Microbial lipoamide dehydrogenase purification and some characteristics of the enzyme derived from selected microorganisms

Scouten, William H.; McManus, I. Rosabelle

doi:10.1016/0005-2744(71)90058-1

Cited by 18 publications

(19 citation statements)

References 35 publications

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“…Like thioredoxin reductase (16,28), two redox-active centers (the disulfide and FAD) and the two binding domains (for NADPH and the lipoamide-containing protein P2) must be accommodated on each enzyme subunit of 34.5 kDa, in contrast to 50 kDa for the well-known type examined thus far. The low specific activities of about 9 and 28 U/mg determined with the NADPH-lipoamide and dihydrolipoamide-NADP test systems, respectively, were another difference in the isolated lipoamide dehydrogenase of Eubacterium acidaminophilum compared with the usual lipoamide dehydrogenases (35). The activity of the lipoamide dehydrogenase of Pseudomonas putida (glucose lipoamide dehydrogenase) reaches 65 U/mg (39); that of Peptostreptococcus glycinophilus is at least 85 U/mg (1), whereas the enzyme from Escherichia coli exhibits activity of 140 U/mg (13).…”

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confidence: 88%

“…The nucleotide sequence predicts a somewhat smaller monomer (Mr, 51,274) (44) than that which is usually found by biochemical methods (Mr, 52,000 to 59,000) (13,54). The DNA sequences for lipoamide dehydrogenase of yeast, porcine, and human origin contain, in addition, a leader sequence coding for 21 to 35 amino acids (5,30,33). In general, the enzyme contains 473 to 478 amino acids; thus, the calculated Mr without flavin adenine dinucleotide (FAD) varies between 49,436 and 51,556 regardless of its procaryotic or eucaryotic origin (4,5,14,18,30,33,50,53,55).…”

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confidence: 90%

“…The molecular masses of native lipoamide dehydrogenases from archaebacterial, eubacterial, and eucaryotic organisms are about 110,000 when determined by biochemical methods (4,8,13,18,35,50,53,54). The only exception is the valine lipoamide dehydrogenase from Pseudomonas putida (Mr, 98,000) (40), which comes close to the molecular mass of 2 x 50,225 to 52,278 (100,450 to 104,556) determined from the nucleotide sequence of the enzymes of procaryotic and eucaryotic (without the leader peptide) origin (5,30,33,44,53).…”

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confidence: 99%

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Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum

et al. 1989

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The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-'4C]riboflayin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The of its procaryotic or eucaryotic origin (4,5,14,18,30,33,50,53,55). The multiplicity of isoenzymes observed from eucaryotic origin was found to be a conformational isomerism (19). Immunological studies suggest that lipoamide dehydrogenase is identical in the pyruvate, 2-oxoglutarate, and branched-chain 2-oxoacid dehydrogenases of the rat heart (26).In enzyme assays pig heart diaphorase is able to replace lipoamide dehydrogenase, protein L, from the chicken liver glycine decarboxylase (12). The lipoamide dehydrogenase of glycine decarboxylase from pea leaf mitochondria exhibits a monomer with an Mr of about 59,000, which is similar to that of the enzyme that is involved in the pyruvate dehydrogenase complex, and monoclonal antibodies raised against lipoamide dehydrogenase inhibit both enzymes to the same extent (51). Therefore, being a conservative enzyme, lipoamide dehydrogenase has also been used to calculate evolutionary relationships (9, 27) to other pyridine nucleotide disulfide oxidoreductase flavoproteins such as glutathione reductase, thioredoxin reductase, and mercuric reductase (11,16,36,54).As the only exception Pseudomonas putida and Pseudomonas aeruginosa produce two lipoamide dehydrogenases during growth on valine, which differ to some degree in molecular masses and kinetic parameters. One is part of 2-oxoglutarate dehydrogenase and probably pyruvate dehydrogenase; the other is part of branched-chain 2-oxoacid dehydrogenase (27,39). During glycine oxidation by P. putida, only the lipoamide dehydrogenase of 2-oxoglutarate dehydrogenase is involved, which has a monomer molecular mass of 56 kilodaltons (kDa), however, rather than the enzyme specific for valine, which has a monomer molecular mass of 49 kDa (38).From all these data it seemed that no specific lipoamide dehydrogenase is involved in glycine oxidation. However, during the isolation of the glycine decarboxylase proteins

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confidence: 88%

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confidence: 90%

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confidence: 99%

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Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum

et al. 1989

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“…In vitro, DLDH can act as a diaphorase (Massey, et al, 1960) that is capable of transferring electrons from NADH to electron acceptors such as cytochrome c (Igamberdiev, et al, 2004) and ubiquinone (Olsson, et al, 1999, Xia, et al, 2001, and to electron-accepting dyes such as 2,6-dichlorophenolindophenol (DCPIP) (Patel, et al, 1995) and nitroblue tetrazolium (NBT) (Scouten andMcManus, 1971, Sokatch, et al, 1981). While DLDH itself may be a source of reactive oxygen species (Bando and Aki, 1991, Gazaryan, et al, 2002, Sreider, et al, 1990, Tahara, et al, 2007, it is also capable of scavenging nitric oxide (Igamberdiev, et al, 2004) and can serve as an antioxidant by protecting other proteins against oxidative inactivation by 4-hydroxyl-2-nonenal (Korotchkina, et al, 2001).…”

