|Moreno, J - U OF MO, COLUMBIA, MO|
|David, N - U OF MO, COLUMBIA, MO|
|Randall, D - U OF MO, COLUMBIA, MO|
Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only
Publication Acceptance Date: July 16, 1999
Publication Date: N/A
Technical Abstract: The E1 component of the plant mitochondrial pyruvate dehydrogenase complex (mtPDC) has been expressed in the cytoplasm of the yeast Pichia pastoris by co-expression of the E1a and E1b subunits. The E1a subunit contains a poly-His affinity tag at the C-terminus while E1b does not. This affinity tag provides an effective means of obtaining a highly enriched fraction of the recombinant E1 using Ni+NTA affinity chromatography. The highly enriched E1 fraction was capable of decarboxylating pyruvate in a Mg2+-thiamin pyrophosphate dependent manner which could be further enhanced by the oxidant, potassium ferricyanide. Purified monoclonal antibodies against E1a subunit was able to immunoprecipitate the pyruvate decarboxylating activity again supporting the conclusion that the purified recombinantly expressed protein is a functional pyruvate dehydrogenase. Initial results using a mitochondrial fraction containing native pyruvate dehydrogenase kinase showed that this kinase fraction was capable to phosphorylate the recombinant E1 with concomitant inhibition of decarboxylation activity. Additional characteristics of the recombinant E1 sub-complex will be presented. This research was supported by NSF, the MoAES, and the Food for 21st Century Program.