[32] Assay for protein lipoylation reaction

Publisher Summary This chapter discusses the assay for protein lipoylation reaction. Lipoate attaches to the ɛ -amino group of the specific lysine residue of the proteins via an amide linkage. The lipoyllysine residue functions as a carrier of intermediates of the reactions and reducing equivalents between the active sites of the components of the complexes. The attachment of lipoate to the proteins involves two consecutive reactions. In mammals, the two reactions are catalyzed by separate enzymes in mitochondria. The first reaction is catalyzed by lipoate-activating enzyme in the presence of MgCl 2 and results in the formation of lipoyl-AMP. The second reaction is catalyzed by lipoyl-AMP: Ne-lysine lipoyltransferase (lipoyltransferase) and protein is lipoylated. The chapter describes two methods using bovine apo-H-protein as an acceptor of the lipoyl group. In the first method, Holo-H-protein formed during the lipoylation reaction is determined quantitatively by the glycine– 14 CO 2 exchange reaction. This method is simple but unsuitable for the assay with the crude enzyme preparations containing the glycine cleavage system. In the second method, the introduction of a lipoyl group to Lys-59 of H-protein by the lipoylation reaction neutralizes a positive charge of apo-H-protein and increases the mobility on nondenaturing polyacrylamide gel electrophoresis. This method is applicable to the assay with the crude mitochondrial extract.