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)

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

The bidirectional, NAD+-dependent hydrogenase from cyanobacteria is encoded by the structural genes hoxFUYH, which have been found to be clustered, though interspersed with different open reading frames (ORFs), in the heterocystous, N2-fixing Anabaena variabilis and in the unicellular Synechocystis PCC 6803. In another unicellular, non N2-fixing cyanobacterium, Anacystis nidulans, hoxF has now been identified as being separated by at least 16 kb from the residual structural genes hoxUYH. An ORF (termed hoxE gene) is located immediately upstream of hoxF in A. nidulans and in Synechocystis. Its deduced amino acid sequence shows similarities to the NuoE subunit of NADH dehydrogenase I of E. coli, to the homologous subunit of respiratory complex I in mitochondria, and also to the first 104 amino acids of HoxF in A. nidulans and Synechocystis. The diversity in the arrangement of hydrogenase genes in cyanobacteria is puzzling. The subunits HoxE, HoxF, and HoxU of the diaphorase part of the bidirectional hydrogenase have been discussed to be shared both by respiratory complex I and bidirectional hydrogenase in cyanobacteria. Different hoxU mutants were obtained by inserting a lacZKmR cassette into the gene both in A. nidulans and Anacystis PCC 7942. Such mutants showed reduced H2-evolution activities catalyzed by the bidirectional hydrogenase, but had nonimpaired respiratory O2-uptake. A common link between respiratory complex I and the diaphorase part of the bidirectional hydrogenase in cyanobacteria may still exist, but this hypothesis could not be verified in the present study by analyzing defined mutants impaired in one of the diaphorase genes.
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PMID:Unusual gene arrangement of the bidirectional hydrogenase and functional analysis of its diaphorase subunit HoxU in respiration of the unicellular cyanobacterium anacystis nidulans 954 59

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins.
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PMID:Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato. 972 64

The fdsGBACD operon encoding the four subunits of the NAD+-reducing formate dehydrogenase of Ralstonia eutropha H16 was cloned and sequenced. Sequence comparisons indicated a high resemblance of FdsA (alpha-subunit) to the catalytic subunits of formate dehydrogenases containing a molybdenum (or tungsten) cofactor. The NH2-terminal region (residues 1-240) of FdsA, lacking in formate dehydrogenases not linked to NAD(P)+, exhibited considerable similarity to that of NuoG of the NADH:ubiquinone oxidoreductase from Escherichia coli as well as to HoxU and the NH2-terminal segment of HndD of NAD(P)+-reducing hydrogenases. FdsB (beta-subunit) and FdsG (gamma-subunit) are closely related to NuoF and NuoE, respectively, as well as to HoxF and HndA. It is proposed that the NH2-terminal domain of FdsA together with FdsB and FdsG constitute a functional entity corresponding to the NADH dehydrogenase (diaphorase) part of NADH:ubiquinone oxidoreductase and the hydrogenases. No significant similarity to any known protein was observed for FdsD (delta-subunit). The predicted product of fdsC showed the highest resemblance to FdhD from E. coli, a protein required for the formation of active formate dehydrogenases in this organism. Transcription of the fds operon is subject to formate induction. A promoter structure resembling the consensus sequence of sigma70-dependent promoters from E. coli was identified upstream of the transcriptional start site determined by primer extension analysis.
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PMID:Structural analysis of the fds operon encoding the NAD+-linked formate dehydrogenase of Ralstonia eutropha. 975 65

The flavoenzyme glutathione reductase catalyzes the NADPH-dependent reduction of glutathione disulfide, yielding two molecules of glutathione. The oxidation-reduction potentials, Eox/EH2 (two-electron reduced enzyme), for yeast, Escherichia coli, and human glutathione reductase have been determined between pH 6.0 and 9.8 relative to the nonphysiological substrate couple NAD+/NADH and were found to be -237, -243, and -227 mV (+/-5 mV) at pH 7.0 and 20 degreesC, respectively. The potential as a function of pH demonstrated slopes of -51, -45, and -42 mV/pH unit, respectively, at low pH and -37, -31, and -34 mV/pH unit, respectively, at high pH. The change in slope indicated pKa values of 7.4, 8.5, and 7.6, respectively. The slopes indicate that two protons are associated with the two-electron reduction of Eox at low pH and that only one proton is involved with the two-electron reduction of Eox at high pH, provided that the effects of nearby titratable residues are considered in the data analysis. The influence of four such groups, Cys50, Cys45, His456', and either Tyr107 or the flavin-(N3), has been included (residue numbering refers to the yeast sequence). The enzyme loses activity upon deprotonation of the acid-base catalyst at high pH. Since the pKa ascribed to the EH2-to-EH- ionization is lower than the pKa of the acid-base catalyst, both the EH2 and EH- forms of glutathione reductase must be catalytically active, in contrast to the closely related enzyme lipoamide dehydrogenase, for which only EH2 is active.
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PMID:Redox potentials for yeast, Escherichia coli and human glutathione reductase relative to the NAD+/NADH redox couple: enzyme forms active in catalysis. 979 22

