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1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.

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