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)

We postulated that Captopril may be capable of acting as a scavenger of free radicals, and performed in vitro studies using harvested human neutrophils. We studied the effect of Captopril on the reduction of Fe3+ cytochrome c by stimulated PMN's. Captopril acts as a reducing agent in this system, and is capable of reducing Fe3+ cytochrome c by itself. NADPH oxidase was harvested from PMA-stimulated human PMN's. Captopril inhibited the activity of this enzyme as assessed by the disappearance of NADPH determined spectrophotometrically. Since similar inhibition could be demonstrated with the superoxide scavenger superoxide dismutase, further studies were conducted using a DTNB assay of the terminal sulfhydryl group of Captopril, in the presence of a biochemical generator of superoxide (hypoxanthine/xanthine oxidase). We were unable to demonstrate disappearance of the thiol group in this system, suggesting that reaction of the SH group with 02- is unlikely under our conditions. We conclude that Captopril may interfere with human PMN NADPH oxidase in vitro.
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PMID:Captopril--a potential free radical scavenger: inhibition of PMN NADPH oxidase. 284 20

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O2- and H2O2, the latter being presumed to arise by spontaneous dismutation of the former. If H2O2 were indeed derived exclusively from released O2- according to the equation 2O2- + 2H+----H2O2 + O2, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H2O2 should not form when cytochrome c is present to scavenge O2- before it can dismutate. Although H2O2 cannot be measured directly in the presence of cytochrome c because it is consumed in reoxidizing reduced cytochrome c, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochrome c. We found that the relative amounts of extracellular H2O2 and O2- that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochrome c from H2O2 oxidation when some stimuli were used but not with others. For example, the ratio of O2- to H2O2 produced by neutrophils exposed to PMA was about 2:1, the expected result if H2O2 were derived from O2-. However when cytochalasin B was added to the cells before the stimulus, the yield of H2O2 was reduced but not the yield of O2-. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O2- and H2O2. Coating the plastic with IgG doubled cytochrome c reduction without effecting H2O2. In contrast, coating with albumin reduced H2O2 without effecting cytochrome c reduction. Soluble IgG aggregates induced production of mostly O2- whereas immune complexes resulted in release of both metabolites. FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O2- than H2O2. The addition of catalase to the cytochrome c solution improved the yield of reduced cytochrome c when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H2O2 release is not a linear function of the amount of O2- generated and that either a variable fraction of O2- spontaneously dismutates to H2O2 or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H2O2 as well as O2-.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Neutrophils may directly synthesize both H2O2 and O2- since surface stimuli induce their release in stimulus-specific ratios. 300 Sep 43

Maintenance of an acidic intralysosomal compartment may be relevant to multiple aspects of neutrophil function. The effect of lysosomal alkalinization on the neutrophil respiratory burst was studied by measuring cytochrome c reduction in response to soluble stimuli in the presence of lysosomotropic weak bases. The weak bases chloroquine, ammonium chloride, methylamine, and clindamycin all raised the intralysosomal pH and inhibited neutrophil oxidative metabolism at concentrations ranging from 0.1 to 100 mmol/L. Inhibition was dose dependent for each base and correlated significantly with the degree of lysosomal alkalinization. Concentrations that did not alkalinize the lysosome did not inhibit the respiratory burst. Inhibition by weak bases was seen when oxidative metabolism was stimulated by phorbol myristate acetate, calcium ionophore A23187, formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, or sodium fluoride. Increasing the stimulus concentration (from 5 ng/mL to 5 micrograms/mL phorbol myristate acetate and from 0.5 to 1 mumol/L A23187) diminished or abolished inhibition by weak bases. Washing the cells after incubation with bases and before stimulation substantially reversed the inhibition. None of the bases impaired detection of superoxide in a cell-free xanthine-xanthine oxidase assay. Other indexes of oxidative metabolism, including oxygen consumption and hydrogen peroxide release, were also inhibited by weak bases. Analysis of particulate NADPH oxidase activity from neutrophils stimulated in the presence of bases suggested that these cells assemble a subnormal amount of an enzyme complex with normal kinetic characteristics. Lysosomotropic weak bases alkalinized the neutrophil lysosome and produced inhibition of oxidative metabolism that was dose related, was not stimulus specific, and was largely reversed by washing the cells before stimulation. A possible explanation would be altered assembly of the enzyme complex involved in respiratory burst activation as a consequence of impaired granule/plasma membrane fusion in the presence of diminished transmembrane pH gradients.
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PMID:Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases. 300 23

