Mammalian α-Keto Acid Dehydrogenase Complexes: VI. NATURE OF THE MULTIPLE FORMS OF PIG HEART LIPOAMIDE DEHYDROGENASE

Abstract

Abstract Lipoamide dehydrogenase (reduced nicotinamide adenine dinucleotide:lipoamide oxidoreductase, EC 1.6.4.3) has been isolated from the pig heart 2-oxoglutarate dehydrogenase complex, the pig heart pyruvate dehydrogenase complex, and a pig heart extract which was devoid of the two complexes. Two major molecular forms, designated as Fp-I and Fp-II in order of increasing anodic mobility, were detected in both electrophoretic patterns and triethylaminoethyl-cellulose column chromatograms of these three flavoprotein preparations. Fp-I is the main flavoprotein component of the 2-oxoglutarate dehydrogenase complex, and Fp-II is the main flavoprotein component of the pyruvate dehydrogenase complex. The uncomplexed form of lipoamide dehydrogenase appears to be a mixture of Fp-I and Fp-II. Ultracentrifugal analyses, absorption spectra, flavin contents, amino acid analyses, end group analyses, peptide mapping, and immunological experiments showed no significant differences between the two forms (Fp-I and Fp-II) of lipoamide dehydrogenase, thereby suggesting the identity of these two molecular forms of the enzyme. Comparison of enzymic activities, thiol contents, electrophoretic patterns after incubation with NADH and arsenite, and optical rotatory dispersion and circular dichroism spectra of the two forms suggests that these multiple forms may result from conformational differences around the active site, involving thiol groups and FAD. The two forms of lipoamide dehydrogenase are interchangeable with respect to both their function and ability to form the complexes; however, the enzymic, electrophoretic, and circular dichroism properties of OGDC-Fp-I were altered with respect to those of PDC-Fp-II when bound to the specific binding site in lipoate acetyltransferase molecule.