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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.
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PMID:Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria. 650 55

NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.
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PMID:Purification and characterization of NADH dehydrogenase from Bacillus subtilis. 681 92

Highly purified preparations of the cholate-solubilized respiratory NADH dehydrogenase, isolated from genetically amplified Escherichia coli strains [Jaworowski, A., Campbell, H. D., Poulis, M. I., & Young, I. G. (1981) Biochemistry 20, 2041-2047], have been characterized. Enzyme preparations were shown to contain 70% (w/w) lipid, predominantly phosphatidylethanolamine. One mol of noncovalently bound FAD and approximately 1 mol of ubiquinone/mol of enzyme subunit were detected. The purified enzyme was shown to contain only low levels of Fe and acid-labile S, indicating the absence of iron-sulfur clusters. No Cu, Mo, W, or covalently bound P was detected, and no evidence for other chromophores was obtained from visible and ultraviolet absorption spectra of the purified enzyme or of the delipidated polypeptide prepared by gel filtration in sodium dodecyl sulfate. Protein chemical studies verified that the enzyme consists of a single polypeptide species of Mr 47 000, and the N- and C-terminal cyanogen bromide peptides were identified. The pure enzyme was shown to reconstitute membrane-bound, cyanide-sensitive NADH oxidase activity in membrane vesicles prepared from ndh mutant strains.
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PMID:Characterization of the respiratory NADH dehydrogenase of Escherichia coli and reconstitution of NADH oxidase in ndh mutant membrane vesicles. 702 Jul 57

1. Chronic marginal riboflavin deficiency was induced in groups of weanling rats by feeding a deficient diet supplemented with 0, 0.5, 1.0 and 1.5 mg riboflavin/kg diet. Ad lib.- and pair-fed controls received 3.0 and 15 mg riboflavin/kg diet respectively. 2. Serial measurement of erythrocyte NAD(P)H2 glutathione oxidoreductase (glutathione reductase; EC 1.6.4.2) and its activation coefficient revealed that after 12 weeks a steady-state of deficiency had been reached following initial fluctuations in status; the animals were then killed, and their tissues analysed. 3. Food intake, growth rate and the appearance of pathological signs were directly proportional to riboflavin content; however relative liver weight was increased above control levels only in the most-severely-deficient group, and anaemia was not detected in any group. 4. The activation coefficient of glutathione reductase in erythrocytes and liver was closely related to dietary riboflavin content; that of skin responded maximally even in the least-severely-depleted animals. 5. Hepatic and renal flavin contents were directly proportional to dietary riboflavin, FAD being conserved at the expense of riboflavin and FMN. ATP:riboflavin 5-phosphotransferase (flavokinase; EC 2.7.1.26) activity was reduced, even in the least-severely-deficient animals; ATP:FMN adenylyltransferase(FAD pyrophosphorylase; EC 2.7.7.2) was increased in liver, but only in the most-severely-deficient animals. 6. Hepatic succinate:(acceptor) oxidoreductase (succinate dehydrogenase; EC 1.3.99.1) activity fell sharply between 1.5 and 0.5 mg riboflavin/kg diet, producing an S-shaped dose-response curve; it showed smaller or less specific changes in other tissues such as brain, skin and intestine. NADH:(acceptor) oxidoreductase (NADH dehydrogenase; EC 1.6.99.3) activity declined in liver and intestine, but not in skin or brain. 7. The activation coefficient of glutathione reductase was correlated strongly with nearly all the riboflavin-sensitive variables measured, once equilibrium had been reached in this chronic deficiency model, and it was particularly strongly correlated with hepatic and renal FAD levels. Under equilibrium conditions, therefore, it appears to represent a good index of the extent of riboflavin deficiency, and significant changes in flavin levels and enzymes in the internal organs were detected even under conditions of marginal deficiency, associated with relatively small increases in the activation coefficient.
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PMID:A biochemical evaluation of the erythrocyte glutathione reductase (EC 1.6.4.2) test for riboflavin status. 2. Dose-response relationships in chronic marginal deficiency. 747 Apr 38

The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1. Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD.
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PMID:The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN. 764 53

The effect of galactosamine on liver mitochondrial functions was studied in vivo in rats at 12hr, 24hr and 36hr after the administration of the drug. State 3 respiration decreased significantly with both NAD+ linked and FAD linked substrates. Respiratory control ratio, an index of membrane integrity and P/O ratio which is a measure of phosphorylation efficiency decreased significantly. There was a significant decrease in the activities of NADH dehydrogenase, succinate dehydrogenase and cytochrome oxidase. A significant decrease was also seen on membrane potential, cytochrome aa3, cytochrome b, cytochrome c and on phospholipids of mitochondria. The observed mitochondrial dysfunctions were related to increased lipid peroxidation, which could cause loss of membrane integrity and a decreased rate of phosphorylation. It is proposed that increased lipid peroxidation was responsible for the inhibition on both oxidation and phosphorylation in mitochondria in galactosamine treated rats.
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PMID:Effect of administration of galactosamine hydrochloride on rat liver mitochondria. 942 49

Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2 delta mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction. The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle. However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol. Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g. the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane. By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1.2, whereas the shuttle gave low rates but a high P/O ratio of 1.7. Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2 delta strain proved dependent on GPD1 but not on GPD2.
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PMID:The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae. 955 43

The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) from Megasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction. The cloned genes are arranged in tandem with the coding sequence for the beta-subunit in the position 5' to the alpha-subunit coding sequence. Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the alpha-subunit and one on the beta-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria. The recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.
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PMID:Cloning and analysis of the genes for a novel electron-transferring flavoprotein from Megasphaera elsdenii. Expression and characterization of the recombinant protein. 969 53

The fitness of organisms depends upon the rate at which they generate superoxide (O-2) and hydrogen peroxide (H2O2) as toxic by-products of aerobic metabolism. In Escherichia coli these oxidants arise primarily from the autoxidation of components of its respiratory chain. Inverted vesicles that were incubated with NADH generated O-2 and H2O2 at accelerated rates either when treated with cyanide or when devoid of quinones, implicating an NADH dehydrogenase as their source. Null mutations in the gene encoding NADH dehydrogenase II averted autoxidation of vesicles, and its overproduction accelerated it. Thus NADH dehydrogenase II but not NADH dehydrogenase I, respiratory quinones, or cytochrome oxidases formed substantial O-2 and H2O2. NADH dehydrogenase II that was purified from both wild-type and quinone-deficient cells generated approximately 130 H2O2 and 15 O-2 min-1 by autoxidation of its reduced FAD cofactor. Sulfite reductase is a second autoxidizable electron transport chain of E. coli, containing FAD, FMN, [4Fe-4S], and siroheme moieties. Purified flavoprotein that contained only the FAD and FMN cofactors had about the same oxidation turnover number as did the holoenzyme, 7 min-1 FAD-1. Oxidase activity was largely lost upon FMN removal. Thus the autoxidation of sulfite reductase, like that of the respiratory chain, occurs primarily by autoxidation of an exposed flavin cofactor. Great variability in the oxidation turnover numbers of these and other flavoproteins suggests that endogenous oxidants will be predominantly formed by only a few oxidizable enzymes. Thus the degree of oxidative stress in a cell may depend upon the titer of such enzymes and accordingly may vary with growth conditions and among different cell types. Furthermore, the chemical nature of these reactions was manifested by their acceleration at high temperatures and oxygen concentrations. Thus these environmental parameters may also directly affect the O-2 and H2O2 loads that organisms must bear.
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PMID:The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli. 1018 94

Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties.
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PMID:Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain. 1051 Feb 71


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