The pyruvate dehydrogenase complex (PDC) functions at an important branch point for energy metabolism in regard to the utilization and storage of cellular fuels--carbohydrate and fat. Mammalian PDC is regulated by interconversion between an active and an inactive form by a specific kinase and phosphatase. The roles of 6 subunits of this multienzyme system are known in part, but there is sparse information on the roles of 3 other subunits. The latter include: 1) a lipoyl-bearing subunit (protein X) tightly bound to but distinct from the transacetylase (E2) that undergoes rapid acetylation and appears to associate with the kinase; 2) a basic (high pI) subunit (Kb) that is tightly associated with the catalytic subunit of the kinase but appears not to be required for known regulatory effects; and 3) an FAD-containing subunit (PF) associated with the catalytic subunit of the phosphatase. By characterizing the molecular properties of these subunits we expect to learn how the kinase and phosphatase attach to the complex and how a large variety of metabolites modulate the regulatory enzymes. Examples of mechanisms of control that should be elucidated are: whether acetylation of protein X and/or E2 mediates activation of the kinase; whether NADH inhibition of the phosphatase is synonymous with dihydrolipoamide inhibition and whether PF or protein X contribute to inhibition by those effectors; and the site of binding of polyamines leading to activation of phosphatase activity. Our general approach will continue to be purification and resolution of the complex and subcomplexes, coupled with characterization of kinetic-regulatory properties and specific component associations. We have begun using polyclonal and monoclonal antibodies to aid in these efforts. Immunological techniques will be used for purifying, selectively assaying and analyzing the structural and functional properties of protein X, Kb and PF. Finally, we will evaluate whether a large (120 kdal) polypeptide detected in rat fat cells, but not rat heart mitochondria using antibodies prepared against the purified (but smaller) phosphatase subunits contributes to the persistent activation of fat cell PDC by insulin.
