|Hirani, T - UNIV OF MISSOURI|
|Tovar-Mendez, A - UNIV OF MISSOURI|
|Randall, D - UNIV OF MISSOURI|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 3, 2002
Publication Date: N/A
Technical Abstract: The pyruvate dehydrogenase complex (PDC) is a very large multi-component structure that catalyzes decarboxylation of pyruvate, yielding CO2, NADH, and acetyl-CoA as products. The decarboxylation reaction is catalyzed by pyruvate dehydrogenase (E1). The PDC occupies a key position in intermediary metabolism, and is subject to multiple layers of regulation. A prominent component of the control of mitochondrial PDC activity is reversible phosphorylation. Mitochondrial E1 is an a2b2 heterotetramer. The E1a subunit can be phosphorylated by an intrinsic E1-kinase (PDK), inactivating the complex. Reactivation is catalyzed by the intrinsic P-E1-phosphatase. The coding sequences for the Arabidopsis thaliana mitochondrial E1a and E1b subunits were cloned, individually and in combination, into pET vectors for expression in Escherichia coli BL21(DE3). Concomitant overexpression of the chaperonins GroESL resulted in a higher yield of soluble E1. A His6 tag was engineered at the N-terminus of E1a, to allow affinity purification using immobilized NiSO4. Gel permeation FPLC plus immunoblotting were used to demonstrate that part of the recombinant E1 assembled into a heterotetramer. When recombinant E1 was incubated with 32P-ATP plus recombinant AtPDK, there was phosphotransfer to E1a. Further data on purification of recombinant E1, measurement of catalytic activity using the 14C-pyruvate decarboxylation assay, and regulatory phosphorylation will be presented.