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)

Activation of superoxide-generating NADPH oxidase system of human neutrophils involves phosphorylation-dependent translocation of p47phox and other cytosolic components to the plasma membrane. In contrast to the stimulation of the NADPH oxidase in intact cells, however, the activation of cell-free system requires the addition of anionic amphiphiles such as sodium dodecyl sulfate (SDS) and arachidonate. In this system, translocation of p47phox is also an essential step for activation, but phosphorylation is not required. The basis of this difference in oxidase activation is not yet clear. We now report that in a cell-free oxidase system, phosphorylated recombinant p47phox can be translocated to the membrane in the absence of SDS or arachidonate. These findings suggest that both phosphorylation and SDS could cause a common change in conformation or charge of p47phox that may result in the association of p47phox with the plasma membrane.
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PMID:Translocation of recombinant p47phox cytosolic component of the phagocyte oxidase by in vitro phosphorylation. 779 51

A photoactivable derivative of FAD, 4-[N-(4-azido-2-nitrophenyl)amino]butyryl-FAD (NAP4-FAD), was synthesized in a tritiated form with tritium placed in the NAP4 moiety of the photoprobe. [3H]NAP4-FAD was used to photolabel the putative flavin binding site of the O2(-)-generating NADPH oxidase located in the plasma membrane of bovine neutrophils. Effective photolabeling required partial deflavination of membranes, which was achieved by mild treatment with ammonium sulfate added to 50% saturation and 0.05% Triton X-100 for 30 min at 2-4 degrees C. Under these conditions, 40-50% of the oxidase activity was lost, but it could be fully recovered by the addition of nanomolar amounts of FAD (KM = 10-20 nM). Added FAD could be substituted by [3H]NAP4-FAD in photolabeling experiments. In the dark, [3H]NNAP4-FAD bound reversibly with high affinity to deflavinated neutrophil plasma membranes (Kd = 50 nM), did not transport electrons, and efficiently inhibited the FAD-dependent restoration of oxidase activity (Ki = 60 nM). Upon photoirradiation of neutrophil plasma membranes in the presence of [3H]NAP4-FAD, the nitrene derivative formed bound covalently to a 80-120 kDa protein that was identified as the beta-subunit of cytochrome b558 by immunodetection and enzymatic deglycosylation. The amount of [3H]NAP4-FAD covalently incorporated into the beta-subunit of cytochrome b558 was 80-90% of the amount of photoprobe specifically bound to neutrophil plasma membranes. A linear relationship between the extent of specific photolabeling by [3H]NAP4-FAD and the percentage of NADPH oxidase inactivation was observed for percentages of inactivation of up to 70-80%, extrapolating to 0.5 mol of covalently bound [3H]NAP4-FAD per mol of heme b558.
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PMID:Photoaffinity labeling and photoinactivation of the O2(-)-generating oxidase of neutrophils by an azido derivative of FAD. 784 36

Interactions between glycosaminoglycans (GAGs) and low density lipoprotein (LDL) are thought to influence the progression of atherogenesis. In an effort to gauge whether macrophages mediate GAG-LDL interaction by GAG modification, we have investigated the endocytosis, degradation and retro-endocytosis of the GAG heparan sulfate (HS) by mouse peritoneal macrophages. Radiolabelled HS was produced by derivatization with sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate and radio-iodination by the chloramine T method. The amount of 125I-HS internalized by cultures of thioglycollate-elicited macrophages rose over a 24 h time period in proportion to the amount of tracer added to the wells (2-2500 ng ml-1). Analysis of GAG molecular weight was performed using gel filtration chromatography and polyacrylamide gel electrophoresis. After a 24 h pulse period, the 125I-HS in the intracellular fraction of the cultured cells was of smaller molecular weight than for control material. During a 24 h cold chase, fragments of 125I-HS were released into the medium. These fragments had lower affinity for Polybrene-Sepharose but did not appear significantly N-desulfated as determined by low pH nitrous acid treatment. The NADPH oxidase inhibitor diphenylene iodonium, although minimizing basal and phorbol ester-triggered radical output, did not inhibit 125I-HS depolymerization. These data indicate that elicited macrophages can interact with and reduce the polymer length of HS without extensively desulfating the molecule. They are consistent with a mechanism by which the macrophage internalizes and partially degrades HS by endoglucuronidase activity rather than NADPH oxidase-generated free radicals, followed by release of the products into the extracellular milieu.
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PMID:Endocytosis, partial degradation and release of heparan sulfate by elicited mouse peritoneal macrophages. 789 29

