Turnover of Pigeon Breast Muscle Pyruvate Dehydrogenase Complex
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Abstract:
The pigeon breast muscle pyruvate dehydrogenase complex was resolved into three component enzymes: lipoate acetyltransferase, pyruvate dehydrogenase, and lipoamide dehydrogenase. The antibodies against each component enzyme were prepared. All of the antibodies against component enzymes precipitated the pyruvate dehydrogenase complex. The enzyme complex was recovered as the immunoprecipitate from the extract of breast muscle of a pigeon that had received a single injection of L-[4,5-3H]leucine. The immunoprecipitate was separated into each component enzyme by SDS-polyacrylamide gel electrophoresis. The relative isotopic leucine incorporations per mg of protein into each component enzyme 4 h after the injection were 1.0 : 0.9 : 1.4 : 2.7 for lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase, respectively. The half-lives of lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase were 7.7, 2.5, 2.6, and 1.8 days, respectively. These results indicate that the component enzymes of the pyruvate dehydrogenase complex were synthesized and degraded at different rates.Keywords:
Dihydrolipoyl transacetylase
Oxoglutarate dehydrogenase complex
Dihydrolipoamide dehydrogenase
The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.
Dihydrolipoyl transacetylase
Oxoglutarate dehydrogenase complex
Dihydrolipoamide dehydrogenase
Polyclonal antibodies
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Dihydrolipoamide dehydrogenase
Dihydrolipoyl transacetylase
Flavoprotein
Oxoglutarate dehydrogenase complex
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Enzyme systems that catalyze the lipoic acid-linked oxidative decarboxylation of pyruvate and α-ketoglutarate have been isolated as multienzyme complexes with molecular weights of several million from bacterial, avian and mammalian cells. These multienzyme complexes have a distinct morphology and catalyze a coordinated sequence of reactions. The Eschcrichia coli pyruvate dehydrogenase complex has been separated into three enzymes-pyruvate dehydrogenase, dihydrolipoyl transacetylase, and a flavoprotein, dihydrolipoyl dehydrogenase. The complex has been reconstituted from the isolated enzymes. The E. coli α-ketoglutarate dehydrogenase complex also has been separated into three enzymes, analogous to those obtaind from the pyruvate dehydrogenase complex and it, too, has been reassembled from the isolated enzymes. The three enzymes are α-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase. Pyruvate and α-ketoglutarate dehydrogenase complexes isolated from pig heart muscle and from beef kidney mitochondria appear to be composed of three enzymes, analogous to those comprising the corresponding bacterial complexes. The macromolecular organization of the bacterial and mammalian α-keto acid dehydrogenase complexes has been elucidated to a large extent by correlated biochemical and electron microscopic studies.
Dihydrolipoyl transacetylase
Dihydrolipoamide dehydrogenase
Oxoglutarate dehydrogenase complex
Oxidative decarboxylation
Decarboxylation
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Oxoglutarate dehydrogenase complex
Dihydrolipoyl transacetylase
Dihydrolipoamide dehydrogenase
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A previously reported deficiency of total pyruvate dehydrogenase complex activity is further characterized. Dihydrolipoyl transacetylase (E2) and lipoamide dehydrogenase (E3) activities in the patient's fibroblasts were normal. Pyruvate dehydrogenase activity (El) was 33% ofthat in fibroblasts from an age-matched control. The amounts of each of the components of pyruvate dehydrogenase complex were analyzed using an immunoblot technique and specific antibodies. Levels of components E2 and E3 were the same in fibroblasts from the patient and control, confirming the activity measurements. However, the levels of Ela and Elft were reduced markedly in fibroblasts from the patient. Thus, impairment in the pyruvate dehydrogenase complex activity was due to a reduction in the amount ofthe El component of the complex.
Dihydrolipoyl transacetylase
Oxoglutarate dehydrogenase complex
Dihydrolipoamide dehydrogenase
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The structure and functional properties of pyruvate dehydrogenase complex (PDHC) have been reviewed briefly.PDHC is a mitochondrial multienzyme complex including pyruvate dehydrogenase(E_1),dihydrolipoamide acetyltransferase(E_2),dihydrolipoamide dehydrogenase(E_3),pyruvate dehydrogenase,pyruvate dehydrogenase phosphatase,five cofactor and protein X.Pyruvate dehydrogenase complex converts pyruvate to aceryl-CoA that is subatrate for the citric acid cycle.Lactic acidosis can arise from the deficiency of PDHC.
Oxoglutarate dehydrogenase complex
Dihydrolipoyl transacetylase
Dihydrolipoamide dehydrogenase
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Dihydrolipoyl transacetylase
Oxoglutarate dehydrogenase complex
Dihydrolipoamide dehydrogenase
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The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.
Dihydrolipoyl transacetylase
Oxoglutarate dehydrogenase complex
Oxidative decarboxylation
Dihydrolipoamide dehydrogenase
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Dihydrolipoyl transacetylase
Oxoglutarate dehydrogenase complex
Dihydrolipoamide dehydrogenase
Flavoprotein
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A previously reported deficiency of "total" pyruvate dehydrogenase complex activity is further characterized. Dihydrolipoyl transacetylase (E2) and lipoamide dehydrogenase (E3) activities in the patient's fibroblasts were normal. Pyruvate dehydrogenase activity (E1) was 33% of that in fibroblasts from an age-matched control. The amounts of each of the components of pyruvate dehydrogenase complex were analyzed using an immunoblot technique and specific antibodies. Levels of components E2 and E3 were the same in fibroblasts from the patient and control, confirming the activity measurements. However, the levels of E1 alpha and E1 beta were reduced markedly in fibroblasts from the patient. Thus, impairment in the pyruvate dehydrogenase complex activity was due to a reduction in the amount of the E1 component of the complex.
Dihydrolipoyl transacetylase
Oxoglutarate dehydrogenase complex
Dihydrolipoamide dehydrogenase
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