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)

Superoxide generation in the NADPH oxidase reaction of NADPH-cytochrome P-450 reductase, demonstrated using the ESR spin trap, 5,5-dimethyl-1-pyrroline-1-oxide, increased on the addition of lactoferrin. The NADPH-lactoferrin reductase activity was assessed in terms of NADPH oxidation and oxygen consumption. From Lineweaver-Burk plots, the Km and Vmax for lactoferrin were estimated to be 13 microM and 0.5 S-1, respectively. The liberation of iron from lactoferrin was proven with the use of bathophenanthroline and by the demonstration of bleomycin-dependent DNA degradation; lactoferrin was reduced by the enzyme in the presence of NADPH. During the reaction, the ESR spectrum of the spin trap adduct changed from one characteristic of DMPO-OOH to that of DMPO-OH. The conversion was ascribed to the reaction of hydrogen peroxide with reduced lactoferrin.
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PMID:Lactoferrin-mediated formation of oxygen radicals by NADPH-cytochrome P-450 reductase system. 169 25

Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.
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PMID:Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols. 203 43

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

The NADPH oxidase in neutrophils was specifically solubilized from membrane vesicles of porcine blood neutrophils and rapidly concentrated by immunoprecipitation with cross-reacting anti-P-450 reductase IgG. The precipitates from both myristic acid-stimulated and resting cells contained one third of the cytochrome b-558 and were slightly contaminated with myeloperoxidase. The immunoprecipitate from stimulated cells gave rhombic high-spin ESR signals of a heme at g = 6.47 and 5.49, which were insensitive to KCN, whereas the preparation from resting cells did not give these signals. The rhombic high-spin signals are discussed in view of the participation of cytochrome b-558 in the NADPH oxidase system.
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PMID:ESR signals from stimulated and resting porcine blood neutrophils. 303 84

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

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

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

Nitric oxide (NO) reacts with heme-containing enzymes, including certain isoforms of cytochrome P450. Cytochrome P4502E1 (CYP2E1) is induced by ethanol and plays an important role in the toxicity of ethanol and other hepatotoxins. CYP2E1 is also very effective in generating reactive oxygen intermediates such as superoxide radical and H2O2, oxidizing ethanol to the 1-hydroxyethyl radical, and has a high NADPH oxidase activity. The effect of NO on CYP2E1 catalytic activity and generation of reactive oxygen intermediates was evaluated. Incubating liver microsomes isolated from rats treated with pyrazole to induce high levels of CYP2E1, with gaseous NO or NO released from a variety of NO donors such as SNAP, DEA/NO, spermine/NO, and GSNO, resulted in a loss of CYP2E1 catalytic activity with specific substrates such as p-nitrophenol or dimethylnitrosamine. Trapping of NO with hemoglobin resulted in protection of CYP2E1 activity against the inactivation by NO. There was no effect by analogues of the donors which do not release NO nor was there any effect by NO on NADPH-cytochrome P450 reductase activity. Inactivation of CYP2E1 by NO was not prevented by superoxide dismutase or catalase, suggesting that superoxide, H2O2, or peroxynitrite were not responsible for the actions of NO. The inactivated CYP2E1 was not degraded nor did it lose its epitope sites as shown by Western blot analysis. Associated with loss of CYP2E1 catalytic activity was a decrease in the formation of superoxide radical and H2O2, in microsomal lipid peroxidation catalyzed by low, but not high concentration of iron, and in consumption of NADPH. Oxidation of ethanol to the 1-hydroxyethyl radical was also inhibited by NO. ESR experiments indicated the formation of stable heme-NO complexes with CYP2E1. NO appears to compete with O2 and CO for binding to CYP2E1 as incubation with gaseous NO, or NO donors inhibited formation of the characteristic CO binding spectrum of P450. Microsomes isolated from a stably transfected HepG2 cell line expressing only CYP2E1 were also inactivated by NO, validating interaction of NO with this isoform of P450. These results indicate that NO inhibits CYP2E1 catalytic activity and generation of reactive radical intermediates. NO may prevent toxicity of agents which require bioactivation by P450 isoforms such as CYP2E1 and in generation of reactive intermediates by CYP2E1.
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PMID:Inhibition of rat and human cytochrome P4502E1 catalytic activity and reactive oxygen radical formation by nitric oxide. 901 19

