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)

Hepatic microsomes from rats fed a crude or a purified diet were compared by measureing their contents of protein, cytochrome P-450, and cytochrome b5, their rates of activity of NADPH- and NADH-cytochrome c reductases, NADPH-cytochrome P-450 reductase, NADPH oxidase, lipid peroxidase, ethylmorphine N-demethylase, aniline hydroxylase, benzpyrene hydroxylase, and their substrate-binding spectra (ethylmorphine, hexobarbital, aniline, and ethyl isoyanide). With the exception of lipid peroxidase activity, which was much higher in microsomes from animals fed the crude diet, little or no consistent diet-related differences in these measurements were observed over a 4-week experimental period, nor were results significantly less variable with one or the other diet. No consistent significant differences were observed with two strains of rats. The lower lipid peroxidase activity seen with the purified diet appeared to be due to the high vitamin E intake when that diet was employed; rats fed the crude diet and an oral supplement of alpha-tocopherol yielded microsomes with low lipid peroxidase activities similar to those seen in microsomes from rats fed the purified diet. A gradual temporal increase in benzpyrene hydroxylase activity was observed with both diets. This was interpreted to be due to environment inducing agents other than those present in the diet.
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PMID:Comparison of hepatic microsomal drug-metabolizing systems from rats fed crude and purified diets. 0 25

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.
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PMID:Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16. 18 26

Activity of aryl hydrocarbon hydroxylase (AHH), cytochrome c-reductase, and NADPH oxidase, and epinephrine oxidation to adrenochrome were determined in lung microsomes from intact, adrenalectomized, and adrenalectomized cortisol-treated female rats under ambient and hyperoxic conditions. Microsomal adrenochrome formation, which is initiated by superoxide anion or other free radicals, was increased by adrenalectomy and decreased by cortisol treatment. Exposure of animals to 100% oxygen caused a further increase in adrenochrome formation. NADPH-cytochrome c-reductase and AHH activities were increased in incubations of microsomes from animals which had received cortisol in vivo while adrenalectomy led to decreases activity. NADPH oxidase activity was increased by cortisol in lung microsomes in the presence of either epinephrine or cytochrome c. Epinephrine conversion to adrenochrome in the presence of lung microsomes was blocked by SOD, but NADPH-cytrochrome c-reductase and AHH activity were unaffected.
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PMID:An effect of corticosteroids and 100% oxygen on aryl hydrocarbon hydroxylase, cytochrome-c reductase, and free radical formation by rat lung microsomes. 21 Mar 49

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.
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PMID:Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ. 130 41

C1q, a plasma glycoprotein and the recognition component of the classical complement pathway, interacts with specific cells of the immune system resulting in the enhancement of cell function. For example, interaction of C1q with its cell-surface receptor on neutrophils induces the activation of the respiratory burst, a finding previously documented using a chemiluminescent assay to detect oxygen radical formation. In an alternative approach we have now used a modified cytochrome c reduction assay to characterize C1q-mediated production of superoxide anion (O2-) in more detail. C1q coated to microtiter wells induced O2- release, which occurred microtiter wells induced O2- release, which occurred after a lag period of 10 to 20 min, and was then sustained over approximately 1 h. O2- production could be triggered by the purified pepsin-resistant, collagen-like fragment of C1q, but not by mannose-binding protein and pulmonary surfactant protein A, proteins that also contain collagen-like domains. Concentrations of C1q which promoted a vigorous O2- generation did not induce release of neutrophil primary granules and caused little or no secondary granule release. Investigation of the biochemical events mediating C1q stimulated O2- production by neutrophils revealed that the response invoked two biochemical pathways with distinct sensitivities to previously described inhibitors. A role for Ca2+ in initiation of the response was suggested by the inhibitory effect of EGTA, the calmodulin antagonist W7, and the intracellular Ca2+ chelator BAPTA. The protein kinase inhibitor staurosporine did not inhibit the induction of the response, but did block that component of the response occurring after approximately 30 min. Neither phase of C1q-mediated O2- production was inhibited by pertussis toxin, a strong inhibitor of the G-protein-coupled FMLP-mediated response. In summary, C1q-triggered O2- production is relatively unique both in terms of the kinetics of the response and the biochemical pathways evoked. These data support the hypothesis that more than one biochemical pathway induced by ligand-receptor interaction can activate the neutrophil NADPH oxidase.
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PMID:Signal transduction mechanisms of C1q-mediated superoxide production. Evidence for the involvement of temporally distinct staurosporine-insensitive and sensitive pathways. 131 35

