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)

Polymorphonuclear leukocytes (PMNL) release superoxide anions formed by a membrane-bound NADPH oxidase induced by stimulations. Properties of the inducers and their antagonists indicate that Ca2+, GTP-binding protein (G-protein), phospholipase C and Ca2+, phospholipid-dependent protein kinase (C-kinase) are mainly associated with the stimulation of receptors. Low concentrations of ATP induce the oxidase accompanied by the increase in the intracellular Ca2+ due to the flux from the medium and the storage site. ATP-gamma-S, UTP and ITP are effective but mononucleotides, dinucleotides, GTP and CTP are not. Leukotriene B4 (LTB4) which acts as a chemotactic agent and the inducer of the NADPH oxidase is catabolized. It is hydroxylated by a specific cytochrome P450 and then oxidized to a carboxy derivative by a cytosolic alcohol dehydrogenase and a microsomal aldehyde dehydrogenase in PMNL. Active NADPH oxidase was obtained by incubating membrane and cytosolic components of resting PMNL in the presence of sodium dodecyl sulfate (SDS). Two cytosolic components were obtained by an affinity chromatography on 2',5'-ADP Sepharose. One component is active in the presence of GTP or GTP-gamma-S and the other component in the presence of another cytosolic fraction.
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PMID:Metabolism of stimulated polymorphonuclear leukocytes. 254 77

The subcellular localization of the microbicidal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and associated b-cytochrome was investigated in human neutrophils. In unperturbed neutrophils 85% of b-cytochrome and the major part of membrane-bound components of the NADPH oxidase co-sedimented with markers for specific granules and gelatinase. Using cytochrome b559 as a marker for membrane-bound components of the NADPH oxidase in quantitative studies we observed that, of the remaining 15%, the vast majority co-sedimented with latent alkaline phosphatase, a marker for a newly identified mobilizable intracellular compartment. Only a small fraction co-localized with the plasma membranes. Azurophil granules contained a protease activity which rapidly inactivated the NADPH oxidase components present in other membranes. Stimulation of the neutrophils with formyl-methionyl-leucyl-phenyl-alanine and leukotriene B4 which caused minimal degranulation of specific granules, resulted in translocation of b-cytochrome to the plasma membrane, concomitant with incorporation of alkaline phosphatase into the plasma membrane.
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PMID:Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils. 254 92

Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.
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PMID:The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils. 255 84

The formation of oxygen-derived free radicals by the phagocytes (neutrophils, eosinophils, monocytes and macrophages) is catalysed by a membrane-bound NADPH oxidase which is dormant in resting cells and becomes activated during phagocytosis or following interaction of the cells with suitable soluble stimulants. This enzyme is under investigation in many laboratories but its molecular structure remains to be clarified. Possible components such as flavoproteins, cytochrome b558, and quinones have been proposed on the basis of enzyme purification studies, effects of inhibitors, kinetic properties and analysis of genetic defects of the oxidase. An extensive discussion of the evidence for the participation of these constituents is reported. On the basis of the available information on the structure and the catalytic properties of the NADPH oxidase, a series of possible models of the electron-transport chain from NADPH to O2 is presented. Finally, the triggering mechanism of the respiratory burst is discussed, with particular reference to the stimulus-response coupling and the final modification(s) of the oxidase (phosphorylation, assembly, change of lipid environment, etc.) which are involved in its activation.
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PMID:The superoxide-forming enzymatic system of phagocytes. 283 75

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.
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PMID:Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase. 283 20

The vast majority of extracellular signals alters cell function by activating cell surface receptors. The transmembranous signalling process initiated by an activated receptor leads to the generation of an intracellular signal and eventually to a cellular response. In contrast to receptors that are permanently coupled to an enzyme or an ion channel representing the effector, a large number of surface receptors for hormones, neurotransmitters and receptors for exogenous chemical or physical stimuli reversibly interacts with membranous signal transduction components which, in turn, regulate intracellular messenger-generating effectors. The transducer molecules isolated so far form a family of guanine nucleotide-binding proteins (G- or N-proteins). All isolated G-proteins are composed of three different subunits (alpha, beta, gamma). The alpha-subunit, which is specific for the individual G-protein, binds and hydrolyzes GTP and is target of ADP-ribosylating bacterial toxins. Hormone-induced activation of a receptor causes interaction with the alpha-subunit of a G-protein and the exchange of bound GDP with GTP. The GTP-bound form of the alpha-subunit represents the active form of the G-protein, which is capable of stimulating or inhibiting the respective effector. The active state of the alpha-subunit is terminated by its inherent GTPase activity causing hydrolysis of bound GTP. The beta gamma-complexes of G-proteins are structurally very similar and functionally interchangeable; they appear to dissociate from the alpha-subunits during receptor activation of the G-protein. Possible functions of the beta gamma-complex are to anchor the non-activated G-protein in the membrane, to facilitate G-protein-receptor interaction, and to promote the inactive state of the alpha-subunit. G-protein-regulated effectors include enzymes, ion channels and probably transporters. The best studied G-protein-regulated enzyme is the retinal cyclic GMP-phosphodiesterase which is activated by bleached rhodopsin via the tissue-specific G-protein, termed transducin. The ubiquitously occurring membrane-bound adenylate cyclase is under dual control by families of stimulatory and inhibitory receptors, acting via G-proteins called Gs and Gi, respectively. Moreover, the receptor control of phospholipases A2 and C and probably of phospholipase D most likely involves G-proteins which have not yet been identified. Finally, the activity of NADPH oxidase of neutrophils and that of cyclic AMP phosphodiesterases in liver and fat cells may be regulated via G-proteins. Modulations of non-enzymatic effectors are reviewed elsewhere.
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PMID:[Guanidine nucleotide binding proteins as membrane signal transduction components and regulators of enzymatic effectors]. 284 11

