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)

In kidney and liver, fibroblasts and fibroblast-like cells, respectively, are sources of erythropoietin (Epo) formation, and these cells also bear a number of other similarities. Renal Epo expression is localized in peritubular type 1 fibroblasts of the cortical labyrinth, and in the liver, apart from parenchymal cells, transcription is found in Ito cells. Both the renal peritubular cells and Ito cells contain ecto-5'-nucleotidase (5'NT). It had been suggested that 5'NT is involved in the oxygen sensing mechanism via a hydrolysis of AMP to adenosine, which in turn may stimulate EPO synthesis. However, the molecular mechanism of the cellular response to hypoxia is currently not well understood. Based on the notion that a heme protein probably acts as the oxygen sensor, it has recently been proposed that a b-type cytochrome as part of the neutrophil NADPH oxidase may influence intracellular superoxide levels depending on local oxygen tension. Superoxide levels were otherwise shown to determine the EPO production in hepatoma cell lines. By double immunofluorescence labeling the alpha-subunit of cytochrome b558 (alpha-SU) and 5'NT were simultaneously localized in rat kidney and liver, and in the kidney Epo mRNA and alpha-SU were double-labeled. Positive signal for alpha-SU was found in the majority of renal peritubular fibroblasts in the cortex and outer medulla, and in Ito cells. In both organs, the cells that coexpress 5'NT and Epo mRNA also contain an immunoreactivity for alpha-SU. In these cells, cytochrome b558 as part of an NADPH oxidase may be involved in a presumptive oxygen sensing mechanism using H2O2 as a possible second messenger for EPO gene regulation.
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PMID:Immunohistochemical colocalization of the alpha-subunit of neutrophil NADPH oxidase and ecto-5'-nucleotidase in kidney and liver. 902 26

We tested the relative efficacy of free dexamethasone, dexamethasone containing liposomes and free liposomes in preventing superoxide anion, O-2 generation by neutrophils. O-2 production by 5 x 10(5) neutrophils, whether primed or not with lipopolysaccharide, was stimulated by phorbol 12-myristate 13-acetate (PMA) to 13.4 +/- 1.3 nmoles after 15 minutes compared to 1.2 +/- 0.3 nmoles with nonstimulated cells. Free liposomes but not dexamethasone (dexa) decreased non-stimulated as well as PMA-induced O-2 generation. Dexa-containing phosphatidylcholine from egg yolk: phosphatidylserine from bovine brain (PC:PS 7:3) liposomes, unlike free dexa, diminished PMA-stimulated O-2 production in a dose-dependent manner with a maximal effect at 37.5 micrograms/ml phospholipid (6.6 +/- 1.6 nmoles). The kinetics of cytochrome-c reduction revealed that decreased O-2 production resulted from an extended lag-time of release to almost 8 minutes with PMA induction and consequently led to the conclusion that liposomes modified the activity of NADPH oxidase as well as that of protein kinase C. Liposomes prepared with PC and PS of natural origin had a greater inhibitory effect on O-2 generation by neutrophils than dipalmitoylphosphatidylcholine (DPPC) and phosphatidylethanolamine from egg yolk (PE):PC (3:1) liposomes. When 100 microM of Ca2+ was added to the medium, the inhibitory action of liposomes prepared with egg yolK PC and DPPC was increased by 30 and 60% respectively, while that of PS and PE:PC was prevented. We also verified that liposomes by themselves, even if phagocytized, did not induce O-2 generation or its concentration was too low to be detected by this technique. From the clinical point of view, some formulations delayed non-induced and PMA-induced O-2 generation, thus adding to the anti-inflammatory effect of the glucocorticoid they transported.
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PMID:Modulation of noninduced and phorbol ester-induced generation of superoxide anion by free liposomes and liposomes containing dexamethasone. 904 63

The elicitation of an oxidative burst in phagocytes rests on the assembly of a multicomponental complex (NADPH oxidase) consisting of a membrane-associated flavocytochrome (cytochrome b559), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O-2), two cytosolic components (p47(phox), p67(phox)), and the small GTPase Rac (1 or 2). We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine protease inhibitor, prevented the elicitation of O-2 production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a cell-free system, consisting of solubilized membrane or purified cytochrome b559 combined with total cytosol or a mixture of recombinant p47(phox), p67(phox), and Rac1. AEBSF acted at the activation step and did not interfere with the ensuing electron flow. It did not scavenge oxygen radicals and did not affect assay reagents. Five other serine protease inhibitors (three irreversible and two reversible) were found to lack an inhibitory effect on cell-free activation of NADPH oxidase. A structure-function study of AEBSF analogues demonstrated that the presence of a sulfonyl fluoride group was essential for inhibitory activity and that compounds containing an aminoalkylbenzene moiety were more active than amidinobenzene derivatives. Exposure of the membrane fraction or of purified cytochrome b559, but not of cytosol or recombinant cytosolic components, to AEBSF, in the presence of a critical concentration of the activating amphiphile lithium dodecyl sulfate, resulted in a marked impairment of their ability to support cell-free NADPH oxidase activation upon complementation with untreated cytosol or cytosolic components. Kinetic analysis of the effect of varying the concentration of each of the three cytosolic components on the inhibitory potency of AEBSF indicated that this was inversely related to the concentrations of p47(phox) and, to a lesser degree, p67(phox). AEBSF also prevented the amphiphile-elicited translocation of p47(phox) and p67(phox) to the membrane. These results are interpreted as indicating that AEBSF interferes with the binding of p47(phox) and/or p67(phox) to cytochrome b559, probably by a direct effect on cytochrome b559.
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PMID:Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)-benzenesulfonyl fluoride and related compounds. 914 50