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.
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PMID:Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. 132 36

The influence of folic acid and several antagonists of the folic acid metabolism on neutrophil superoxide generation was investigated with the cytochrome c reduction assay. The compounds were found to be partial competitive inhibitors of the NADPH oxidase, their activity apparently increasing with larger substituents at the 10 position. There is evidence that compounds with a 4-oxo substituent are taken up more slowly by neutrophils than those with a 4-amino functionality. Scavenging properties could be excluded from control measurements with the xanthine/xanthine oxidase assay.
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PMID:Inhibition of the neutrophil NADPH oxidase by folic acid and antagonists of the folic acid metabolism. 132 93

The oxidative metabolic status of blood monocytes (BM) and alveolar macrophages (AM) in patients with active pulmonary tuberculosis (TB) (n = 40) and in successfully treated patients (n = 40) was assessed and compared with that of healthy control subjects (n = 40). Oxygen free radical (OFR) generation, measured by chemiluminescence (CL) and cytochrome c reduction assay and confirmed by using scavengers of different OFR, was suppressed in AM of the pulmonary TB group compared with healthy controls, whereas it was enhanced in BM. Successfully treated patients showed partial recovery of CL and cytochrome c reduction in AM. There was no significant change in BM of patients after having been treated. The overall capacity to generate OFR was markedly suppressed upon in vitro stimulation with latex in both BM and AM of TB patients. The observed suppressed oxidative metabolic activity in BM and AM was further elucidated by studying the molecular mechanism of respiratory burst. The activities of NADPH oxidase and enzymes of the hexose monophosphate (HMP) shunt were significantly (p less than 0.05) decreased in BM and AM of pulmonary TB patients compared with healthy controls. Patients who had been treated showed marked recovery of NADPH oxidase and HMP shunt activity. The present study suggests that tubercle bacilli escape the microbicidal action of macrophages as a result of suppressed OFR generation caused by decreased activity of HMP shunt, leading to decreased levels of NADPH, thereby preventing NADPH oxidase from working at its full capacity.
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PMID:Oxidative metabolic status of blood monocytes and alveolar macrophages in the spectrum of human pulmonary tuberculosis. 158 98

Effects of non-steroidal anti-inflammatory drugs (NSAID: amfenac sodium, diclofenac sodium, indomethacin and ketoprofen) on the generation of superoxide anion (O2-) by isolated rat polymorphonuclear leukocytes (PMN) were studied spectrophotometrically using cytochrome c. The effects of these drugs were also studied on O2- production by the xanthine-xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-NADPH oxidase systems. Amfenac sodium, at 0.1 mM, inhibited significantly O2- generation in rat PMN induced by opsonized zymosan. At 0.5 mM, diclofenac sodium and indomethacin inhibited the O2- generation in rat PMN. All of the above drugs slightly inhibited O2- production by the xanthine-xanthine oxidase system. On the other hand, O2- production by the NADPH-NADPH oxidase system was significantly inhibited by the addition of amfenac sodium, ketoprofen or indomethacin. These results suggest that non-steroidal anti-inflammatory drugs do not work as an O2- scavenger and block O2- production by the NADPH-NADPH oxidase system of rat PMN. It is concluded that amfenac sodium and the other drugs are able to inhibit granulocyte O2- production by blocking the activation of NADPH-oxidase.
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PMID:Inhibitory effects of non-steroidal anti-inflammatory drugs on superoxide generation. 165 19

Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.
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PMID:Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system. 165 5


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