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)

Mitochondria may be isolated from various types of leukocyte (neutrophil polymorphs and lymphocytes from human blood, neutrophil polymorphs and macrophages from peritoneal exudates of the guinea pig) after destruction by heparin of the cell membrane. This procedure is very simple and less traumatic for these subcellular structures than the usual mechanical procedures. The enzyme activities of the respiratory chain and oxygen consumption may be measured in these mitochondrial preparations. The oxygen consumption is determined using oxyhemoglobin which serves both as oxygen donor, as in the respiratory system in vivo, and as indicator of the reaction at 435.8 nm. The integrity of the mitochondria may be demonstrated by determination of the "acceptor control index", the existence of ADP phosphorylation coupled with oxygen consumption (phosphorylating oxidation) was proved in all the cells studied even if the ADP/O ratio can only be calculated for certain of them (lymphocytes, macrophages). In these cases, the ratios obtained are close to theoretical values whatever the oxidation substrate used. The mitochondria of leukemic cells have a higher oxidation activity than the corresponding reference cells. Determination of leukocyte coenzymes by enzyme cycling (NAD, NADH, NADP, NADPH) showed the following facts: -- Generally, the NAD concentrations remain constant, those of NADH increase whilst those of NADP and NADPH fall during incubation of neutrophil polymorphs in Dulbecco's medium. -- The metabolic changes observed during S. albi heat-induced endocytosis are in favour of simultaneous stimulation of NADH oxidase and NADPH oxidase in human polymorphs, and of NADPH oxidase in the corresponding cells of peritoneal exudates in guinea pigs.
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PMID:[Enzyme system and coenzymes involved in the energy metabolism of leukocytes. Function and metabolism of polymorphonuclear neutrophils]. 0 34

The effect of hyperoxia (1-14 days, 85% O2) on rat alveolar macrophage and alveolar type II cell oxidant and antioxidant characteristics was investigated. Unstimulated control macrophages (2 h ex vivo) released hydrogen peroxide at a rate of 3.5 +/- 1.3 nmol/min mg protein-1, which was a cyanide-sensitive process. H2O2 release from alveolar macrophages decreased slightly but not significantly after 1 day in hyperoxia and increased significantly after 3 days (180%, p less than .05) and 14 days (380%, p less than .01). When H2O2 release was expressed as nmol from total macrophages per animal, the increase after 14 days in hyperoxia was 760%. H2O2 generation by hyperoxic macrophages was cyanide resistant, indicating the involvement of active NADPH oxidase. In both control and hyperoxic macrophages H2O2 release could be significantly stimulated with phorbol myristate acetate (PMA). Comparisons of H2O2 release by freshly isolated alveolar macrophages and alveolar type II cells must be cautiously interpreted because some cell functions may change during the isolation procedure. Freshly isolated (6 h ex vivo) control alveolar type II cells were found to generate H2O2 at a rate of 0.26 +/- 0.05 nmol/min mg protein-1. In type II cells H2O2 release, calculated as nmol/mg protein, decreased during the first 7 days of hyperoxia to 10% (p less than .01) of the control value and then returned back up to the control level after 14 days. A similar decrease was observed if H2O2 release was calculated as nmol/cell number. H2O2 release from control and hyperoxic type II cells was cyanide sensitive. The decrease in H2O2 release in type II cells was associated with cell membrane injury (as assessed by electron microscopy), while biochemical markers of cellular injury (trypan blue exclusion and cellular high-energy phosphates ATP, ADP) were unchanged. The ability of type II cells to scavenge extracellular H2O2 did not change in acute hyperoxia, but it increased significantly during the second week in hyperoxia. These results indicate that macrophages but not type II cells are stimulated to produce H2O2 during prolonged exposure to hyperoxia.
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PMID:Hydrogen peroxide release from alveolar macrophages and alveolar type II cells during adaptation to hyperoxia in vivo. 139 11

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the participation of both membrane and cytosolic components. We reported that ammonium sulfate fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143, 4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of cytosol in two fractions (sigma 1 and sigma 2) that support O2- production by solubilized membrane synergistically. We now describe the purification of sigma 1 to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction sigma 2. Sigma 1 was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether sigma 1 was added to cell-free systems composed of: (a) solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b) purified cytochrome b559, incorporated in liposomes, and purified sigma 2. Sigma 1 was purified by a sequence comprising ammonium sulfate fractionation, hydrophobic chromatography on phenyl-Superose, absorption with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sigma 1 of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the presence of both 24- and 22-kDa bands. We suggest that, in its native form, sigma 1 might represent a complex of the 24- and 22-kDa proteins. The specific roles of each molecule in NADPH oxidase function remain to be determined.
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PMID:Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages. 166 Aug 77

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E2 but did not affect the zymosan-induced release of arachidonic acid, PGD2, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A2, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.
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PMID:Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages. 166 39

When a particulate NADPH oxidase prepared from phorbol ester-activated human neutrophils was treated with pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), the superoxide anion-producing activity was inhibited according to affinity labeling kinetics. NADPH afforded a protection against inactivation which was competitive with respect to PLP-AMP; 2',5'-ADP and 2'-phospho-5' diphosphoadenosine (ATP ribose) appeared to be as potent as NADPH as protecting agents. NADP+ and ATP were less effective, while ADP and GTP-gamma-S did not protect significantly. These results suggest that PLP-AMP can be used, in conjunction with tritiated cyanoborohydride, to identify the elusive NADPH-dependent flavoprotein which is part of the electron transfer chain of NADPH oxidase.
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PMID:Inactivation of NADPH oxidase from human neutrophils by affinity labeling with pyridoxal 5'-diphospho-5'-adenosine. 176 75

