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)

Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytes prepared from pig blood was carried out with concentrated cellular and subcellular fractions to characterize EPR signals of cytochrome b-558 (cyt b-558). A thick cell suspension (approximately 2 x 10(9) cells/ml), containing mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not only these signals but also low spin heme signals at g values of 2.86, 2.13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130-132). MPO and eosinophil peroxidase (EPO) were released from the membrane fractions with 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and then were highly concentrated, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O2(-)-forming NADPH oxidase associated in the membranes was solubilized with heptyl-thio-glucoside at 0 degree C and concentrated up to 45 microM cyt b-558 with no modification of the heme moiety confirmed by its O2(-)-generating activity and lack of carbon monoxide-binding capacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 +/- 0.05, which was cyanide-insensitive and reducible with reductants. The signal intensity was concentration dependent, suggesting that the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.
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PMID:Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pig blood granulocytes. 132 37

Incubation of the isolated mouse diaphragm with a high rate of oxygenation (10 ml s-1, 95% O2 + 5% CO2) causes a characteristic cellular damage with widely-separated myofibrils and swollen sarcotubular system within 10 min. This damage was ameliorated by inhibitors of the hydroxyl radical (.OH), desferrioxamine, dimethyl thiourea and 120 mM mannitol, and by incubation at 8 degrees C. It was not prevented either by inhibitors of the pathway leading to sarcolemma damage (nordihydroguaiaretic acid, alpha-tocopherol, butylated hydroxytoluene) nor by agents and treatments that inhibit the oxygen paradox of cardiac muscle (glucose, omission of extracellular calcium, incubation at 30 degrees C, superoxide dismutase and catalase). Nevertheless there are similarities between these two types of damage triggered by O2 and the possibility that in both an NAD(P)H oxidase is stimulated and cytotoxic oxygen radicals are generated is discussed.
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PMID:Cytotoxic effect of oxygen on the skeletal muscle of mouse diaphragm. 256 50

The present experiments were designed to evaluate the effect of lead on the capacity of macrophages to respond to activating signals by increased respiratory-burst activity. When mouse peritoneal macrophages were exposed for 24 h to macrophage-activating factor (MAF) and/or bacterial lipopolysaccharide in the presence of lead acetate, a marked inhibition of their oxidative metabolism was observed. The hexosemonophosphate-shunt (HMPS) activity and the release of oxygen derivatives upon triggering by phorbol myristate acetate (PMA) were impaired. Treatment with the metal for 1 h led, however, to stimulation rather than inhibition of the PMA-triggered superoxide production, suggesting that the metal interfered with neither the triggering steps nor the activity of the NADPH oxidase. Moreover, the lead-induced inhibition of macrophage oxidative metabolism did not result from blockade of enzymes of the HMPS pathway. Glucose-6-phosphate dehydrogenase in macrophage extracts, as well as CO2 production from glucose, remained unaffected by the presence of lead, and extracts of lead-treated macrophages were as active as extracts from control cells in those two assays. Lead appeared to interfere with an early event in the MAF-induced activation process. In addition, lead decreased the uptake of 2-deoxyglucose by macrophages, suggesting that the metal might inhibit trans-membrane glucose-transport systems, a phenomenon that might explain in part the metabolic inhibition observed in lead-treated cells.
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PMID:Lead inhibits oxidative metabolism of macrophages exposed to macrophage-activating factor. 266 32

Using nitrogen cavitation and Percoll density gradient centrifugation for subcellular centrifugation of human neutrophils, approximately 90% of the low potential b-cytochrome, unique for phagocytes, as well as 50% of the flavoproteins in normal neutrophils were found in a granule fraction which co-sedimented with the specific granules. Upon stimulation of the intact cells with phorbol myristate acetate, both the b-cytochrome and the flavoprotein translocated from this granule fraction to the fractions which contained the plasma membranes and the NADPH oxidase activity. In neutrophils from two patients with chronic granulomatous disease, both the b-cytochrome and the flavoprotein of the granules were absent, but flavoprotein was present in normal amounts in the membrane and cytosol fractions. Taken together, these findings suggest that the specific granules, or granules co-sedimenting with the specific granules, are important stores for the components of the NADPH oxidase, which is responsible for the respiratory burst. Analysis of the stoichiometry of CO2 generation, H+ secretion and O2 consumption by stimulated neutrophils indicated that the hexose monophosphate shunt is the source of both protons and electrons for the NADPH oxidase activity, as well as of the extra protons secreted during the respiratory burst.
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PMID:The respiratory burst of phagocytosis: biochemistry and subcellular localization. 393 81

