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)

Human fibroblasts in primary culture released reactive oxygen species upon exposure to synovial fluid obtained by joint aspiration from twelve patients suffering from rheumatoid arthritis. The primary radical produced was O2- as determined by ESR spin trapping and cytochrome c reduction. In contrast to the oxidative burst in granulocytes and monocytes, radical formation proceeded continuously for at least four hours. Low-level chemiluminescence was increased upon exposure to inflammatory human synovial fluids. Spectral characteristics and effects of azide and 1,4-diazabicyclo-(2,2,2)-octane led to the conclusion that the photoemissive species were excited carbonyls. Radical production and light emission were not altered either by xanthine or allopurinol, nor by azide, cyanide or rotenone. The O2- production increased in the presence of NADH or NADPH, making an NAD(P)H oxidase a likely source. The liberation of reactive oxygen species correlated with the number of leukocytes present in the inflammatory joint fluids, but not with the concentrations of immunoglobulins and complement factor C3.
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PMID:Human fibroblasts release reactive oxygen species in response to treatment with synovial fluids from patients suffering from arthritis. 215 76

Phorbol myristate acetate (PMA, 10(-7) M) activation of adherent neutrophils (PMNs) led to a markedly attenuated release of superoxide anion (O2-) per cell when PMNs were activated at high density (2.85 fmol O2-/PMN at 2 million in 0.1 ml) in comparison with cells activated at low cell density (12.0 fmol O2-/PMN at 250,000 in 0.1 ml). This "autoregulatory" phenomenon was not due to a defect in the superoxide anion assay employed, to a differential adherence of neutrophils at high vs. low density, or to substrate (cytochrome c) or cell stimulus (PMA) limitation. It was associated with an inhibition of apparent NADPH oxidase activity and a leftward shift (toward a lower level of activation) in the activation profile of PMNs (as determined by FACS analysis using PMNs preloaded with 2'7'-dichlorofluorescin diacetate in which H2O2 production results in the production of the fluorescent product 2'7'-dichlorofluorescein intracellularly). Other aspects of the neutrophil activation response including arachidonic acid mobilization, phospholipid metabolism, and perhaps phosphatidylinositol turnover were also attenuated when PMNs were activated at high cell density. Studies with cells in solution, cells treated with cycloheximide, and cells treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid suggest that PMN contact with a surface, neutrophil protein synthesis, and an increased surface expression of the heterodimer CD11b/CD18 on PMNs all were not required for autoregulation. Finally, morphometric and morphologic examination of PMNs activated at low vs. high density revealed histologic and structural correlates associated with the attenuated PMN activation response of cells triggered at high cell density. We conclude that multiple structural and functional aspects of the PMN activation response are modulated by cell density and suggest that this property is important both in the physiologic control of neutrophil activation and in the design of in vitro assays of the neutrophil activation response.
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PMID:"Autoregulation" of human neutrophil activation in vitro: regulation of phorbol myristate acetate-induced neutrophil activation by cell density. 215 15

The effects of quinones (benzoquinone, menadione, and doxorubicin) on the superoxide production in cell free systems (xanthine oxidase and rat liver microsomes) and of polycationic electrolyte- and latex-stimulated rat peritoneal macrophages have been studied. Contradictory results were obtained in cell free systems when two traditional assays for detection of superoxide ion, the cytochrome c reduction and the lucigenin-dependent chemiluminescence (CL), were used: all quinones inhibited the lucigenin-dependent CL at sufficiently large concentrations, but they did not inhibit at all the reduction of cytochrome c. It was proposed that the cytochrome c assay gave erroneous results due to the reversibility of the interaction of semiquinones with dioxygen. The effect of quinones on the superoxide production by peritoneal macrophages was biphasic: all quinones stimulated the O2-. formation at low concentrations and inhibited it at elevated concentrations. It was concluded that among the quinones studied, only menadione was capable of stimulating the superoxide production via a one-electron transfer mechanism in cell free systems, while the stimulatory effect of small concentrations of quinones on the O2-. production in macrophages was possibly due to their action on the activation of NADPH oxidase.
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PMID:Are quinones producers or scavengers of superoxide ion in cells? 216 57

Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.
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PMID:Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity. 217 79

