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:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.
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PMID:Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase. 314 55

Yeast glutathione reductase exists in a single molecular form which exhibits preferred NADPH and weak NADH linked multifunctional activities. Kinetic parameters for the NADPH and NADH linked reductase, transhydrogenase, electron transferase and diaphorase reactions have been determined. The functional preference for the NADPH linked reductase reaction is kinetically related to the high catalytic efficiency and low dissociation constants for substrates. NADP+ and NAD+ may interact with two different sites or different kinetic forms of the enzyme. The active site disulfide and histidine are required for the reductase activity but are not essential to the transhydrogenase, electron transferase and diaphorase activities. Amidation of carboxyl groups and Co(II) chelation of glutathione reductase facilitate the electron transferase reaction presumably by encouraging the formation of an anionic flavosemiquinone.
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PMID:Multifunctional activities of yeast glutathione reductase. 329 44

The spectral properties of ten redox indicator dyes were evaluated with the aim of finding the optimal choice for coupling to enzymatic reactions with high sensitivity for the production of the reduced form. Eight of the dyes were selected for coupling into a reaction cycle formed by yeast alcohol dehydrogenase with substrates ethanol and nicotinamide adenine dinucleotide (NAD+) and diaphorase with substrates reduced nicotinamide adenine dinucleotide (NADH, produced by the prior reaction) and the oxidized form of the respective dye. Two of the dyes exhibited decreased absorption on reduction, whereas all (eight) tetrazolium dyes increased in their absorption substantially upon reduction. Bis-tetrazolium dyes had a significantly higher molar extinction coefficient (up to 23,000 M-1.cm-1) than mono-tetrazolium dyes (down to 8000 M-1.cm-1). Kinetically, most dyes could be reduced with NADH (and diaphorase), but the rate of reduction varied considerably among the dyes with nitroblue tetrazolium (NBT) and tetranitroblue tetrazolium (TNBT) being the fastest. Therefore, NBT and TNBT seem to be the most suitable for fast response.
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PMID:Coupling of redox indicator dyes into an enzymatic reaction cycle. 337 38

In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD+-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%-70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.
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PMID:A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae. 352 55

Lipoamide dehydrogenase (EC 1.6.4.3) from the ketoglutarate dehydrogenase complex of adrenals catalyzes the oxidation of NADH by lipoamide and quinone compounds according to the "ping-pong" scheme. The catalytic constants of these reactions are equal to 220 and 24 s-1, respectively (pH 7.0). The maximal quinone reductase activity is observed at pH 5.6, whereas the lipoamide reductase activity changes insignificantly at pH 7.5-5.5. The maximal dihydrolipoamide-NAD+ reductase activity is observed at pH 7.8. The oxidative constants of quinone electron acceptors vary from 6 X 10(6) to 4 X 10(2) M-1 s-1 and increase with their redox potential. The patterns of NAD+ inhibition in the quinone reductase reaction differ from that of lipoamide reductase reaction. The quinones are reduced by lipoamide dehydrogenase in the one-electron mechanism.
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PMID:[Characteristics of the interaction of adrenal lipoamide dehydrogenase with physiological and quinone electron acceptors]. 357 23

A direct spectrophotometric assay for determination of the serum bile acid concentration in the woodchuck (Marmota monax) has been validated. The assay relies on the conversion of 3-hydroxy bile acids to 3-oxo bile acids by 3 alpha-hydroxysteroid dehydrogenase with concomitant reduction of NAD+ to NADH. Reduction of NAD+ is coupled via a diaphorase catalyst to the formation of a diformazan dye from nitrotetrazolium blue and the diformazan product is measured spectrophotometrically at 540 nm. Interfering endogenous dehydrogenase activity present in woodchuck sera was inactivated with sodium pyruvate. Mean recovery of seven exogenous bile acids added to woodchuck sera was 102.0 +/- 2.2%. Intra-assay precision was determined with ten replicate samples giving a mean +/- standard error of the mean of 1.94 +/- 0.12 micron/L with a coefficient of variation of 3.9%. The mean serum bile acid concentration determined in 33 clinically healthy animals was 5.52 +/- 0.81 micron/L. The serum bile acid concentration increased following surgical ligation of the bile duct from 3.78 +/- 0.58 micron/L to a maximum value of 148.0 +/- 30.7 micron/L and remained increased for the 42 day study period. In woodchucks treated with carbon tetrachloride, the serum bile acid concentration peaked at 16 hours following treatment at 72.7 +/- 29.3 micron/L, and returned to pretreatment concentration within 6 days. The serum bile acid concentration therefore appears to be a sensitive biochemical test of cholestasis and hepatocellular forms of hepatic injury and of potential value in the clinical assessment of hepatic disease associated with woodchuck hepatitis virus infection.
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PMID:Serum bile acid determination for assessment of hepatic injury in the woodchuck. 359 95