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to approximately 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.
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PMID:Metabolic and biosynthetic alterations in cultured astrocytes exposed to hypoxia/reoxygenation. 790 52

The small GTP-binding protein (G protein) Rac1 is an obligatory participant in the assembly of the superoxide (O2-.)-generating NADPH oxidase complex of macrophages. We investigated the effect of synthetic peptides, mapping within the near carboxyl-terminal domains of Rac1 and of related G proteins, on the activity of NADPH oxidase in a cell-free system consisting of solubilized guinea pig macrophage membrane, a cytosolic fraction enriched in p47phox and p67phox (or total cytosol), highly purified Rac1-GDP dissociation inhibitor for Rho (Rho GDI) complex, and the activating amphiphile, lithium dodecyl sulfate. Peptides Rac1-(178-188) and Rac1-(178-191), but not Rac2-(178-188), inhibited NADPH oxidase activity in a Rac1-dependent system when added prior to or simultaneously with the initiation of activation. However, undecapeptides corresponding to the near carboxyl-terminal domains of RhoA and RhoC and, most notably, a peptide containing the same amino acids as Rac1-(178-188), but in reversed orientation, were also inhibitory. Surprisingly, O2-. production in a Rac2-dependent cell-free system was inhibited by Rac1-(178-188) but not by Rac2-(178-188). Finally, basic polyamino acids containing lysine, histidine, or arginine, also inhibited NADPH oxidase activation. We conclude that inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of certain small G proteins is not amino acid sequence-specific but related to the presence of a polybasic motif. It has been proposed that such a motif serves as a plasma membrane targeting signal for a number of small G proteins (Hancock, J.F., Paterson, H., and Marshall, C.J. (1990) Cell 63, 133-139).
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PMID:Inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of small GTP-binding proteins. Lack of amino acid sequence specificity and importance of polybasic motif. 796 67

It is well known that sodium dodecyl sulfate (SDS) activates NADPH oxidase in a cell-free system independently of protein kinase C (PKC). However, in intact neutrophils, direct evidence has never been presented to show that O2- production by SDS is actually due to the NADPH oxidase activation observed in the cell-free system. So, in this paper, we investigated the activation mechanism by SDS in intact guinea pig neutrophils. We previously reported that hypotonic treatment reversibly enhanced O2- production stimulated by PKC activators in intact neutrophils (M. Hiura et al., 1991, Arch. Biochem. Biophys. 291, 31-37). In this paper, SDS also significantly stimulated O2- production in the intact cells under the hypotonic condition. This enhancement was gradual and was PKC inhibitor resistant. Furthermore, phosphorylation of the 46-kDa protein, one of cytosolic activation factors, was not detected by autoradiography of two-dimensional electrophoresis. Translocation of cytosolic activation factors was demonstrated by a decrease in the activity of the factors remained in the cytosol. In the presence of SDS, addition of 1-oleoyl-2-acetylglycerol, a PKC activator, further enhanced O2- production and translocation of the cytosolic activation factors. On the other hand, SDS remarkably increased membrane fluidity in intact neutrophils as well as in the cell-free system. These results indicate that activation of NADPH oxidase by SDS in intact neutrophils seems to be partly due to the same mechanism observed in cell-free activation, and that SDS alone slightly activates the oxidase and other stimulation, such as hypotonic and/or PKC activator treatments, is required for significant activation. The increase in the membrane fluidity may be one of the activation mechanisms of NADPH oxidase by SDS.
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PMID:Activation mechanism of NADPH oxidase by SDS in intact guinea pig neutrophils. 797 93

