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)

The effect of pyridine on the heme environment of cytochrome b558 was studied using ESR and optical absorption spectroscopy in relation to the O2(-)-generating activity in the NADPH oxidase system of stimulated pig neutrophils. As the concentration of pyridine increased, the absorption maxima of the alpha- and gamma-bands of cytochrome b558 shifted which correlated with a concomitant decrease in O2(-)-generating activity. In addition, the g = 3.2 signal of cytochrome b558 decreased with the concomitant appearance of a new ESR spectrum that strikingly resembled that of cytochrome P450. The results suggest that pyridine induces a structural modification in the heme environment of cytochrome b558 by shifting the 5th heme ligand (histidine) to a nearby thiolate group without direct binding of pyridine to the heme. The existence of a reactive thiolate near the heme iron was confirmed by pretreatment of blocked cytochrome b558 with p-chloromercuribenzoate, which completely inhibited the formation of the cytochrome P450-like ESR spectrum. The results provide further evidence that a low-spin heme iron of cytochrome b558 with a g-value of 3.2 is essential to the O2(-)-forming reaction of the NADPH oxidase system. From sequence alignments of cytochrome P450 with those of large and small subunits of cytochrome b558, the heme in cytochrome b558 appears to be specifically associated with the large subunit.
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PMID:Modulation of the heme environment of neutrophil cytochrome b558 to a "cytochrome P450-like" structure by pyridine. 785 3

In previous studies, we showed that interleukin-4 (IL-4) suppressed porcine (p) macrophage superoxide production and that the mechanism of suppression involved down-regulation of the superoxide-generating enzyme NADPH oxidase heavy-chain 91-kDa subunit mRNA (gp91-phox) expression. In order to examine the effect of IL-4 on expression of the gene encoding the porcine NADPH oxidase light-chain 22-kDa subunit (p22-phox), we cloned the p22-phox cDNA from a macrophage library. The p22-phox cDNA is 786 bp in length and contains a 576-bp open reading frame which predicts a primary translation product of 192 amino acids (aa). Comparison of the porcine and human 22-phox cDNAs showed a high degree of similarity between the two species in their nucleotide (85%) and deduced aa (83%) sequences. as well as in their hydropathy profiles. Notable features, including a high proline content and an iron-coordinating His94, are conserved in both the porcine and human 22-Phox. A single species of mRNA of about 1 kb was detected in macrophages. The mRNA levels remained unchanged in cells treated with lipopolysaccharide (LPS) or with IL-4 at various concentrations from 0-50 ng/ml. Prolonged treatment with LPS or IL-4 did not enhance the effect of these substances on p22-phox mRNA expression. The effect of IL-4 on p22-phox mRNA expression was also compared with another immunosuppressive cytokine, transforming growth factor-beta 1 (TGF beta 1). No change in mRNA expression was found in the cells with or without TGF beta 1 treatment. The results indicated that the heavy and light chains of NADPH oxidase are independently regulated by IL-4 in macrophages.
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PMID:Cloning and expression of the gene encoding the porcine NADPH oxidase light-chain subunit (p22-phox). 795 70

Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OHzero) formed in an extracellular Fenton-like reaction. H2O2-induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OHzero scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin-trapped OHzero adduct. The role of OHzero was confirmed in experiments with exogenously added iron; free or EDTA-bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OHzero depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N-formyl-methionyl-leucyl-phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as 'second messengers' during the initial phase of the platelet activation process.
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PMID:Role of hydroxyl radicals in the activation of human platelets. 817 49

Nicotinic acid hydroxylase from Clostridium barkeri contains selenium in an unidentified form that is dissociated as a low molecular weight compound upon denaturation of the enzyme. Other cofactors of this enzyme are molybdopterin, FAD, and iron-sulfur clusters. In the current study, we show that the enzyme, as isolated, exhibits a stable Mo(V) electron paramagnetic resonance (EPR) signal ("resting" signal) and that this signal is correlated with the selenium content and nicotinate hydroxylase activity of the enzyme. Substitution of 77Se for normal selenium isotope abundance results in splitting of the Mo(V) EPR signal of the native protein without affecting the iron signals of the FeS clusters. The Mo(V) EPR signal and nicotinic acid hydroxylase activity of enzyme isolated from cells grown in selenium-deficient medium are barely detectable. In contrast, the EPR signals of the FeS clusters, the electronic absorption spectrum, the NADPH oxidase activity, and the chromatographic behavior are changed little and are typical of active selenium-containing enzyme. An EPR signal indicative of the presence of molybdenum in the selenium-deficient enzyme also is exhibited. From these results, we conclude that a dissociable selenium moiety is coordinated directly with molybdenum in the molybdopterin cofactor and, moreover, this selenium is essential for nicotinic acid hydroxylase activity.
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PMID:Nicotinic acid hydroxylase from Clostridium barkeri: electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. 827 71

