| Abstract: | The dihydrolipoyl transacetylase component, which serves as the structural core of mammalian pyruvate dehydrogenase complexes, is acetylated when treated with either pyruvate or with acetyl-CoA in the presence of NADH. Besides the dihydrolipoyl transacetylase component, we have found that another protein, referred to as protein X, is rapidly acetylated at thiol residues. Protein X remains fully bound to the transacetylase core under conditions that remove the pyruvate dehydrogenase and dihydrolipoyl dehydrogenase components. Mapping of 125I-tryptic peptides indicated that the transacetylase subunits and protein X are structurally distinct; however, under the same mapping conditions, there is considerable similarity in the positions of acetylated peptides derived from these subunits. Affinity-purified rabbit immunoglobulin G prepared against the dihydrolipoyl transacetylase core reacted exclusively with the transacetylase and with both its tryptic-derived inner domain and outer lipolyl-bearing domain. Those results further indicate that protein X is not derived from the transacetylase subunit Affinity-purified mouse antibody to protein X reacted selectively with large tryptic polypeptides derived from protein X and did not react with the inner domain of the transacetylase. However, the anti-protein X antibody did react with the intact transacetylase subunit, the lipoyl-bearing domain of the transacetylase, and weakly with the transsuccinylase component of the alpha-ketoglutarate dehydrogenase complex. This cross-reactivity reflected specificity of a portion of the polyclonal antibodies for a related structural region in the transacetylase and protein X (possibly a similar lipoyl-bearing region). Furthermore, a major portion of that polyclonal antibody was shown to react exclusively with protein X. Thus, protein X subunits differ substantially from transacetylase subunits but the two components have a region of structural similarity. We estimate that there are about 5 mol of protein X per mol of the kidney pyruvate dehydrogenase complex. Under a variety of conditions that result in a wide range of levels of acetylation of sites in the complex, about 1 acetyl group is incorporated into protein X per 10 acetyl groups incorporated into the transacetylase subunits per mol of complex. That ratio is close to the ratio of protein X subunits of transacetylase subunits in the complex, indicating that there are efficient mechanisms for acylation and deacylation of protein X. |
