Molecular cloning of a cDNA for the E1 alpha subunit of rat liver branched chain alpha-ketoacid dehydrogenase

Abstract

We have isolated a cDNA encoding the branched chain alpha-ketoacid dehydrogenase E1 alpha subunit. A rat liver lambda gt11 expression library was screened with antibody reactive with the 2-oxoisovalerate dehydrogenase (lipoamide) component. A positive clone, lambda BZ304, contains a 1.7-kilobase pair cDNA insert with a 1323-base pair open reading frame. Translation of the open reading frame predicts the 24 residues of the previously reported phosphorylation sites 1 and 2 for the bovine kidney and rabbit heart enzymes. The N-terminal sequence of purified E1 alpha was determined, and this sequence was found 40 residues from the beginning of the deduced peptide sequence. Northern blots of rat liver and muscle RNA demonstrate a single mRNA species of approximately 1.8 kilobase pairs in each tissue, suggesting that this cDNA is nearly full length.