Accumulating evidence indicates that protein kinase C plays an essential role in the activation of NADPH oxidase. In the present study, the correlation between superoxide generation, intracellular calcium, activation of purified protein kinase C and stabilized membrane-bound protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) and 1-deacyl-2-acetyl-rac-glycerol (OAG) were found to induce equal activation of purified protein kinase C and translocation of protein kinase C to the membrane fraction, but differed significantly in their ability to induce superoxide generation. Intracellular calcium was varied using calcium ionophores and increasing the intracellular calcium concentration to more than 1 microM was found to induce increased superoxide generation in maximally OAG-stimulated cells; this contrasted to maximally PMA-stimulated leukocytes. Ionomycin and A23187 were both found to induce a translocation of protein kinase C to the membrane fraction. This translocation was highly dependent upon extracellular calcium. In contrast, PMA- and OAG-induced translocation of protein kinase C was not dependent upon extracellular calcium. In conclusion, our results indicate that although PMA, OAG and calcium ionophores seem to activate protein kinase C in human polymorphonuclear leukocytes these activators differ in their ability to induce superoxide generation.
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PMID:A study on the role of protein kinase C and intracellular calcium in the activation of superoxide generation. 284 93

As demonstrated by others, diisopropyl fluorophosphate (DFP) markedly inhibits the O2- generation from guinea-pig polymorphonuclear leukocytes (PMN) stimulated by an antibody complex with ovalbumin (Ag-Ab complex), and also the intracellular uptake of antibody-sensitized erythrocytes by the cells. However, when PMN were treated with DFP and washed to remove the inhibitor, they again became able to exhibit the O2- -generating and phagocytic activities. The [3H]DFP-labeling of intact PMN followed by solubilization with Triton N101, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the existence of several [3H]DFP-labeled proteins with different mol. wts, which disappeared on pretreatment of cells with cold DFP. However, stimulation of DFP-pretreated PMN with Ag-Ab complex in the presence of [3H]DFP resulted in the appearance of a [3H]DFP-labeled, membrane-bound protein with a mol. wt of 40,000. This protein was isolated by affinity chromatography of the solubilized PMN and phagosomes on anti-Ig antibody-Sepharose 4B. Although the enzymatic properties of the protein are not clear, the results so far obtained suggest that it is a putative, stimulus-activated serine protease participating in the triggering events leading to the activation of NADPH oxidase responsible for the respiratory burst and the formation of phagosomes.
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PMID:Isolation of a protein labeled with diisopropyl fluorophosphate on stimulation of polymorphonuclear leukocytes with immune complexes. 299 80

Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.
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PMID:Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase. 301 89

The reduction with dithionite of neutrophil cytochrome b-558, implicated in superoxide generation by activated neutrophils, was investigated by a stopped-flow technique in non-ionic-detergent extracts of the membranes and in crude membrane particles. The dependence of the pseudo-first-order rate constants on the concentration of dithionite was consistent with a mechanism of reduction that involves the dithionite anion monomer SO2.- as the reactive species. The estimated second-order rate constant was 7.8 X 10(6) M-1 X S-1 for Lubrol PX-solubilized cytochrome b-558 and 5.1 X 10(6) M-1 X S-1 for the membrane-bound protein. The similarity of the kinetic constants suggests that solubilization did not introduce gross changes in the reactive site. Imidazole and p-chloromercuribenzoate, known as inhibitors of NADPH oxidase, did not affect significantly cytochrome b-558 reduction rates. The reaction rate of cytochrome b-558 with dithionite exhibited a near-zero activation energy. The first-order rate constant for reduction decreased with increasing ionic strength, indicating a positive effective charge on the reacting protein.
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PMID:Kinetic studies of the reduction of neutrophil cytochrome b-558 by dithionite. 302 24


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