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)

Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.
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PMID:Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO. 308 53

The binding protein-dependent galactose transport of Salmonella typhimurium has been reconstituted in proteoliposomes made from a partially purified protein fraction (containing the three membrane protein implicated in this transport and a lipoamide dehydrogenase activity) and soybean phospholipids. The reconstitution of galactose transport requires the addition of the purified galactose binding protein. Transport is energized either by reduced lipoamide and NAD or by the membrane potential and is inhibited by ATP.
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PMID:[Reconstitution of high-affinity galactose transport of Salmonella typhimurium in proteoliposomes: energization by lipoamide and NAD or by the membrane potential; inhibition by ATP]. 311 76

The thioredoxin peptide Trp-Cys-Gly-Pro-Cys-Lys, which contains the redox active dithiol, was found to be reduced by lipoamide in a coupled reaction with lipoamide dehydrogenase and NADH. The reduced peptide in turn was shown to reduce insulin, oxidized lens protein and glyceraldehyde-3-phosphate dehydrogenase. While the peptide is not as effective a catalyst for utilizing pyridine nucleotides to reduce protein disulfides as thioredoxin, it offers a system which may be developed to provide more efficient disulfide reduction. This is particularly relevant since no thioredoxin peptides have been found to be active with thioredoxin reductase.
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PMID:Thioredoxin fragment 31-36 is reduced by dihydrolipoamide and reduces oxidized protein. 312 52

A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18 degrees C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 degrees C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pH-dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography. 320 89

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-[(bromoacetyl)amino]phenyl arsenoxide (BrCH2 14CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG. This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.
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PMID:The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide. 352 30

Lipoamide dehydrogenase (EC 1.6.4.3) from the ketoglutarate dehydrogenase complex of adrenals catalyzes the oxidation of NADH by lipoamide and quinone compounds according to the "ping-pong" scheme. The catalytic constants of these reactions are equal to 220 and 24 s-1, respectively (pH 7.0). The maximal quinone reductase activity is observed at pH 5.6, whereas the lipoamide reductase activity changes insignificantly at pH 7.5-5.5. The maximal dihydrolipoamide-NAD+ reductase activity is observed at pH 7.8. The oxidative constants of quinone electron acceptors vary from 6 X 10(6) to 4 X 10(2) M-1 s-1 and increase with their redox potential. The patterns of NAD+ inhibition in the quinone reductase reaction differ from that of lipoamide reductase reaction. The quinones are reduced by lipoamide dehydrogenase in the one-electron mechanism.
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PMID:[Characteristics of the interaction of adrenal lipoamide dehydrogenase with physiological and quinone electron acceptors]. 357 23

The effect of rat hepatic cytosolic constituents on microsomal 5'-deiodinase (5'-DI) activity in the presence of the dihydrolipoamide-generating system or dihydrolipoamide itself has been studied. Reconstitution experiments have demonstrated for the first time the essential role of cytosol as well as lipoamide and NADH in the activation of 5'-iodothyronine DI to a level equivalent to that attained in the presence of 5 mM dithiothreitol. Omission of NADH, lipoamide, or cytosol reduced the DI activity by 86%. By gel filtration chromatography of the cytosol, a fraction of intermediate mol wt (approximately 13,000) was shown to be highly stimulatory of microsomal DI in washed microsomes, provided either a dihydrolipoamide-generating system (lipoamide dehydrogenase, as well as NADH and lipoamide) or dihydrolipoamide alone was added. These observations suggest a possible role for a heretofore unreported reducing system influencing hepatic 5'-deiodination, wherein cytosolic cofactors appear to function as efficient intermediaries which enhance the action of a dihydrolipoamide in the activation of isolated hepatic microsomal 5'-DI.
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PMID:Cytosolic cofactors and dihydrolipoamide stimulate hepatic microsomal 5'-deiodination. 392 68

Pseudomonas putida grown on valine produces two lipoamide dehydrogenases, LPD-glu (Mr, 56,000 and LPD-val (Mr, 49,000). The 49,000-dalton protein is used by P. putida for branched-chain keto acid dehydrogenase, whereas the 56,000-dalton protein is presumably used for pyruvate and 2-ketoglutarate dehydrogenases. The objective of this study was to isolate and characterize mutants of P. putida with mutations affecting lipoamide dehydrogenases in order to study the relationship of these two proteins. Mutant JS287 lacked LPD-val, the lipoamide dehydrogenase which is induced by growth on valine and is specific for branched-chain keto acid dehydrogenase, and had normal amounts of LPD-glu, the lipoamide dehydrogenase which is formed during growth on glucose and which is probably used by both pyruvate and 2-ketoglutarate dehydrogenases. Mutant JS94 was a pleiotropic mutant with defects in 2-ketoglutarate, branched-chain, and lipoamide dehydrogenases. Proteolysis of LPD-glu and LPD-val produced completely different digestion products, suggesting that these two proteins are products of separate structural genes. Antisera prepared against LPD-glu reacted only with LPD-glu, whereas antisera prepared against LPD-val reacted with LPD-val and cross-reacted with LPD-glu. Although mutant JS94 did not produce active lipoamide dehydrogenase, cell-free extracts of this mutant contained a protein which cross-reacted with anti-LPD-val.
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PMID:Mutations affecting lipoamide dehydrogenases of Pseudomonas putida. 618 68

Pseudomonas putida produces two lipoamide dehydrogenases, LPD-glc and LPD-val. LPD-val is specifically required as the lipoamide dehydrogenase of branched-chain keto acid dehydrogenase and LPD-glc fulfills all other requirements for lipoamide dehydrogenase. Both proteins are dimers with one FAD per subunit. LPD-glc has an absorption maximum at 455 nm, but LPD-val has a maximum at 460 nm. Comparison of amino acid compositions revealed that LPD-glc was more closely related to Escherichia coli and pig heart lipoamide dehydrogenase than to LPD-val. LPD-val did not appear to be closely related to any of the proteins compared with the possible exception of mercuric reductase.
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PMID:Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases. 637 65

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.
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PMID:Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties. 640 48


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