Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a⁴⁵Ca exchange technique in the presence of magnesium, phosphate, and ATP at 37°C

The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2 mm inorganic phosphate, 3 mm ATP, 0.05 or 1.1 mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a⁴⁵Ca exchange technique. The amounts of⁴⁰Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of⁴⁵Ca exchange. At 0.05 mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 μm, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 μm. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The⁴⁵Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the⁴⁵Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value of K_0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and⁴⁵Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.