Energy-dependent Accumulation of Calcium and Phosphate by Purified Inner Membrane Vesicles of Rat Liver Mitochondria

Abstract 1. Inner membrane vesicles of rat liver mitochondria prepared by treating inner membrane + matrix particles with Lubrol WX to remove matrix (Chan, T. L., Greenawalt, J. W., and Pedersen, P. L. (1970) J. Cell Biol. 45, 291) are impermeable to sucrose and exhibit a rapid phase of swelling in ammonium phosphate but not in ammonium chloride. The mercurials mersalyl and p-mercuribenzoate, inhibitors of the phosphate transport system in intact mitochondria, markedly inhibit swelling of these vesicles. Ruthenium red, an inhibitor of the Ca++ transport system of intact mitochondria, has no effect on the swelling process. 2. When phosphate and a respiratory substrate are present, these vesicles accumulate up to 450 nmoles of Ca++ per mg of protein in 2 min. When arsenate, ATP, and ADP replace phosphate as anion a much lower level of Ca++ uptake is observed. The following anions when substituted for phosphate have essentially no supporting capacity: AMP, acetate, malate, citrate, α-ketoglutarate, glutamate, sulfate, nitrate, and thiocyanate. Unlike intact mitochondria, inner membrane vesicles neither support Ca++ uptake when ATP replaces respiratory substrates as an energy source, nor require addition of ADP or ATP to support maximal uptake in the presence of substrates. 3. Ca++ uptake is inhibited by uncoupling agents, respiratory inhibitors, ruthenium red, and the mercurials mersalyl and p-mercuribenzoate. Similar to its action on intact mitochondria ruthenium red inhibits Ca++ uptake catalyzed by inner membrane vesicles without significantly inhibiting respiration or phosphorylation. 4. Phosphate is accumulated together with Ca++ by a process also inhibited by uncoupling agents, mercurials, and ruthenium red. Phosphate accumulation is prevented by omission of Ca++ from the incubation medium. Calculation of Ca++:Pi accumulation ratios gives values near 1.50 corresponding to Ca3(PO4)2. 5. Two conclusions seem evident from these studies. First, the process of Ca++ uptake catalyzed by inner membrane vesicles, although similar, is not identical with the Ca++ uptake process catalyzed by intact mitochondria. Secondly, the active uptake of Ca++ and the active uptake of phosphate by these vesicles are interdependent processes which require both mercurial- and ruthenium red-sensitive components.