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)

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

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.
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PMID:Characterization of a Rac1- and RhoGDI-associated lipid kinase signaling complex. 944 72

Reactive oxygen species contribute to glomerular damage and proteinuria. In this study, we show that cultured human podocytes produce superoxide in response to extracellular adenosine triphosphate (ATP), and we identified the oxidases involved in this process. Adenosine triphosphate (10-4 M for 4 hr) raised superoxide production from 1.28 +/- 0.15 to 2.67 &/- 0.34 nmol/mg protein/min. Studies with podocyte homogenates revealed activation of both nicotinamide adenine dinucleotide (NADH; from 2.65 +/- 0.23 to 7.43 +/- 0.57) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidases [from 1.74 +/- 0.13 to 4.05 +/- 0.12 (nmol O2/mg protein/min)] by ATP. Activity of xanthine-oxidases was low and unchanged by ATP. Activation of the plasma-membrane bound NAD(P)H oxidases by ATP was time and dose dependent. Reverse transcribed-polymerase chain reaction (RT-PCR) studies with primers derived from monocyte sequences amplified mRNA for the NADPH oxidase subunits p22phox, p47phox, gp91phox, and p67phox, and the latter was transiently increased by ATP. Experiments with actinomycin D and cycloheximide suggested that ATP modulates enzyme activity at the transcriptional and translational levels. In conclusion, NAD(P)H dependent, membrane associated oxidases represent the major superoxide source in human podocytes. Activation of NAD(P)H oxidase by ATP might be secondary to increased mRNA expression of the NADPH oxidase subunit gp67phox.
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PMID:NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate. 950 11

Nitric oxide (NO.) has a complex role in the inflammatory response. In this study, we modified the levels of endogenous NO. in vivo in an acute model of inflammation and evaluated the interactions between NO. and superoxide anion (O2-.) produced by polymorphonuclear leukocytes (PMNs) accumulated in the inflamed area. We injected phosphate-buffered saline (control group), 6 mumol of L-N5-(1-iminoethyl)ornithine (L-NIO group), or 6 mumol of L-arginine (L-arginine group) into the granuloma pouch induced by carrageenan in rats. NO2- plus NO3- (indicative of NO. generation) was 188 nmol in the exudate of the control group, but it decreased in the L-NIO group (P < 0.05) and increased in the L-arginine group (P < 0.05). When PMNs from treated rats were incubated in vitro, the production of superoxide anion (O2-.) decreased by approximately 46% in the L-arginine group. Furthermore, O2-. was inhibited in PMNs when L-arginine was added to the incubation medium before phorbol 12-myristate 13-acetate stimulation but not when added simultaneously. Our results suggest a protective role for NO. in inflammation, through the inactivation of NADPH oxidase and the consequent impairment of O2-. production for cell-mediated injury.
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PMID:Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes. 953 Jan 15

The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen and NADPH. During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2 production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2 production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when expressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated. In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2 production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2 production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.
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PMID:Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47PHOX on serine 303 or 304. 954 83

In this study we tested the hypothesis that the pentose phosphate pathway (PPP) participates in the meiotic induction of mouse oocytes. The electron acceptors methylene blue, phenazine ethosulfate (PES), and pyrroline-5-carboxylate (P5C) oxidize NADPH to NADP and activate the NADP-dependent enzymes of the PPP. Each of these compounds triggered a dose-dependent increase in meiotic maturation in hypoxanthine-arrested cumulus cell-enclosed oocytes during 17- to 18-h cultures. More than 96% of the oocytes underwent germinal vesicle breakdown (GVB) at the highest concentrations of P5C and PES tested (250 and 1 microM, respectively) as compared to only 45-52% of control oocytes. P5C was also stimulatory to denuded oocytes. Analysis of energy substrates in microdrop cultures revealed a 3.6-fold increase in glucose consumption by PES-treated oocyte-cumulus cell complexes that was associated with stimulation of GVB. On the other hand, 2-deoxyglucose, which interferes with glucose utilization, prevented the induction of maturation brought about by P5C. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase, prevented meiotic maturation in the presence or absence of FSH. Gonadotropin-induced maturation was also prevented by 6-aminonicotinamide (6-AN) and dehydroepiandrosterone (DHEA), inhibitors of the two NADP-dependent enzymes of the PPP, and this was accompanied by suppression of glucose consumption. Phosphoribosyl-pyrophosphate (PRPP) is an important compound required in purine metabolism and can be formed from the end product of the oxidative arm of the PPP, ribose-5-phosphate. Ribose, which can be metabolized to PRPP, increased PRPP synthesis in complexes and induced meiotic maturation when added to hypoxanthine-arrested cumulus cell-enclosed oocytes in glucose-free medium in both the presence and absence of FSH. PRPP levels within complexes were also increased by glucose and FSH, but were reduced by hypoxanthine, 6-AN, and DHEA. In addition, exogenous PRPP stimulated maturation in hypoxanthine-arrested oocytes. These results support the proposition that glucose metabolism through the PPP is important in the meiotic induction mechanism and may involve the generation of PRPP that acts, at least in part, through the purine metabolizing pathways.
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PMID:Meiotic induction in cumulus cell-enclosed mouse oocytes: involvement of the pentose phosphate pathway. 954 44

