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:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. NADH oxidase was extracted from the membranes of Acholeplasma laidlawii with buffer containing 3% Triton X-100 and subsequently purified by several chromatographic steps. The final preparation was essentially homogeneous as judged by gel electrophoresis under nondenaturing conditions. 2. The enzyme appears to be a copper-containing iron-sulfur flavoprotein (FMN:CU:Fe:labile S = 1:1:6:6). The enzyme, containing a high fraction of hydrophobic amino acids, is composed of three subunits of molecular weight 65 000, 40 000 and 19 000. 3. When oxygen is used as electron acceptor the purified enzyme demonstrates a specific activity of 58.0 IU/mg of protein and catalyzes the formation of H2O2 in nearly stoichiometric amount. The apparent Km value for NADH is estimated to be 0.4 mM (pH 7.4). NADPH cannot serve as a substrate for the enzyme. In addition to the NADH oxidase activity, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (ferricyanide, dichloroindophenol, cytochrome c). Metal-chelating agents and mercurials are shown to inhibit the activity of the enzyme. 4. From electron paramagnetic resonance and optical absorption measurements evidence was obtained that the flavin semiquinone radical in the NADH oxidase has a high air-stability, and that the flavin shuttles between the fully reduced and the semiquinone state upon electron transport from NADH to the electron acceptors. Inhibition of the NADH oxidoreductase activities by superoxide dismutase indicates that O-2 serves as an intermediate in the electron transfer from NADH to all electron acceptors used in this work. In addition to electron transfer via the superoxide radical O-2, an alternative pathway probably involving Fe-S centers is operative. From these results and literature data we present a reaction scheme for electron transport from NADH to the various electron acceptors.
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PMID:Purification and characterization of NADH oxidase from membranes of Acholeplasma laidlawii, a copper-containing iron-sulfur flavoprotein. 731 30

The flavoprotein NADP+ reductase from spinach chloroplasts may form a ternary complex with one molecule of NADP+ and one molecule of ferredoxin. Spectroscopic titration studies show that the NADP+ binding site and the ferredoxin binding site are totally independent, that is previous binding of ferredoxin does not modify binding of NADP+, and conversely. Since NADP+ reductase conditions the diaphorase reaction, that is an electron transfer between NADPH and various acceptors such as ferricyanide, the binding of ferrocyanide and its possible interaction with NADP+ and ferredoxin has been studied. Ferrocyanide behaves as a competitive inhibitor with respect to both NADP+ and ferredoxin. This seems paradoxical since NADP+ and ferredoxin are independently bound at two different non-overlapping sites of the flavoprotein. This apparent paradox may be resolved by a theoretical analysis of the interactions between either ferrocyanide and NADP+, or ferrocyanide and ferredoxin. Theory shows that if ferrocyanide is non-specifically bound at two independent sites, namely the NADP+ and the ferredoxin binding sites, it appears competitive with respect to both NADP+ and ferredoxin, although ternary flavoprotein-ferredoxin-ferrocyanide and flavoprotein-NADP+-ferrocyanide complexes are formed. The binding constants of NADP+, ferredoxin and ferrocyanide for the enzyme have been determined. These results are discussed in connection with the possible mechanism of the diaphorase reaction.
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PMID:Complex-forming properties of spinach NADP+ reductase with ferredoxin, ferrocyanide and NADP+. 740 54