The stimulation-specific NADPH-dependent reduction of ubiquinone-1 (Q-1) in guinea-pig macrophages was studied. The activity was due neither to any modified product of the phagocytosis-specific NADPH oxidase nor to non-specific diaphorases of the cells, since the activity was measured in sonicated or detergent-disrupted cells by subtracting the activity in the resting cells from that in cells activated by phorbol 12-myristate 13-acetate. The activity was not mediated by superoxide anions, since strict anaerobic conditions were employed. The anaerobic reduction of Q-1 was NADPH-specific, like superoxide formation under aerobic conditions, and its maximal velocity was also essentially the same as that of superoxide formation. The oxidase does not directly reduce Q-1 under aerobic conditions [Nakamura, Murakami, Umei & Minakami (1985) FEBS Lett. 186, 215-218], and the electron transfer from NADPH to cytochrome c by the oxidase under aerobic conditions was not enhanced by the addition of Q-1. The observations indicate that the phagocytosis-specific NADPH oxidase reduces Q-1 and that oxygen competes with the reduction of Q-1. Q-1 seems to accept electrons not from the intermediary electron carriers of the oxidase but from the terminal oxygen-reducing site of the enzyme.
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PMID:NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone-1 under anaerobic conditions. 302 22

Alpha,beta-unsaturated aldehydes such as acrolein (ACR) and crotonaldehyde (CRO) have been shown previously in our laboratory to inhibit the production of superoxide anion radical (O2-) by stimulated phagocytic cells in vitro in a dose-related manner. Based on the known reactivity of these compounds towards cellular sulfhydryls (SH), the present studies were aimed at investigating cellular SH status in relation to O2- production. Plasma membrane surface SH groups were measured using carboxypyridinedisulfide and monitoring the resultant formation of mixed disulfides through assay of thione released into the supernatant fraction. Intracellular non-protein sulfhydryls were measured using 5,5'-dithiobis-2-nitrobenzoic acid. In both human polymorphonuclear leukocytes (PMN) and rat pulmonary alveolar macrophages (PAM) there was a dose-related decrease in surface SH and soluble SH after ACR and CRO treatment. Propionaldehyde, a three-carbon saturated aldehyde, was without effect. The decrease in surface SH was greater than the decrease in soluble SH. In addition, in PMN and PAM preincubated with 5-40 microM ACR, there was a dose-related inhibition in the rate of O2- production with no effect on the lag time as measured by cytochrome c reduction. In stimulated PMN, there was a dose-related decrease in the rate after addition of 5-40 microM ACR. These data suggest that changes in SH status by reactive aldehydes can modulate the activity of the plasma membrane NADPH oxidase responsible for O2- production.
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PMID:Inhibition by reactive aldehydes of superoxide anion radical production from stimulated polymorphonuclear leukocytes and pulmonary alveolar macrophages. Effects on cellular sulfhydryl groups and NADPH oxidase activity. 303 Mar 33

The 2,6-dichlorophenolindophenol (DCIP)-reducing activity of the phagocytosis-associated NADPH oxidase was investigated using homogenates and a membrane fraction (F2) of elicited guinea pig peritoneal macrophages stimulated by phorbol myristate acetate. Essentially all of the stimulation-specific DCIP reduction under aerobic conditions could be inhibited when high concentrations of superoxide dismutase (SOD), about 10 times those usually used to inhibit the superoxide (O-2)-mediated cytochrome c reduction, were used. SOD inhibited the DCIP reduction by chemically generated O2- in the same manner as the stimulation-specific DCIP reduction by the macrophage F2, and the concentration of SOD necessary for 50% inhibition was about 10 times that for the reduction of cytochrome c. Under anaerobic conditions, however, the NADPH oxidase could reduce DCIP, though the rate was slow because we could not use a sufficiently high DCIP concentration. The observations indicate that the NADPH oxidase preferentially reduces oxygen under aerobic conditions, though the oxidase can reduce DCIP in the anaerobic state.
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PMID:2,6-Dichlorophenolindophenol-reducing activity of phagocytosis-associated NADPH oxidase. 303 17

