Proton Movements across the Mitochondrial Membrane Supported by Hydrolysis of Adenosine Triphosphate

Abstract

ATP may undergo substantial oligomycin‐insensitive breakdown in rat liver mitochondria, leading to production of two molecules of phosphate and H+ per molecule of ATP disappearing. Use of rotenone instead of malonate or antimycin A as respiratory inhibitor prevented this side‐reaction, and thus permitted quantitative study of the relationships between H+ ejection and ATP hydrolysis. H+ ejection and ATP hydrolysis were greatly inhibited by previous depletion of the mitochondria of endogenous Ca++ or by addition of EDTA to the test system; addition of Ca++ restored these activities. Rat liver mitochondria preloaded with small amounts of 45Ca++ quickly lost the 45Ca++ to the medium on incubation in the presence of rotenone; subsequent addition of ATP caused reaccumulation of the Ca++ and hydrolysis of ATP accompanied by H+ ejection. Similarly, labeled mitochondria isolated from rats preinjected with carrier‐free 45Ca++ also lost 45Ca++ to the medium during anaerobic incubation; the Ca++ was re‐accumulated and H+ was ejected during subsequent ATP hydrolysis. In Ca++‐dependent ATP hydrolysis about 2 H+ are ejected into the medium and titratable alkalinity equivalent to about 1.2 OH− ions is accumulated by the mitochondria per high‐energy bond utilized. It is concluded that the H+ ejection accompanying ATP hydrolysis in respiration‐inhibited rat liver mitochondria in the absence of added Ca++ is in reality caused by the endogenous presence of small amounts of Ca++ (or other bivalent cations) in the mitochondria. The endogenous Ca++ can leak into the medium in the absence of energy‐providing reactions; on addition of ATP the subsequent hydrolysis of ATP and re‐accumulation of Ca++ is accompanied by ejection of H+. Such H+ ejection is in all probability the direct result of a Ca++–H+ exchange across the mitochondrial membrane, rather than the result of a cation‐independent asymmetric ATP hydrolysis as postulated by the chemiosmotic hypothesis. Copyright © 1967, Wiley Blackwell. All rights reserved