Purification and acetylation of protein X subunit of pyruvate dehydrogenase complex (PDC) from bovine kidney

Abstract

Protein X is one of the subunits of pyruvate dehydrogenase complex. The biological role of this protein has not been fully elucidated, mainly because of the difficulty in its dissociation from the tightly bound dihydrolipoamide acetyltransferase-protein X subcomplex. We have found that the detachment of protein X from acetyltransferase subunit can be easily accomplished by the cycles of freezing and thawing process. Several lines of evidence including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis and acetylation with [2-14C] pyruvate confirmed that the purified protein is protein X. The purified intact form of protein X was acetylated by [2-14C] pyruvate in the presence of pyruvate dehydrogenase subunit. The acetylation efficiency of this protein was lower than that of acetyltransferase and was not affected by the presence of acetyltransferase.