In this report, a protocol for the preparation of the hydrochloride of S-nitroso-L-cysteine ethyl ester (SNCEE.HCl; 2) is presented. The synthesis of 2 has been targeted because S-nitroso-L-cysteine (SNC; 2b), which is extensively used for trans-S-nitrosation of thiol-containing proteins, has a limited ability of crossing cellular membranes. The nitrosothiol 2 was prepared via direct S-nitrosation of the hydrochloride of L-cysteine ethyl ester (CEE.HCl; 1a) with ethyl nitrite. 2 is relatively stable in crystal form and when neutralized to SNCEE (2a) in aqueous solutions treated with chelators of metal ions. Traces of metal ions, however, triggered the decomposition of 2a to nitric oxide and a S-centered radical, which were detected by ESR spectrometry. In contrast to 2b, 2a is a lipophilic compound that was taken up by human neutrophils. The latter process was paralleled by inhibition of the NADPH oxidase-dependent generation of superoxide anion radicals, presumably via reaction(s) of intracellular trans-S-nitrosation. Intracellular accumulation of S-nitrosothiols was observed with 2a but not with 2b. It is expected that the use of 2a will be advantageous when intracellular reactions of trans-S-nitrosation are to be studied.
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PMID:Preparation and properties of S-nitroso-L-cysteine ethyl ester, an intracellular nitrosating agent. 1138 48

Previous studies have shown that a constitutively active isoform of Ras is able to produce superoxide radical (O2(-)). The present study investigate the mechanisms by which O2(-) radical mediates signals from Ras protein to the nucleus, leading to cellular responses such as apoptosis in Cr(VI)-stimulated cells. Two human prostate tumor cell lines, Ras(+), which overexpresses Ras, and Ras(-), which has a normal Ras level, were utilized. Compared to Ras(-) cells, Ras(+) cells exhibited higher susceptibility to apoptosis induced by Cr(VI). Catalase, sodium formate, and deferoxamine inhibited Cr(VI)-induced apoptosis. Similar differences were observed in both cellular DNA damage and the activation of p53 protein. The differences in Cr(VI)-induced cell responses in Ras(+) and Ras(-) cells were due to differences in the generation of free radicals between these two cells. ESR spin trapping measurements showed that Ras(+) cells generated more hydroxyl radical ((.)OH), O2(-) radical, and Cr(V) than Ras(-) cells following Cr(VI) stimulation. The generation of the reactive oxygen species (ROS) can be abolished by the addition of superoxide dismutase (SOD) or if the experiment were carried out in an argon atmosphere. Catalase inhibited spin adduct signals but was much less potent than SOD. The mechanism of ROS generation in Cr(VI)-stimulated Ras(+) cells involves the reduction of molecular oxygen to O2(-) radical by a flavoenzyme-containing NADPH oxidase complex as shown by oxygen consumption and diphenylene iodonium (DPI) inhibition. Results shown above support the following conclusions: (a) Ras protein mediates O2(-) radical generation through reduction of molecular oxygen by NADPH oxidase in Cr(VI)-stimulated cells. (b) The O2(-) radical and Cr(VI) produce other reactive species, including H2O2, OH radical, and Cr(V) through O2(-) dismutation and Haber-Weiss type of reactions. (c) Among these reactive species, (.)OH radical is responsible for the further transduction of signals from Ras to the nucleus, leading to various cell responses.
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PMID:Role of reactive oxygen species and Cr(VI) in Ras-mediated signal transduction. 1497 53


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