Botulinum D toxin has been shown to ADP-ribosylate 22-kD proteins in neutrophilic leukocytes, but the function of these GTP-binding proteins remains unknown. In analogy to small GTP-binding proteins like SEC4 to YPT1, it has been suggested that botulinum D toxin substrates might be involved in secretory process of myeloid cells. Three main findings lead to the opposite conclusion. First of all, in human neutrophils, botulinum D toxin does not modify the release of azurophilic and specific granules induced by a chemoattractant (a formylpeptide) or a phorbol ester. Second, botulinum D toxin ADP-ribosylates 24 to 26-kD proteins that are only present in plasma membranes of human neutrophils. The membrane location of these substrates differs largely from that of the GTP-binding proteins involved in exocytosis and located in granules. Finally, since the same quantity of the toxin substrates is present in neutrophils as in their precursors, HL60 cells (which are devoid of specific granules and characterized by immature azurophilic granules and NADPH oxidase), it is unlikely that endogenous botulinum D toxin substrates are directly involved in the secretory responses of neutrophils.
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PMID:Effect of botulinum D toxin on human neutrophilic leukocytes and localization of its substrates. 196 13

The superoxide generating NADPH oxidase was studied in an SDS-activated cell-free system. This system requires the participation of both membranal and cytosolic components. Cytosol derived from elicited peritoneal guinea pig macrophages was fractionated by several nucleotide affinity chromatography procedures. Various such fractionations led to the separation of two distinct factors, both of which are necessary for the activation and/or activity of the superoxide-forming NADPH oxidase. One factor (sigma 2), bound to octyl, 2',5'-ADP-, 5'-ATP-, 5'-GTP-agarose and carboxymethyl-Sepharose but did not bind to hexyl, 5'-AMP-, 5'-ADP- and 5'-GDP-agarose. The other factor (sigma 1) did not bind to any of the above matrices. Subsequent elution of sigma 2 from 2',5'-ADP-agarose was effected by ATP, GTP and NADPH but not by NADH. Elution from GTP-agarose was by ATP and GTP but not by NADPH. Elution from ATP-agarose was by ATP, GTP and also, albeit weakly, by NADPH. The above results suggest that sigma 2 contains a site which recognizes the phosphate group at the ribose 2' position in adenosine, and a site that recognizes purine nucleotide triphosphates.
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PMID:Nucleotide binding properties of cytosolic components required for expression of activity of the superoxide generating NADPH oxidase. 215 58

Neutrophil guanine nucleotide-binding proteins are important components of receptor-mediated cellular responses such as degranulation, chemotaxis, and superoxide production. Because the cytoplasmic granules of neutrophils serve as an intracellular store of receptors and NADPH oxidase components, we investigated the subcellular distribution of substrates for ADP-ribosylation by both pertussis and cholera toxins. Cholera toxin substrates of Mr 43 and 52 kDa were present only in the plasma membrane fraction. A 39-kDa pertussis toxin substrate was present in the plasma membrane, cytosol, and a specific granule-enriched fraction. There were no substrates for either toxin in the primary granules. Quantitative GTP-gamma-5 binding was localized predominantly to the plasma membrane fraction (47%), but significant portions were found in the specific granule-enriched fractions (13%) and cytosol (34%) as well. Two-dimensional gel electrophoresis and chymotryptic digests of the pertussis toxin substrate from these three subcellular fractions suggested that they are highly homologous. Triton X-114 phase partitioning was used to investigate the hydrophobicity of the toxin substrates. The pertussis toxin substrates in the plasma membrane and granule fractions behaved like integral membrane proteins, whereas the cytosolic substrate partitioned into both lipophilic and aqueous fractions. ADP-ribosylation converted the substrates to a somewhat less lipophilic form. These data suggest that the specific granules or an organelle of similar density serve as an intracellular store of a G protein with a 39-kDa alpha-subunit and that the cytosolic fraction of neutrophils contains free alpha-subunits of the same size.
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PMID:Subcellular distribution and membrane association of human neutrophil substrates for ADP-ribosylation by pertussis toxin and cholera toxin. 249 61

Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.
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PMID:Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins. 254 Sep 69

Human neutrophils and HL-60 leukaemic cells possess an NADPH oxidase which catalyses superoxide (O2-) formation and is activated by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). In dibutyryl cyclic AMP-differentiated HL-60 cells, ATP and UTP in the presence of cytochalasin B activated O2- formation with EC50 values of 5 microM and efficacies amounting to 30% of that of fMet-Leu-Phe. The potency order of purine nucleotides in activating O2- generation was ATP = adenosine 5'-O-(3-thiotriphosphate) greater than ITP greater than dATP = ADP. Pyrimidine nucleotides activated NADPH oxidase in the potency order UTP greater than dUTP greater than CTP = TTP = UDP. Pertussis toxin completely prevented activation of NADPH oxidase by fMet-Leu-Phe and UTP, whereas the effect of ATP was only partially inhibited. ATP and UTP enhanced O2- generation induced by fMet-Leu-Phe by up to 8-fold, and primed the cells to respond to non-stimulatory concentrations of fMet-Leu-Phe. Activation of NADPH oxidase by UTP but not by ATP was inhibited by various activators of adenylate cyclase. In dimethyl sulphoxide-differentiated HL-60 cells and in human neutrophils, ATP and UTP per se did not activate NADPH oxidase, but they potentiated the effect of fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via purino- and novel pyrimidinoceptors respectively, which are coupled to guanine nucleotide-binding proteins leading to the activation of NADPH oxidase. As ATP and UTP are released from cells under physiological and pathological conditions, these nucleotides may play roles as intercellular signal molecules in the activation of O2- formation.
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PMID:Activation of NADPH oxidase by purine and pyrimidine nucleotides involves G proteins and is potentiated by chemotactic peptides. 254 70


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