The polyamines putrescine, spermidine and spermine, at concentrations of 10 microM, stimulated superoxide generation by human polymorphonuclear leukocytes induced by fMet-Leu-Phe in the presence of Ca2+. This positive effect was not evident in the absence of Ca2+ or when the polymorphonuclear leukocytes were stimulated by phorbol myristate acetate. Spermidine in the range of 10-100 microM showed a dose-dependent stimulatory effect on the superoxide generation induced by fMet-Leu-Phe, whilst at doses above 25 mM it produced an inhibitory effect. At this concentration, spermidine did not reduce the phorbol myristate acetate-neutrophil-induced O2-. generation, while an inhibitory effect by the polyamine was evident at concentrations above 50 mM. In addition, 100 microM spermidine increased the amount of superoxide generated and enhanced the ability of the chemotactic peptide to stimulate superoxide generation. The polyamines in the range of 10 microM-25 mM did not modify the activity of purified NADPH oxidase, nor the rate of reduction of cytochrome c as supported by the xanthine/xanthine oxidase reaction. These results indicate that physiological concentrations of polyamines can stimulate superoxide formation by polymorphonuclear leukocyte cells produced by the chemotactic peptide fMet-Leu-Phe, probably by increasing the availability of external calcium.
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PMID:Polyamines stimulate superoxide production in human neutrophils activated by N-fMet-Leu-Phe but not by phorbol myristate acetate. 304 Jan 18

Limited proteolysis of brewer's yeast old yellow enzyme (OYE) was carried out with bovine pancreatic alpha-chymotrypsin. The reaction proceeded with a decrease of the NADPH oxidase activity, generating specifically two peptides (designated as 34K and 14K fragments) with apparent molecular weights of 34,000 and 14,000, respectively. The same proteolytic treatment of apo OYE resulted in rapid and complete digestion of the protein. The 34K and 14K fragments are so intimately associated with each other that the isolation of each peptide from the other in the native form was unsuccessful. However, the complex of the two fragments was separated from the intact OYE and termed "nicked OYE." Nicked OYE still retained FMN and showed a visible-absorption spectrum slightly modified from that of intact OYE. Nicked OYE showed decreased affinity toward rho-bromophenol as compared to intact OYE. Nicked OYE exhibited lower Km and Vmax values than intact OYE in the NADPH oxidase reaction. The 34K and 14K fragments could be separated from each other by reversed-phase HPLC under denaturing conditions and the amino acid sequences of the two fragments and intact OYE in the amino terminal regions were determined. The N-terminal sequence of the 34K fragment coincided with that of intact OYE, indicating that the 34K fragment lies in the N-terminal side of OYE. The N-terminal sequence of the 14K fragment was found to show homology with the site of flavodoxin where it forms an electron-transfer complex with cytochrome c. The characteristic feature of this region is the presence of acidic residues and is shared by the FMN domain of NADPH-cytochrome P-450 reductase. We interpret these findings as indicating that OYE has a physiological role as an electron transfer component.
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PMID:On the structure of old yellow enzyme studied by specific limited proteolysis. 312 66

Homogenates of all rat tissues examined, except brain, catalyze reduction of N,N-dimethyl-p-aminoazobenzene N-oxide (DMAB N-oxide) to N,N-dimethyl-p-aminoazobenzene by NADPH. Liver is the most active, and about one third of the homogenate activity of this tissue is recovered in the cytosol fraction. The purified cytosol enzyme has the properties of a tetrameric protein (Mr 370,000) consisting of identical subunits free from chromophores that absorb in the visible spectrum and from metals or other detectable prosthetic groups. The purified reductase is also free from NADPH oxidase and from cytochrome c or azo reductase activities. The enzyme is quite specific for NADPH as reductant and DMAB N-oxide as the electron acceptor. Reduction of other N,N-dimethyl-arylamine or alkylamine oxides as well as N-methylheterocyclicamine oxides could not be detected. Analysis of kinetic data indicate that, at saturating concentrations of the other substrate, 21 microM NADPH and 700 microM DMAB N-oxide are required for half maximal velocity. At infinite concentrations of both substrates the turnover is 150 min-1 at 37 degrees C.
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PMID:Properties of an N,N-dimethyl-p-aminoazobenzene oxide reductase purified from rat liver cytosol. 315 68

Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.
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PMID:The respiratory burst oxidase of neutrophils. Separation of an FAD enzyme and its characterization. 362 61


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