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:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A flavoprotein dehydrogenase assayed for the activity of electron transfer from NADPH to cytochrome c was highly purified from the cytosolic fraction of differentiated human promyelocytic leukemia HL-60 cells. The purified enzyme had an apparent molecular mass of 68 kDa by sodium dodecyl sulfate gel electrophoresis and an equimolar amounts of flavin mononucleotide and flavin-adenine dinucleotide. The purification factor of the enzyme with respect to the cytosolic fraction was close to 1100 and the recovery of activity was approximately 18%. Reduction of cytochrome c by NADPH indicated Michaelis-Menten kinetics with a Km value of 1.50 microM for NADPH. When cytochrome c was the varied substrate, a Km value of 4.10 microM was obtained. NADH was not an effective electron donor for cytochrome c reduction and NADPH-dependent reduction of nitroblue tetrazolium was negligibly small. The purified enzyme alone did not exhibit superoxide production, and NADPH oxidase activity was not markedly stimulated upon incubation of the reductase with cytochrome b558 purified from porcine neutrophils. The purified flavoprotein gave a positive cross-reactivity to polyclonal antibodies raised to microsomal NADPH-cytochrome P450 reductase, indicating structural homology between these enzymes. The catalytic properties of the purified NADPH-cytochrome c reductase have similarities to those of liver NADPH-cytochrome P450 reductase.
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PMID:Characterization of superoxide dismutase-insensitive cytochrome c reductase activity in HL-60 cytosol as NADPH-cytochrome P450 reductase. 848 36

The latent NADPH oxidase activity of purified cytochrome b(558) from rabbit peritoneal neutrophils was expressed in a cell-free system consisting of either gel-filtrated cytosol from resting neutrophils, or a mixture of the three cytosolic activation factors, namely p47, p67 and the G protein Rac1. The cell-free system was supplemented with arachidonic acid and GTPgammaS. With gel-filtrated cytosol, the oxidase activity was relatively high (22 moles O(2)(-)/s/mole heme b in the absence of added FAD), and enhanced by less than one fourth upon addition of FAD. In contrast, with the purified cytosolic activation factors the rate of O(2)(-) production was low (8 moles O(2)(-)/s/mole heme b), and enhanced more than two-fold by a saturating concentration of FAD. The specificity of FAD was demonstrated by the lack of effect of FMN. FAD was determined together with heme b and the oxidase activity in eluates from a Sepharcryl column at the last step of the purification of cytochrome b(558). In the eluted fraction that contained both the maximal inducible oxidase activity and the highest amount of heme b, the molar amount of FAD was 20 times less than that of heme b. It is concluded that cytochrome b(558) is an NADPH-dependent flavocytochrome oxido-reductase (NADPH oxidase) in which one part of FAD is firmly bound and another, loosely attached. On the other hand, there may exist a parallel pathway of electron transfer from NADPH via distinct FAD dehydrogenase(s) to the heme b component of the NADPH oxidase.
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PMID:Assessment of the flavoprotein nature of the redox core of neutrophil NADPH oxidase. 864 81

Plasma membrane preparations from strains of the yeast Saccharomyces cerevisiae gave a reduced minus oxidized spectrum characteristic of a b-type cytochrome and very similar to the spectrum of flavocytochrome b558 of human neutrophils. The magnitude of the signal correlated with the level of ferric reductase activity and the copy number of the FRE1 gene, indicating that the FRE1 protein is a cytochrome b. Sequence similarities with the flavin binding site of flavocytochrome b558 and other members of the ferredoxin-NADP reductase family, together with increased levels of noncovalently bound FAD and iodonitrotetrazolium violet reductase activity in membranes from a yeast strain overexpressing ferric reductase, suggested that the FRE1 protein may also carry a flavin group. Potentiometric titrations indicated that FRE1, like neutrophil NADPH oxidase, has an unusually low redox potential, in the region of -250 mV, and binds CO.
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PMID:The FRE1 ferric reductase of Saccharomyces cerevisiae is a cytochrome b similar to that of NADPH oxidase. 866 73

