Heat production and proton release during the atp driven calcium uptake by fragmented sarcoplasmic reticulum from bull frog and rabbit skeletal muscle

Kodama, T.; Kurebayashi, N.; Ogawa, Y.

Journal of Biochemistry 88(5): 1259-1266


ISSN/ISBN: 0021-924X
Accession: 005551368

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Measurements were made of heat production and proton release during ATP hydrolysys by fragmented sarcoplasmic reticulum (FSR) prepared from bullfrog and rabbit skeletal muscle. A microcalorimeter with an improved time resolution was used. The FSR was incubated under ATP-limiting conditions in a medium containing 100 mM KCl, 4 mM MgCl2, 0.1-0.3 mM CaCl2, 60 .mu.M P1,P5-di(adenosine-5')pentaphosphate and 20 mM MOPS[3-(N-Morpholino)propanesulfonic acid]-KOH (pH 7.1) at 8 and 20.degree. C. In the presence of 6 .mu.M 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 hearts of ATP hydrolysis (.DELTA.HATP) obtained were -25.2 .+-. 1.5 and -26.8 .+-. 0.9 kJ mol at 8 and 20.degree. C, respectively, after correction for the heat (-18.7 kJ/mol) due to the interaction of MOPS with 0.89 .+-. 0.01 mol of H+ released/mol of ATP hydrolyzed. The .DELTA.HATP at pH 8.0 determined at 8 and 20.degree. C under conditions otherwise the same as above were -18.5 .+-. 1.8 and -19.7 .+-. 1.3 kJ/mol, respectively. These values are in good agreement with those calculated according to the Alberty's method. 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 ATP hydrolysis. 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 at 8 and 20.degree. C, respectively. Corresponding values for frog FSR were +4.8 and +3.0 kJ/mol. The ATP-driven Ca uptake by FSR apparently is an endothermic process accompanied by the release of a fraction of a proton in exchange for a Ca ion sequestered.