Lipoyl content and other properties of the protein X and the transacetylase components of the pyruvate dehydrogenase complex

Previous work demonstrated by structural and immunological techniques that protein X (X) was distinct from the transacetylase (E2) but that regions of X and E2 (specifically including the portions acetylated) were similar. Trypsin cleaved X and E2 into large domains giving acetylated portions with apparent M/sub r/ values of ∼20 kdal and ∼38 kdal, respectively. Purified (denatured) E2 and X subunits were prepared and amino acid compositions determined. Reduced subunits were reacted with FDNB and acid hydrolyzed. Bis(DNP)dihydrolipoic was extracted into ethylacetate and quantitated by HPLC (epsilon = 25 O.D.mM-1cm-1 at 340 nm) and the levels normalized based on the amino acid analysis of the acid hydrolysates. E2 and X were estimated to contain about 1 lipoyl moiety per subunit. Following dihydrolipoyl dehydrogenase-dependent NADH reduction of E2-X, 1.5-2 14C-NEM per subunit were incorporated into E2 and X consistent with reduction of one lipoyl moiety per subunit. Incorporation into E2-X subcomplex of > 90 acetyl groups per molecule of subcomplex led to > 1.5 acetyl group incorporated per X and per E2 subunit and nearly eliminated NADH-dependent incorporation of 14C-NEM into these subunits suggesting diacetyl moieties were formed. Consistent with that possibility, acetylation to high levels yielded rapid and slowly exchanging acetyl groups on both E2 and X