Respiratory burst activity of murine microglial cells was investigated in vitro under normoxic and hypoxic conditions with a chemoluminometric assay. Hypoxia for 24 hours reduced the release of extracellular reactive oxygen intermediates (ROIs), whereas reoxygenation increased the chemoluminescence more than sevenfold. Blockade of potassium channels inhibited the increase of oxidative burst after reoxygenation, indicating that potassium ions, which were increased in the supernatant of hypoxic microglial cells, were involved in this activation process. Also, blockade of voltage-gated calcium channels with nifedipine attenuated the increased release of ROIs. With fura-2 analysis, it was shown that the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by potassium ions was mediated by calcium influx via voltage-gated calcium channels. Thus, influx of calcium ions through voltage-gated channels activates the NADPH oxidase in microglial cells during reoxygenation. By the increased production of ROIs, microglial cells may add to the reperfusion injury after ischemia in vivo.
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PMID:Reoxygenation increases the release of reactive oxygen intermediates in murine microglia. 962 91

Osteoclasts use a variety of chemical agents to degrade bone. One important component of this process is the generation of superoxide. It has been reported that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the enzyme responsible for superoxide production in phagocyte; however, the NADPH oxidase present in osteoclasts has not been studied in detail. One of the membrane-bound subunits of the NADPH oxidase is gp91(phox) which represents the rate-limiting component for the formation of the NADPH oxidase complex. This study was designed to demonstrate the presence of gp91(phox) in individual osteoclasts using the RT-PCR technique developed for limited numbers of cells. Compared with white cells, 1.8 times the amount of gp91(phox) mRNA was found in osteoclasts. This difference may be related to the size of the osteoclast and the multiple nuclei present. The presence of gp91(phox) in osteoclasts was confirmed at protein level by immunocytochemistry. Osteoclastic superoxide generation is inhibited by diphenylene iodonium, a specific inhibitor of the NADPH oxidase. These studies suggest that superoxide generation by osteoclasts correlates with the activity of NADPH oxidase.
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PMID:Nicotinamide adenine dinucleotide phosphate oxidase in the formation of superoxide in osteoclasts. 974 95

Human neutrophils have a short half-life and are believed to die by apoptosis or programmed cell death both in vivo and in vitro. We found that caspases are activated in a time-dependent manner in neutrophils undergoing spontaneous apoptosis, concomitant with other characteristic features of apoptotic cell death such as morphologic changes, phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs) significantly accelerated this process. However, in cells treated with the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA), caspase activity was only evident after pharmacologic inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Similarily, inhibition of the NADPH oxidase in constitutive and Fas/APO-1-triggered apoptosis resulted in increased rather than suppressed levels of caspase activity, suggesting that reactive oxygen species may prevent caspases from functioning optimally in these cells. Moreover, oxidants generated via the NADPH oxidase were essential for PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an important component of constitutive and Fas/APO-1-triggered neutrophil apoptosis. However, these redox sensitive enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate PS exposure and neutrophil clearance under these conditions.
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PMID:Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species. 984 48

A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2--forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the cytochrome b558, of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of cytochrome b558.
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PMID:A novel mutation at a probable heme-binding ligand in neutrophil cytochrome b558 in atypical X-linked chronic granulomatous disease. 985 76


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