Branched-chain α-ketoacid dehydrogenase complex was isolated from rat heart, bovine kidney, and rabbit liver, heart, kidney, brain, and skeletal muscle. Phosphorylation to approximately 1 mol Pi/mol α-subunit of the α-ketoacid decarboxylase component was linearly associated with 90-95% inactivation. The complex from some tissues (i.e., from rabbit kidney and heart, and rat heart) showed 30-40% more phosphate incorporation for an additional 5-10% inactivation. Reverse-phase HPLC analysis of tryptic digests of 32P-labeled complexes from all of the above tissues revealed two major (peaks 1 and 2) and one minor (peak 3) phosphopeptide which represent phosphorylation sites 1, 2, and a combination of 1 and 2, respectively. These phosphopeptides, numbered according to the order of elution from reverse-phase HPLC, had the same elution time regardless of the tissue or animal source of the complex. The amino acid sequence of site 1 from rabbit heart branched-chain α-ketoacid dehydrogenase was IleGlyHisHisSer(P)ThrSer AspAspSerSerAlaTyrArg. Regardless of the source of the complex, both sites were almost equally phosphorylated until total phosphorylation was approximately 1 mol Pi/mol of α-subunit and the rate of inactivation was correlated with the rate of total, site 1, or site 2 phosphorylation. Phosphorylation beyond this amount was associated with greater site 2 than site 1 phosphorylation. α-Chloroisocaproate, a potent inhibitor of branched-chain α-ketoacid dehydrogenase kinase activity, greatly reduced total phosphorylation and inactivation; however, phosphorylation of site 2 was almost abolished and inactivation was directly correlated with phosphorylation of site 1. Thus, the complex isolated from different tissues and mammals had an apparent conservation of amino acid sequence adjacent to the phosphorylation sites. Both sites were phosphorylated to a similar extent temporally although site 1 phosphorylation was directly responsible for inactivation.
ASJC Scopus subject areas
- Molecular Biology