Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].
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PMID:Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase. 1 31

It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th
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PMID:Piericiden A sensitivity, site 1 phosphorylation, and reduced nicotinamide adenine dinucleotide dehydrogenase during iron-limited growth of Candida utilis. 16 85

The binding of specific inhibitors to the ubiquinol oxidation pocket ("QP center") of cytochrome c reductase was analyzed before and after removal of bound phospholipid and the "Rieske" iron-sulfur protein using optical spectroscopy and fluorescence quench binding assays. The enzyme lacking iron-sulfur protein showed almost unchanged, tight binding of the E-beta-methoxyacrylate inhibitors oudemansin A and MOA-stilbene, whereas binding of the chromone inhibitor stigmatellin was almost completely abolished. The affinity of the weak inhibitor 3-undecyl-2-hydroxy-naphthoquinone was decreased. Oudemansin A binding to the defective pocket of the iron-sulfur protein-depleted enzyme was lowered by added phospholipid. It was deduced from these results that the QP center is a spacious pocket formed by domains of cytochrome b, bearing the E-beta-methoxcyacrylate binding site, and the iron-sulfur protein, bearing the stigmatellin binding site. Moreover, removal of the iron-sulfur protein leaves this pocket defective but essentially unchanged in its remaining binding capability. The affinity of three preparations of cytochrome c reductase, the complete, the delipidated, and the iron-sulfur depleted enzyme for E-beta-methoxyacrylate-stilbene, was analyzed for different redox states of the catalytic centers of cytochrome c reductase. The apparent Kd values for the different redox states were interpreted in terms of two conformational states. It is suggested that these changes reflect the two states of the "catalytic switch" proposed recently for the QP pocket of cytochrome c reductase (Brandt, U., and von Jagow, G. (1991) Eur. J. Biochem. 195, 163-170). According to the refined model presented in this work, changeover to the "b" state is triggered by reduction of the iron-sulfur cluster, and changeover back to the "FeS" state is triggered by electron transfer from the low potential onto the high potential heme b center. Our interpretation implies that the stability of the two states is affected by the redox states of the enzyme, but that additionally changing the redox states of the two centers is required for "switching" on a catalytic time scale.
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PMID:Significance of the "Rieske" iron-sulfur protein for formation and function of the ubiquinol-oxidation pocket of mitochondrial cytochrome c reductase (bc1 complex). 165 9

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
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PMID:The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4. 189 80

Menadione (2-methyl-1,4-naphthoquinone) was used as a model compound to test the hypothesis that thioether conjugates of quinones can be toxic to tissues associated with their elimination through a mechanism involving oxidative stress. Unlike menadione, the glutathione (2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone; MGNQ) and N-acetyl-L-cysteine (2-methyl-3-(N-acetylcysteine-S-yl)-1,4-naphthoquinone; M(NAC)NQ) thioether conjugates were not able to arylate protein thiols but were still able to redox cycle with cytochrome c reductase/NADH and rat kidney microsomes and mitochondria. Interestingly, menadione and M(NAC)NQ were equally toxic to isolated rat renal epithelial cells (IREC) while MGNQ was nontoxic. The toxicity of both menadione and M(NAC)NQ was preceded by a rapid depletion of soluble thiols and was associated with a depletion of soluble thiols and was associated with a depletion of protein thiols. Treatment of IREC with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, potentiated the thiol depletion and toxicity observed with menadione and M(NAC)NQ indicating the involvement of oxidative stress in this model of renal cell toxicity. The lack of MGNQ toxicity can be attributed to an intramolecular cyclization reaction which destroys the quinone nucleus and therefore eliminates its ability to redox cycle. These findings have important implications with regard to our understanding of the toxic potential of quinone thioether conjugates and of quinone toxicity in general.
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PMID:The toxicity of menadione (2-methyl-1,4-naphthoquinone) and two thioether conjugates studied with isolated renal epithelial cells. 199 Sep 78

The naphthoquinone moiety was proven to be essential to the biological activities of sakyomicin A using various naphthoquinone derivatives. Among the naphthoquinones tested, juglone (5-hydroxy-1,4-naphthoquinone) which resembles the partial structure of sakyomicin A was the most active in cytotoxicity against murine lymphosarcoma L5178Y cells, electron acceptor function in the oxidation of NADH by Clostridium kluyveri diaphorase or rat liver mitochondria and inhibition against avian myeloblastosis virus reverse transcriptase. The significantly lower cytotoxicity of sakyomicin A as compared with juglone was attributable to its poor membrane transport. The inhibition of reverse transcriptase activity may result from the interaction between a sulfhydryl group in the active center of the enzyme and quinone groups of the naphthoquinones and sakyomicin A.
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PMID:Role of the naphthoquinone moiety in the biological activities of sakyomicin A. 242 91

