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)

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) of the rat brain, apparently identical with nitric oxide (NO) synthase, was demonstrated at the electron microscopic level by means of the tetrazolium salt 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)tetrazolium chloride (BSPT). BSPT is a non-osmiophilic compound that yields an insoluble, osmiophilic, and lipophobic formazan on reduction. The reaction product was deposited sharply on membranes of the endoplasmic reticulum including the nuclear envelope. Other membrane structures were, as a rule, free of reaction product, likewise mitochondria. Occasionally, however, the outer membrane of mitochondria was labeled, and their contents displayed a homogeneous, medium electron density. The findings suggest that NADPH-d, i.e. neuronal NO synthase, is a predominantly membrane-bound enzyme, which is ubiquitously distributed in cells of brain tissue, but highly concentrated in nerve cells described as 'NADPH-d-positive' at the light microscopic level.
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PMID:Nitric oxide synthase in rat brain is predominantly located at neuronal endoplasmic reticulum: an electron microscopic demonstration of NADPH-diaphorase activity. 128 94

We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons and provided a Golgi-like image of the neurons exhibiting this activity. The NADPH-diaphorase reaction appeared to be enzyme catalyzed, since it was abolished by pre-treatment with proteases, heat, and acid or alkaline denaturation. Under anaerobic conditions, any tetrazolium salt with a redox potential more positive than NADPH could be reduced by the enzyme. NADPH-diaphorase activity was sensitive to inhibition by sulfhydryl reagents but was unaffected by metal chelators, superoxide dismutase, and catalase. Therefore, the enzyme is unlikely to be a metalloenzyme or to reduce tetrazoliums by producing superoxide anions or hydrogen peroxide. Various analogues of beta-NADPH could be used by the enzyme; however, beta-NADH, which can be used by DT-diaphorase, was ineffective. The enzyme was also resistant to dicumarol, an inhibitor of DT-diaphorase activity. Electron microscopy indicated that the NADPH-diaphorase reaction resulted in staining of various membranous organelles. We conclude that neuronal NADPH-diaphorase is a membrane-bound enzyme distinct from DT-diaphorase and other known enzymes with diaphorase activity. The histochemical characteristics presented here should now enable meaningful biochemical studies of neuronal NADPH-diaphorase to be undertaken.
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PMID:Histochemical characterization of neuronal NADPH-diaphorase. 270 1

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplast completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W10BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.
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PMID:Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase. 312 Jul 72

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

The influence of storage of bovine and porcine muscle at -20 degrees C for 12 months and of thawing on the total extractable activity and subcellular distribution (activities in the supernatant of a phosphate buffer extract and in the press juice of the thawed tissue) of the mitochondrial enzymes lipoamide dehydrogenase (LIPDH), citrate synthase (CS) and beta-hydroxyacyl-CoA-dehydrogenase (HADH) was studied. The total activity of LIPDH decreased during frozen storage whereas the activities of CS and HADH did not change appreciably. From the increase of CS and HADH activities in the muscle press juice it was concluded that--additionally to the effect of freezing and thawing itself--frozen storage results in further damage to the inner membrane of muscle mitochondria, which may be recognized from the increased release of membrane-bound enzymes. In this respect porcine muscle mitochondria seem to be more sensitive than bovine muscle mitochondria.
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PMID:[Lipoamide dehydrogenase, citrate synthase, and beta-hydroxyacyl-CoA-dehydrogenase in skeletal muscles. X. The influence of frozen storage of bovine and porcine muscle on activity and subcellular distribution]. 384 Sep 39

Frozen storage at -20 degrees C for three months and thawing of muscles from sheep, hare and deer, and of the breast and leg muscles from chicken and duck did not result in significant changes in the extractable total activities of the mitochondrial enzymes citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase; however there was a decrease in the total activity of lipoamide dehydrogenase except in the chicken leg muscle, where such a decrease did not occur. From the increase in the activities of the three enzymes in the muscle press juice it was concluded that - additionally to the effect of freezing and thawing itself - frozen storage results in further damage to the inner membrane of muscle mitochondria which is signalled by the release of membrane-bound enzymes. Chicken muscle mitochondria seem to be more stable against frozen storage of the tissue than the mitochondria in the muscles of the other species studied (including bovine and porcine muscle).
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PMID:[Lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase of skeletal muscle. 9. Influence of frozen storage of musculature of sheep, game and poultry on activity and subcellular distribution]. 384 Sep 40

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.
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PMID:Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex. 641 56

Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+ reductase of spinach chloroplasts. Either NADP+ or NADPh afforded complete protection against modification. Ki and the apparent Kd for protection afforded by NADP+ depended on the ionic strength of the medium. Nucleophylic displacement of reagent bound to the soluble enzyme by [14C]glycine ethyl ester showed that 5 to 6 carboxyl groups/flavin were modified when the diaphorase activity was completely inhibited. In differential labeling experiments using NADP+ as protective agent, it was shown that enzyme inactivation was due to blocking of only 1 carboxyl group/mol. Derivatized reductase did not bind pyridine nucleotides. Protection by NADP+ of the membrane-bound reductase was higher, and the apparent Kd for NADP+ lower, in the light than in the dark. Inactivation increased abruptly with the external pH, indicating a progressive exposure of the carboxyl group as the pH was raised. The results presented suggest (a) the existence of a light-driven conformational change and a pH-dependent transition in membrane-bound ferredoxin-NADP+ reductase; (b) the presence of an essential carboxyl residue in the nucleotide binding site of the reductase.
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PMID:An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase. 689 98

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.
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PMID:Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. 769 24

A dihydrolipoamide dehydrogenase (LPD; dihydrolipoamide:NAD oxidoreductase, EC 1.8.1.4.) activity has been detected in the cyanobacterium Synechocystis PCC 6803. The enzyme was isolated from the membraneous fraction after detergent solubilization and shown to be homogenous on the basis of SDS-PAGE and N-terminal sequencing. The isolated enzyme had a specific activity of 75 U (mg protein)(-1) and was shown to be a homodimer with an apparent molecular mass of 104 kDa for the dimer and 55 kDa for the subunits. The enzyme contains 1.75 mol noncovalently bound FAD (mol enzyme)(-1) suggesting that each subunit contains 1 mol FAD and that the FAD is fairly tightly associated with the enzyme. N-terminal sequencing gave a contiguous amino acid sequence of 17 residues and showed that the N-terminus of the LPD from Synechocystis PCC 6803 has significant homologies to other LPDs sequenced so far. Immunoblot experiments indicated that the enzyme is mainly present in the membrane fraction, and immunocytochemical investigations gave evidence that the LPD in Synechocystis PCC 6803 is located in the periplasma space between the cytoplasma membrane and the peptidoglycan layer. This is the first report on an extracellular, membrane-bound LPD in a cyanobacterium.
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PMID:Isolation, partial characterization and localization of a dihydrolipoamide dehydrogenase from the cyanobacterium Synechocystis PCC 6803. 921 12


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