Heat Production and Proton Release during the ATP-Driven CaUptake by Fragmented Sarcoplasmic Reticulum from Bullfrog and Rabbit Skeletal Muscle12

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Abstract
Measurements were made of heat prcxluction and proton release during ATP hydrolysis by fragmented sarcoplasmic reticulum (FSR) prepared from bullfrog and rabbit skeletal muscle. A microcalorimeter with an improved time resolution was used (Kodma, T. & Woledge, R.C. (1979) J. Biol. Chem.254, 6382-6386). The FSR was incubated under ATP-limiting conditions in a medium containing 100 mM KCI, 4 mM MgCl2 0.1–0.3 mM CaCl2 60 μM P1, P5 and 20 mM M0PS-KOH (pH 7.1) at 8 and 20°C. In the presence of 6 μM A23187, which abolishes the Ca uptake by FSR without impairing its ATPase activity, the results with frog and rabbit FSR were very similar at each temperature. The heats of ATP hydrolysis (δHATP) thus obtained were -25.2±1.5 and -26.8±0.9kJ mol−1 at 8 and 20°C, respectively, after correction for the heat (−18.7 kJ mol−1)due to the interaction of MOPS with 0.89 ±0.01 mol of H+ released per mol of ATP hydrolyzed. The δHATP at pH 8.0 determined at 8 and 20°C under conditions otherwise the same as above were −18.5±1.8 and −19.7±1.3 kJ·mol−1, respectively. These values are in good agreement with those calculated according to the method of Alberty (J. Biol. Chem.244, 3290–3302 (1969)). When the ATP hydrolysis was coupled to the Ca uptake, some protons (0.26–0.30 mol for rabbit FSR; 0.16–0.25 mol for frog FSR) were released in addition to those derived from the ATP hydrolysis itself, but the heat produced was not much different from that of the ATP hydrolysis uncoupled from the Ca uptake. Using values for the stoichiometry of Ca uptake and ATP hydrolysis determined in separate experiments, the heat of Ca uptake was estimated. The values for rabbit FSR were +13.1 and +5.6 kJ·mol−1 at 8 and 20°C, respectively. Corresponding values for frog FSR were +4.8 and +3.0 kJ·mol−1. It can be con cluded that the ATP-driven Ca uptake by FSR is an endothermic process accompanied by the release of a fraction of a proton in exchange for a Ca ion sequestered.

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