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)

The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was found to be 1.8:1.7:1[pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)]. This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. The above ratio is the average of four separate experiments with two different enzyme preparations. The average molecular weights of the individual enzymes were found to be 96,000, 76,000, and 55,000 for E1, E2, and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine . HCl. The molecular weight of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide. The molecular weights of the complex, E1, and E3 were found to be 4,800,000, 182,000, and 104,000, respectively, with low-angle laser light scattering. Both E1 and E3 are dimeric under the conditions employed. If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the molecular weight of the pyruvate dehydrogenase complex.
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PMID:Subunit stoichiometry and molecular weight of the pyruvate dehydrogenase multienzyme complex from Escherichia coli. 38 35

A rapid enzymatic method was developed for the assay of serum argininosuccinate lyase (ASAL: EC 4.3.2.1) which is a useful marker enzyme for diagnosis of parenchymal liver diseases. Fumarate, liberated from argininosuccinate in the lyase-mediated reaction, was converted to pyruvate via L-malate by the actions of fumarase and malic enzyme in the presence of NADP+. The NADPH formed was then oxidized with a diaphorase-resazurin system to give a highly fluorescent resorufin. All the enzymatic reactions proceeded continuously in 0.1 M Tris-HCl buffer (pH 7.5) and allowed direct assay of ASAL in serum by monitoring the increase in the fluorescence intensity due to resorufin. The method is rapid and sensitive; only 50 microliter of serum is required. This method was used to detect increases in the activities in sera from patients with liver diseases.
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PMID:An enzymatic method for the assay of serum argininosuccinate lyase. 367 95

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.
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PMID:Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils. 384 37

1. Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (s20,w 77 S) and flavin content (1.3--1.4 nmol/mg) to the complexes from mammalian mitochondria. 2. The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58 000; lipoamide dehydrogenase (E3), 56 000; pyruvate dehydrogenase (E1), alpha-subunit, 45 000, and beta-subunit, 35 000. Gel chromatography in the presence of 6 M guanidine . HCl gave a value of 52 000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes. Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria. 3. The complex was resolved into its component enzymes by the following methods. E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution. E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea. The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.
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PMID:Pyruvate dehydrogenase complex from baker's yeast. 2. Molecular structure, dissociation, and implications for the origin of mitochondria. 703 Jul 41

A TiO(2)/polymer film on a quartz plate fabricated by a layer-by-layer method was employed for NADH production from NAD(+) with lipoamide dehydrogenase and methyl viologen in Tris-HCl buffer on irradiation with UV light, and the ultra thin film prevented from enzyme deactivating effectively.
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PMID:Photosensitized NADH formation system with multilayer TiO2 film. 1504 77

An enzymatic method for determining L-malic acid in wine based on an L-malate sensing layer with nicotinamide adenine dinucleotide (NAD+), L-malate dehydrogenase (L-MDH) and diaphorase (DI), immobilized by sol-gel technology, was constructed and evaluated. The sol-gel glass was prepared with tetramethoxysilane (TMOS), water and HCl. L-MDH catalyzes the reaction between L-malate and NAD+, producing NADH, whose fluorescence (lambdaexc=340 nm, lambdaem=430 nm) could be directly related to the amount of L-malate. NADH is converted to NAD+ by applying hexacyanoferrate(III) as oxidant in the presence of DI. Some parameters affecting sol-gel encapsulation and the pH of the enzymatic reaction were studied. The sensing layer has a dynamic range of 0.1-1.0 g/L of L-malate and a long-term storage stability of 25 days. It exhibits acceptable reproducibility [sr(%) approximately 10] and allows six regenerations. The content of L-malic acid was determined for different types of wine, and polyvinylpolypyrrolidone (PVPP) was used as a bleaching agent with red wine. The results obtained for the wine samples using the sensing layer are comparable to those obtained from a reference method based on UV-vis molecular absorption spectrometry, if the matrix effect is corrected for.
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PMID:Fluorescent sensing layer for the determination of L-malic acid in wine. 1720 64

With a view to their use in the kinetic resolution of racemic non-natural amino acids, five variants of the enzyme L-phenylalanine dehydrogenase, the wild-type enzyme from Bacillus sphaericus and four active-site mutants, have been tested with a range of amino acids. In each case, the rates of reaction with 0.2 mM L-amino acid and with the racemic mixture at 0.4 mM were compared, so that the starting concentration of the active substrate was kept constant. Although the D-amino acids are not substrates, they were inhibitory in all cases. The extent of inhibition, however, varied greatly from compound to compound and among the mutants. With the N145L mutant and DL 4-O-Me-Phe, the equimolar D-enantiomer gave 83.2% inhibition, and with the wild-type enzyme there was 86.7% inhibition with racemic norleucine. By contrast, with these same substrates the N145V mutant showed less than 9% and 24% inhibition respectively. The N145A mutant was selected for use with DL-4-Cl-Phe. The pH was decreased from the enzyme's optimum of 10.4 to 9.5 to minimise breakdown of the coenzyme NAD(+), and the coenzyme was recycled by molecular oxygen with the assistance of a commercial diaphorase. Reaction on a 200 micromole scale in 20 ml ethanolamine HCl buffer, pH 9.5, with 25 microg N145A enzyme and 100 microg diaphorase, was monitored by chiral HPLC. The L-isomer was removed to an extent of >99% after 40 h, with the D-isomer peak undiminished. The pure D-isomer was isolated from the reaction mixture in 85% overall yield after ion-exchange chromatography.
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PMID:Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the D-enantiomer from racemic mixtures. 1908 64