Uptake and incorporation of ³⁵S-lipoic acid in the presence of Lpa.

<p>(A) KER176 cells that are auxotrophic for lipoic acid were transformed with plasmids encoding wild type Lpa (Lpa) or the active site nucleophile mutant Lpa S259A and were grown in minimal medium supplemented with ³⁵S-lipoic acid. After a 10 hour induction at 20°C, cell samples were normalized by OD₆₀₀ and protein extracts were separated by SDS-PAGE and analyzed by autoradiography. The assignment of the labeled species to the three lipoylated proteins in <i>E. coli</i>, the PDH, KDH, and H-protein, is indicated. (B) Scintillation counting was used to quantify ³⁵S-lipoic acid taken up by the KER176 cultures shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007392#pone-0007392-g002" target="_blank">Figure 2A</a>. Counts per minute (CPM) correspond to the uptake of cells from 5 µl of culture with an OD₆₀₀ of 1.1.