Mammalian a-Keto Acid Dehydrogenase Complexes

1. The pig heart pyruvate dehydrogenase complex was separated into lipoamide hydrogenase (&I,~ = 5.55 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 (7.45 S) and partially resolved lipoate acetyltransferase, with ammonium sulfate. Highly purified lipoate acetyltransferase (28.2 S) was isolated by the resolution of the yellow fraction with 4 M urea, which was separated from the complex in the presence of 0.02 M ethanolamine (pH 9.4) on the calcium phosphate gel-cellulose column. 2. The molecular weights of the original complex, pyruvate dehydrogenase, lipoate acetyltransferase, lipoamide dehydrogenase, and the reconstituted complex were approximately 7.4 million, 110,000, 150,000, 2.0 million, and 7.5 million, respectively. 3. All three isolated constituent enzymes were required to reconstitute coenzyme Aand nicotinamide adenine dinucleotide-linked oxidative decarboxylation of pyruvate. Reconstitution of these constituent enzymes to produce a large unit resembling the original complex in composition, hydrodynamic parameters, and enzymic activities was achieved. Lipoate acetyltransferase appears to possess specific binding sites for pyruvate dehydrogenase and lipoamide dehydrogenase. 4. Morphological resolution and reconstitution of the complex through electron microscopic data have been shown. The macromolecular organization of the complex is discussed.