Studies of the Energy-dependent Uptake of Divalent Metal Ions by Beef Heart Mitochondria

The energy-dependent uptake of calcium and magnesium by beef heart mitochondria was investigated using a combination of direct measurement of metal cation uptake and the interaction of divalent metal cations with the membrane-associated chelate probe, chlorotetracycline. Maximal rates of calcium and magnesium uptake in energized heart mitochondria were observed when the incubations contained a permeant anion. However, maximal changes in the chlorotetracycline-associated fluorescence were observed during divalent cation uptake in the absence of a permeant counter ion. Following rapid energy-dependent calcium or magnesium uptake by the mitochondrial membranes in the absence of a permeant anion, the addition of uncoupler or EDTA caused a release of the divalent metal ions from the membrane. Also, after the initial association of the divalent metal ions with the membrane, the addition of a permeant anion caused not only a dramatic decrease in the chlorotetracycline-associated fluorescence of the mitochondrial suspension but also an enhanced uptake of the metal ion. When phosphate was added as the permeant anion, the chlorotetracycline-associated fluorescence decrease was prevented by the inclusion of mersalyl, an inhibitor of phosphate transport in isolated mitochondria. Increasing the potassium concentration of the reaction medium caused a significant decrease in the fluorescence change during the energized uptake of either magnesium or calcium by the mitochondria. However, potassium was without effect on the initial base-line fluorescence prior to substrate addition. Furthermore, the addition of the ionophorous antibiotic, A23187, to deplete the mitochondrial membranes of their divalent metal ions, resulted in a complete prevention of the chlorotetracycline-associated fluorescence changes which occurred upon energization of the membranes. These observations indicate that calcium and magnesium transport across mitochondrial membranes consists of at least three phases: (a) an energy-independent binding of the divalent metal to the surface of the membrane, (b) an energy-dependent movement of the metal ion in or on the membrane to a site which is accessible to the membrane-associated chelate probe, chlorotetracycline, and (c) an anion-dependent transfer of the metal ion from the membrane into the matrix space.