|Mooney, B - UNIV OF MISSOURI-COLUMBIA|
|Henzl, M T - UNIV OF MISSOURI-COLUMBIA|
|Randall, D - UNIV OF MISSOURI-COLUMBIA|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 19, 2000
Publication Date: N/A
Technical Abstract: Pre-dihydrolipoyl acyltransferase, the E2 subunit of the branched-chain ketoacid dehydrogenase complex (BCE2), from Arabidopsis thaliana, was imported into etiolated pea seedling mitochondria. The in vitro synthesized precursor (Mr 64,000) was processed to a mature form (Mr 55,000). Import was ATP-dependent and inhibited by valinomycin. Mature BCE2 was expressed in Escherichia coli. The highly soluble recombinant protein was purified using a Sepharose 6 size exclusion column to >90% homogeneity with a specific activity of 91 nmoles 14C-acetyl-dihydrolipoamide formed per minute per mg protein. The recombinant protein had a native molecular mass of 1.1 MDa and a S coefficient of 21.4, consistent with the formation of a 24-mer. Negative-staining transmission electron microscopy of the recombinant protein confirmed that BCE2 forms a core with octagonal symmetry. This is the first detaild report on plant dihydrolipoyl acyltransferase.