Lazaroids (21-aminosteroids and 2-methylaminochromans) are a new series of drugs designed and demonstrated to protect against tissue damage after trauma and/or ischemia. It has been suggested that the protective effects of lazaroids are derived from their potent actions to inhibit iron-dependent lipid peroxidation, but whether this is sufficient to explain their therapeutic effects is unknown. In an effort to better understand their mechanism of action, these drugs were tested for other modes of antioxidant activity such as scavenging superoxide and hydroxyl radicals and inhibition of production of oxygen free radicals by human neutrophils stimulated with phorbol myristate acetate. Using an ESR spin-trapping technique, with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for superoxide and hydroxyl radicals, we found that the lazaroids U74500A and U78518F are, at best, weak scavengers of superoxide radicals whereas U78518F is a strong scavenger of hydroxyl radicals. In addition, lazaroids were found to be strong inhibitors (60-80% inhibition at 50 microM) of the superoxide-generating NADPH oxidase of human neutrophils. Inhibition of NADPH oxidase by lazaroids in cell-free systems suggested the action to be on the activated enzyme rather than on the process of activation. This may represent an important mode action of lazaroids and suggests their potential use in ischemic/inflammatory conditions involving oxygen free radical production by activated phagocytic cells.
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PMID:Inhibition of superoxide-generating NADPH oxidase of human neutrophils by lazaroids (21-aminosteroids and 2-methylaminochromans). 838 Oct 5

We have studied the relationships between in vivo (whole cells) and in vitro (plasma membranes) ferrireductase activity in Saccharomyces cerevisiae. Isolated plasma membranes were enriched in the product of the FRE1 gene and had NADPH dehydrogenase activity that was increased when the cells were grown in iron/copper-deprived medium. The diaphorase activity was, however, independent of Fre1p, and Fre1p itself had no ferrireductase activity in vitro. There were striking similarities between the yeast ferrireductase system and the neutrophil NADPH oxidase: oxygen could act as an electron acceptor in the ferrireductase system, and Fre1p, like gp91, is a glycosylated hemoprotein with a b-type cytochrome spectrum. The ferrireductase system was sensitive to the NADPH oxidase inhibitor diphenylene iodonium (DPI). DPI inhibition proceeded with two apparent Ki values (high and low affinity binding) in whole wild-type and Deltafre2 cells and with one apparent Ki in Deltafre1 cells (high affinity binding) and in plasma membranes (low affinity binding). These results suggest that the Fre1-dependent ferrireductase system involves at least two components (Fre1p and an NADPH dehydrogenase component) differing in their sensitivities to DPI, as in the neutrophil NADPH oxidase. A third component, the product of the UTR1 gene, was shown to act synergistically with Fre1p to increase the cell ferrireductase activity.
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PMID:Evidence for the Saccharomyces cerevisiae ferrireductase system being a multicomponent electron transport chain. 866 26

Unlike other cytochrome P450-dependent oxygenase inhibitors, ketoconazole has been shown to suppress the murine macrophage-mediated oxidative modification of human low-density lipoproteins (LDL) in a dose-dependent manner. The benzo[alpha]pyrene-induced microsomal monooxygenase activity was accomplished by a 1,5-fold increase in LDL oxidation by macrophages, ketokonazole (20 mu), methoxalene (20 mu), and alpha-naphthaflavone (50 mu). Ketoconazole was also effective in inhibiting macrophageal NADPH oxidase and LDL autooxidation induced by Fe2+ rather than Cu+, which is likely to be associated with its ability to act as a chelator of free and heme-bound iron ions.
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PMID:[Effect of cytochrome P-450 inhibitors on oxidative modification of low-density lipoproproteins by macrophages]. 878 70

