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Stoichiometric Relationships in Mitochondrial Accumulation of Calcium and Phosphate Supported by Hydrolysis of Adenosine Triphosphate

From the ¹ From the Department of Physiological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Addition of small amounts of Ca⁺⁺ (80 nmoles per mg of protein) to rat liver mitochondria in the presence of adenosine triphosphate, Mg⁺⁺, and fluoride caused stimulation of ATPase activity, accumulation of Ca⁺⁺, ejection of H⁺ ions into the medium, and accumulation of phosphate. The molar ratio of Ca⁺⁺ taken up-H⁺ ejected-phosphate accumulated-ATP hydrolyzed was found to be approximately 2.0:2.0:1.2:1.0. Together with earlier evidence, these findings indicate that utilization of a single hypothetical high energy intermediate (or its equivalent), whether generated by electron transport or ATP utilization, can cause accumulation of 2 Ca⁺⁺ ions, ejection of 2 H⁺ ions, and accumulation of 1.2 phosphate ions.

ATP-supported Ca⁺⁺ uptake was blocked by 2,4-dinitrophenol, Dicumarol, azide, and oligomycin, but not by aurovertin. Other nucleoside 5'-triphosphates, such as guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, failed to support Ca⁺⁺ uptake. The stoichiometry between Ca⁺⁺ uptake and H⁺ ejection was not significantly influenced by changes in pH between 7.2 and 8.2 or by varying the salt concentration from 80 to 320 mm. At pH 6.5, ATP-supported ejection of H⁺ in the presence of Ca⁺⁺ underwent rebounds and oscillations.

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