1. Paraquat and diquat produce only a slight increase in the oxygen uptake of rat liver mitochondria, and it is likely that they do not penetrate the mitochondrial membrane. 2. In mitochondrial fragments inhibited by antimycin A or by Amytal, both substances stimulate oxygen uptake with NADH or beta-hydroxybutyrate as substrate but not with succinate. The NADH dehydrogenase of the respiratory chain appears to be involved, at a site only partially inhibited by Amytal. 3. An NADPH oxidase activity is stimulated in rat liver microsomes by diquat, and to a smaller extent by paraquat; diquat also causes an NADH oxidase activity to develop. The effect is not inhibited by carbon monoxide or p-chloromercuribenzoate, and it is probable that a flavoprotein is involved by a mechanism not requiring thiol groups. 4. One molecule of oxygen can oxidize two molecules of NADPH in the stimulated microsomal system, the hydrogen peroxide produced being broken down by a catalase activity in the microsomes. 5. Diquat can stimulate NADH oxidase and NADPH oxidase activity in the postmicrosomal soluble fraction; the enzyme involved may be DT-diaphorase. 6. The mechanism of these reactions and their significance in relation to the toxicity of the dipyridilium compounds are discussed.
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PMID:The action of paraquat and diquat on the respiration of liver cell fractions. 438 31

Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation. Addition of carbonic anhydrase and its inhibitors had no effect on either the quantities of CO2 measured or the quantities of protons secreted. Therefore, the CO2 generated in the respiratory burst of stimulated neutrophils is hydrated to form H2CO3, which then dissociates, accounting for the observed proton secretion. Furthermore, the CO2 generated corresponds to the O2 consumed with a respiratory quotient of nearly 1. We conclude on the basis of this and previous studies that the HMPS activity is the source of both the electrons for the NADPH oxidase and of protons secreted in association with the respiratory burst.
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PMID:Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst. 643 Sep 61

Intraperitoneal administration of chloramphenicol (100 mg/kg) to phenobarbital-treated rats causes 50% inhibition of liver microsomal 7-ethoxycoumarin and 1,1,2,2 tetrachloroethane metabolism but has no effect on the level of cytochrome P-450 detectable as its carbon monoxide complex or on the NADPH-cytochrome c reductase (EC 1.6.2.4) activity. Both the endogenous NADPH oxidase activity and the enzymatic reduction of cytochrome P-450 are inhibited by chloramphenicol treatment, whereas the Km and Ks for ethoxycoumarin and the cumene hydroperoxide- or iodosobenzene-supported deethylation of ethoxycoumarin are unaffected, suggesting that impaired electron transport to cytochrome P-450 may be the cause of the loss of enzymatic activity. Administration of [14C]chloramphenicol (100 mg/kg) leads to the covalent binding of 0.7 nmole of metabolite(s) per nanomole of the major cytochrome P-450 isozyme. Alkaline hydrolysis of a cytochrome P-450 fraction obtained by chromatography of solubilized 14C-labeled microsomes on octylamino-Sepharose releases oxalic acid and chloramphenicol oxamic acid, whereas enzymatic digestion releases N-epsilon-chloramphenicol oxamyl lysine in addition. These data obtained with radiolabeled chloramphenicol suggest that the same metabolic pathways which lead to the inactivation of cytochrome P-450 in vitro are also operative in vivo.
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PMID:Suicide inactivation of rat liver cytochrome P-450 by chloramphenicol in vivo and in vitro. 660 Dec 33

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme,' NAD(P)H:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase, hydrogenase, acetate:succinate CoA transferase and succinate thiokinase leading to the formation of H2,CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role--especially in zoospores--as H2-evolving, ATP-generating and O2-scavenging organelles.
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PMID:Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2. 825 82

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


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