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

---

Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.
...
PMID:The mechanism of oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart. 2 68

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.
...
PMID:Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16. 18 26

The actions of Dexon on the NADH-ferricyanide oxidoreductase and the NADPH oxidase system of electron transfer particles (ETP) from beef heart as well as on the NADPH-cytochrome c oxidoreductase from brewer's yeast (Saccharomyces carlsbergensis Hansen) were investigated. The inhibition of the NADH dehydrogenase activity of ETP and that of the yeast enzyme correspond with respect to the following characteristics: 1) increase in the inhibition, 2) enhancement of the Dexon sensitivity by one order of magnitude after preincubation in the presence of NAD(P)H, 3) irreversibility of the inhibition, 4) no detectable changes in the spectral properties and in coenzyme activity of FMN after acid extraction from Dexon-treated enzyme. The inhibition of the NADH dehydrogenase activity of ETP is diminished by both NAD+ and FMN. However, no interaction of Dexon with NAD(P)H or FMN could be detected in the absence of enzyme or apoenzyme. The concentration of half-inhibition by Dexon for the yeast enzyme corresponds with its FMN concentration. It is proposed that both apoenzyme, NAD(P)H and FMN are involved in the interaction with Dexon. Possible mechanisms of binding are both complanar complexations of the ring systems and a triazene formation between FMNH2 and Dexon. The NADPH oxidase activity of the ETP is partly inhibited; the share inhibited by Dexon may represent the pathway via the transhydrogenase reaction.
...
PMID:[Mechanism of action of the inhibition of pyridine-nucleotide-dependent flavine enzymes using the systemic fungicide Dexon]. 41 38

Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.
...
PMID:Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system. 165 5

Brain nitric oxide synthase is a Ca2+/calmodulin-regulated enzyme which converts L-arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme-bound H4biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non-heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin-dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin.
...
PMID:Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase. 171 90

The cytosolic component of macrophage-derived superoxide generating NADPH oxidase was partially purified by affinity chromatography on 2',5'-ADP-agarose. Elution was nonspecific by elevated phosphate molarity. A single step attains at least 40-fold enrichment of specific activity, the recovery being over 20%. Elution with various ligands in the concentration range 2-3.5 mM was also tested. The most effective ligands were: ATP, dATP, GTP, NADPH and 2',5'-ADP. Ineffective were AMP, 2'-AMP, FMN, FAD and NADH. ADP was of medium potency. On the basis of the above and other results, we infer that the molecule (or complex) purified by us may contain the enzymatic NADPH binding site. This component is fully retained by a 100 kDa cutoff membrane and is labile at room temperature, the lability being cancelled by 2-mercaptoethanol.
...
PMID:Macrophage-derived superoxide-generating NADPH oxidase in an amphiphile-activated, cell-free system; partial purification of the cytosolic component and evidence that it may contain the NADPH binding site. 282 78

The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O2 of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6-6.5.
...
PMID:Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH. 301 6

Limited proteolysis of brewer's yeast old yellow enzyme (OYE) was carried out with bovine pancreatic alpha-chymotrypsin. The reaction proceeded with a decrease of the NADPH oxidase activity, generating specifically two peptides (designated as 34K and 14K fragments) with apparent molecular weights of 34,000 and 14,000, respectively. The same proteolytic treatment of apo OYE resulted in rapid and complete digestion of the protein. The 34K and 14K fragments are so intimately associated with each other that the isolation of each peptide from the other in the native form was unsuccessful. However, the complex of the two fragments was separated from the intact OYE and termed "nicked OYE." Nicked OYE still retained FMN and showed a visible-absorption spectrum slightly modified from that of intact OYE. Nicked OYE showed decreased affinity toward rho-bromophenol as compared to intact OYE. Nicked OYE exhibited lower Km and Vmax values than intact OYE in the NADPH oxidase reaction. The 34K and 14K fragments could be separated from each other by reversed-phase HPLC under denaturing conditions and the amino acid sequences of the two fragments and intact OYE in the amino terminal regions were determined. The N-terminal sequence of the 34K fragment coincided with that of intact OYE, indicating that the 34K fragment lies in the N-terminal side of OYE. The N-terminal sequence of the 14K fragment was found to show homology with the site of flavodoxin where it forms an electron-transfer complex with cytochrome c. The characteristic feature of this region is the presence of acidic residues and is shared by the FMN domain of NADPH-cytochrome P-450 reductase. We interpret these findings as indicating that OYE has a physiological role as an electron transfer component.
...
PMID:On the structure of old yellow enzyme studied by specific limited proteolysis. 312 66

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.
...
PMID:The heterogeneity of brewer's yeast old yellow enzyme. 351 95

The mammalian mitochondrial electron transport chain catalyzes the oxidation of NADH at pH 8.0 and pH 6.5, and the oxidation of NADPH at pH 6.5. The pH-dependencies of the rate of steady-state oxidation of NADPH and NADH by Complex I as well as by its flavoprotein fraction have been extensively studied by the laboratory of Hatefi. One model to explain these pH-dependent oxidations was proposed by Bakker and Albracht (Biochim. Biophys. Acta 850 (1986) 413-422 and 423-428, modified by Van Belzen and Albracht (Biochim. Biophys Acta 974 (1989) 311-320), which predicts that Complex I is a heterodimer with promoter B, containing FMN and Fe-S clusters 1-4 in stiochiometric amounts, catalyzing NADH oxidation at pH 8, and Protomer A, containing FMN and Fe-S clusters 2, 4, catalyzing NAD(P)H oxidation at pH 6.5. A pH-dependent transfer of electrons from protomer A Fe-S clusters 2, 4 to protomer B Fe-S clusters 2, 4 is an obligate step in the oxidation of NAD(P)H at low pH. Strict interpretation of this model allows for only three types of inhibitor: one which inhibits all three oxidase activities (type 1); one which inhibits NADH oxidase, pH 8.0 (type 4) and a third which inhibits NAD(P)H oxidase, pH 6.5 (type 5). Another possibility is that there are three separate pathways of oxidation of NAD(P)H, which would allow for a total of seven different types of inhibitor, e.g., the three types above plus type 2 inhibiting NADH oxidase pH 8.0 and pH 6.5; type 3 inhibiting NADH oxidase pH 8.0, and NADPH oxidase pH 6.5; type 6 inhibiting NADH oxidase pH 6.5; and type 7 inhibiting NADPH oxidase pH 6.5. Using a series of thirteen inhibitors of Complex I activity and the chemical modification reagent ethoxyformic anhydride (EFA), four different inhibitor types were found: seven inhibitors of type 1, four inhibitors of type 2, one inhibitor of type 3 and one inhibitor of type 4. Treatment of submitochondrial particles (SMP) with EFA abolished NADH-dependent reduction of coenzyme Q at both pH 8.0 and 6.5, while inhibiting NADPH-dependent reduction of coenzyme Q at pH 6.5 by only 30%. These results do not support the heterodimer model of Complex I electron transport of Bakker and Albracht, but do support three separate electron flow pathways through complex 1 from reduced pyridine nucleotides to coenzyme Q. A new model of electron flow through Complex I based on these finding is proposed.
...
PMID:Evidence for three separate electron flow pathways through Complex I: an inhibitor study. 761 35


1 2 3 Next >>

---