Methods Pyruvate dehydrogenase phosphate phosphatase activity was assayed by the release of [32p] pl from pig heart pyruvate dehydrogenase [32P] phosphate and in a number of experiments by the associated increase in pyruvate dehydrogenase activity, which was measured as described by Coore et al.(1971). Incubations were made at 30 C in a total volume of 60, l containing pyruvate dehydrogenase phosphate (equivalent to 0.15-0.30 unit of pyruvate dehydro-genase) or pyruvate dehydrogenase [32P] phosphate (equivalent to 0.15-0.30 unit of pyruvate dehydro-genase; 70-28OnCi of 32P) and other additions given* Abbreviation: EGTA, ethanedioxybis (ethylamine)-tetra-acetate. Vol. 128 in the text and Table 1 in 20mM-potassium phosphate buffer, pH7. 0, containing 5mM-2-mercaptoethanol (phosphate-mercaptoethanol buffer). Samples were taken for assay of pyruvate dehydrogenase activity or [32P] PI at 0, 5 and10min. For the assay of [32p] p1, samples (20, ul) were added to 1OO, ul of 1%(w/v) bovine plasma albumin in Beckman 152 Microfuge tubes and protein was precipitated at 0 C with lOO1ul of 12%(w/v) trichloroacetic acid. After a freezing and thawing to aggregate the precipitatedprotein, the tubes were centrifuged for 5min and samples (1OO, ul) of supematant assayed for radioactivity by liquid-scintillation spectrometry. Reaction rateswere constant over this time-period and proportional to the amount of heart pyruvate dehydrogenase phos-phate phosphatase or mitochondrial extract added; there was close correspondence between amounts of pyruvate dehydrogenase and [32P] P, released. Tri-chloroacetic acid-soluble 32P radioactivity in samples taken at zero time corresponded to less than 2% of the pyruvate dehydrogenase [32P] phosphate added. The effects of Ca2+ were investigated by using CaEGTA buffers. The concentrations of free Ca2+ and Mg2+ were calculated from the association constants given by Portzehl et al.(1964) by using a computer program provided by Dr. PJ England. Mitochondria were prepared by using methods given by Martin & Denton (1970)(fat-cells) and Chappell & Hansford (1969)(rat liver and pig kidney cortex). Extracts were made by freezing andthawing three times in phosphate-mercaptoethanol buffer. Pig heart pyruvate dehydrogenase, pyruvate dehydrogenase phosphate and pyruvate dehydrogenase phosphate phosphatase were prepared as follows. Pyruvate dehydrogenase and pyruvate dehydrogenase phosphate were extracted by freezing and thawing (three times) a pig heart mitochondrial fraction prepared by the method of Sanadi et al.(1952). The phosphorylated form was then converted into active pyruvate dehydrogenase by incubation at 30 C with lOmM-MgCI2 in 20mM-phosphate-mercaptoethanol buffer, pH7. 0 (complete in 30min, tested by pyruvate dehydrogenase assay). Pyruvate dehydrogen-ase was then precipitated by addition of saturated (NH4) 2SO4 to 35%(v/v) at pH7. 0. The precipitate was redissolved in phosphate-mercaptoethanol