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH-ferricyanide reductase activity was shown to belong to the H2O2-generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well-defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH-K3Fe(CN)6 reductase activity associated with the H2O2-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.
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PMID:Solubilization and characterization of a thyroid Ca(2+)-dependent and NADPH-dependent K3Fe(CN)6 reductase. Relationship with the NADPH-dependent H2O2-generating system. 885 87

Macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherosclerosis, is the result of enhanced cellular uptake of plasma low density lipoprotein (LDL). Native LDL, has to undergo oxidative modifications in order to be taken up at an enhanced rate by macrophages, leading to foam cell formation. Macrophage uptake of oxidized LDL involves its binding to scavanger receptors (including cellular proteoglycans) and this is followed by an impaired cellular cholesterol metabolism. Cells of the arterial wall including macrophages can oxidize LDL in a process that involves activation of cellular oxygenases, such as NADPH oxidase and 15-lipoxygenase. This process, however, also depends on the macrophage antioxidant environment, where glutathione peroxidase and reduced glutathione play an important protective role against cell-mediated oxidation of LDL. Macrophage phospholipids peroxidation under oxidative stress can also contribute to macrophage-mediated oxidation of LDL. Evidence for the occurrence of oxidized LDL in vivo is as follows: 1) In the atherosclerotic lesion [in humans, as well as in the transgenic, apolipoprotein E-deficient mice], LDL is oxidized (and as a result, it is also aggregated), in comparison to plasma LDL which is normally not oxidized. 2) Plasma LDL from patients at high risk for atherosclerosis (such as hypercholesterolaemic, hypertensive, diabetic and renal failure patients), as well as from the apolipoprotein E-deficient mice, demonstrates increased susceptibility to oxidation in comparison to normal LDL. In some groups of these patients LDL is minimally oxidized already in plasma. 3) Supplementation of nutritional antioxidants, which are rich in polyphenols (red wine, licorice, olive oil), or of selenium to humans or to the apolipoprotein E-deficient mice, as well as therapy with beta-hydroxy-beta-methyl-glutaryl-CoA reductase inhibitors (so-called "statins") in hyperocholesterolaemic patients, were shown to reduce the susceptibility of LDL to oxidation. This effect could be associated with a reduction in the size of the atherosclerotic lesion and may thus contribute to attenuation of the atherosclerotic process.
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PMID:Interaction of oxidized low density lipoprotein with macrophages in atherosclerosis, and the antiatherogenicity of antioxidants. 887 34

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91(phox) protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.
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PMID:Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. 894 93

Recent reports suggest that excess amounts of sugar alcohol are linked to leukocyte dysfunctions associated with diabetes. As the polyol pathway has not been firmly established in leukocytes, we have investigated NADPH-dependent reductases and sugar alcohol formation in dog leukocytes. NADPH-dependent reductase activity was observed with DL-glyceraldehyde as substrate in both mononuclear and polymorphonuclear leukocytes isolated from dog. By chromatofocusing, this activity corresponded primarily to aldehyde reductase rather than aldose reductase. The enzymatic conversion of glucose to the sugar alcohol sorbitol in leukocytes was confirmed in vitro by 19F nuclear magnetic resonance (NMR) spectroscopy using 3-deoxy-3-fluoro-D-glucose as substrate. The NMR spectrum obtained after incubation with 10 Mm 3-deoxy-3-fluoro-D-glucose at 37 degrees C for 24 h displayed newly formed 3-deoxy-3-fluoro-D-sorbitol and 3-deoxy-3-fluoro-D-fructose peaks with both mononuclear and polymorphonuclear leukocytes. Sugar alcohol production in leukocytes from galactose-fed dogs was also observed in vivo. Galactitol accumulation was consistently observed by gas chromatography to occur in mononuclear cells while only trace amounts of galactitol were observed in polymorphonuclear leukocytes. Activation of NADPH oxidase activity in neutrophils isolated from galactose-fed dogs by zymosan was also significantly reduced compared to that of nongalactosemic control dogs. These results indicate that glucose is converted to fructose through sorbitol in both mononuclear and polymorphonuclear leukocytes despite the observations that these cells primarily contain aldehyde reductase rather than aldose reductase. In vivo, sugar alcohol accumulation in mononuclear cells is greater than in polymorphonuclear leukocytes.
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PMID:Polyol pathway and NADPH-dependent reductases in dog leukocytes. 897 81

