Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase

The metal-ion requirement of extracted and partially purified pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat-pads was investigated with pig heart pyruvate dehydrogenase [³²P]phosphate as substrate. The enzyme required Mg²⁺ (K_m 0.5mm) and was activated additionally by Ca²⁺ (K_m 1μm) or Sr²⁺ and inhibited by Ni²⁺. Isolated fat-cell mitochondria, like liver mitochondria, possess a respiration- or ATP-linked Ca²⁺-uptake system which is inhibited by Ruthenium Red, by uncouplers when linked to respiration, and by oligomycin when linked to ATP. Depletion of fat-cell mitochondria of 75% of their total magnesium content and of 94% of their total calcium content by incubation with the bivalent-metal ionophore A23187 leads to complete loss of pyruvate dehydrogenase phosphate phosphatase activity. Restoration of full activity required addition of both MgCl₂ and CaCl₂. SrCl₂ could replace CaCl₂ (but not MgCl₂) and NiCl₂ was inhibitory. The metal-ion requirement of the phosphatase within mitochondria was thus equivalent to that of the extracted enzyme. Insulin activation of pyruvate dehydrogenase in rat epididymal fat-pads was not accompanied by any measurable increase in the activity of the phosphatase in extracts of the tissue when either endogenous substrate or ³²P-labelled pig heart substrate was used for assay. The activation of pyruvate dehydrogenase in fat-pads by insulin was inhibited by Ruthenium Red (which may inhibit cell and mitochondrial uptake of Ca²⁺) and by MnCl₂ and NiCl₂ (which may inhibit cell uptake of Ca²⁺). It is concluded that Mg²⁺ and Ca²⁺ are cofactors for pyruvate dehydrogenase phosphate phosphatase and that an increased mitochondrial uptake of Ca²⁺ might contribute to the activation of pyruvate dehydrogenase by insulin.