Reversible phosphorylation of pyruvate dehydrogenase in rat skeletal-muscle mitochondria. Effects of starvation and diabetes

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Abstract
The total activity of pyruvate dehydrogenase (PDH) complex in rat hind-limb muscle mitochondria was 76.4 U/g of mitochondrial protein. The proportion of complex in the active form was 34% (as isolated), 8-14% (incubation with respiratory substrates) and > 98% (incubation with respiratory substrates). Complex was also inactivated by ATP in the presence of oligomycin B and carbonyl cyanide-m-chlorophenylhydrazone. Ca2+ (which activates PDH phosphatase) and pyruvate or dichloroacetate (which inhibit PDH kinase) each increased the concentration of active PDH complex in a concentration-dependent manner in mitochondria oxidizing 2-oxoglutarate/L-malate. Values giving half-maximal activation were 10 nM-Ca2+, 3 mM-pyruvate and 16 .mu.M-dichloroacetate. Activation by Ca2+ was inhibited by Na+ and Mg2+. Mitochondria incubated with [32P]Pi/2-oxoglutarate/L-malate incorporated 32P into 3 phosphorylation sites in the .alpha.-chain of PDH; relative rates of phosphorylation were sites 1 > 2 > 3, and of dephosphorylation, sites 2 > 1 > 3. Starvation (48 h) or induction of alloxan-diabetes had no effect on the total activity of PDH complex in skeletal-muscle mitochondria, but each decreased the concentration of active complex in mitochondria oxidizing 2-oxoglutarate/L-malate and increased the concentrations of Ca2+, pyruvate or dichloroacetate required for half-maximal reactivation. In extracts of mitochondria the activity of PDH kinase was increased 2- to 3-fold by 48 h starvation or alloxan-diabetes, but the activity of PDH phosphatase was unchanged.