The small molecular weight GTP-binding protein Rac (1 or 2) is an obligatory participant in the activation of the superoxide-generating NADPH oxidase. Active NADPH oxidase can be reconstituted in a cell-free system, consisting of phagocyte-derived membranes, containing cytochrome b559, and the recombinant cytosolic proteins p47-phox, p67-phox, and Rac, supplemented with an anionic amphiphile as an activator. The cell-free system was used before for the analysis of structural requirements of individual components participating in the assembly of NADPH oxidase. In earlier work, we mapped four previously unidentified domains in Rac1, encompassing residues 73-81 (a), 103-107 (b), 123-133 (c), and 163-169 (d), as important for cell-free NADPH oxidase activation. The domains were defined by assessing the activation inhibitory effect of a series of overlapping peptides, spanning the entire length of Rac1 [Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082]. We now used the construction of Rac1/H-Ras chimeras, domain deletion, and point mutations, to ascertain the functional relevance of three domains (b, c, and d) predicted by "peptide walking" and to determine the importance of specific residues within these domains. This methodology firmly establishes the involvement of domains b and d in the activation of NADPH oxidase by Rac1 and identifies H103 and K166, respectively, as residues critical for the effector function of these two domains. The functional significance of domain c (insert region) could not be confirmed, as shown by the minor effect of deleting this domain on NADPH oxidase activation. Analysis of the three-dimensional structure of Rac1 reveals that residues H103 and K166 are exposed on the surface of the molecule. Modeling of the activity-impairing point mutations suggests that the effect on the ability to activate NADPH oxidase depends on the side chains of the mutated amino acids and not on changes in the global structure of the protein. In conclusion, we demonstrate the existence of two novel effector sites in Rac1, necessary for supporting NADPH oxidase activation, supplementing the canonical N-terminal effector region.
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PMID:Mutational analysis of novel effector domains in Rac1 involved in the activation of nicotinamide adenine dinucleotide phosphate (reduced) oxidase. 958 26

The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47(phox) participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47(phox), p67(phox), and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. These were (N- and C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119); c (149-159); d (193-207); e (253-267); f (305-319); g (325-339), and h (373-387). Four of these domains (c, d, e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, whereas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overlaps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47(phox), and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to the identification of domains in p47(phox) mediating binding to p67(phox). This was done by quantifying, by enzyme-linked immunosorbent assay, the binding of p67(phox), in solution, to a series of 95 overlapping biotinylated p47(phox) peptides, attached to streptavidin-coated 96-well plates. A single proline-rich domain (residues 357-371) was found to bind p67(phox) in the absence and presence of lithium dodecyl sulfate.
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PMID:Mapping of functional domains in p47(phox) involved in the activation of NADPH oxidase by "peptide walking". 962 28

The phagocyte NADPH oxidase flavocytochrome b558 is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91(phox), and a nonglycosylated subunit, p22(phox). It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O2), resulting in the generation of superoxide ion (O2-). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91(phox), p22(phox), or both polypeptides to examine the relative role of each flavocytochrome b558 subunit in heme binding and O2- formation. A similar membrane localization was observed when gp91(phox) and p22(phox) were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91(phox) or both gp91(phox) and p22(phox) showed a b-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome b558. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22(phox). Furthermore, redox titration studies suggested that two heme groups were contained in gp91(phox) expressed in COS7 membranes, with midpoint potentials of -264 and -233 mV that were very similar to those obtained for neutrophil flavocytochrome b558. These results provide strong support for the hypothesis that gp91(phox) is the sole heme binding subunit of flavocytochrome b558. However, coexpression of gp91(phox) and p22(phox) in COS7 membranes was required to support O2- production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b558.
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PMID:Gp91(phox) is the heme binding subunit of the superoxide-generating NADPH oxidase. 965 28

We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K+ and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K+. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 micromol/L PR-39. ROS production in lungs exposed to high K+ or ischemia was essentially abolished in mice with a "knockout" of gp91(phox), a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control. PR-39 also inhibited ischemia and the high K+-mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47phox, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K+ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.
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PMID:Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+. 975 43

