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)

The oxidation-reduction potential, E2, for the couple oxidized lipoamide dehydrogenase/2-electron reduced lipoamide dehydrogenase has been determined by measurement of equilibria of these enzyme species with lipoamide and dihydrolipoamide or with oxidized and reduced azine dyes. E2 is -0.280 V at pH 7, and deltaE2/deltapH is -0.06 V in the pH range 5.5 to 7.6. Values for E1, the oxidation-reduction potential for the couple 2-electron reduced enzyme/4-electron reduced enzyme, were obtained from measurements of the extent of dismutation of 2-electron reduced enzyme to form mixtures containing oxidized and 4-electron reduced enzyme. E1 is -0.346 V at pH 7, and deltaE1/deltapH is -0.06 V in the pH range 5.7 to 7.6. Spectra of oxidized enzyme and 4-electron reduced enzyme do not show variations with pH over this range, but the spectrum of the 2-electron reduced enzyme is pH-dependent, with the molar extinction at 530 nm changing from 3250 M-1 cm-1 at pH 8 to 2050 M-1 cm-1 at pH 5.2. The pH-dependent changes which are observed in the absorption properties of the 2-electron reduced enzyme are consistent with the disappearance of a charge transfer complex between an amino acid side chain and the oxidized flavin at the lower pH values, with the apparent pK of the side chain at pH 5. It has been suggested that the 530 nm absorbance of 2-electron reduced enzyme is due to a charge transfer complex between thiolate anion and oxidized flavin, and we propose that the thiolate anion is stabilized by interaction with a protonated base. The thermodynamic data predict that the amount of 4-electron reduced enzyme formed when the enzyme is reduced by excess NADH will be pH-dependent, with the greatest amounts seen at low pH values. These data support earlier evidence (Matthews, R.G., Wilkinson, K.D., Ballou, D,P., and Williams, C.H., Jr. (1976) in Flavins and Flavoproteins (Singer, T.P., ed) pp. 464-472; Elsevier Scientific Publishing Co., Amsterdam) that the role of NAD+ in the NADH-lipoamide reductase reaction catalyzed by lipoamide dehydrogenase is to prevent accumulation of inactive 4-electron reduced enzyme by simple reversal of the reduction of 2-electron reduced enzyme by NADH.
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PMID:Measurement of the oxidation-reduction potentials for two-electron and four-electron reduction of lipoamide dehydrogenase from pig heart. 0 67

A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.
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PMID:Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart. 3 86

1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.
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PMID:Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography. 16 34

A covalently bound adduct of nicotinamide adenine dinucleotide (NAD) with alginic acid has been found to be enzymatically active and to undergo electrochemical oxidation or reduction without significant loss of its enzymatic activity. The preparation of the adduct itself (from NAD+, alginic acid, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate) is also accomplished with substantially complete retention of enzymatic activity. This adduct has been converted from the oxidized to the reduced form by controlled potential electrolysis using mercury and stainless-steel electrodes. This electrolytically produced NADH complex could be oxidized again to the enzymatically active NAD+ complex by enzymatic reaction with the proton acceptor, 2,6-dichlorophenol indophenol, as catalyzed by diaphorase. Using this electrolytic method with immobilized NAD, it is now possible to carry out redox reactions in which NADH is enzymatically oxidized to NAD+, with the simultaneous electrolytic regeneration of the reduced form, NADH, from the oxidized form, NAD+, produced in the enzymatic reaction.
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PMID:Electrolytic regeneration of the reduced from the oxidized form of immobilized NAD. 17 64

Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have been obtained in homogeneous state from asparagus mitochondria. They are flavin enzymes with 1 mol of FAD/mol of protein. Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have s20,w of 6.22 S, 6.39 S, and 5.91 S, respectively, and molecular weights of 111,000, 110,000, and 95,000 (sedimentation equilibrium) or 112,000, 112,000, and 92,000 (gel filtration). They are slightly acidic proteins with isoelectric points of 6.75, 5.75, and 6.80. Both asparagusate dehydrogenases catalyzed the reaction Asg(SH)2 + NAD+ equilibrium AsgS2 + NADH + H+ and exhibit lipoyl dehydrogenase and diaphorase activities. Lipoyl dehydrogenase is specific for lipoate and has no asparagusate dehydrogenase activity. NADP cannot replace NAD in any case. Optimum pH for substrate reduction of the three enzymes are near 5.9. Asparagusate dehydrogenases I and II have Km values of 21.5 mM and 20.0 mM for asparagusate and 3.0 mM and 3.3 mM for lipoate, respectively. Lipoyl dehydrogenase activity of asparagusate dehydrogenases is enhanced by NAD and surfactants such as lecithin and Tween 80, but asparagusate dehydrogenase activity is not enhanced. Asparagusate dehydrogenases are strongly inhibited by mercuric ion, p-chloromercuribenzoic acid, and N-ethylmaleimide. Amino acid composition of the three enzymes is presented and discussed.
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PMID:Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria. Physical, chemical, and enzymatic properties. 18 3

A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.
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PMID:Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase. 18 94

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) isolate from pig heart and Escherichia coli was covalently coupled by both diazonium and amide bonds to controlled pore glass beads (96% silica). When the enzyme was immobilized in the presence of NAD+, the enzyme no longer exhibited its normal requirement for NAD+ for full activity. If the immobilized enzyme was then treated with NADase, the requirement for NAD+ was restored. Enzyme immobilized in the absence of NAD+ exhibited normal NAD+ dependence both prior to an after NADase treatment. These results are discussed in terms of co-immobilization of NAD+ at or near the allosteric site of the enzyme.
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PMID:Lipoamide dehyrogenase immobilized on porous glass. 19 68

We describe a highly sensitive and accurate automated continuous-flow method for determining bile acids in serum. The bile acids are first liberated from serum protein by dialysis at alkaline pH and then measured fluorometrically after the following enzymic reaction. Bile acids are converted to 3-oxo bile acids with 3alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD+ to NADH. The hydrogen in the generated NADH is transferred by diaphorase (EC 1.6.4.3) to resazurin to yield resorfin, the fluorophore. Only 100 microliter of serum is required and 40 determinations can be done per hour. The CV for 20 replicate determinations in serum with a mean bile acid concentration of 9.8 mumol/liter was 2.6%. The CV for day-to-day variation for another serum on 27 successive days was 3.0% (mean concentration, 10.0 mumol/liter). We applied this method to 826 sera from various diseases; 29% exceeded the upper limit of normal, 10 mumol/liter, and abnormally high values (greater than 20 mumol/liter) were almost exclusively limited to sera from hepatobiliary and enteric disorders.
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PMID:Continuous-flow determination of bile acids in serum, and its clinical application. 65 94

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.
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PMID:The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 120 21

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.
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PMID:The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution. 132 38


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