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)

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

Genes encoding dihydrolipoamide dehydrogenase (E3) and the E3-binding protein (E3BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E3BP-E3 complex, thereby minimizing proteolysis of E3BP and facilitating its purification. The 2 genes were linked into a single transcriptional unit separated by a 31-nucleotide segment containing a ribosome-binding sequence. The E3BP-E3 complex was highly purified and then separated into E3 and E3BP by chromatography on hydroxylapatite in the presence of 5 M urea. The E3BP-E3 complex combined rapidly with a pyruvate dehydrogenase (E1)-dihydrolipoamide acetyltransferase (E2) subcomplex (E1-E2 subcomplex) to reconstitute a functional PDH complex, with pyruvate oxidation activity similar to that of PDH complex from bakers' yeast. The stoichiometry of binding of E3BP and E3BP-E3 complex to the 60-subunit pentagonal dodecahedron-like E2 was determined with a truncated form of E2 (tE2, residues 206-454) lacking the lipoyl domain and the E1-binding domain, and with E1-E2 subcomplex, which contains intact E2. Mixtures containing tE2 or E1-E2 subcomplex and excess E3BP or E3BP-E3 complex were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or E3BP-E3, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that the E1-E2 subcomplex binds about 12 E3BP monomers attached to 12 E3 homodimers. Similar results were obtained by analysis of highly purified PDH complex from bakers' yeast.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression, purification, and characterization of the dihydrolipoamide dehydrogenase-binding protein of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae. 794 91

We have previously demonstrated that ibotenate (IBO) injected into the pedunculopontine tegmental nucleus (PPTg) damages all neurones there while quinolinate (QUIN) makes relatively selective lesions of cholinergic neurones. We now compare the effects of two anaesthetics, sodium pentobarbitone and Avertin (tribromoethanol/tert-amylalcohol dissolved in ethanol, saline and phosphate buffer) on three doses of IBO and QUIN in the PPTg. Diaphorase-positive cell loss after QUIN was attenuated under barbiturate, the relative selectivity of QUIN for diaphorase-positive neurones was lost and lesion volumes were uniformly small compared with lesions made under Avertin anaesthesia. IBO toxicity was unaffected by anaesthesia. These data are discussed with reference to the actions of excitotoxins at glutamate receptor subtypes and interactions of barbiturates with the GABAA receptor.
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PMID:Barbiturate anaesthesia reduces the neurotoxic effects of quinolinate but not ibotenate in the rat pedunculopontine tegmental nucleus. 841 94

The activities of the enzyme components of the pyruvate dehydrogenase complex are affected to different extents by changes in ionic strength and pH. At pH 7.4 the optimum activity of pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) occur in the ranges of ionic strengths of 0.06-0.08, 0.01-0.02, and 0.10-0.15 M, respectively. The activity of dihydrolipoamide dehydrogenase is least sensitive to changes in the ionic strength of the assay medium. At constant ionic strength (0.15 M) the optimum activity of E1, E2, and E3 occur at pH 7.4, 7.0, and 8.0, respectively. Changes in pH mostly affect the dihydrolipoamide acetyltransferase activity. Cl- and HCO3- anions inhibit the activity of pyruvate dehydrogenase. In the presence of 80 mM Cl- or HCO3- ions the activity of E1 is inhibited by 25 and 10% respectively. K+, Na+, and HPO4(2-) ions affect the activity of dihydrolipoamide acetyltransferase. The activity of this enzyme component is stimulated by 28 and 25% in the presence of 80 mM K+ and Na+ cations, respectively. HPO4(2-) stimulates the dihydrolipoamide acetyltransferase in a calcium-dependent manner. In the presence of 20 mM HPO4(2-) the activity of the dihydrolipoamide acetyltransferase increases 20 and 40% in the absence and presence of 0.1 mM Ca2+, respectively. The activity of dihydrolipoamide dehydrogenase is not affected by K+, Na+, HPO4(2-), Cl-, or HCO3-.
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PMID:The regulatory properties of kidney pyruvate dehydrogenase complex components. 842 84

Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COSI cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductaseI (NQOI or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQOI because of a lack of correlation between NQOI (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADH-cytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreductase) and its activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH).
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PMID:Non-enzymatic and enzymatic activation of mitomycin C: identification of a unique cytosolic activity. 856 27

Selenium is a potent chemopreventive agent; however, the mechanisms for its chemopreventive activities remain elusive. Selenium binds to several proteins, some of which require selenium for functional activity. In this study, two 58kDa selenium-labeled proteins were identified in mouse kidney using a 75Se labeling method. The proteins were partially purified using Sephadex G.150 gel filtration, DEAE-Sephadex A-50 ion-exchange chromatography and one- / two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-/2D-SDS-PAGE). The two proteins migrated at 58kDa on 2D-SDS-PAGE and differed only slightly in their pI values; i.e., 6.2 and 6.6, respectively. The polyclonal antibodies raised in rabbits against the 58kDa proteins electro-eluted from the 1D-SDS-PAGE of the DEAE purified fraction, recognized both protein spots on 2D-SDS-PAGE gel. The in situ enzymatic digestion of the two proteins separated in 2D-SDS-PAGE gels, followed by microsequencing of the peptides, resulted in the identification of these two proteins as related to human lipoamide dehydrogenase and thiol: protein disulfide oxidoreductase (TPDO). In common, both these proteins have a bis (cysteinyl) sequence motif cys-X-X-cys (for lipoamide dehydrogenase it is cys-X-X-X-X-cys) which is also an integral part of several other proteins such as thioredoxin, protein disulfide isomerase, endoplasmic reticulum protein (ERp72), selenoprotein W, 56kDa acetaminophen binding protein and formate dehydrogenase. This sequence motif acts as an active redox center for majority of the proteins mentioned above, that may be controlling the oxidation/reduction of proteins in vivo. How and why selenium is binding to proteins with this common sequence motif needs further investigation.
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PMID:Isolation and identification of selenium-labeled proteins in the mouse kidney. 871 83