Our laboratory has previously reported evidence that tone responses of isolated endothelium-removed calf pulmonary arteries elicited by changes in PO2 appear to be mediated via changes in H2O2 and guanosine 3',5'-cyclic monophosphate, and that the PO2 sensor mechanism is hypothesized to involve changes in superoxide anion (O2-.) production by a microsomal NADH-oxidoreductase, which is the major source of O2-. detected by lucigenin-elicited chemiluminescence (CL) in this tissue. In this study we examined if the flavoprotein-directed inhibitor of O2-. producing NAD(P)H oxidoreductases, diphenyliodonium (DPI), could be employed as an inhibitor of O2-. production by NADH oxidoreductase, which functions as a selective probe for PO2-elicited tone responses in calf pulmonary arterial smooth muscle. It was found that 1 microM DPI inhibited NADH-dependent production of CL in the arterial smooth muscle homogenate by 49% (n = 10). DPI reduced basal CL from endothelium-removed pulmonary arteries by 41% (n = 15). In endothelium-removed pulmonary arteries precontracted with U-46619, the hypoxic contraction of 2.3 +/- 0.5 g was reduced to 0.1 +/- 0.4 g (n = 7) by DPI, and the reoxygenation relaxation of 32.7 +/- 7.5% was decreased to 4.4 +/- 1.4% (n = 7). DPI did not have any significant effect on U-46619- or K(+)-elicited tone generation. DPI also did not alter the relaxation to H2O2 (1 microM-0.1 mM, n = 6), nitric oxide (0.42 nM-420 nM, n = 12), or isoproterenol (1 nM-1 microM, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Potential role of NADH oxidoreductase-derived reactive O2 species in calf pulmonary arterial PO2-elicited responses. 749 83

The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1. Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD.
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PMID:The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN. 764 53

The ability of the mussel postmitochondrial fraction (S9) to activate benzo[a]pyrene (BaP) and 2-aminoanthracene (2AA) to mutagenic metabolites towards Salmonella typhimurium strain TA98 was tested. The mechanisms involved in this activation were investigated and mussel cytochrome P-450-dependent monooxygenases and its NADPH cytochrome c reductase were found to contribute to the activation of BaP. This activation was improved by treating the mussel with 4,5,4',5'-tetrachlorobiphenyl (TCB) (a 3-methylcholanthrene-type inducer of cytochrome P-450-dependent monooxygenase in marine fish) and was inhibited by alpha-naphthoflavone (ANF), a cytochrome P-450 inhibitor. However, both BaP activation and cytochrome P-450-related metabolic activities are much weaker in mussels than in vertebrates. Mussel S9 activates aromatic amines more effectively than BaP. Pretreatment of mussels with TCB or addition of ANF in the incubation medium has no effect on 2AA activation. As suggested by Kurelec (1985), aromatic amine metabolism may be supported by a flavoprotein mixed-function amine oxidase which is NADPH-dependent.
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PMID:Activation of benzo[a]pyrene and 2-aminoanthracene to bacteria mutagens by mussel digestive gland postmitochondrial fraction. 767 68

EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.
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PMID:EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm). 774 2

The effects of BRB-I-28 and its derivatives (GLG-V-13, SAZ-VII-22 and SAZ-VII-23), a novel group of antiarrhythmic agents, were investigated on the rat heart mitochondrial respiratory chain. The results indicate that BRB-I-28 and its derivatives have concentration-dependent inhibitory effects on NADH oxidase and NADH-CoQ reductase (complex I), but they have no significant effects on succinate oxidase, succinate dehydrogenase (complex II), CoQ-cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and NADH-K3Fe(CN)6 reductase. The site of inhibition of BRB-I-28 and its derivatives on the respiratory chain was localized between flavoprotein n (FPn) and CoQ, which is similar to the effect of rotenone and several other antiarrhythmic drugs such as amiodarone, propranolol, etc. BRB-I-28 and its derivatives also have significant inhibitory effects on mitochondrial ATPase activity as reported for other antiarrhythmic drugs such as amiodarone, propranolol, quinidine, and lidocaine. However, BRB-I-28 and its derivatives have no direct effects on sarcoplasmic reticulum Ca(2+)-ATPase activity. The inhibitory effects of BRB-I-28 and its derivatives on mitochondrial oxidative phosphorylation may result in the depletion of ATP. This effect, in combination with their effects on Na+,K(+)-ATPase, could possibly produce an increase in Ca2+ concentration in cytosol. This may be another mechanism by which these DHBCN derivatives produce an increase in systemic arterial blood pressure and contractile force of isolated cardiac muscle. On the other hand, inhibition on mitochondrial respiration may account for some of the potential toxic effects of these diheterabicyclo[3.3.1]nonane derivatives.
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PMID:Effects of novel antiarrhythmic agents, BRB-I-28 and its derivatives, on the heart mitochondrial respiratory chain and sarcoplasmic reticulum Ca(2+)-ATPase. 799 64

