Mammalian α-Keto Acid Dehydrogenase Complexes

• Published in: The Journal of biological chemistry Vol. 247; no. 12; pp. 4043 - 4049
• Main Authors: Tanaka, Nobuyuki, Koike, Kichiko, Hamada, Minoru, Otsuka, Kin-Ichi, Suematsu, Tadashi, Koike, Masahiko
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 25.06.1972
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)45136-3

The nonionic detergent Triton X-100 has been found to solubilize readily the pig heart 2-oxoglutarate dehydrogenase complex from the protamine precipitate eluate. A highly active preparation was obtained from the Triton X-100 extract by calcium phosphate gel-cellulose column chromatography. The complex was first separated into lipoamide dehydrogenase and a colorless fraction by fractionation on a calcium phosphate gel-cellulose column in the presence of 2.5 m urea. Then, the latter fraction was further dissociated into two additional components, 2-oxoglutarate dehydrogenase and lipoate succinyltransferase, by Sepharose 6B gel chromatography at pH 7.0 in the presence of 0.7 m guanidine hydrochloride, 0.5% Triton X-100, and 2 mm dithiothreitol. When mixed at neutral pH, the three isolated enzymes reassociated spontaneously to produce a large unit functionally and structurally resembling the native complex. The complex was also reconstituted from the colorless fraction and lipoamide dehydrogenase. Reconstitution experiments indicated that 2-oxoglutarate dehydrogenase and lipoamide dehydrogenase did not combine with each other but each of these enzymes did combine with lipoate succinyltransferase. Electron micrographs of the complex negatively stained with 0.25% sodium phosphotungstate (pH 7.2) showed a polyhedral structure with a diameter of about 260 A. This suggests symmetrical distribution of morphological subunits around the core of a polyhedron with dimensions and appearance similar to that of the isolated lipoate succinyltransferase molecule, which is 117 A in diameter. Biochemical and electron microscopic data indicate that the complex is a mosaic comprising 1 molecule of lipoate succinyltransferase, 6 molecules of 2-oxoglutarate dehydrogenase, and 6 molecules of lipoamide dehydrogenase.