Dihydrolipoamide dehydrogenase has been discovered in the bloodstream form of the eukaryotic African parasite, Trypanosoma brucei. The enzyme catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+ and exhibited a hyperbolic dependence of catalytic activity on the concentrations of both dihydrolipoamide and NAD+. Chemical modification with the tervalent arsenical reagent p-aminophenyldichloroarsine indicates the involvement in catalysis of a reversibly reducible disulphide bond. Plasma-membrane sheets were purified from T. brucei, and it was shown that virtually all the dihydrolipoamide dehydrogenase remained closely associated with this membrane preparation. T. brucei apparently lacks the 2-oxoacid dehydrogenase multienzyme complexes of which dihydrolipoamide dehydrogenase is usually an integral component. In the context of this absence, the possible function of trypanosomal dihydrolipoamide dehydrogenase is discussed, with particular reference to its cellular location in the plasma membrane.
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PMID:Dihydrolipoamide dehydrogenase from Trypanosoma brucei. Characterization and cellular location. 366 96

The kinetic behavior and spectroscopic characteristics of the nucleotide site(s) of lipoamide dehydrogenase have been investigated. Both subunits of the dimeric enzyme interact with NAD+. The binding of NAD+ is associated with a negative trough around 420-450 nm and a positive peak at 507 nm of the difference spectrum. The transhydrogenation between NADH and thionicotinamide nucleotide or acetylpyridine nucleotide is shown to proceed via a Ping Pong or an ordered Bi Bi mechanism, respectively, at pH above 7.0. Lowering pH or acetamidation lose the spectral characteristic of the positive peak of the enzyme-NAD+ complex with a concurrent change in the kinetic mechanism in the NADH+-acetylpyridine nucleotide transhydrogenation.
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PMID:Kinetic and spectroscopic studies of transhydrogenase activity and nucleotide site of lipoamide dehydrogenase. 384 Jan 1

Lipoamide dehydrogenase reacts irreversibly with arsonous acids, RAs(OH)2, and arsonic acids, RAs(O)(OH)2, to form enzyme-inhibitor complexes. The formation of inactive enzyme requires NADH and is kinetically first order in the presence of excess arsonous acid. The second-order rate constant for formation of the enzyme-inhibitor complex was 545 min-1 M-1 for phenylarsonous acid, C6H5As(OH)2, and 5640 min-1 M-1 for methanearsonous acid, CH3As(OH)2. The kinetics of formation of inactive enzyme in the presence of arsonic acids was found to obey a rate law predicted by a two-step mechanism in which a rate-limiting reduction of an arsonic acid to the corresponding arsonous acid by reduced enzyme, E(SH)2, preceded formation of an inactive binary complex of reduced enzyme and arsonous acid: ES2 + NADH + H+ = E(SH)2 + NAD+; E(SH)2 + RAs(O)(OH)2 = ES2 + RAs(OH)2 + H2O; and E(SH)2 + RAs(OH)2 = ES2AsR + 2H2O. GSSG reductase reacts reversibly with C6H5As(OH)2 to form an inactive binary addition compound in the presence of NADPH. The value of the association constant for formation of enzyme inhibitor complex at pH 7.0 was 119 M-1. The initial rate of the GSSG reductase-catalyzed oxidation of NADPH by GSSG was insensitive to MeAs(OH)2. The kinetics of inhibition of GSSG reductase by arsenite and C6H5As(O)(OH)2 were found to obey the rate law described for lipoamide dehydrogenase and arsonic acids. GSSG reductase catalyzed the oxidation of NADPH by p-arsanilic acid. The initial rate of oxidation of NADPH was linearly dependent on enzyme concentration. The turnover number for GSSG reductase with p-arsanilic acid as an oxidant was 0.13 mol NADPH mol FAD-1 min-1.
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PMID:Reactions of lipoamide dehydrogenase and glutathione reductase with arsonic acids and arsonous acids. 384 Mar 44

When the pyruvate dehydrogenase complex of Escherichia coli is reduced by NADH and alkylated by N-[14C]ethylmaleimide, 19-20 nmol of N-[14C]ethylmaleimide are bound per mg of complex. This is in accord with the presence of 10 nmol of functional lipoyl moieties per mg of complex as previously reported. Thus the lipoyl groups are all coupled via dihydrolipoyl dehydrogenase (E3) to reduction by NADH. As previously reported, the complex reductively acetylated by pyruvate and containing 10 nmol of acetyldihydrolipoyl groups per mg of complex produces about 5 nmol of NADH/mg of complex when challenged with CoA and NAD+ in a fast burst. Under anaerobic conditions a slow secondary process extending over 1 h produces another 5 nmol of NADH/mg of complex. The relationship between the two classes of acetyldihydrolipoyl groups is unknown but could reflect either intrinsic structural inequivalence of lipoyl groups (2/subunit of dihydrolipoyl transacetylase, E2). Alternatively, the acetyldihydrolipoyl groups may undergo reversible isomerization to structurally distinct forms. The purified complex catalyzes the cleavage of acetyl-CoA by two processes. The trace contaminant phosphotransacetylase catalyzes cleavage by phosphate to acetyl-P. The complex itself catalyzes hydrolysis of acetyl-CoA in a reaction that requires all three enzymes, NADH, thiamin pyrophosphate, and the lipoyl groups of E2. The hydrolytic pathway evidently involves overall reversal of the reaction, leading ultimately to the formation of acetyl-thiamin pyrophosphate, which undergoes hydrolysis to acetate.
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PMID:Pyruvate dehydrogenase complex of Escherichia coli. Thiamin pyrophosphate and NADH-dependent hydrolysis of acetyl-CoA. 390 34


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