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)

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

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment caused a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH:cytochrome c reductase remained in the peroxisomal 'ghosts'. At the same time, treatment by Triton X-100 led to solubilization of the membrane-bound NADH:cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehyde. The enzyme Km values lie in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells is discussed.
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PMID:Intraparticulate localization and some properties of a clofibrate-induced peroxisomal aldehyde dehydrogenase from rat liver. 399 98

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

1. Induction of the formation of lipid peroxide in suspensions of liver microsomal preparations by incubation with ascorbate or NADPH, or by treatment with ionizing radiation, leads to a marked decrease of the activity of glucose 6-phosphatase. 2. The effect of peroxidation can be imitated by treating microsomal suspensions with detergents such as deoxycholate or with phospholipases. 3. The substrate, glucose 6-phosphate, protects the glucose 6-phosphatase activity of microsomal preparations against peroxidation or detergents. 4. The loss of glucose 6-phosphatase activity is not due to the formation of hydroperoxide or formation of malonaldehyde or other breakdown products of peroxidation, all of which are not toxic to the enzyme. 5. All experiments lead to the conclusion that the loss of activity of glucose 6-phosphatase resulting from peroxidation is a consequence of loss of membrane structure essential for the activity of the enzyme. 6. In addition to glucose 6-phosphatase, oxidative demethylation of aminopyrine or p-chloro-N-methylaniline, hydroxylation of aniline, NADPH oxidation and menadione-dependent NADPH oxidation are also strongly inhibited by peroxidation. However, another group of enzymes separated with the microsomal fraction, including NAD(+)/NADP(+) glycohydrolase, adenosine triphosphatase, esterase and NADH-cytochrome c reductase are not inactivated by peroxidation. This group is not readily inactivated by treatment with detergents. 7. Lipid peroxidation, by controlling membrane integrity, may exert a regulating effect on the oxidative metabolism and carbohydrate metabolism of the endoplasmic reticulum in vivo.
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PMID:Effects of lipid peroxidation on membrane-bound enzymes of the endoplasmic reticulum. 439 3

The effects of an oral administration of carbon tetrachloride on various liver microsomal and supernatant components were studied 1hr. and 2hr. after dosing. The modifications of such early changes resulting from a concomitant administration of promethazine together with the carbon tetrachloride were also investigated. The microsomal components studied were: cytochromes P-450 and b(5); inorganic pyrophosphatase; NADH- and NADPH-cytochrome c reductases; NADH- and NADPH-neotetrazolium reductases; a lipid-peroxidation system associated with the oxidation of NADPH and stimulated by ADP and Fe(2+). NAD- and NADP- DT-diaphorases were measured in the supernatant solution remaining after isolation of liver microsomes, and the distribution of RNA phosphorus between the microsomes and supernatant solution was also determined. Carbon tetrachloride produced a rapid fall in inorganic pyrophosphatase activity, a rather slower decrease in cytochrome P-450 content of the microsomes and small increases in the activities of NADH-cytochrome c reductase and neotetrazolium reductases. The activities of NADPH-cytochrome c reductase, the NADPH-ADP/Fe(2+)-linked lipid-peroxidation system, DT-diaphorases and the content of cytochrome b(5) in the microsomes were unchanged. There was also a loss of RNA phosphorus from the microsomes into the supernatant solution. The RNA phosphorus redistribution, the decrease in inorganic pyrophosphatase and the increases in neotetrazolium reductase activities were at least partially prevented by a concomitant dosing with promethazine. However, the decrease in cytochrome P-450 was not affected by promethazine treatment. These early changes are discussed in terms of the liver necrosis produced by carbon tetrachloride and which is greatly retarded in its onset by the administration of promethazine.
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PMID:The effects of carbon tetrachloride on rat liver microsomes during the first hour of poisoning in vivo, and the modifying actions of promethazine. 576 54

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

NADPH-cytochrome c reductase in Hepa-1 cells was induced 2-fold by phenobarbital, but was not induced by benz[a]anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The apparent Km of the enzyme for NADPH was 0.57 microM; the activity was inhibitable by NADP; and segregated primarily to the microsomal fraction. Cytoplasm of Hepa-1 cells bound antibody to rabbit cytochrome P-450 reductase. 3T3 cells, which possessed one sixth of the cytochrome c reductase activity of Hepa-1 cells, bound correspondingly less cytochrome P-450 reductase antibody. This supports the notion that cytochrome P-450 reductase was responsible for the cytochrome c reductase activity that was measured.
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PMID:Characterization of NADPH-cytochrome P-450 reductase in a mouse hepatoma cell line. 630 57

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP+ reductase, in a competitive manner with respect to NADPH. The Ki values were 1.5 and 0.2 microM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP+ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP+ reductase than Cibacron blue. The Kd values were 1.9 and 5 microM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP+ oxidoreductase from spinach leaves with good yields.
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PMID:Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography. 682 90


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