1. Brief exposure of cultured rat glomerular mesangial cells (GMC) to H2O2 in nominally bicarbonate-free solution induced a rapid dose dependent, dantrolene-inhibitable increase in intracellular free Ca2+ from 65 +/- 6 to 203 +/- 14 nmol/L and a prolonged release of [14C]-arachidonic acid [14C]-AA which preceded the onset of cell membrane damage assessed by trypan-blue uptake. 2. Ca2+ responses were potentiated in HCO3-/CO2 containing buffers and reached values of 1145 +/- 100 nmol/L at 1 mmol/L H2O2. In HCO3-/CO2 solutions, but not HEPES buffer, H2O2-induced Ca2+ increases were markedly attenuated by verapamil (100 mumol/L) or removal of extracellular calcium. 3. Enhanced release of [14C]-AA was partially attenuated by inhibitors of key intracellular signalling mechanisms including the phospholipase-A2 (PLA2) inhibitor mepacrine (100 mumol/L), the NADPH oxidase inhibitor diphenyliodonium (10 mumol/L), the mitochondrial calcium-cycling inhibitor ruthenium red (10 mumol/L) and the iron chelator dipyridyl (100 mumol/L). Release was unaffected by protein kinase C inhibition with H7 (100 mumol/L), inositol triphosphate antagonism with neomycin (1 mmol/L) or overnight treatment with the G-protein antagonist pertussis toxin (5 micrograms/mL). 4. Several structurally diverse lipoxygenase inhibitors, including esculetin, baicalein and phenidone, over the dose range 1-100 mumol/L, also prevented [14C]-AA release and markedly protected against cell membrane damage. No drug directly scavenged H2O2 assessed by UV absorption. 5. These results indicate that H2O2 activates in GMC a complex series of interrelated pathological mechanisms which in turn contribute to a prolongation of oxidative damage beyond the time of the initial exposure. These include an increase in intracellular calcium which, depending upon conditions, appears to be mediated by release from intracellular stores as well as Ca2+ entry from the extracellular space. In turn there is a sustained release of arachidonic acid, which may partly depend on prolonged activation of PLA2 but not phospholipase C. 6. Release of [14C]-AA could be attenuated by inhibitors of NADPH oxidase, mitochondrial calcium-cycling, iron chelators and a structurally diverse range of lipoxygenase inhibitors in association with protection from H2O2-mediated cell membrane damage.
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PMID:Role of intracellular signalling pathways in hydrogen peroxide-induced injury to rat glomerular mesangial cells. 884 14

The NADPH oxidase complex of activated neutrophils consists of a membrane-bound flavocytochrome b and cytosolic activation factors. Despite its ability to react with O2, the heme b component of the flavocytochrome is insensitive to cyanide and CO2, and slowly reactive to butyl isocyanide. We report here that arachidonic acid, an anionic amphophil which elicits oxidase activation in a cell-free system induces a transition of the heme iron of the neutrophil flavocytochrome b from a low-spin hexacoordinated state to a high-spin pentacoordinated state and promotes the binding of butyl isocyanide to the heme b. Low-temperature EPR spectra of air-oxidized flavocytochrome b either purified or in its membrane-bound form showed a low-spin signal at g = 3.26 and a high-spin signal at g = 6.0. Upon addition of arachidonic acid, the g = 3.26 signal vanished; a low-spin signal at g = 2.23 appeared, and the signal at g = 6.0 progressively increased. The subsequent addition of butyl isocyanide resulted in the decrease of the g = 6.0 and g = 2.23 signals and in the appearance of a new low-spin signal at g = 2.33. Consistent with the EPR results, upon addition of arachidonic acid to oxidized flavocytochrome b, a 2.5 nm blue shift of the Soret peak was detected in low-temperature optical spectra. The subsequent addition of butyl isocyanide resulted in the emergence of a peak at 432 nm reflecting the formation of a butyl isocyanide-oxidized heme b complex. In the case of sodium dithionite-reduced flavocytochrome b, arachidonic acid promoted the binding of butyl isocyanide to the reduced heme b, as shown by the emergence of a peak at 434 nm and the decrease of the alpha band at 558 nm. The same promoting effect was encountered with sodium dodecyl sulfate, an anionic amphophil capable of eliciting oxidase activation like arachidonic acid. In contrast to arachidonic acid, arachidonic acid methyl ester was ineffective and counteracted the effect of arachidonic acid. Butyl isocyanide added to intact neutrophils was found to bind to heme b, only after the cells have been activated. These data demonstrate the transient accumulation of a pentacoordinated form of the heme iron of flavocytochrome b under in vitro and in vivo conditions; the pentacoordinated form of the reduced heme b is postulated to react with O2 to generate the superoxide anion.
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PMID:Electron transfer across the O2- generating flavocytochrome b of neutrophils. Evidence for a transition from a low-spin state to a high-spin state of the heme iron component. 887 8

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91(phox) protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.
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PMID:Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. 894 93


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