Pyruvate Dehydrogenase Complexes from Ricinus communis Endosperm

Abstract

Summary The pyruvate dehydrogenase complex was isolated, partially purified and characterized from mitochondria of germinating and developing endosperm of castor oil seeds ( Ricinus communis var. N5). The complex required coenzyme A, thiamine pyrophosphate, NAD + and a divalent cation for activity which was optimal at pH 7.0–7.4. The Michaelis constants were 51, 3 and 90μM for NAD + , coenzyme A and pyruvate, respectively. There was competitive inhibition between NADH (K i = 15μM) and NAD + , as well as acetyl-CoA (K i = 20μM) and CoA. Incubation of lysed mitochondria with ATP phosphorylated and inactivated the complex. A pyruvate dehydrogenase-kinase transfers the γ-phosphate of ATP to the complex. Pyruvate, dichloroacetate and EDTA inhibited this phosphorylation. Reactivation (dephosphorylation) occurred after treatment with Mg 2+ . Dephosphorylation was inhibited by fluoride. Western blots revealed no detectable difference in subunit structure of PDC from mitochondria of both germinating and developing tissue. The plastid enzyme subunit structure was significantly different from the mitochondrial complex. Results of these studies indicate that the pyruvate dehydrogenase complex from mitochondria of the two tissue types exhibit similar characteristics and regulation, but are very distinct from the plastid complex.