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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.
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PMID:Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. 255 Apr 23

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.
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PMID:[The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae]. 267 96

Biosynthesis of ferredoxin-NADP+ reductase in higher plants was investigated in relation with the mechanism of formation of the holoenzyme. The putative precursor of the flavoprotein, obtained after cell-free translation on a wheat germ extract primed with poly(A)-rich mRNA, was able to spontaneously bind free FAD, rendering a functional prereductase. The newly synthesized preholoenzyme showed diaphorase and cytochrome c reductase activities, an apparent molecular mass of 45 kDa, and contained FAD as the only flavin cofactor. It gave a positive reaction towards antisera against mature ferredoxin-NADP+ reductase. On the other hand, intracellular distribution of flavin-synthesizing enzymes indicates that FAD formation occurs in the cytoplasm; that is, in the same compartment as the site of reductase synthesis. On the basis of the preceding data a model is presented for the biosynthesis of the enzyme in vivo, involving conjugation of the apoprotein with FAD in the cytoplasm, followed by transport of the preholoreductase across the chloroplast envelope to reach its final destiny in the thylakoid membrane.
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PMID:Biosynthesis of ferredoxin-NADP+ oxidoreductase. Evidence for the formation of a functional preholoenzyme in the cytoplasmic compartment. 286 41

NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.
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PMID:Purification and characterization of the human neutrophil NADH-cytochrome b5 reductase. 299 39

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplast completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W10BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.
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PMID:Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase. 312 Jul 72

An enzyme (NADPH-dependent diaphorase) present in rat brain microsomes has been solubilised and shown to utilise both nitrobluetetrazolium and cytochrome c as electron acceptors, when reduced by NADPH. The kinetics of the enzyme have been determined using cytochrome c (Km = 1.3 microM), NADPH (Km = 1.4 microM) and the Vmax (4.7 nmol/min/mg solubilised microsome protein). The subunit Mr is approximately 73,000 D and that of the native enzyme is 170,000-180,000 D, indicating that the enzyme is probably a dimer. Evidence is also provided to show that the enzyme is a flavoprotein, and that it has equimolar amounts of FAD and FMN with respect to the subunit concentration. It seems a possibility that the rat brain diaphorase enzyme may be cytochrome P450 reductase, EC 1.6.2.4.
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PMID:Rat brain NADPH-dependent diaphorase. A possible relationship to cytochrome P450 reductase. 313 10

The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s). The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes. The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580-600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme. The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.
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PMID:The redox interconversion mechanism of Saccharomyces cerevisiae glutathione reductase. 389 86

Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors.
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PMID:Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. 405 27

Crude extracts of Methanospirillum hungatei strain GP1 contained NADH and NADPH diaphorase activities. After a 483-fold purification of the NADH diaphorase the enzyme was further separated from contaminating proteins by polyacrylamide disc gel electrophoresis. Two distinct activity bands were extracted from the acrylamide, each one having oxygen, 2,6-dichlorophenolindophenol, and cytochrome c linked activities. In these preparations NADPH could not replace NADH as electron donor. During the initial purification steps all activity was lost due to the removal of a readily released cofactor. Enzyme activity was restored by either FAD or a FAD fraction isolated from M. hungatei. Oxidase activity exhibited a broad pH optimum from 7.0 to 8.5 and apparent Km values of 26 microM for NADH and 0.2 microM for FAD. Superoxide anion, formed in the presence of oxygen, accounted for all of the NADH consumed in the reaction. The molecular weight of the diaphorase was about 117 500 by sodium dodecyl sulfate gel electrophoresis. Sulfhydryl reagents and chelating agents were inhibitory. Inactivation, which occurred during storage in phosphate buffer at 4 degrees C, was delayed by dithiothreitol. The isolated NADH diaphorase lacked NADPH:NAD transhydrogenase and NAD reductase activities.
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PMID:Isolation and characterization of a FAD-dependent NADH diaphorase from Methanospirillum hungatei strain GP1. 626 28

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.
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PMID:Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties. 640 48


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