A quantitative analysis of super stoichiometric H+ ejection and Ca2+ uptake in respiring rat liver mitochondria

Experiments described in this paper provide an explanation for super-stoichiometry of respiration-dependent uptake of Ca²⁺ and ejection of H⁺ by mitochondria, i.e. Ca²⁷2e^- ratios greatly exceeding 2.0 per site and correspondingly increased H⁺/2e^- ratios when the medium is devoid of phosphate. In rat liver mitochondria super-stoichiometry is evoked by Ca²⁺ concentrations in excess of 60 to 80 ng-ions/mg of protein, in an otherwise normal reaction medium of 120 mM KCl, pH 7.2. Super-stoichiometric H⁺ ejection and Ca²⁺ uptake is caused by a distinctly different process than the stimulation of oxygen consumption. Kinetic analysis shows that respiration-dependent Ca²⁺ stimulated H⁺ ejection under conditions yielding super-stoichiometry is biphasic. It consists of a large early burst of H⁺ release, exceeding 1400 ng ions of H⁺/mg/min, in which the H⁺/2e^- ratio may be infinitely high; and a slow phase stoichiometric with electron transport. Simultaneously, there is a large early burst of respiration-dependent Ca²⁺ binding, which may exceed 1000 ng ions/mg/min, again with a very high Ca²⁺/2e^- ratio, and a slow phase of Ca²⁺ uptake stoichiometric with respiration. Super-stoichiometric Ca²⁺/2e^- and H⁺/2e^- ratios are thus due to a rapid and large energy-dependent Ca²⁺ binding and H⁺ ejection superimposed on the slower, normal uptake of Ca²⁺ and ejection of H⁺ that is stoichiometric with electron transport. The extra Ca²⁺ uptake and H⁺ ejection in the early burst require a finite preceding period of State 4 electron transport, presumably for energization. Some 40 to 50 ng-ions of Ca²⁺ may be bound and up to 60 ng-ions of H⁺ may be ejected/mg of protein in the early burst under optimal conditions. Similar results are observed with Sr²⁺ uptake, but Mn²⁺ yields only the slow stoichiometric phase. Since the superstoichiometry occurs in the absence of permeant anions and with mersalyl present to prevent cycling of mitochondrial phosphate, it is concluded that the fast extra Ca²⁺ binding and H⁺ ejection involve specific membrane-binding sites. It is postulated that State 4 energization of the membrane causes changes in the affinity of these binding sites, leading to decreased affinity for H⁺ and increased affinity for Ca²⁺.