Plant Physiology 59:842-848 (1977)
© 1977 American Society of Plant Biologists
Articles
Pyruvate Dehydrogenase Complex from Higher Plant Mitochondria and Proplastids 1
E. Ellen Reid,
Paul Thompson,
C. Richard Lyttle2 and
David T. Dennis
a Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6
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,w 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.
2 Present address: University of Chicago, Ben May Laboratories, Chicago, III.
1 This research was supported by a grant from the National Research Council of Canada.
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