The leukocyte iodonitrotetrazolium violet (INT) reductase activity of disrupted bovine polymorphonuclear neutrophils is closely associated with the activation of the O2(-)-generating NADPH oxidase in a cell-free system. It is dependent upon NADPH, cytosolic factors, and amphiphiles (such as arachidonate), the same factors required for O2- generation. Both O2- generation and INT reductase activity are inhibited by phenylarsine oxide, an inhibitor of the activation of the NADPH oxidase [Li, J., & Guillory, R. J. (1997) J. Biochem. Mol. Biol. Biophys. (in press)]. In this report, the INT diaphorase activity of disrupted bovine polymorphonuclear neutrophils is shown to be resolved by DEAE-Sepharose chromatography into two fractions: an NADPH-cytochrome c reductase-containing fraction and a cytochrome b558-associated fraction. The diaphorase activity in the NADPH-cytochrome c reductase-containing portion is not dependent upon the presence of an amphiphile or phospholipid and is not associated with O2- generation. Upon incorporation into liposomes, the cytochrome b558-containing fraction demonstrates high O2- and INT reductase activities in the presence of cytosolic factors. Both O2- generation and INT reductase activities are SDS and FAD dependent and further stimulated by GTPgammaS. Phenylarsine oxide inhibits both O2- generation and INT reductase activities when added prior to activation by SDS. With the cytochrome b-containing liposomes, the Km values (O2- formation) for NADPH and NADH are 27.2 microM and 810 microM, and for INT reductase the Km values are 27.5 microM and 1017 microM, respectively. Under anaerobic conditions and thus in the absence of O2- formation, the NADPH-dependent INT reductase activity does not change, indicating that the dye reduction is not due to its direct reduction by O2 anion but is an intrinsic property of the superoxide-generating NADPH oxidase. Cytochrome b558 is the essential component of the NADPH oxidase and contains all the redox centers necessary for electron flow between NADPH and oxygen. The correlation of the activation and inhibition patterns for O2- generation and INT reduction by cytochrome b558 incorporated into artificial liposomes strongly indicates that the two activities are associated with the same membrane protein, cytochrome b558.
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PMID:Purified leukocyte cytochrome b558 incorporated into liposomes catalyzes a cytosolic factor dependent diaphorase activity. 915 36

The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN.
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PMID:Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica. 949 88

Thioredoxin reductase (TR, EC 1.6.4.5) was purified 5800-fold from the livers of adult male B6C3F1 mice. The estimated molecular mass of the purified protein was about 57 kDa. The activity of the purified enzyme was monitored by the NADPH-dependent reduction of 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB); this activity was fully inhibited by 1 microM aurothioglucose. Arsenicals and arsinothiols, complexes of As(III)-containing compounds with L-cysteine or glutathione, were tested as inhibitors of the DTNB reductase activity of the purified enzyme. Pentavalent arsenicals were much less potent inhibitors than trivalent arsenicals. Among all the arsenicals, CH(3)As(III) was the most potent inhibitor of TR. CH(3)As(III) was found to be a competitive inhibitor of the reduction of DTNB (K(i) approximately 100 nM) and a noncompetitive inhibitor of the oxidation of NADPH. The inhibition of TR by CH(3)As(III) was time-dependent and could not be reversed by the addition of a dithiol-containing molecule, 2,3-dimercaptosuccinic acid, to the reaction mixture. The inhibition of TR by CH(3)As(III) required the simultaneous presence of NADPH in the reaction mixture. However, unlike other pyridine nucleotide disulfide oxidoreductases, there was no evidence that mouse liver TR was inactivated by exposure to NADPH. Treatment with CH(3)As(III) did not increase the NADPH oxidase activity of the purified enzyme. Thus, CH(3)As(III), a putative intermediate in the pathway for the biomethylation of As, is a potent and irreversible inhibitor of an enzyme involved in the response of the cell to oxidative stress.
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PMID:Methylarsenicals and arsinothiols are potent inhibitors of mouse liver thioredoxin reductase. 1052 67


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