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid dependent or independent manner. The alpha-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of alpha-lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, alpha-lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid, coenzyme A, and pyruvate or alpha-ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an alpha-lipoic acid (K0.5 = 1.4 +/- 0.8 mM) and lipoamide (K0.5 = 0.9 +/- 0.3 mM) dependent manner.
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PMID:alpha-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria. 878 42

Lipoamide dehydrogenase from Mycobacterium smegmatis was purified to homogeneity over 60-fold. Of 20 amino acid residues identified at the amino terminus of the enzyme, 18 and 17 were identical to the sequences of Mycobacterium leprae and Pseudomonas fluorescens lipoamide dehydrogenases, respectively. The visible spectrum of the isolated enzyme was characteristic of a flavin in apolar environment. Reduction of the enzyme with dithionite results in the appearance of an absorbance shoulder at 530-550 nm, suggesting that reducing equivalents of the two-electron reduced enzyme reside predominantly on the redox-active disulfidedithiol. The kinetic mechanism of the forward (NAD+ reducing) and reverse (NADH oxidizing) reactions proved difficult to study due to severe substrate inhibition by NAD+ and NADH. The rate of lipoamide reduction was found to depend upon the NAD+/NADH ratio, with the reaction being activated at low ratios and inhibited at high ratios. The use of 3-acetylpyridine adenine dinucleotide allowed initial velocity kinetics to be performed and revealed that the kinetic mechanism is ping pong. In addition to catalyzing the reversible oxidation of dihydrolipoamide, the enzyme displayed high oxidase activity (30% of the lipoamide reduction rate), hydrogen and t-butyl peroxide reductase activity (10% of the lipoamide reduction rate), and both naphthoquinone and benzoquinone reduction (approximately 200% of the lipoamide reduction rate). The enzyme failed to catalyze the redox cycling of nitrocompounds, but could anaerobically reduce nitrofurazone. The lipoamide-reducing reaction was reversibly inactivated by sodium arsenite, but no decrease in diaphorase activity was observed under these conditions.
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PMID:Catalytic properties of lipoamide dehydrogenase from Mycobacterium smegmatis. 914 18

Human spermatozoa possess a specialized capacity to generate reactive oxygen species (ROS) that is thought to be of significance in the redox regulation of sperm capacitation (De Lamirande and Gagnon, 1993; Aitken et al., 1995). However, the mechanisms by which ROS are generated by these cells are not understood. In this study we have examined the possible significance of NADPH as a substrate for ROS production by human spermatozoa. Addition of NADPH to viable populations of motile spermatozoa induced a sudden dose-dependent increase in the rate of superoxide generation via mechanisms that could not be disrupted by inhibitors of the mitochondrial electron transport chain (antimycin A, rotenone, carbonyl cyanide m-chlorophenylhydrazone [CCCP], and sodium azide), diaphorase (dicoumarol) xanthine oxidase (allopurinol), or lactic acid dehydrogenase (sodium oxamate). However, NADPH-induced ROS generation could be stimulated by permeabilization and was negatively correlated with sperm function. Both NADH and NADPH were active electron donors in this system, while NAD+ and NADP+ exhibited little activity. Stereo-specificity was evident in the response in that only the beta-isomer of NADPH supported superoxide production. The involvement of a flavoprotein in the electron transfer process was indicated by the high sensitivity of the oxidase to inhibition by diphenylene iodonium and quinacrine. These results indicate that NAD(P)H can serve as an electron donor for superoxide generation by human spermatozoa and present a simple strategy for the production of motile populations of free radical generating cells with which to study the significance of these molecules in the control of normal and pathological sperm function.
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PMID:Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. 921 32

To investigate the involvement of neuronal nitric oxide (NO) in the response of the brain to changes in blood pressure, we studied the activation of putative NO-producing neurons in the paraventricular nucleus of the hypothalamus (PVN) in rats whose mean arterial pressures (MAPs) were decreased by 40-50% with hemorrhage (HEM) or infusion of sodium nitroprusside (NP). Activation was assessed on the basis of expression of the immediate early gene, c-fos; putative NO-producing neurons were identified with the histochemical stain for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d); and the proportions of neurons projecting to the nucleus of the tractus solitarius (NTS) and/or caudal ventrolateral medulla (CVLM) were determined with retrograde tracing techniques. No differences were found for results obtained from HEM and NP animals. Three to four percent of activated PVN neurons projected to the NTS or CVLM. Conversely, approximately 33% and 16% of neurons projecting to the NTS and CVLM, respectively, were activated. About 43% of NADPH-d neurons in the PVN were activated. Of PVN neurons projecting to the NTS or CVLM, 38% and 32%, respectively, were NADPH-d positive. About 11% of NADPH-d PVN neurons projected to the NTS or CVLM. An average of 3 NADPH-d neurons per section were activated and projected to either target. Finally, 7 PVN cells per section sent collateral branches to the NTS and CVLM; 2 or 3 of these cells per section were also activated by decreases in arterial pressure. No NADPH-d cells were found that sent collateral branches to the NTS and CVLM. This study shows that decreases in MAP activate PVN neurons that project, singly and through collaterals, to the NTS and CVLM. A relatively high proportion of the singly projecting neurons is NADPH-d positive. These results support the contention that descending projections from the PVN to the brainstem play an important role in the physiological response to decreases in arterial pressure and suggest that NO may participate in this response.
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PMID:Activation by hypotension of neurons in the hypothalamic paraventricular nucleus that project to the brainstem. 926 28


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