Action of Calcium on Corn Mitochondria

Our laboratory has been investigating membranelinked functions of plant mitochondria which might help explain active ion transport. Corn mitochondria will actively accumulate calcium + phosphate in a process very like that of animal mitochondria (9) ; i.e., a high energy intermediate derived from substrate oxidation or ATP furnishes the energy. With the substrate-powered system, there appears to be a competition between uptake and ATP formation, for the addition of ADP + hexokinase trap diminished calcium uptake. However, unlike animal mitochondria, the corn mitochondria proved to require phosphate for calcium uptake. Furthermore, the accumulation of magnesium and phosphate could not be demonstrated unless some calcium was present. These experiments were done with low concentrations of calcium (0.1-0.2 mM) in order to avoid uncoupling the mitochondria. We had observed that concentrations of calcium of 1 mm and higher reduced P/O ratios, and we assumed that calcium was acting as an uncoupling agent, a view commonly expressed on the basis of experiments with mammalian mitochondria. Calcium has been long known to stimulate acceptorless respiration of animal mitochondria (16, 17) and to induce adenosine triphosphatase activity (13), thus mimicking the action of the uncoupling agent, 2,4-dinitrophenol (DNP). Calcium also accelerates the swelling of animal mitochondria, which is thought to reflect a calcium-stimulated release of fatty acids, or U-factor (10). More recent work suggests that the action of calcium is more complex than initially visualized. Chance (2) has shown that low concentrations of calcium do not really uncouple; rather, calcium simulates the effect of ADP as an energy acceptor. In the presence of phosphate, calcium activates a rapid energy-linked process which stimulates respiration. and highly oxidizes the respiratory carriers. This process terminates with the binding of calcium in a form that no longer stimulates respiration, and the carriers return to the reduced state. High concentrationis of calcium damage the mitochondria, causing pyridine nucleotide to be highly oxidized and respiratioln to be inhibited. Rossi and Lehniinger (15) have examined the stoichiometry of calcium uptake and respiration in rat liver mitochondria. They conclude that the release of acceptorless respiration by small amounts of calcium is associated with a reaction at energy conserving sites of the electron transport chain. No phosphate is required for the reaction, but only a small amount of calcium can be bound or accumulated. The stoichiometry of binding and respiration is abolished by phosphate, and complete uncoupling of respiration results. However, further addition of ATP + magnesium will lead to calcium + phosphate uptake. When calcium and ADP + phosphate are added, both accumulation of calcium + phosphate and phosphorylation of ADP occur. The affinity of calcium for the energy conserving sites appears to be higher than that of ADP. There is very little comparable information on the effect of calcium on plant mitochondria. Calcium has been reported to both accelerate (5) and inhibit (14) adenosine triphosphatase. The swelling of cauliflower mitochondria is unaffected or slightly retarded by 1 mM calcium (11). Hackett (6) found calcium and other divalent ions to accelerate the NADH-oxidase of sweet potato mitochondria. It seemed to us that before we went further in studies of calcium + phosphate uptake we should study the effect of calcium on other energy-linked processes in corn mitochondria. The experiments are reported here, and comparisons are made with the action of 2,4-dinitrophenol (DNP). Contrary to our initial assumption, we find that calcium is not a true uncoupling agent for corn mitochondria. Calcium does not mimic the action of DNP as it does with animal mitochondria. However, calcium definitely reacts with corn mitochondria. At concentrations of ImM or less, calcium diverts phosphate from ATP formation to phosphate uptake and promotes the contraction of swollen mitochondria. Concentrations of calcium above 1 mm are very inhibitory to electron transport and phosphorylation.