1991 Volume 37 Issue 3 Pages 257-267
Pyruvate dehydrogenase was separated from pig heart pyruvate dehydrogenase complex by gel-permeation chromatography on a TSK G4000SWG column in the presence of 4M urea, followed by chromatography on a calcium phosphate gel-cellulose column. The pyruvate dehydrogenase was further separated into two nonidentical subunits, α and β, by high-performance liquid chromatography on a Synchropak CM-300 column in the presence of 8 M urea. The complete amino acid sequences of two subunits of pyruvate dehydrogenase were determined. The peptide fragments of S-carboxymethylated subunits were generated by treatment with endoproteinase Lys-C, endoproteinase Asp-N, trypsin, and cyanogen bromide. The subunits α and β contain 361 amino acid residues (Mr 40, 294) and 329 residues (Mr 35, 787), respectively. The amino acid sequences of subunits α and β in the pig were, respectively, 98 and 96% identical to those in humans. Hydropathy analysis and prediction of the secondary structure of two subunits suggest that the subunit α contains the thiamin pyrophosphate-binding domain and that the subunit β contains segments with a high hydrophobicity.
