Abstract
Polyclonal antibodies directed against the dihydrolipoyl transacylase (E2) and alpha subunit of branched-chain alpha-keto acid decarboxylase (E1 alpha) components of the bovine branched-chain keto acid dehydrogenase complex were shown to cross-react with the E2 and E1 alpha polypeptides of the enzyme complex of different rat tissues. Phosphorylation of the branched-chain keto acid dehydrogenase complex resulted in inhibition of enzyme activity concomitant with phosphate incorporation into the E1 alpha polypeptide. Phosphorylation of E1 alpha slowed its rate of migration through sodium dodecyl sulfate-polyacrylamide gels. This permitted resolution of the phosphorylated and unphosphorylated forms of E1 alpha on immunoblots. Liver and skeletal muscle mitochondria were prepared from rats consuming 6, 20, or 50% casein diets. The enzyme complex in mitochondria was measured by radioisotopic enzyme assay and immunoassay. Liver branched-chain keto acid dehydrogenase was 25% active in rats consuming 6% casein diets; whereas in rats consuming 20 or 50% casein diets, the liver enzyme was 82 or 100% active, respectively. Branched-chain keto acid dehydrogenase of muscle was 10, 13, and 22% active, respectively, in rats consuming 6, 20, and 50% casein diets. The amount of protein consumed by rats did not affect the total amount of the enzyme complex per unit of mitochondrial protein as measured by either the radioisotopic assay (enzyme activity) or the immunoassay. However, the protein intake of rats did affect activity of the enzyme kinase in liver. Liver branched-chain keto acid dehydrogenase kinase was more active in rats consuming 6% casein than in those fed chow or 50% casein diets. The amount of protein consumed by rats thus influences the enzyme activity in liver and muscle by affecting the reversible phosphorylation mechanism and not by induction of branched-chain keto acid dehydrogenase.
Highlights
From the DeDartments of Nutritional Sciences and Biochemistry, College of Agricultural and Life sciences, University of
Polyclonal antibodies directed against the dihydro- The branched-chain a-keto acid dehydrogenase complex, lipoyl transacylase ( E z ) and a subunit of branched- an intramitochondrial multienzyme complex, catalyzes the chain a-keto acid decarboxylase ( E l a )components of oxidative decarboxylation of the branched-chain a-keto acids the bovine branched-chain keto acid dehydrogenase formed through transamination of the branched-chain amino complex were shown to cross-react with thEez and El, polypeptides of the enzyme complex of different rat tissues
Antiserum alsoreacted with E2 and E, polypeptides in preparationsof rat mitochondria from several different tissues: kidney,liver, skeletal muscle, and heart(Fig. protein immunoblot of mitochondrial proteins was performed using
Summary
From the DeDartments of Nutritional Sciences and Biochemistry, College of Agricultural and Life sciences, University of. The enzyme complex is composed of three separate enzymes, branched-chain a-keto acid decarboxylase (El)d,ihydrolipoyl transacylase ( E 2 )a, nd dihydrolipoamide dehydrogenase ( E J [1].El is composed of two subunits denoted as a ( M .= 46,000) and p (Mr= 38,000); phosphorylation of the Erapolypeptide causes inactivation of sulfate-polyacrylamidegels. Liver branched-chain keto acid dehydrogenase kinase was more active inrats consuming 6%casein than inthose fed chow or 50% casein diets. The extent of phosphorylation and the degree of activation of the branched-chain keto acid dehydrogenase complex appear to differ among tissues of the rat[10,11,12]. Consumption of different amounts of protein by rats has been shown to alter significantly the degree of activation of branched-chain keto acid dehydrogenase in liver and kidney, but has little or no effect on the activation state of the heart enzyme [10, 12]. Activation state and total amount of the enzyme complex were measured by a radioisotope enzyme assay as well as by a method based on the use of polyclonal antibodies which permits detection of immunoreactive E2 and E,, components of the branched-chain ketoacid dehydrogenase complex
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