Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.8.1.4 (diaphorase)
 2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Up to now, more than 40.000 determinations of urinary estrogens (E1 + E2) have been carried out in routine clinical analysis by the enzymatic method using estradiol dehydrogenase. This method makes use of the transhydrogenating activity of the placental enzyme: this enzyme transfers hydrogen from NADP to NAD with recycling of the specific substrate (E1 + E2). For several years the necessary reagents have been commercially available in the form of a kit. Nonetheless, various improvements have been made to the measurement of reduced NAD, which accumulates in the reaction medium and is directly proportional to the concentration of the two estrogens. Three protocols are available at present: Spectrophotometric measurement at 340 nm (initial technique); Colorimetric measurement at 492 nm. The pink colour measured arises from the reduction of a tetrazolium salt (INT) by reduced NAD in a coupled system using diaphorase; Measurement by bioluminescence of the light energy liberated on the reduction of flavin derivatives by NADH. The reaction is mediated by various enzymes isolated from marine bacteria (FMN oxidoreductase and luciferase) in the presence of an aliphatic aldehyde (decanal). The procedure for each of these protocols is described as well as the means for controlling the linearity of the reaction. The choice of protocol is determined by the biological fluid available, the speed of response desired and the cost of the analysis.
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PMID:[Various protocols for determining estrogens by the enzymatic method using estradiol dehydrogenase. Respective procedures and advantages]. 386 35

The complex between ferredoxin-NADP+ oxidoreductase and its proposed membrane-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051) was isolated from spinach thylakoids and compared with isolated cytochrome b/f complex containing associated ferredoxin NADP+ oxidoreductase (Clark, R. D., and Hind, G. (1983) J. Biol. Chem. 258, 10348-10354). There was no immunological cross-reactivity between the 17.5-kDa binding protein and an antiserum raised against the 17-kDa polypeptide of the cytochrome complex. Association of ferredoxin-NADP+ oxidoreductase with the binding protein or with the thylakoid membrane gave an allotopic shift in the pH profile of diaphorase activity, as compared to the free enzyme. This effect was not seen in enzyme associated with the cytochrome b/f complex. Identification of the 17.5-kDa binding protein as the 17-kDa component of the cytochrome b/f complex is ruled out by these results.
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PMID:The ferredoxin-NADP+ oxidoreductase-binding protein is not the 17-kDa component of the cytochrome b/f complex. 390 86

Among the three closely related enzymes, lipoamide dehydrogenase, mercuric reductase, and glutathione reductase only the latter is inhibited by 2,4,6-trinitrobenzenesulfonate (TNBS). On the other hand, all three enzymes exhibit high rates of TNBS-dependent NADPH oxidation. In the case of glutathione reductase and mercuric reductase this TNBS-dependent activity displays substrate inhibition by excess of NADPH and is strongly stimulated by NADP+. The stimulation is especially pronounced with mercuric reductase, 25-fold under some conditions. Neither substrate inhibition nor stimulation by NAD+ is observed with lipoamide dehydrogenase.
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PMID:The effect of 2,4,6-trinitrobenzenesulfonate on mercuric reductase, glutathione reductase and lipoamide dehydrogenase. 391 36

Ferredoxin-NADP+ oxidoreductase (FNR, EC 1.18.1.2) was purified to molecular homogeneous form as judged by regular and sodium dodecyl sulfate (SDS)-electrophoresis using EDTA extraction of spinach thylakoids, followed by anion exchange on DEAE-cellulose, Procion Red HE 3B dye-ligand chromatography, and hydroxyapatite chromatography. By this procedure, within 1 week approx 7.5 mg of pure FNR, starting from 1 kg of spinach leaves, could be routinely obtained. By comparison with commercially available FNR and with aged preparations two different molecular forms of the enzyme were observed in SDS-electrophoresis. FNR prepared according to the described procedure revealed an apparent molecular mass of 36,000 Da, whereas all other tested preparations showed molecular masses of 3000 Da smaller. Migration in regular gel electrophoresis was the same for all preparations and zymogram stain indicated similar diaphorase activity of both the smaller and the larger forms.
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PMID:Rapid procedure for the preparation of ferredoxin-NADP+ oxidoreductase in molecularly pure form at 36 kDa. 408 75

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

Erythrocytic NADH methemoglobin diaphorase acquires NADH-dichlorophenolindophenol diaphorase activity when enzyme-associated NAD is removed. This transformation is reversible and can be mediated by membrane NAD glycohydrolase (EC 3.2.2.5) in hemolysates as well as in intact cells exposed to hydrogen peroxide. It is abolished either in NADH methemoglobin diaphorase deficiency or in NAD(P) glycohydrolase (EC 3.2.2.6) deficiency which is common in Afro-American but not in European-American adults. Activities of erythrocytic NADP glycohydrolase and NAD glycohydrolase appear to depend on a single membrane enzyme.
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PMID:NAD(P) glycohydrolase deficiency in human erythrocytes and alteration of cytosol NADH-methemoglobin diaphorase by membrane NAD-glycohydrolase activity. 436 76

1. NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least 70-fold and separated into two enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper. 2. Purified nitrite reductase accepted electrons from ferredoxin (the natural donor) or from reduced dyes. Ferredoxin was reduced by illuminated chloroplasts or dithionite, or by NADPH when diaphorase was present. The purified enzyme did not accept electrons directly from NADPH. 3. Ferredoxins purified from maize, spinach or Clostridium were interchangeable in the nitrite-reductase system. 4. Nitrite reductase had K(m) 0.15mm for nitrite. The pH optimum varied with plant and method of assay. The preparation had low sulphite-reductase activity. Ammonia was the product of nitrite reduction. 5. For some plants, the assay of crude preparations with NADPH was limited by diaphorase and the addition of diaphorase gave a better estimate of nitrite-reductase activity. A simple method of assay is described that uses dithionite with benzyl viologen as electron donor.
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PMID:The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin. 438 17

The activity of some dehydrogenases and hydrolases was studied by cytochemical methods in the peripheral blood neutrophils of germ-free guinea pigs infected with adenoviruses. The gnotobiotic animals were obtained by hysterotomy in an operation isolation room after which they were transferred into manipulation isolation room and infected with human adenovirus type 1. A depression of enzymes of alpha-glycerophosphate shunt and NADP-H2-diaphorase in neutrophils two days after infection and activation of lactate dehydrogenase and acid phosphatase at 4 days were demonstrated. The pattern of changes in the enzymatic status of intact and infected gnotobiotic animals allowed a diagnosis of adenovirus infection in most cases.
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PMID:[Cytochemical study of granulocyte enzymes in germ-free animals with adenovirus infections]. 626 24

Rapid reaction studies presented herein show that ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) catalyzes electron transfer from spinach ferredoxin (Fd) to NADP+ via a ternary complex, Fd X FNR X NADP+. In the absence of NADP+, reduction of ferredoxin:NADP+ reductase by Fd was much slower than the catalytic rate: 37-80 s-1 versus at least 445 e-s-1; dissociation of oxidized spinach ferredoxin (Fdox) from one-electron reduced ferredoxin:NADP+ reductase (FNRsq) limited the reduction of FNR. This confirms the steady-state kinetic analysis of Masaki et al. (Masaki, R., Yoshikaya, S., and Matsubara, H. (1982) Biochim. Biophys. Acta 700, 101-109). Occupation of the NADP+ binding site of FNR by NADP+ or by 2',5'-ADP (a nonreducible NADP+ analogue) greatly increased the rate of electron transfer from Fd to FNR, releiving inhibition by Fdox. NADP+ (and 2',5'-ADP) probably facilitate the dissociation of Fdox; equilibrium studies have shown that nucleotide binding decreases the association of Fd with FNR (Batie, C. J. (1983) Ph.D. dissertation, Duke University; Batie, C. J., and Kamin, H. (1982) in Flavins and Flavoproteins VII (Massey, V., and Williams, C. H., Jr., eds) pp. 679-683, Elsevier, New York; Batie, C.J., and Kamin, H. (1982) Fed. Proc. 41, 888; and Batie, C.J., and Kamin, H. (1984) J. Biol. Chem. 259, 8832-8839). Premixing Fd with FNR was found to inhibit the reaction of the flavoprotein with NADP+ and with NADPH; thus, substrate binding may be ordered, NADP+ first, then Fd. FNRred and NADP+ very rapidly formed an FNRred X NADP+ complex with flavin to nicotinamide charge transfer bands. The Fdred X NADP+ complex then relaxed to an equilibrium species; the spectrum indicated a predominance of FNRox X NADPH charge-transfer complex. However, charge-transfer species were not observed during turnover; thus, their participation in catalysis of electron transfer from Fd to NADP+ remains uncertain. The catalytic rate of Fd to NADP+ electron transfer, as well as the rates of electron transfer from Fd to FNR, and from FNR to NADP+ were decreased when the reactants were in D2O; diaphorase activity was unaffected by solvent. On the basis of the data presented, a scheme for the catalytic mechanism of catalysis by FNR is presented.
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PMID:Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex. 648 May 92

The elucidation of the primary structure of the Escherichia coli lipoamide dehydrogenase (EC 1.8.1.4) by sequencing the corresponding structural gene (lpd) has enabled a detailed structural comparison between lipoamide dehydrogenase and the related disulphide oxido-reductase, human erythrocyte glutathione reductase (EC 1.6.4.2). Some 28% of the amino acid residues were found to be identical and a striking degree of homology was apparent throughout the polypeptide chains. It was concluded that the two enzymes possess very similar three-dimensional structures with particularly strong conservation of residues around the FAD and NAD(P) binding sites and at the redox centres of the molecules. Significant amino acid substitutions occur in the substrate binding pocket and these include an extra 18 amino acid residues at the C terminus of lipoamide dehydrogenase. Under physiological conditions, lipoamide dehydrogenase and glutathione reductase act in opposite directions, passing reducing equivalents to NAD+ or from NADPH (respectively), and two key substitutions near the redox centre could be associated with this difference in function. This study represents the first direct structural comparison between two related enzymes that are NADP+-linked (glutathione reductase) and NAD+-linked (lipoamide dehydrogenase). The differential recognition of these two cofactors could be explained in terms of amino acid substitutions. A divergent evolutionary relationship between the two enzymes including their NAD and NADP binding domains is fully supported by this analysis.
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PMID:Structural relationship between glutathione reductase and lipoamide dehydrogenase. 654 54


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