Open AccessProton Movements across the Mitochondrial Membrane Supported by Hydrolysis of Adenosine Triphosphate
Author(s) -
Rossi C. S.,
Siliprandi N.,
Carafoli E.,
Bielawski J.,
Lehinger A. L.
Publication year - 1967
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1967.tb00143.x
Subject(s) - oligomycin , atp hydrolysis , antimycin a , chemistry , mitochondrion , adenosine triphosphate , chemiosmosis , rotenone , biochemistry , atp synthase , uncoupling agents , hydrolysis , atpase , respiratory chain , biophysics , enzyme , biology
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 45 Ca ++ quickly lost the 45 Ca ++ 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 45 Ca ++ also lost 45 Ca ++ 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.