Stoichiometry of Respiratory Stimulation, Accumulation of Ca++ and Phosphate, and Oxidative Phosphorylation in Rat Liver Mitochondria

Abstract

Isolated mitochondria of various tissues have been found to accumulate large amounts of Ca++ and phosphate from the medium in a process stoichiometrically dependent on high energy intermediates generated by the energy-conserving mechanisms of the respiratory chain (cf. References l-18). In most of the cited investigations (e.g. References 2, 3, 5, 11, 13), the mitochondria were exposed to relatively high concentrations of Ca* (2 to 4 mM), which are known to cause complete uncoupling of oxidative phosphorylation (cj. References 2, 19, 20), mitochondrial swelling (cf. Reference 21), and stimulation of the formation of endogenous, free fatty acids (22). Nevertheless, nearly exact stoichiometry of Ca++ uptake with electron transport could be demonstrated under these conditions (10, 11). Adenosine triphosphate is necessary in such experiments to support Ca++ uptake, for reasons which are not understood (1, 2, 59-11, 23). There is another set of observations in which much lower concentrations of Ca++ (i.e. 100 to 400 PM) have been employed. Siekevitz and Potter (19) and Potter, Siekevitz, and Simonson (20) showed that low concentrations of Ca+f greatly stimulated respiration of rat liver mitochondria. Chance showed that the addition of Ca++ to resting mitochondria in the absence of ADP produced a transient stimulation of respiration; the oxygen uptake returned to its original rate after a period of stimulation in a manner resembling the activation of respiration by ADP (23, 24). The addition of about 2.2 moles of Ca++ was required to stimulate the same amount of extra oxygen uptake as given by 1.0 mole of ADP. Chance (24) assumed that Ca++ was accumulated in the mitochondria stoichiometrically during the period of respiratory stimulation, but he reported no actual measurements of Ca++ uptake to support this important conclusion. A second finding made with low concentrations of Ca* is that phosphate does not necessarily enter the mitochondria with Ca++ (4, 6, 7, 25) ; in contrast, experiments carried out with high concentrations of Ca++ showed that phosphate is required for Ca++ uptake and is accumulated with Ca(2, 5, 11). Apparently, ATP is not required for accumulation of low concentrations of Ca++ (4, 6, 7; 23-25), again in contrast with the experiments in which high concentrations of Gaff were studied. It has been rather diffi-