20] Lipoate addition to acyltransferases of α-keto acid dehydrogenase complexes and H-protein of glycine cleavage system

Abstract

Publisher Summary This chapter presents the method employed for the purification of lipoyltransferase from bovine liver mitochondria using lipoyl-AMP and apoH-protein as donor and acceptor of the lipoyl group, respectively. Two isoforms were separated by chromatography on a hydroxylapatite column. The lipoyltransferase that eluted at about 220 mM phosphate was purified to homogeneity and termed “lipoyltransferase II.” Lipoyltransferase I eluted at about 200 mM phosphate and was partly purified. The molecular masses and optimal pH values of lipoyltransferase I and II were both 40 kDa and pH 7.9, respectively. The Km values for lipoyl-AMP obtained with lipoyltransferase I and II were 13 and 16 μM and for apoH-protein were 0.29 and 0.17 μM, respectively. The Vmax values of lipoyltransferase I and II were 135 and 144 nmol/min/mg of protein, respectively. Thus, the biochemical properties of both isoforms were similar. The chapter describes the assay methods for and properties of the lipoylation reaction of lipoyl domains of mammalian acyltransferases (E2s) and H-protein, using purified lipoyltransferase I and II.