The effects of prostaglandin (PG) E1 and I2 analogs (OP-41483 and OP-2507) on the superoxide generation of human neutrophil NADPH oxidase (EC 1.6.99.6) in both whole-cell and cell-free systems were investigated. In a whole-cell system, OP-2507 inhibited the superoxide generation by neutrophils exposed to phorbol myristate acetate concentration-dependently through its superoxide-scavenging action. The concentration of the drug required for 50% inhibition of the oxidase (IC50) was 21 microM. In a cell-free system, however, the drug in concentrations of < 100 microM did not inhibit the activation of NADPH oxidase by sodium dodecyl sulfate because of its inactivation by the detergent. Although PGE1 and OP-41483 did not inhibit the superoxide production by stimulated neutrophils in a whole-cell system, they both inhibited the activation of NADPH oxidase in a cell-free system concentration-dependently, with IC50 values of 44 and 170 microM, respectively. In addition, in the cell-free system, the Km value for NADPH of the oxidase was unchanged by PGE1. The results suggest that the PGI2 analog, OP-2507, is a possible superoxide-scavenger and that PGE1 inhibits the NADPH oxidase activation by sodium dodecyl sulfate in a cell-free system concentration-dependently.
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PMID:Prostaglandin E and analogs of prostacyclin influencing superoxide production by the human neutrophil NADPH oxidase system. 808 10

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.
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PMID:Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. 820 90

The effect of an inhibitor of protein kinase, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine HCl], and its hydroxylated metabolite, HA1100, on the activation of NADPH oxidase in human neutrophils were studied. Cells were preincubated with each drug for 10 min and then activated by treatment with phorbol myristate acetate (PMA) or formylmethionyl leucyl phenylalanine (FMLP). After activation, the rate of superoxide dismutase-inhibitable reduction of cytochrome c was estimated. HA1077 and HA1100 inhibited the PMA-induced production of O2- by neutrophil NADPH oxidase in a concentration-dependent manner (IC50 = 15 and 24 microM, respectively). The sensitivity of the FMLP-induced production of O2- to these drugs was similar. The production of O2- in 1,25-dihydroxyvitamin D3-treated HL-60 cells, which differentiated to macrophage-like cells, was also inhibited by the drugs. The extent of inhibition by HA1077 was almost the same as that by a calmodulin inhibitor (W-7) and by inhibitors of protein kinase (H-7 and H-8). In a cell-free lysate of neutrophils, the NADPH-dependent production of O2- can be induced by sodium dodecyl sulfate (SDS). HA1077 at 100 microM had only a weak inhibitory effect on the cell-free, SDS-induced production of O2-, an indication that HA1077 inhibits the activation of NADPH oxidase, not the actual activity. The effects of H-7 and H-8 were similar to that of HA1077, whereas W-7 inhibited the production of O2- by the cell-free extract of HL-60 cells. This action of HA1077 could explain, in part, its ability to protect neuronal cells from death after ischemia.
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PMID:Inhibition by the protein kinase inhibitor HA1077 of the activation of NADPH oxidase in human neutrophils. 824 Apr

The signal transductional mechanisms regulating the activation of NADPH oxidase, the respiratory burst enzyme in phagocytic cells, are not completely understood. Receptors for most physiologic stimuli trigger the activation of various phospholipases, including phospholipases A2, C, and D. The lipid mediators formed (arachidonic acid, 1,2-diacylglycerol, and phosphatidic acid) have been implicated as second messengers in the induction of the respiratory burst. In intact cells, we have correlated phospholipase D activation and the production of phosphatidic acid with the activation of NADPH oxidase, using the drug propranolol. Phosphatidic acid activated NADPH oxidase in a cell-free system, but the level of activation was low. 1,2-Diacylglycerol markedly enhanced NADPH oxidase activation by phosphatidic acid. The synergistic effect required the diacyl species, since mono- or tri-acylglycerols were ineffective. Phosphatidic acid could be replaced by either lysophosphatidic acid or phosphatidylserine, but not by phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol, suggesting specificity for an anionic phospholipid. Since other cell-free activators of NADPH oxidase (arachidonic acid, sodium dodecyl sulfate) are also anionic amphiphiles, phosphatidic acid may directly interact with an enzyme component(s). The targets for phosphatidic acid and diacylglycerol in the cell-free system are currently under investigation. These results emphasize the critical importance of phospholipases, particularly phospholipase D, in the regulation of the respiratory burst.
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PMID:Phospholipases and activation of the NADPH oxidase. 828 91


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