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)

The hepatic microsomal cytochromes P-450 and b5, as well as the enzymes of the hepatic microsomal electron-transport system (HMETS), including NADPH oxidase and NAPDH cytochrome c reductase, were monitored in male ICR mice (25 - 30 g) over a six-day period following repeated oral administration of methadone hydrochloride 12.5, 25, or 50 mg/kg per day, or an equivalent volume of water. Cytochrome P-450 content, when expressed per milligram of microsomal protein, was elevated as early as day 1 of administration. This increase in cytochrome P-450, which lasted throughout the period of administration, appeared to correlate with the previously reported increase in the hepatic microsomal enzyme methadone N-demethylase and tolerance to methadone lethality. The activities of the enzymes NADPH cytochrome c reductase and NADPH oxidase were both elevated significantly by day 2 of administration. However, these increases returned to control levels by day 6 of treatment. The only other cytochrome in the HMETS, cytochrome b5, showed no significant change following repeated oral methadone administration. Further, methadone administration depressed the hepatic microsomal protein content following two days of treatment and no elevation above control values was noted. The significance of these findings with respect to the role of the HMETS in the development of tolerance is discussed in some detail for methadone, as well as the findings previously reported by this laboratory for its acetylated congener, l-alpha-acetylmethadol.
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PMID:The role of the hepatic microsomal electron-transport system in the development of metabolic tolerance from repeated oral methadone administration in mice. 676 37

Spironolactone pretreatment (10mg/100g, twice daily for 4 days, orally) caused a significant decrease in cytochrome P-450 levels in the liver microsomes in female rats but male rats were unaffected. NADH oxidase activity was significantly decreased in both sexes by this pretreatment but NADPH oxidase and NADH cytochrome C reductase activities were not altered. NADPH cytochrome c reductase activity was increased more markedly in female rats. Despite the decrease in P-450 levels, aminopyrine N-demethylase activity was increased in female rats, while it remained unchanged in males. 7-Ethoxycoumarin O-deethylase activity was markedly increased in male and slightly decreased in female rats. The azoreductase activity was slightly reduced in treated male rats and remained unaltered in female rats when it was expressed in activity per mg microsomal protein, but the activity did show a significant increase in female rats when it was expressed as a P-450 specific rate. Sex associated differences in the effect of spironolactone on the rat liver microsomal drug metabolizing enzyme system demonstrated in the present study cannot be simply explained by the previously reported effect on adrenal and testicular steroids in male rats. It also seems unlikely that these effects were caused by an alteration in P-450 quality by selective destruction of certain species of P-450.
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PMID:Effect of spironolactone on hepatic microsomal monooxygenase and azoreductase activities. 707 90

Cytochrome b558 isolated from human neutrophils was inactive and contained no detectable FAD. However, high NADPH oxidase activity was seen upon reconstitution of the cytochrome with either native FAD or 8-mercapto-FAD in the presence of phospholipids (phosphatidylcholine/phosphatidylethanolamine/phosphatidylinositol/ sphingomyelin/cholesterol, 4:2:1:3:3 (w/w)). Their cell-free superoxide-generating activities were 40.5 and 35.5 mol/s/mol of heme, respectively, which corresponded to 70 and 61% of the original activity of the plasma membranes. Both flavins co-eluted with heme and protein on gel exclusion chromatography. The respective specific flavin content was 6.45 and 7.93 nmol/mg of protein and corresponded to a flavin:heme molar ratio of 0.41 and 0.51 consistent with a 2:1 ratio of heme to flavin. Mixing of 8-mercapto-FAD with flavin-depleted cytochrome b558 caused a red-shift of the flavin absorption maximum from 520 nm to around 560 nm, as has been seen when a variety of other apoflavoprotein dehydrogenases bind this analog. The 8-mercapto-FAD reconstituted into the cytochrome reacted readily with either iodoacetamide (k = 38.8 M-1.min-1) or iodoacetic acid (k = 12.1 M-1.min-1) to give a fluorescence spectrum characteristic of a 8-mercaptoflavin derivative, 8-SCH2CONH2 FAD or 8-SCH2COOH FAD. These results indicate that position 8 of FAD bound to the protein is freely accessible to solvent. These studies support the idea that cytochrome b558 is a flavocytochrome.
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PMID:Reconstitution of flavin-depleted neutrophil flavocytochrome b558 with 8-mercapto-FAD and characterization of the flavin-reconstituted enzyme. 760 14