Lipoamide dehydrogenase was purified around 22-fold relative to the crude extracts of Streptomyces seoulensis with an overall yield of 9. 5%. The enzyme was composed of two identical subunits with a molecular mass of 54 kDa and contained 1 mol of FAD per mol of subunit. The absorption spectra of the enzyme revealed the absorption maxima of flavoprotein at 272, 349, and 457 nm. Catalytically active two-electron reduced lipoamide dehydrogenase was produced by anaerobic reduction with one equivalent of NADH. Addition of excess amount of NADH led to the four-electron reduced lipoamide dehydrogenase. The reaction of the enzyme in the reduction reaction of lipoamide or lipoic acid could be explained by a ping-pong mechanism like many other lipoamide dehydrogenases reported earlier. The enzyme also catalysed the reduction of various quinone compounds with NADH as electron donor via a ping-pong mechanism. The enzyme can catalyse a single electron transfer in case of quinone-reducing process, evidenced by the production of 1, 4-naphthosemiquinone radical anion. The quinone-reducing activity of the enzyme was dramatically inhibited by NAD+, indicating the involvement of four-electron reduced form. The structural gene for the enzyme was cloned using a DNA fragment PCR-amplified with the primers designed from N-terminal and internal amino acid sequences. The deduced amino acid sequence shared striking similarity with those of lipoamide dehydrogenases from prokaryotes and eukaryotes. The gene was named lpd. All tested Streptomyces contained one homologue of the lpd gene, which is consistent with the fact that most organisms contain only one lipoamide dehydrogenase.
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PMID:Lipoamide dehydrogenase from streptomyces seoulensis: biochemical and genetic properties. 985 75

Electroenzymatic reduction of NAD+ to NADH for subsequent use in enzymatic synthesis has been carried out at carbon electrodes bearing lipoamide dehydrogenase (LiDH) immobilized under a Nafion film. The self-mediated electron transfer was made possible by an excess of flavin adenine dinucleotide (FAD) entrapped together with LiDH. Results were compared to those obtained with a similar electrode containing both LiDH and a polymeric form of FAD (pFAD) prepared by anodic polymerization of FAD.
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PMID:FAD-mediated enzymatic conversion of NAD+ to NADH: application to chiral synthesis of L-lactate. 987 29

The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased (<5% wild-type). In the case of K54E E3, the Km for lipoamide in the reverse reaction was increased by about twofold. Surprisingly, for both SK-KS and E192Q E3s, the Kms for both dihydrolipoamide (forward reaction) and lipoamide (reverse reaction) were markedly reduced. The catalytic rate constants (kcat/Km) for both reactions for SK-KS E3 were significantly lower than wild-type, indicating that K54 is crucial for the catalytic efficiency of the enzyme. Fluorescence spectral analyses showed that the FAD in E3s were reduced by the addition of dihydrolipoamide, and that its reoxidation by NAD+ in the mutant E3s was slower than wild-type E3. Interestingly, in K54E E3 dihydrolipoamide reduced FAD efficiently only when NAD+ was present, indicating that K54 stabilizes the thiolate-FAD interaction. The lack of the formation of thiolate-FAD intermediate in the absence of NAD+ in K54E E3 was also confirmed by CD spectra. The SK-KS mutation demonstrates that the correct sequence of residues is as critical as the nature of the amino acid residues. These results suggest that K54 plays an important role in stabilizing the thiolate-FAD intermediate during the electron transfer in the reaction, and E192 is involved in maintaining correct orientation of K54 during catalysis.
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PMID:Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate. 1033 57

This study had as its purpose to assess the effects of acute diabetes induced by streptozotocin (35 mg/kg body weight) on the number and size of the myenteric neurons of the duodenum of adult rats considering equally the antimesenteric and intermediate regions of the intestinal circumference. Experimental period extended for a week. Neuronal counts were carried out on the same number of fields of both regions of the duodenal circumference and measurements of neuronal and nuclear areas on equal numbers of cells. Number and size of the myenteric neurons stained with Giemsa were not significantly different between groups. On the other hand, the proportion of NADH-positive neurons increased from 18.54% on the controls to 39.33% on the diabetics. The authors discuss that this increased reactivity probably results from a greater NADH/NAD+ ratio, described in many tissues of diabetic animals, which has consequences on the modulation of the enzymes that use these cofactors and whose activity is detected by the NADH-diaphorase technique.
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PMID:Number and size of myenteric neurons of the duodenum of adult rats with acute diabetes. 1075 7

In preparation for the development of a xylitol biosensor, the xylitol dehydrogenase of Candida tropicalis IFO 0618 was partially purified and characterized. The optimal pH and temperature of the xylitol dehydrogenase were pH 8.0 and 50 degrees C, respectively. Of the various alcohols tested, xylitol was the most rapidly oxidized, with sorbitol and ribitol being reduced at 65% and 58% of the xylitol rate. The enzyme was completely inactive on arabitol, xylose, glucose, glycerol, and ethanol. The enzyme's xylitol oxidation favored the use of NAD+ (7.9 U/mg) over NADP+ (0.2 U/mg) as electron acceptor, while the reverse reaction, D-xylulose reduction, favored NADPH (7.7 U/mg) over NADH (0.2 U/mg) as electron donor. The K(m) values for xylitol and NAD+ were 49.8 mM and 38.2 microM, respectively. For the generation of the xylitol biosensor, the above xylitol dehydrogenase and a diaphorase were immobilized on bromocyan-activated sephallose. The gel was then attached on a dissolved oxygen electrode. In the presence of vitamin K3, NAD+ and phosphate buffer, the biosensor recorded a linear response to xylitol concentration up to 3 mM. The reaction was stable after 15 min. When the biosensor was applied to a flow injection system, optimal operation pH and temperature were 8.0 and 30 degrees C, respectively. The strengths and limitations of the xylitol biosensor are its high affinity for NAD+, slow reaction time, narrow linear range of detection, and moderate affinity for xylitol.
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PMID:Development of a xylitol biosensor composed of xylitol dehydrogenase and diaphorase. 1077 71


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