Abstract
A cDNA for branched-chain alpha-ketoacid dehydrogenase kinase was cloned from a rat heart cDNA library. The cDNA had an open reading frame encoding a protein of 382 amino acid residues with a calculated molecular weight of 43,280. The clone codes for the branched-chain alpha-ketoacid dehydrogenase kinase based on the following: 1) the deduced amino acid sequence contained the partial sequence of the kinase determined by direct sequencing; 2) expression of the cDNA in Escherichia coli resulted in synthesis of a 43,000-Da protein that was recognized specifically by kinase antibodies; and 3) enzyme activity that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex was found in extracts of E. coli expressing the protein. Northern blot analysis indicated the mRNA for the branched-chain alpha-ketoacid dehydrogenase kinase was more abundant in rat heart than in rat liver, as expected from the relative amounts of kinase activity expressed in these tissues. The deduced sequence of the kinase aligned with a high degree of similarity within subdomains characteristic of procaryotic histidine protein kinases. This first mitochondrial protein kinase to be cloned appears more closely related in sequence to procaryotic histidine protein kinases than to eucaryotic serine/threonine protein kinases.
Highlights
A cDNA for branched-chaian-ketoacid dehydrogenase kinase was cloned froma rat heart cDNAlibrary
Degenerate, inosine-containing oligonucleotides corresponding to the amino-terminal sequence of BCKDH kinase were used as polymerase chain reaction (PCR) primers to amplify rat heart mRNA
One clone contained an insert of 2,250 bp with a single 1,257-bp open reading frame, a 120-bp 5"noncoding region, an ATG initiation signal, and a 873-bp 3'-noncoding region containing a polyadenylation signal and a poly(A) tail (Fig. 1)
Summary
A cDNA for branched-chaian-ketoacid dehydrogenase kinase was cloned froma rat heart cDNAlibrary. Rat heart BCKDH kinase was cloned in thepresent study to determine its relationship to other protein kinases and to facilitate future work on the mechanisms responsible for the regulation of its activity. Internal amino acid sequence analysis was obtained by in situ trypsin digestion on nitrocellulose and separation of the peptide fragments by narrow-bore reverse phase high performance liquid chromatography (9).Degenerate, inosine-containing oligonucleotides synthesized according to the amino-terminal sequence of BCKDH kinase were used to amplify rat heartmRNA by reverse transcriptase PCR.
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