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The ATP-dependent inactivation of the pyrvate dehydrogenase complex (PDC) was examined using ruptured mitochondria and partisaly prilfed pyrvate dehydrogenase complex isolated from broccoli and caulfllower (Brassica oeracca) bud mitochondria. The ATP-dependent iactivation was temperature-and pH-dependent.[ The complex is composed of three enzymes operating sequentially: pyruvate dehydrogenase (decarboxylating), dihydrolipoate transacetylase, and dihydrolipoate dehydrogenase.The PDC2 has been purified from numerous nonplant tissues (3,7,16) and recently from plants (18,19,20). The properties of the complex from nonplant tissues have been reviewed recently by Reed et al. (16), Denton et al. (7), and Hucho (9). PDC has been shown to be regulated by product or feedback inhibition by NADH and acetyl-CoA (7, 9, 16). Regulation by other metabolites such as GTP, PEP, fructose bis-P, citrate, glyoxylate, and hydroxypyruvate have also been reported (7,9,16).A second level of regulation which involves interconvertible forms (phosphorylated and dephosphorylated) of the complex has been established in mammalian tissues and Neurospora

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ATP inactivated plant pyruvate dehydrogenase complex (PDC) from broccoli (Brassica oleracca) mitochondria. ATP inactivation of the complex was time-dependent and proportional to the ATP concentration. rime-dependent incorporation of 32P from [y32PJATP into trichloroacetic acid-predpitable protein corresponded to the inactivation of the PDC. It is conduded that plant PDC is phosphorylated and inactivated by a PDC kinase.The pyruvate dehydrogenase complex (PDC)2 catalyzes the oxidative decarboxylation of pyruvate by a sequence of reactions. The complex consists of three enzymes: (a) pyruvate dehydrogenase; (b) lipoyl transacetylase; and (c) lipoyl dehydrogenase, acting sequentially in that order. The essentially irreversible nature of the first reaction in the complex and the pivotal position that PDC occupies in metabolism make PDC a logical candidate for regulation.Reed and colleagues (6, 7) established that mammalian PDC can also be regulated by a phosphorylation-dephosphorylation mechanism. Phosphorylation and the concomitant loss of PDC activity require a Mg-ATP2--dependent kinase (5). Dephosphorylation and reactivation of PDC is catalyzed by a Mg2+-and Ca2+-requiring phosphatase. The phosphorylation-dephosphorylation of PDC has been demonstrated in numerous mammalian tissues using both purified PDC (4, 5) and intact mitochondria (1, 11). This regulatory mechanism has also been demonstrated in Neurospora crassa PDC (14).Characterization of the plant PDC has been very limited (2, 8-10) and there have been few reports describing the regulation of the plant PDC. There have been no reports to date of inactivation or reactivation of plant PDC by a phosphorylation or dephosphorylation mechanism. We report here our finding of phosphorylation and concomitant inactivation of PDC isolated from the floral bud mitochondria of broccoli. Preparation of Mitochondria. Mitochondria were isolated from floral buds of broccoli (Brassica oleracea var. italica) purchased at local markets. The floral buds were shaved from the broccoli heads and crude mitochondria suspensions were prepared by homogenizing floral buds in 2.5 volumes of medium containing 0.5 M sucrose, 0.1 M sodium phosphate buffer (pH 7.8), 0.1% BSA, 5 mm EGTA, and 13.5 mm 2-mercaptoethanol. The brei was filtered through two layers of cheesecloth and two layers of Miracloth (Chicopee Mills, Milltown, N. J.). The filtrate was centrifuged at 400g for 15 min. The resulting supernatant was centrifuged for 30 min at 14,500g to pellet the mitochondria. The mitochondria were gently resuspended in extraction medium, diluted 1:1 with 25 mm K-phosphate buffer (pH 6.8) containing 13.5 mm 2-mercaptoethanol. The mitochondria were repelleted and resuspended as above. This mitochondrial suspension was centrifuged as above and the pellet was resuspended in a minimal amount of the diluted extraction medium, but without 2-mercaptoethanol, and stored on ice until used. MATERIALS AND METHODSInactivation of PDC. ATP-dependent inactivation of PDC was performed by incubating 0.4...