Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.
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PMID:The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme. 0 8

A covalently bound adduct of nicotinamide adenine dinucleotide (NAD) with alginic acid has been found to be enzymatically active and to undergo electrochemical oxidation or reduction without significant loss of its enzymatic activity. The preparation of the adduct itself (from NAD+, alginic acid, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate) is also accomplished with substantially complete retention of enzymatic activity. This adduct has been converted from the oxidized to the reduced form by controlled potential electrolysis using mercury and stainless-steel electrodes. This electrolytically produced NADH complex could be oxidized again to the enzymatically active NAD+ complex by enzymatic reaction with the proton acceptor, 2,6-dichlorophenol indophenol, as catalyzed by diaphorase. Using this electrolytic method with immobilized NAD, it is now possible to carry out redox reactions in which NADH is enzymatically oxidized to NAD+, with the simultaneous electrolytic regeneration of the reduced form, NADH, from the oxidized form, NAD+, produced in the enzymatic reaction.
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PMID:Electrolytic regeneration of the reduced from the oxidized form of immobilized NAD. 17 64

Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have been obtained in homogeneous state from asparagus mitochondria. They are flavin enzymes with 1 mol of FAD/mol of protein. Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have s20,w of 6.22 S, 6.39 S, and 5.91 S, respectively, and molecular weights of 111,000, 110,000, and 95,000 (sedimentation equilibrium) or 112,000, 112,000, and 92,000 (gel filtration). They are slightly acidic proteins with isoelectric points of 6.75, 5.75, and 6.80. Both asparagusate dehydrogenases catalyzed the reaction Asg(SH)2 + NAD+ equilibrium AsgS2 + NADH + H+ and exhibit lipoyl dehydrogenase and diaphorase activities. Lipoyl dehydrogenase is specific for lipoate and has no asparagusate dehydrogenase activity. NADP cannot replace NAD in any case. Optimum pH for substrate reduction of the three enzymes are near 5.9. Asparagusate dehydrogenases I and II have Km values of 21.5 mM and 20.0 mM for asparagusate and 3.0 mM and 3.3 mM for lipoate, respectively. Lipoyl dehydrogenase activity of asparagusate dehydrogenases is enhanced by NAD and surfactants such as lecithin and Tween 80, but asparagusate dehydrogenase activity is not enhanced. Asparagusate dehydrogenases are strongly inhibited by mercuric ion, p-chloromercuribenzoic acid, and N-ethylmaleimide. Amino acid composition of the three enzymes is presented and discussed.
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PMID:Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria. Physical, chemical, and enzymatic properties. 18 3

We describe a highly sensitive and accurate automated continuous-flow method for determining bile acids in serum. The bile acids are first liberated from serum protein by dialysis at alkaline pH and then measured fluorometrically after the following enzymic reaction. Bile acids are converted to 3-oxo bile acids with 3alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD+ to NADH. The hydrogen in the generated NADH is transferred by diaphorase (EC 1.6.4.3) to resazurin to yield resorfin, the fluorophore. Only 100 microliter of serum is required and 40 determinations can be done per hour. The CV for 20 replicate determinations in serum with a mean bile acid concentration of 9.8 mumol/liter was 2.6%. The CV for day-to-day variation for another serum on 27 successive days was 3.0% (mean concentration, 10.0 mumol/liter). We applied this method to 826 sera from various diseases; 29% exceeded the upper limit of normal, 10 mumol/liter, and abnormally high values (greater than 20 mumol/liter) were almost exclusively limited to sera from hepatobiliary and enteric disorders.
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PMID:Continuous-flow determination of bile acids in serum, and its clinical application. 65 94

