High Affinity and Low Affinity Binding of Ca++ by Rat Liver Mitochondria

Abstract Respiration-inhibited rat liver mitochondria possess two major classes of Ca++-binding sites, which have widely different affinity. At pH 7.4, the low affinity sites can bind about 40 nmoles of Ca++, whereas the high affinity sites bind about 1.2 nmoles of Ca++ per mg of protein and are half-saturated at about 0.025 µm. High affinity Ca++ binding is inhibited by 2,4-dinitrophenol and other uncoupling agents, but is not influenced by the respiratory inhibitors, oligomycin, aurovertin, valinomycin, or gramicidin. Uncoupling agents cause discharge of some of the endogenous Ca++ of mitochondria. No H+ ejection accompanies binding of Ca++ at either the high affinity or low affinity sites if respiration is inhibited. Binding of Ca++ is extremely rapid and apparently temperature-independent. Mn++ and Sr++ are also bound at the high affinity sites, but Mg++ and K+ are not. Aging of the mitochondria caused loss of high affinity Ca++-binding capacity, but not of low affinity binding. Although sonic particles and inner membrane preparations retain the high affinity sites, they are lost in water-lysed mitochondrial ghosts or in mitochondria treated with the nonionic detergent Lubrol WX. The properties of the high affinity Ca++-binding sites suggest they may be contributed either by hypothetical high energy intermediates generated in electron transport or by a specific divalent cation carrier molecule in the membrane.