukocytes lysed by membrane-damaging agents. 300 Sep 40

Citrulline formation by Ca(2+)-calmodulin (CaM)-dependent nitric oxide synthase from bovine brain is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC50 values of 2.3 mM, 1.15 mM, 40 microM, and 2.5 microM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a Ki value of 0.16 microM and was competitive versus both arginine substrate and (6R)-5,6,7,8-tetrahydrobiopterin cofactor. The NADPH oxidase activity of bovine brain CaM-dependent nitric oxide synthase was inhibited by 7-nitroindazole with an IC50 value of 0.6 microM. Citrulline formation by the interferon-gamma/lipopolysaccharide-inducible nitric oxide synthase of murine macrophages (264.7 cell line) is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC50 values of 470, 240, 56, and 20 microM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a Ki value of 1.6 microM and was noncompetitive versus arginine substrate but competitive versus (6R)-5,6,7,8-tetrahydrobiopterin cofactor. None of the indazoles tested inhibited the cytochrome c reductase activity of either nitric oxide synthase isoform at concentrations up to 1000-fold higher than their IC50 values for inhibition of citrulline formation. These observations are consistent with the proposal that the indazoles exert their inhibitory actions by interaction with the heme-iron of nitric oxide synthase such that oxygen does not bind.
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PMID:The inhibition of the constitutive and inducible nitric oxide synthase isoforms by indazole agents. 751 13

The dynamics and mechanisms of extracellular release of hydrogen peroxide (H2O2) from bovine pulmonary artery endothelial cells (EC) subjected to anoxia, hypoxia, and hypoxia followed by reoxygenation were examined using various inhibitors of enzymatic systems in intact cells and by direct measurement of H2O2 production from isolated EC plasma membranes. Extracellular H2O2 was measured with a fluorometric assay. EC exposed to hypoxia (3% O2) and anoxia (0% O2) released less H2O2 (29.6 +/- 1.3% and 4.2 +/- 0.7%, respectively) compared with EC exposed to normoxia (20% O2). The extracellular release of H2O2 from EC previously exposed to hypoxia for 24 h increased immediately after reoxygenation (20% O2) to 272 +/- 48%, as compared with EC exposed continuously to normoxia (100% release). Inhibition of xanthine oxidase (XO) by allopurinol did not reduce the release of H2O2 from cells exposed to normoxia or hypoxia followed by reoxygenation. Furthermore, inhibitors of cyclooxygenase (indomethacin), phospholipase A2 (quinacrine and chlorpromazine), nitric oxide synthase (L-arginine analogs), the mitochondrial electron transport chain (rotenone and cyanide), and cytochrome P-450 (methoxypsoralen) had no or minimal effect on this release. On the other hand, inhibitors of protein kinase C (calphostin and staurosporine) and NADPH oxidase (diphenyliodonium) reduced the release of H2O2 from EC in a dose-dependent manner in both exposure groups. In separate experiments, plasma membranes isolated from EC were found to produce H2O2 in the presence of NADH or NADPH as electron donors. This was inhibited by diphenyliodonium but not by allopurinol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane. 752 30

Aminoguanidine produces a time-dependent inactivation of the citrulline forming activity of all three nitric oxide synthase isoforms that is blocked by arginine. Aminoguanidine inactivates both the NADPH oxidase and citrulline forming activities of GH3 pituitary constitutive nitric oxide synthase (cNOS) but does not alter its cytochrome c reductase activity. GH3 pituitary cells contain an NOS isoform identical physically, kinetically, and immunologically to cerebellar neuronal NOS (Wolff and Datto, Biochemical J. (1992) 285, 201-206). The inactivation of GH3 cNOS NADPH oxidase activity, as measured without added tetrahydrobiopterin cofactor, is saturable, is inhibited by arginine, and follows pseudo-first-order kinetics with an inactivation rate constant of 0.25 min-1 and a Ki value of 0.83 mM aminoguanidine. The inactivation of the citrulline forming activity of GH3 cNOS by aminoguanidine was not saturable by aminoguanidine. Aminoguanidine, at concentrations in the millimolar range, inhibited the citrulline forming activity of endothelial cNOS by an apparently nonsaturable mechanism. Aminoguanidine inactivates the citrulline forming activity of murine macrophage iNOS. The inactivation is saturable and follows pseudo-first-order kinetics with an inactivation rate constant of 0.46 min-1 and a Ki value of 16 microM. The inactivation of the constitutive isoforms of nitric oxide synthase by aminoguanidine required the concurrent presence of Ca2+, calmodulin, NADPH, tetrahydrobiopterin, and oxygen in preincubations and was not reversed either by dilution or dialysis. These observations support the assertion that aminoguanidine is a mechanism-based inactivator of the nitric oxide synthase isoforms and exhibits marked specificity for the inactivation of the inducible isoform.
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PMID:Aminoguanidine is an isoform-selective, mechanism-based inactivator of nitric oxide synthase. 753 Sep 37

Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product, (12S)-hydroxyeicosatetraenoic acid, was also significantly protective. As this analogue can act as a substrate for certain lipoxygenases, this effect may be due to prevention of generation of physiological products.
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PMID:Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-alpha-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain. 764 35


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