Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a 45Ca exchange technique in the presence of magnesium, phosphate, and ATPase at 370C

The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37 degrees 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 a 45Ca exchange technique. The amounts of 40Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of 45Ca exchange. At 0.05 mM free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 microM, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 microM. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The 45Ca 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 the 45Ca 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 K0.5 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 and 45Ca 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