PLANT MITOCHONDRIAL PYRUVATE-DEHYDROGENASE COMPLEX - PURIFICATION ANDIDENTIFICATION OF CATALYTIC COMPONENTS IN POTATO

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberos um cv, Romano) tuber mitochondria was purified 40-fold to a specific a ctivity of 5.60 mu mol/min per mg of protein. The activity of the comp lex depended on pyruvate, divalent cations, NAD(+) and CoA and was com petitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed th e complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing re vealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2 ), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N- terminal sequencing of the 55 kDa protein band yielded two protein seq uences: one was another E3; the other was similar to the sequence of E 2 from plant and yeast sources but was distinctly different from the s equence of the 78 kDa protein. Incubation of the mPDC with [2-C-14]pyr uvate resulted in the acetylation of both the 78 and 55 kDa proteins.