Recent studies have shown that, in cell free systems, Mg2+, but not Ca2+, is absolutely required for the generation of reducing equivalents by neutrophil NADPH oxidase. Apparently Mg2+ is required for binding of cytosolic protein(s) to the plasma membrane in forming a functional NADPH oxidase complex. Using intact neutrophils made permeable to Mg2+ and Ca2+ by A23187 it was found that Mg2+ was required for the generation of reducing equivalents by PMA stimulated cells while Ca2+ inhibited cytochrome c reduction. On the other hand, FMLP-induced neutrophils were unable to generate reducing equivalents unless both Ca2+ and Mg2+ were present in the media. Sequential addition of these cations indicated that Ca2+ was required for transduction of the FMLP-receptor generated signal to a point in the pathway where Mg2(+)-dependent synthesis of reducing equivalents occurred. This step, presumably, is the Mg2(+)-dependent completion of the NADPH oxidase complex. Finally, approximately 50% of the neutrophil's 2.19 fmoles (6.32 mM) of Mg2+ was found to be in a readily exchangeable pool and that pretreatment of neutrophils with either FMLP or PMA increased this pool by only 4-5%. This suggests there is an adequate pool of Mg2+ available to support completion of NADPH oxidase complex and that a stimulus-induced rise in free Mg2+ is not required to assist in the formation of the complex.
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PMID:Potential roles of Mg2+ and Ca2+ in NADPH oxidase dependent superoxide anion synthesis by human neutrophils. 217 63

1. A microtechnique for quantitating human neutrophil NADPH oxidase in a cell-free system is described. 2. This spectrophotometric discontinuous (fixed time) method is less material-consuming than existing methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples. 3. Measurement of NADPH oxidase using the new method can be accomplished in a final vol of 0.15 ml. 4. In the assay, neutrophil membranes solubilized with deoxycholate were incubated for 3 min with cytosolic fractions, magnesium, sodium dodecyl sulfate, and cytochrome c in the absence of NADPH to preincubate the oxidase before the addition of the reducing agent. 5. The reaction was started by adding NADPH and 2 min later terminated by adding superoxide dismutase. 6. The apparent Km for NADPH obtained by the new method was almost the same as that by the authorized method (39.2 +/- 3.1 SD vs 36.8 +/- 1.6). Activation of neutrophil NADPH oxidase was characterized using the new assay method.
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PMID:A microtechnique for neutrophil respiratory burst oxidase in a cell-free system--characterization of oxidase activation system. 239 Aug 59

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.
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PMID:NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles. 253 93

It is well known that Fc gamma R mediate the rapid release of agents of inflammation and, in addition, play an important role in the uptake of stimulatory antibody complexes. Activation of the FcR for human IgG1 (Fc gamma RI) on human monocytic cells triggers a transient activation of the NADPH oxidase. In this study, we tested the possibility that transience of the NADPH oxidase activation might have been the result of rapid internalization of cross-linked Fc gamma RI. Stimulatory receptor moieties were formed by cross-linking Fc gamma RI with receptor-specific mAb that are known to trigger superoxide anion release. The formation of the stimulatory receptor units was determined by quantitating the rate of superoxide anion production through its reduction of cytochrome c. This rate has been found to correlate with the rate of binding of cross-linking antibody and, therefore, the rate of formation of the stimulatory moieties (receptor aggregates). Internalization of cross-linked Fc gamma RI was measured by quantitation of cell-associated FITC-labeled Fc gamma RI-specific mAb resistant to acid elution. We found that cross-linking antibody bound to Fc gamma RI continued to be taken up by the cells well after cessation of oxidase activity. The constant rate of uptake and the differential effect of temperature on these two functions suggested that they are separately regulated. Quantitation of cross-linked receptors that were inactive, i.e., no longer stimulating superoxide anion production, indicated that 50% of internalizable, and therefore cross-linked, Fc gamma RI remained on the surface after oxidase activity had ceased. This evidence of cessation of oxidase activity before the endocytic uptake of mAb/R stimulatory units indicates that the activated state of surface cross-linked Fc gamma RI is of brief duration and that occupation of the receptors by cross-linking-ligand does not sustain the activated state of the receptor. Thus, Fc gamma RI-mediated oxidase activation is temporally limited to the formation of the stimulatory receptor moiety.
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PMID:Transient activation of the NADPH oxidase through Fc gamma RI. Oxidase deactivation precedes internalization of cross-linked receptors. 255 66

Human fibroblasts in primary culture released reactive oxygen species upon stimulation with cytokines such as interleukin-1 alpha (IL-1) or tumour necrosis factor-alpha (TNF). The primary radical produced was O2.- as determined by e.s.r. spin trapping and cytochrome c reduction. In contrast to the oxidative burst in granulocytes and monocytes, radical formation took place continuously for at least 4 h. Low-level chemiluminescence was increased by stimulation with IL-1 and TNF. Spectral characteristics and tests with azide led to the conclusion that the photoemissive species were excited carbonyls and not singlet oxygen. Further, there was a liberation of ethane from the cells. Radical production and light emission were not altered by either xanthine or allopurinol, nor by azide, cyanide or rotenone. O2.- production increased in the presence of NADH or NADPH, making an NAD(P)H oxidase a likely source.
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PMID:Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha. 255 98

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


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