Pyruvate Dehydrogenase Complex from Higher Plant Mitochondria and Proplastids

Abstract

The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s20,,0, of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD+ and pyruvate; NADP+ and other keto acids give no reaction.Mg2+, thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria.Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg2+, cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations. The sequence of reactions catalyzed by these enzymes is:

Coimmunopurification of Phosphorylated Bacterial- and Plant-Type Phosphoenolpyruvate Carboxylases with the Plastidial Pyruvate Dehydrogenase Complex from Developing Castor Oil Seeds

“…Native PDC pl is difficult to purify because of subunit dissociation (Mooney et al, 2002). Although PEPC and PDC pl are believed to be localized in different metabolic compartments, there are only two enzymatic steps between them (malate dehydrogenase and malic enzyme), and PEPC and PDC pl have both been implicated in playing an important role in supporting fatty acid synthesis in developing COS (Reid et al, 1977;Sangwan et al, 1992;Smith et al, 1992). This raises the interesting question of whether PEPC and PDC pl might interact in a multienzyme complex (metabolon) that could channel carbon from PEP to acetyl-CoA and/or recycle CO 2 from PDC pl to PEPC in developing COS.…”

“…Plant cells are unique in containing two different PDC isozymes: mitochondrial PDC (PDC mt ) which links cytosolic glycolysis with the citric acid cycle, and PDC pl , which produces acetyl-CoA and NADH for plastidic fatty acid synthesis (Reid et al, 1977;TovarMendez et al, 2003). PDC pl was originally discovered in leucoplasts isolated from developing COS (Reid et al, 1977) and has since been found in all plants Figure 7.…”

Cross Talk among Calcium, Hydrogen Peroxide, and Nitric Oxide and Activation of Gene Expression Involving Calmodulins and Calcium-Dependent Protein Kinases in Ulva compressa Exposed to Copper Excess

“…PDH activity was determined as described by Reid et al (1977). PDH activity was detected in 1 mL of reaction mixture containing 100 mM Tris-HCl (pH 7.0), 20 mM magnesium chloride, 0.5 mM thiamine pyrophosphate, 1 mM lipoic acid, 0.13 mM CoA, 1 mM Cys, 2 mM sodium pyruvate, 1 mM NAD, and 5 mg of protein extract.…”

The Role of Acetyl-Coenzyme A Synthetase in Arabidopsis

“…Historically, two reactions have been proposed to supply this acetyl-CoA, the plastidic pyruvate dehydrogenase complex (PDHC; Reid et al, 1977) and plastidic acetyl-CoA synthetase (ACS; Smirnov, 1960). ACS was the first enzyme implicated in acetyl-CoA synthesis in plastids.…”