NADH was metabolized both by serum components and at the cell surface. The metabolism by serum was either oxidation to NAD+, or hydrolysis of the pyrophosphate to yield nicotinamide mononucleotide (reduced) (NMNH) and AMP. NMNH was further hydrolysed to yield nicotinamide riboside (reduced) (NRH), which was stable. NAD+ was hydrolysed (although at a slower rate than was NADH), but was also reduced to yield NADH. The reduction of NAD+ was catalysed by the enzyme serum L(+)lactate dehydrogenase (EC 1.1.1.27) and was dependent on the concentration of L(+)lactate in the serum. NADPH was hydrolysed in a similar manner to NADH but not oxidized by serum. NADH generated from NAD+ by serum derived from human, foetal calf and horse sources was capable of driving the bioreductive activation of CB 1954 by the enzyme DT diaphorase. Cell surfaces oxidized NADH to NAD+, but did not oxidize NADPH or NRH. These observations suggest that NAD(P)H would be unsuitable as a source of reducing equivalents for the bioreductive activation of prodrugs by a reductase enzyme in Antibody Directed Enzyme Prodrug Therapy (ADEPT). In contrast, NAD+ (which could act as a source of NADH) and NRH could avoid the shortcomings of NAD(P)H, and act as suitable cofactors for an enzyme in an ADEPT system.
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PMID:Metabolism of NAD(P)H by blood components. Relevance to bioreductively activated prodrugs in a targeted enzyme therapy system. 138 14

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.
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PMID:Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii. 152 76

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.
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PMID:Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases. 157 98

All four life cycle stages (bloodstream, procyclic, epimastigote, and metacyclic) of Trypanosoma congolense IL 3000 were assayed with an oxygen electrode (polarograph) for the presence of terminal oxidases and carbon-source preference. In addition, these stages were used for histochemical analysis of mitochondrial activity using rhodamine 123, nitroblue tetrazolium, and diaminobenzidine. Morphometry was used to compare mitochondrial volumes and surface area among the different life cycle stages. It was found that in contrast to epimastigote forms, which were metabolically almost identical to procyclic forms, metacyclic forms showed characteristics of, and seemed preadapted to, differentiation into the bloodstream stage. While mitochondrial NAD+ diaphorase activity and an electrochemical potential were detected in all life cycle stages, metacyclic metabolism was glucose-based and terminal oxidase activity was primarily dependent upon the trypanosome alternative oxidase with the contribution of cyanide-sensitive respiration accounting for only 20-30% of the total respiratory capacity.
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PMID:Trypanosoma (Nannomonas) congolense: changes in respiratory metabolism during the life cycle. 172 Mar 94

Lipoamide dehydrogenase (E.C. 1.6.4.3) was found in Trypanosoma cruzi, Tulahuen strain, stocks Tul-2 and Q501, and CA-1 strain. After differential centrifugation of epimastigote homogenates, ammonium sulfate fractionation of the 105,000 g supernatant yielded a partially purified preparation which precipitated between 0.40 and 0.80 ammonium sulfate saturation. The enzyme (a) catalyzed the oxidation of dihydrolipoamide by NAD+ and the reduction of lipoamide by NADH, the forward reaction being 2.5-fold faster than the reverse reaction; (b) exhibited hyperbolic dependence on substrate concentration and (c) possessed diaphorase activity which was less than 5% of the lipoamide reductase activity. The NADH-reduced enzyme was inhibited by arsenite, cadmium and p-chloromercuribenzoate in a concentration-dependent manner. Substrate specificity allowed lipoamide dehydrogenase to be differentiated from T. cruzi trypanothione reductase and other NADPH-dependent flavoenzymes. After cell disruption, lipoamide dehydrogenase was found mostly in the cytosolic fraction and no evidence for association with the plasma membrane was obtained.
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PMID:Lipoamide dehydrogenase from Trypanosoma cruzi: some properties and cellular localization. 176 55

NADH acts as an incomplete competitive inhibitor for 5,8-dioxy-1,4-naphtoquinone during its rotenone-insensitive reduction by mitochondrial NADH:ubiquinone reductase. NAD+ and ADP-ribose act as incomplete mixed-type inhibitors. Ki of NAD+ and NADH towards quinone are about one order less than towards ferricyanide. The bimolecular rate constant of the reduction of the enzyme by NADH in the quinone reductase reaction is about 2 times less than that of ferricyanide reductase reaction. These data indicate that the reduction site of 5,8-dioxy-1,4-naphtoquinone is close to NAD+/NADH and ferricyanide binding site. It seems that during the steady-state reduction of ferricyanide and 5,8-dioxy-1,4-naphtoquinone these oxidizers react with NADH:ubiquinone reductase reduced to different extents.
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PMID:On the mechanism of rotenone-insensitive reduction of quinones by mitochondrial NADH:ubiquinone reductase. The high affinity binding of NAD+ and NADH to the reduced enzyme form. 190 49


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