Mammalian α-Keto Acid Dehydrogenase Complexes

Pig heart pyruvate dehydrogenase complex was separated into lipoamide dehydrogenase (s20,w = 5.74 S) and a colorless fraction by the fractionation on a calcium phosphate gel-cellulose column in the presence of 4 m urea. Subsequently, in the presence of 0.3 m potassium iodide, the colorless fraction was fractionated into two additional components, pyruvate dehydrogenase (7.84 S) and partially resolved lipoate acetyltransferase, by solid ammonium sulfate. In the presence of 4 m urea, highly purified lipoate acetyltransferase (31.6 S) was prepared by the resolution of the yellow fraction which was separated from the native complex on a calcium phosphate gel-cellulose column in the presence of 0.02 m ethanolamine (pH 9.4). All three components were required to reconstitute the coenzyme A- and nicotinamide adenine dinucleotide-linked oxidation of pyruvate. The sedimentation coefficient (65.5 S), the composition, and enzymatic activities of the reconstituted complex were in good agreement with the values of the native complex.