We have tested the membrane-protein solubilizing properties of two perfluoroalkylphosphocholines. These compounds belong to a series of fluorinated amphiphiles which are being investigated as potential stabilizing agents for a variety of fluorocarbon-based systems. We are particularly interested in cytochrome b558 from phagocytes, the redox component of NADPH oxidase. Its heavy subunit is believed to carry binding sites for NADPH and FAD. Nevertheless, when the cytochrome is purified in the presence of classical detergents, it carries no FAD. This could be due to a delipidating, denaturing effect of these detergents (octyl glucoside, Triton, etc). The first perfluoroalkyphosphocholine, C8F17(CH2)2O-P(O2-)-O(CH2)2N+(CH3)3(F8C2PC), extracted about as much protein from neutrophil plasma membranes into a 100,000 g supernatant as octyl glucoside. The second compound, C8F17(CH2)11O-P(O2-)-O(CH2)2N+(CH3)3(F8C11PC), was less efficient. We found that flavin was still protein-bound in the crude F8C2PC extract at a FAD to heme ratio of about 1, and a good NADPH oxidase activity was obtained without addition of exogenous FAD, even after dialysis or gel filtration, whereas dialysis eliminated most of the FAD from the octyl glucoside extracts. These experiments appeared to make F8C2PC an interesting membrane-solubilizing agent. Nevertheless, no protein in the F8C2PC extract could be adsorbed on the chromatographic supports normally used for purification. After dilution of the extract and addition of 15 mM octyl glucoside, some of the proteins, such as myeloperoxidase, could be adsorbed (and eluted), but not cytochrome b558. Freeze-fracture electron microscopy showed that the F8C2PC extracts contained numerous vesicles and aggregates of small shapeless particles. Higher centrifugal forces sedimented most proteins of the 100,000 g supernatant. As a check, the effect of F8C2PC was tested on sarcoplasmic reticulum vesicles, the behavior of which with respect to the usual non-denaturating detergents has been well studied. There was little, if any, solubilization. We conclude that, although supernatants of F8C2PC extracts of neutrophil membranes are optically clear, proteins are not really solubilized. This result is in keeping with the absence of lytic effects of F8C2PC on erythrocyte membranes.
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PMID:Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes. 978 91

Acute lung injury represents a wide spectrum of pathologic processes, the most severe end of the spectrum being the acute respiratory distress syndrome. Reactive oxygen intermediates have been implicated as important in the pathobiochemistry of acute lung injury. The endogenous sources that contribute to the generation of reactive oxygen intermediates in acute lung injury are poorly defined but probably include the molybdenum hydroxylases, NAD(P)H oxidoreductases, the mitochondrial electron transport chain, and arachidonic acid-metabolizing enzymes. Our laboratory has focused, in particular, on the regulation of two of these enzyme systems, xanthine oxidoreductase (XDH/XO) and NAD(P)H oxidase. We observe that gene expression of XDH/XO is regulatory in a cell-specific manner and is markedly affected by inflammatory cytokines, steroids, and physiologic events such as hypoxia. Posttranslational processing is also important in regulating XDH/XO activity. More recently, the laboratory has characterized an NAD(P)H oxidase in vascular cells. The cytochrome components of the oxidase, gp91 and p22, appear similar to the components present in phagocytic cells that contribute to their respiratory burst. In human vascular endothelial and smooth muscle cells, oncostatin M potently induces gp91 expression. We believe that regulation of gp91 is a central controlling factor in expression of the vascular NAD(P)H oxidase. In summary, the studies support the concept that the oxidoreductases of vascular cells are expressed in a highly regulated and self-specific fashion.
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PMID:Lung injury and oxidoreductases. 978 4

The role of magnesium ions in the activation of NADPH oxidase has been investigated using flavocytochrome b-245 and either neutrophil cytosol or mixtures of recombinant p40phox, p47phox, p67phox and Rac2. Purified flavocytochrome b-245 is highly active (turnover number 120-150 mol of O2(-)/s per mol of cytochrome haem) in the absence of Mg2+, in marked contrast to neutrophil membranes or detergent-solubilized membranes, which have an absolute requirement for Mg2+ for NADPH oxidase activity. It was also found that Mg2+ affected the anionic amphiphile requirement for oxidase activation, and this was dependent on whether neutrophil cytosol or mixtures of recombinant cytosolic proteins were used in the assay. Unexpectedly we found that, using purified flavocytochrome b-245 and recombinant cytosolic proteins, NADPH oxidase undergoes spontaneous activation in the absence of anionic amphiphiles under Mg2+-free conditions. The results suggest that Mg2+ ions play an important role in NADPH oxidase function, perhaps stabilizing the 260 kDa complex of cytosolic phox proteins or the regulation of a guanine nucleotide-binding protein. We provide evidence that if the latter explanation is correct, the identity of the guanine nucleotide-binding protein is unlikely to be Rap1a.
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PMID:Spontaneous activation of NADPH oxidase in a cell-free system: unexpected multiple effects of magnesium ion concentrations. 993 20


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