Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria

Abstract

Evidence is given for 3 sites of phosphorylation in the .alpha.-chains of the decarboxylase component of purified rat heart pyruvate dehydrogenase complex, analogous to those established for procine and bovine complexes. Inactivation of rat heart complex was correlated with phosphorylation of site 1. Relative initial rates of phosphorylation were site 1 > site 2 > site 3.2. Methods are described for measurement of incorporation of 32Pi into the complex in rat heart mitochondria oxidizing 2-oxoglutarate + L-malate (total, sites 1,2 and 3). Inactivation of the complex was related linearly to phosphorylation of site 1 in mitochondria of normal or diabetic rats. The relative inital rates of phosphorylation were site 1 > site 2 > site 3. Rates of site-2 and site-3 phosphorylation may have been closer to that of site 1 in mitochondria of diabetic rats than in mitochondria of normal rats. The concentration of inactive (phosphorylated) complex was varied in mitochondria from normal rats by inhibiting the kinase reaction with pyruvate at concentrations of inactive complex is related linearly to incorporation of 32Pi into site 1. Inhibition of 32Pi incorporations with pyruvate at all concentrations over this range was site 3 > site 2 > site 1. With mitochondria from diabetic rats, pyruvate (0.15-0.4 mM) inhibited incorporations of 32Pi into site 3 but had no effect on the concentration of inactive complex or on incorporations of 32Pi into site 1 or site 2. site-3 phosphorylation is not required for inactivation of the complex in rat heart mitochondria. Evidence is given that phosphorylation of sites 2 and 3 may inhibit reactivation of the complex by dephosphorylation in rat heart mitochondria.