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:1.6.99.1 (NADPH-diaphorase)
3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.
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PMID:Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin. 250 11

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

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.
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PMID:On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions. 283 37

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.
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PMID:On the structure of old yellow enzyme studied by specific limited proteolysis. 312 66

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 biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated. When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant. However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant. Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+. Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type. Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+. The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation. The kinetics of [14C]PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion. The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.
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PMID:Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli. 389 18

Paraquat (PQ++) increased cyanide-resistant univalent respiration in cell suspensions of five strains of obligately thermophilic bacteria. PQ++ was reduced by an NADH: or NADPH:paraquat diaphorase and selectivity for NADH, NADPH, or both electron donors varied among the thermophiles. Superoxide anion production that was dependent on the presence of PQ++ was shown by following the superoxide dismutase-inhibitable reduction of cytochrome c. In addition, the PQ++-dependent formation of hydrogen peroxide from superoxide anion was evident in two of the thermophilic strains. Catalase synthesis was induced by adding hydrogen peroxide to the growth medium of the thermophiles. The induction of catalase to eliminate hydrogen peroxide appears to be an important response of these thermophilic bacteria to oxygen toxicity.
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PMID:Paraquat toxicity and effect of hydrogen peroxide on thermophilic bacteria. 391 5

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

Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP(+) reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP(+) reductase in the photochemical reduction of NADP(+) by blue-green algal particles. The ferredoxin-NADP(+) reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP(+) was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD(+) transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (K(m) = 5.0 x 10(-3)M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase.
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PMID:Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium. 414 48

Bulk membrane fragments were prepared from cells of Bacillus cereus ATCC 4342 harvested at different stages of growth and sporulation and examined for enzymes involved in electron transport functions. The presence of succinate: DCPIP oxidoreductase (EC 1.3.99.1), succinate: cytochrome c oxidoreductase (EC 1.3.2.1), NADH:DCPIP oxidoreductase (EC 1.6.99.1), NADH:cytochrome c oxidoreductase (EC 1.6.2.1), succinate oxidase [succinate: (O(2)) oxidoreductase, EC 1.3.3.1], and NADH oxidase [NADH:(O(2)) oxidoreductase, EC 1.6.3.1] were demonstrated in membrane fragments from vegetative cells, early and late stationary-phase cells, and in cells undergoing sporulation. During the transition from a vegetative cell to a spore, there was a significant increase in the levels of enzymes associated with energy production via the electron transport system. Cytochromes of the a, b, and c type were detected in all membrane preparations; however, there was a marked increase in the level of cytochromes by the end of vegetative growth which remained throughout sporulation; there were no qualitative changes in the cytochromes throughout growth and sporulation. Sporulation was inhibited by cyanide, stressing the significance of the electron transport system. Enzyme activities were partially masked in washed membrane fragments; however, unmasking (stimulation) was achieved by sodium deoxycholate, sodium dodecyl sulfate, or Triton X-100. The degree of enzyme masking was less in vegetative cell membrane fragments than in membranes prepared from stationary-phase or sporulating cells. Results indicate the development of a membrane-bound electron transport system in B. cereus by the end of growth and prior to sporulation, which results in an increased masking of a number of enzymes associated with the terminal respiratory system of the cell.
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PMID:Development of a membrane-bound resiratory system prior to and during sporulation in Bacillus cereus and its relationship to membrane structure. 433 50


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