We have demonstrated that human fibroblasts can release O2-. radicals by an NADPH oxidase system that appears to be functionally similar to the phagocytic system. Further analysis of these systems, however, with respect to the low-potential b-type cytochromes involved suggests that these two O2-.-generating systems are not structurally identical. Immunoblot analysis of fibroblast membranes with six different antibodies directed against both subunits of human neutrophil cytochrome b-558 indicated that the b-type cytochrome molecules involved in these systems were not identical. None of these anti-(neutrophil cytochrome b) antibodies recognized a similar cytochrome in fibroblast membranes, suggesting that the two cytochrome species are immunologically distinct. In addition, fibroblasts obtained from a patient suffering from X-linked chronic granulomatous disease (CGD) had a normal cytochrome b-558 content compared with control fibroblast membranes, whereas the cytochrome b-558 concentration in polymorphonuclear leucocytes (PMNs) from this patient was decreased to 10% of that found in PMNs from healthy controls. Likewise, the stimulated O2-. release in PMNs from this patient was less than 10% of that in control PMNs, whereas the fibroblasts showed stimulated O2-.-release rates that were indistinguishable from those of fibroblasts obtained from healthy persons. Since the genetic mutation responsible for this type of CGD results in the absence of cytochrome b-558 in PMNs, fibroblasts should be affected in the same way if both cytochrome species were identical. These results suggest therefore that the low-potential b-type cytochromes in PMNs and fibroblasts are structurally and genetically distinct.
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PMID:The cytochrome b-558 molecules involved in the fibroblast and polymorphonuclear leucocyte superoxide-generating NADPH oxidase systems are structurally and genetically distinct. 767 34

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91-phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91-phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.
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PMID:p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox. 791 88

Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b558 were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octylagarose, 2',5'-ADP-Sepharose and heparin-Sepharose column chromatographies. The purified cytochrome c reductase with an apparent molecular mass of 68 kDa contained FMN and FAD (FMN/FAD approx. 1). Cytochrome b558 prepared in the presence of phospholipids and FAD showed marked O2-.-producing activity (Vmax., 8.53 mumol of O2-./min per mg of cytochrome; Km for NADPH 58.8 microM) in a cell-free assay system consisting of cytosol, arachidonate and GTP[S]. However, when it was obtained without FAD added to the purification process, it had negligible FAD and little or no O2-.-forming activity in the reconstituted system. The NADPH oxidase activity was not markedly stimulated on incubation of the purified reductase with either flavinated or flavin-depleted cytochrome b558 in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O2-. generation. The purified reductase cross-reacted with polyclonal antibodies against both hepatic NADPH-cytochrome P-450 reductase and a synthetic peptide, ILVGPGTGIAPFRSF, which indicates residues 529-543 located in the glycine-rich NADPH-binding domain of the P-450 reductase, but cytochrome b558 did not produce any immunoreactive bands to these antibodies. These antibodies also produced a positive reaction with a 76 kDa protein from dimethyl sulphoxide-induced HL-60-cell microsomes. After solubilization of the microsomal membranes, the 76 kDa protein was readily converted into a partially proteolysed form (68 kDa) even in the presence of antiproteases. In addition, the microsomal fraction shows a CO difference spectrum with a peak at about 454 nm and a trough at 476 nm in the presence of dithionite, indicating the presence of a cytochrome P-450-like haemoprotein.
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PMID:NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization. 811 Jan 98

In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp91-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H+ translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H+ extrusion. CGD cells expressing diminished amounts of cytochrome displayed H+ conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+. Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity.
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PMID:Activation of H+ conductance in neutrophils requires assembly of components of the respiratory burst oxidase but not its redox function. 816 76

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages. 822 83

Cell stimulation of blood phagocytes activates the superoxide-producing NADPH oxidase. Cytochrome b558, one of the two oxidase redox components, comprises a light (alpha) and a heavy glycosylated (beta) subunit. The other redox component, a flavoprotein, is now thought to be the heavy subunit, on the basis of amino acid sequence comparisons and of reconstitution experiments with purified components. We published that pyridoxal-5'-diphospho-5'-adenosine is an inactivating affinity label for the NADPH-binding site of particulate oxidase from activated neutrophils. We have now radiolabeled the inactivated oxidase by reducing with Na[3H]BH4 the Schiff base formed between proteins and the reagent. Upon SDS-PAGE, the NADPH-inhibitable incorporation is found at the same position as the immunodetectable cytochrome heavy subunit, before and after deglycosylation. Membranes from either activated cells of a cytochrome-deficient X-linked granulomatous disease patient or normal resting cells show no incorporation at this position. Our results provide experimental evidence for the existence on the cytochrome b558 heavy chain of an NADPH-binding site which can only be affinity-labeled by PLP-AMP when the oxidase is active. This suggests the occurrence of a conformational change in the cofactor binding site upon enzyme activation.
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PMID:Affinity-labeling of an NADPH-binding site on the heavy subunit of flavocytochrome b558 in particulate NADPH oxidase from activated human neutrophils. 824 Mar 26

Purified cytochrome b559 relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O2-) producing ability, respectively, in the absence of cytosolic activators [Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57-62]. This system was used as a model for the study of the mechanism of NADPH oxidase activation. It is shown that, depending on the composition of the phospholipid environment, cytochrome b599 binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b559 relipidated with phosphatidylcholine combined with phosphatidic acid is associated with an enhanced NADPH-driven O2- producing capacity. A kinetic study of O2- production by cytochrome b559 reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O2- production by high- and low-activity relipidated and reflavinated cytochrome b559, at varying substrate concentrations, and the determination of steady-state difference spectra of such preparations, reduced by NADPH, indicated that O2- production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.
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PMID:Superoxide production by cytochrome b559. Mechanism of cytosol-independent activation. 830 96


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