The NAD(P)H-flavin oxidoreductase gene from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, was expressed in Escherichia coli, and the enzyme purified using Cibacron Blue 3G-A affinity column chromatography from crude extracts in a single step. The purified enzyme had a typical flavoprotein absorption spectrum and flavin mononucleotide (FMN) was identified as a prosthetic group, non-covalently bound in a molar ratio of 1:1. The enzyme catalyzed the electron transfer from NADH via FMNH2 to various other electron acceptors. Reduced flavin produced by flavin reductase participated non-enzymatically in the following reactions: H2O2-forming NADH oxidase-like, oxygen-insensitive nitroreductase-like, diaphorase (quinone reductase)-like and bacterial luciferase reactions.
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PMID:NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein. 803 96

NADPH-adrenoferredoxin reductase, a flavoprotein from bovine adrenocortical mitochondria, has been investigated to elucidate the equilibrium and dynamic properties of the interaction with NADP+ and adrenoferredoxin (adrenodoxin) using proton NMR spectroscopy. The line width of the signals from NADP+ depends on the presence of the reductase. The off rate constant of NADP+ from the reductase is estimated to be about 15-20 s-1 on the basis of line width measurements. No appreciable difference in off rate is detected between adenine and nicotinamide moieties of NADP+. Transferred nuclear Overhauser effect experiments for NADP+ indicate the time-dependent magnetization transfer profiles with a long lag phase. The proton NMR spectra during the titration of the reductase with adrenodoxin reveal that the reductase possesses distinct binding sites for both NADP+ and adrenodoxin. The sharp resonances in the aromatic region due to His-10 and His-62 of adrenodoxin were utilized as a probe to explore the interaction with the reductase. IN the mixture of adrenodoxin and the reductase at the mol ratio of 6:1, T1 values of the histidine residue in adrenodoxin were measured by the inversion recovery method. At low ionic strength, T1 values of the resonances are not affected in the presence or absence of the reductase. In the presence of the reductase, T1 values of resonances resulting from the histidine residues become shorter as the concentration of KCl increases because of rapid exchange between bound and free states. At low ionic strength (10 mM phosphate buffer), the off rate from the reductase is estimated to be less than about 4 s-1. The off rate of adrenodoxin from the reductase could be the rate-limiting step in cytochrome c reductase activity at low ionic strength.
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PMID:Interaction of NADPH-adrenoferredoxin reductase with NADP+ and adrenoferredoxin. Equilibrium and dynamic properties investigated by proton nuclear magnetic resonance. 813 21

DT diaphorase is a flavoprotein that enzymatically transfers two electrons from quinones as intermediate substrates and has been reported to increase its activity in the liver after exposure to toxicants. In this series of experiments, we tested the hypothesis that DT diaphorase also increases its activity after exposure to oxidants following gradient ischemia in skin. Using dorsal rat flaps, oxidant stress was induced immediately or during a 7-day period of preconditioning as a bipedicle flap before the distal attachment was divided. DT diaphorase activity (delta Abs/min/100 g) or expression of message was measured during the period of preconditioning to determine the relationship between skin survival, enzyme activity, and expression of message. There was 4.7 +/- 0.8 cm of skin necrosis in the distal end of acute flaps while the preconditioned flaps had no skin necrosis after the distal attachment was divided. In the acute flaps, the DT diaphorase activity was equal throughout the flap for the first 6 hr. After 24 hr of ischemia, the DT diaphorase activity was significantly higher in the proximal end of the flap (1.83 +/- 0.21 delta Abs/min/100 g) than that in the distal end (0.005 +/- 0.01 delta Abs/min/100 g), which was significant (P < 0.05). In the preconditioned flaps, enzyme activity did not increase but there was as 50-fold increase in DT diaphorase activity at the distal end 24 hr after they were divided (P < 0.05). Maximal enzyme induction of DT diaphorase activity occurred after 4 days of preconditioning and correlated with the maximal expression of mRNA. These studies provide the first evidence that DT diaphorase enzyme activity is inducible after oxidant stress. The data also suggests that DT activity remains elevated for at least 6 hr of ischemia and may be a potential source of anti-oxidant activity in ischemic skin.
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PMID:dT diaphorase: increased enzyme activity and mRNA expression in oxidant stress of skin. 815 25


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