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confidence: 99%

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

Thangthaeng

Forster

2008

Mechanisms of Ageing and Development

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Brain energy metabolism is increased during postnatal development and diminished in neurodegenerative diseases linked to senescence. The objective of this study was to determine if these conditions could involve postnatal or senescence-related shifts in activity or expression of dihydrolipoamide dehydrogenase (DLDH), a key mitochondrial oxidoreductase. Rats ranging from 10 to 60 days of age were used in studies of postnatal development, whereas rats aged 5 or 30 months were used in the aging studies. The expression of DLDH was determined by Western blot analysis using anti-DLDH antibodies and DLDH diaphorase activity was measured by an in-gel activity staining method using nitroblue tetrazolium (NBT)/NADH. Activity of DLDH dehydrogenase was measured as NAD + oxidation of dihydrolipoamide. When these measures were considered in separate groups of 10-, 20-, 30-, or 60-day-old rats, all three showed an increase between 10 and 20 days of age. However, dehydrogenase activity of DLDH showed a further, progressive increase from 20 days to adulthood, in the absence of any further change in DLDH expression or diaphorase activity. No age-related decline in DLDH activity or expression was evident over the period from 5 to 30 months of age. Moreover, aging did not render DLDH more susceptible to oxidative inactivation by mitochondria-generated reactive oxygen species (ROS). Taken together, results of the present study indicate that (1) brain DLDH expression and activity undergo independent postnatal maturational increases; (2) Senescence does not confer any detectable change in the activity of DLDH or its susceptibility to inactivation by mitochondrial oxidative stress.

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“…Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed by the method of Laemmli (9) by using 15% polyacrylamide gels. Electrophoresis under nondenaturing conditions was done in 5% polyacrylamide gels with a cross-linkage of 1% as described previously (22). Coloration of protein bands was made by silver staining (27).…”

Section: Methodsmentioning

confidence: 99%

Purification of a new dihydrolipoamide dehydrogenase from Escherichia coli

Richarme

1989

J Bacteriol

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I purified a new dihydrolipoamide dehydrogenase from a Ipd mutant of Escherichia coli deficient in the lipoamide dehydrogenase (EC 1.6.4.3) common to the pyruvate dehydrogenase (EC 1.2.4.1) and 2-oxoglutarate dehydrogenase complexes. The occurrence of the new lipoamide dehydrogenase in lpd mutants, including a lpd deletion mutant and the immunological properties of the enzyme, showed that it is'different from the lpd A second important function of the dihydrolipoamide dehydrogenases as a component of branched-chain keto acid dehydrogenase complexes is related to their implication in the oxidative decarboxylation of branched-chain keto acids. These enzyme complexes are encountered in bacteria which can utilize branched-chain amino acids for growth (they perform an essential step in the catabolism of these amino acids after they are transaminated to keto acids). The bacterium Pseudomonas putida, which can use branchedchain amino acids for growth, possesses a branched-chain keto acid dehydrogenase complex which contains (in addition to its dehydrogenase and transacylase components) a specific lipoamide dehydrogenase (lpd Val) that is different from the lipoamide dehydrogenase (lpd Glc) of the pyruvate and 2-oxoglutarate dehydrogenase complexes (24). Branched-chain keto acid dehydrogenase activities have also been found in bacteria, such as Bacillus subtilis, which synthesize branched-chain fatty acids (from branched-chain keto acids) for maintenance of their membrane fluidity (13).A dihydrolipoamide dehydrogenase (lpd Glc) has also been shown to participate in glycine oxidation in P. putida as a component of glycine decarboxylase (23). The bacterium Escherichia coli, which cannot use branched-chain amino acids for growth and does not synthesize significant amounts of branched-chain phospholipids (11), does not appear to contain branched-chain keto acid dehydrogenase activities (28).Dihydrolipoamide dehydrogenases have also been discovered in several microorganisms which do not seem to possess lipoamide-dependent 2-oxo acid dehydrogenase complexes. A dihydrolipoamide dehydrogenase has been purified from the halophilic archaebacterium Halobacterium halobium, although this bacterium catalyzes pyruvate and 2-oxoglutarate oxidation by ferredoxin-linked oxidoreductases (4); similarly, a dihydrolipoamide dehydrogenase has been isolated from the African parasite Trypanosoma brucei (2); these discoveries raise the question of whether dihydrolipoamide dehydrogenases have a cellular function, in addition to their established role in the 2-oxo acid dehydrogenase complexes.In studies concerning the possible implication of lipoamide dehydrogenase in the function of binding protein-dependent transport systems, I have discovered a residual dihydrolipoamide dehydrogenase activity in lpd mutants of E. coli which were deficient in the lipoamide dehydrogenase component of the pyruvate and 2-oxo glutarate dehydrogenase complexes (including the deletion mutant JRG599 [21]). The purification and characterization of this second lipoamid...

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Microbial lipoamide dehydrogenase purification and some characteristics of the enzyme derived from selected microorganisms

Cited by 18 publications

References 35 publications

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

Purification of a new dihydrolipoamide dehydrogenase from Escherichia coli

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