Inhibition of avian myeloblastosis virus (AMV) reverse transcriptase by natural and synthetic quinones including antibiotics could be accounted for by an oxidation-reduction reaction. The quinones were shown to function as electron acceptors as revealed by the catalytic oxidation of NADH by Clostridium kluyveri diaphorase which was in excellent agreement with enzyme inhibition activity. The kinetics of inhibition of AMV reverse transcriptase by three synthetic quinones with different core structures, i.e., 6-methoxy-5,8-dihydroquinoline-5,8- dione, 5,8-dihydroisoquinoline-5,8-dione and 1,4-naphthoquinone, were studied. These quinones inhibited reverse transcriptase in the same manner as streptonigrin (STN) and were shown to act at a single class of reaction site(s) on the enzyme molecule. In contrast, the quinones with bulky substituents, i.e., 7-(2-nitrophenethylamino)-5,8-dihydroisoquinoline-5,8-dione and 7-methoxy-6-methyl-3-piperidino-5,8-dihydroisoquinoline-5,8-dione, were inactive as inhibitors of reverse transcriptase, whereas they retained competent catalytic activities in the oxidation of NADH by C. kluyveri diaphorase. Based on these observations, the existence of a specific site of interaction on the enzyme molecule, referred to as a quinone pocket, was proposed. The quinone pocket might play a crucial role in the early sequence of events leading to the inhibition of reverse transcriptase by quinones including STN and sakyomicin A (SKM). Access of SKM to a quinone pocket might be restricted due to its bulky structure in the vicinity of the quinone group. This is inferred from unsuccessful inhibition of reverse transcriptase by the quinones with bulky substituents, resulting in much poorer inhibition of reverse transcriptase in spite of more potent electron acceptor activity in the oxidation-reduction system as compared with those of STN.
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PMID:Mechanism of inhibition of reverse transcriptase by quinone antibiotics. II. Dependence on putative quinone pocket on the enzyme molecule. 246 54

An NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75 degrees C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.
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PMID:Purification and properties of NADH dehydrogenase from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius. 282 84

The reduction of the following exogenous quinones by succinate and NADH was studied in mitochondria isolated from both wild type and ubiquinone (Q)-deficient strains of yeast: ubiquinone-0 (Q0), ubiquinone-1 (Q1), ubiquinone-2 (Q2), and its decyl analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), duroquinone (DQ), menadione (MQ), vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone), the plastoquinone analogue 2,3,6-trimethyl-1,4-benzoquinone (PQOc1), plastoquinone-2 (PQ2), and its decyl analogue (2,3-dimethyl-6-decyl-1,4-benzoquinone). Reduction of the small quinones DQ, Q0, Q1, and PQOc1 by NADH occurred in both wild type and Q-deficient mitochondria in a reaction inhibited more than 50% by myxothiazol and less than 20% by antimycin. The reduction of these small quinones by succinate also occurred in wild type mitochondria in a reaction inhibited more than 50% by antimycin but did not occur in Q-deficient mitochondria suggesting that endogenous Q6 is involved in their reduction. In addition, the inhibitory effects of antimycin and myxothiazol, specific inhibitors of the cytochrome b-c1 complex, on the reduction of these small quinones suggest the involvement of this complex in the electron transfer reaction. By contrast, the reduction of Q2 and DB by succinate was insensitive to inhibitors and by NADH was 20-30% inhibited by myxothiazol suggesting that these analogues are directly reduced by the primary dehydrogenases. The dependence of the sensitivity to the inhibitors on the substrate used suggests that succinate-ubiquinone oxidoreductase interacts specifically with center i (the antimycin-sensitive site) and NADH ubiquinone oxidoreductase preferentially with center o (the myxothiazol-sensitive site) of the cytochrome b-c1 complex. The NADH dehydrogenase involved in the myxothiazol-sensitive quinone reduction faces the matrix side of the inner membrane suggesting that center o may be localized within the membrane at a similar depth as center i.
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PMID:Direct interaction between yeast NADH-ubiquinone oxidoreductase, succinate-ubiquinone oxidoreductase, and ubiquinol-cytochrome c oxidoreductase in the reduction of exogenous quinones. 282 38

The results presented in this paper reveal the existence of three distinct menadione (2-methyl-1,4-naphthoquinone) reductases in mitochondria: NAD(P)H:(quinone-acceptor) oxidoreductase (D,T-diaphorase), NADPH:(quinone-acceptor) oxidoreductase, and NADH:(quinone-acceptor) oxidoreductase. All three enzymes reduce menadione in a two-electron step directly to the hydroquinone form. NADH-ubiquinone oxidoreductase (NADH dehydrogenase) and NAD(P)H azoreductase do not participate significantly in menadione reduction. In mitochondrial extracts, the menadione-induced NAD(P)H oxidation occurs beyond stoichiometric reduction of the quinone and is accompanied by O2 consumption. Benzoquinone is reduced more rapidly than menadione but does not undergo redox cycling. In intact mitochondria, menadione triggers oxidation of intramitochondrial pyridine nucleotides, cyanide-insensitive O2 consumption, and a transient decrease of delta psi. In the presence of intramitochondrial Ca2+, the menadione-induced oxidation of pyridine nucleotides is accompanied by their hydrolysis, and Ca2+ is released from mitochondria. The menadione-induced Ca2+ release leaves mitochondria intact, provided excessive Ca2+ cycling is prevented. In both selenium-deficient and selenium-adequate mitochondria, menadione is equally effective in inducing oxidation of pyridine nucleotides and Ca2+ release. Thus, menadione-induced Ca2+ release is mediated predominantly by enzymatic two-electron reduction of menadione, and not by H2O2 generated by menadione-dependent redox cycling. Our findings argue against D,T-diaphorase being a control device that prevents quinone-dependent oxygen toxicity in mitochondria.
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PMID:Menadione- (2-methyl-1,4-naphthoquinone-) dependent enzymatic redox cycling and calcium release by mitochondria. 309 56


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