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Abstract

This chapter focuses on the synthesis of ATP from Ca2+ gradient by sarcoplasmic reticulum Ca2+ transport ATPase. Sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle consist of tightly sealed membranes, which only allow a slow passive back-diffusion of Ca2+ taken up previously by a pathway not yet identified. The driving force for ATP formation is the Ca2+ gradient existing between the vesicular volume and the external medium. Conditions are chosen in such a way that nearly all Ca2+ that was released could contribute to ATP synthesis. Loading of the vesicles is usually performed in the presence of acetyl phosphate (AcP) as energy-yielding substrate, and inorganic phosphate (Pi) serving as precipitating anion and substrate for the backward reaction. Ca2+ loads obtained by slow passive diffusion of millimolar concentrations (<100 nmol/mg protein) or by active transport in the absence of a precipitating anion (100–200 nmol/mg protein) set limits to the duration of the two fast-occurring processes, and to the accuracy of the results obtained.