Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases

Carothers, Donna J.; Raefsky-Estrin, Cindy; Pons, Gabriel; Patel, Mulchand S.

doi:10.1016/0003-9861(87)90617-5

Cited by 19 publications

(13 citation statements)

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“…All efforts to detect multiple forms of lipoamide dehydrogenase in mammals have yielded what appear to be conformational isomers of the same protein [44]. However, there is recent evidence that rat liver mitochondria contain two immunologically distinct forms of lipoamide dehydrogenase [16], one of which is thought to be the L-protein of the glycine-oxidation system. We have shown by biochemical evidence that the L-protein of the glycineoxidation system of P. putidu is LPD-glc [lo].…”

Section: Discussionmentioning

confidence: 99%

“…Evidence from the study of lactic acidosis suggests that there is a single lipoamide dehydrogenase in man for pyruvate, 2-oxoglutarate and branched-chainoxoacid dehydrogenases [15]. However, there is immunological evidence for two lipoamide dehydrogenases in mammals [16], one of which may be the L-factor of the glycine oxidation system.The structural genes for the branched-chain-oxoacid-dehydrogenase complex of P. putidu have been mapped on the P. putidu chromosome [1 11 and cloned into the broad-hostrange vector, pKT230, [17]. The structural gene encoding LPD-val, lpdV, has been subcloned and is expressed in E. coli.…”

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Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida

Burns

Brown

Hatter

et al. 1989

European Journal of Biochemistry

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The production of two lipoamide dehydrogenases by Pseudomonus is so far unique. One, LPD-val, is the specific E3 component of the branched-chain-oxoacid dehydrogenase and the second, LPD-glc, is the E3 component of 2-oxoglutarate dehydrogenase and the L-factor of the glycine oxidation system. The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD-val in order to compare its deduced amino acid structure with that of other redox-active disulfide flavoproteins. Northern blots using mRNA isolated from P. putidu grown in media with branched-chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex. The nucleotide sequence of the structural gene for LPD-Val, lpdV, was determined and consists of 459 codons plus the stop codon. The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G + C . Codon usagc is characteristic of moderately strongly expressed genes. There is a ribosome-binding site preceding the ATG start codon and a strong candidate for a rho-independent terminator at the 3' end of the reading frame. The M , of the protein encoded is 48164 and when the M , of FAD is added, the total M , is 48949, which is very close to the value of 49 000 obtained by SDS-polyacrylamide gel electrophoresis.Similarity comparisons of LPD-Val with sequences of three other lipoamide dehydrogenases showed that LPDval was somewhat more distantly related. It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein.Branched-chain-oxoacid dehydrogenase, the second enzyme in the catabolism of valine, leucine and isoleucine [I], is a multienzyme complex composed of three subunits: El, the dehydrogenase-decarboxylase, E2, the transacylase and E3, lipoamide dehydrogenase. The complex has been purified from bovine kidney [2], Pseudomonas putidu [3], rabbit liver [4] and Pseudomonus ueruginosu [5]. In addition, an oxoacid dehydrogenase has been purified from Bacillus subtilis [6] which has activity with pyruvate and branched-chain oxoacids. All of these complexes are composed of four polypeptides including Elcl and Elfl subunits. The mammalian complexes are regulated by phosphorylation [4, 71, while the P.~eudomonus complex is allosterically regulated by L-valine I3, 51.P. putidu and P. ueruginosu contain two lipoamide dehydrogenases, LPD-Val and LPD-glc. LPD-val was so named because it is induced in media containing branched-chain amino acids as carbon sources and is the specific E3 subunit of branched-chain-oxoacid dehydrogenase, which is its only known function [5, 81. LPD-glc is found in cells grown in glucose synthetic medium and is the E3 subunit of 2-oxoglutarate dehydrogenase [9] and the L-factor of the glycine-oxidation system in P. putidu [lo]. LPD-val and LPDglc are specific for their functions and are not interchangeable. Mutants affected in IpdV, the structural gene for LPD-val, lack b...

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Section: Discussionmentioning

confidence: 99%

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Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida

Burns

Brown

Hatter

et al. 1989

European Journal of Biochemistry

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“…However, this system has not been purified as an intact complex. We have recently suggested that this E3 (i.e., L protein) is immunologically different from the E3 common to the three a-ketoacid dehydrogenase complexes (12).Approximately half of the amino acid sequence of pig heart E3 has been previously determined by sequencing overlapping peptide fragments (13). The availability of a cDNA for mammalian E3 would be valuable to complete the primary amino acid sequence of mammalian E3.…”

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Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

Pons

Raefsky-Estrin

Carothers

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACTcDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blothybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.The a-ketoacid dehydrogenase complexes-the pyruvate dehydrogenase multienzyme complex, the a-ketoglutarate dehydrogenase multienzyme complex, and the branchedchain a-ketoacid dehydrogenase multienzyme complexcatalyze the oxidative decarboxylation of pyruvate, aketoglutarate, and the branched-chain a-ketoacids, respectively (1-4). These three complexes occupy key positions in energy metabolism and are involved (i) in connecting glycolysis with the Krebs cycle, (ii) in the Krebs cycle itself, and (iii) in the regulation of the oxidation of branched-chain amino acids, respectively. Each complex consists of at least three catalytic components. The decarboxylase component, which has been designated E1, is specific for each complex, and it most likely represents the rate-limiting step in the overall reaction. The dihydrolipoamide acyltransferase component, designated E2, also substrate specific, is involved in the transfer of the acyl group to CoA. The third component of each complex, dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4), designated E3, is considered to be common to all three complexes. E3, a homodimer (subunit Mr = 55,000), contains a noncovalently bound molecule of FAD per subunit and catalyzes the reoxidation of the disulfhydryl form of the lipoyl residue bound to the E2 molecules of the complexes: E3 E2-Lip-(SH)2 + NAD+ <--E2-LiP(S)2 + NADH + H+ Three lines of evidence indicate that the E3 present in these three complexes is identical; they are reconstitution experiments (5), immunological cross-reactivity (6-9), and genetic disorders of E3, which cause simultaneous increases in the blood levels of pyruvate, a-ketoglutarate, and branched-chain ketoacids (10). The glycine cleavage system also contains a dihydrolipoamide dehydrogenase component known as L protein (11). However, this system has not been purified as an intact complex. We have recently suggested th...

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Gene for lipoamide dehydrogenase maps to human chromosome 7

Otulakowski

Robinson

Willard

1988

Somat Cell Mol Genet

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The gene for lipoamide dehydrogenase (LD) has been assigned to human chromosome 7 based on filter hybridization analysis of genomic DNA from rodent-human somatic cell hybrids using a cDNA probe for human LD. No indication of multiple copies of the gene was found, in accordance with previous evidence that LD in the pyruvate, alpha-ketoglutarate, and branched chain alpha-ketoacid dehydrogenase complexes is genetically as well as biochemically identical.

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Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases

Cited by 19 publications

References 30 publications

Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida

Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

Gene for lipoamide dehydrogenase maps to human chromosome 7

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