Author:

Keywords:

Adenosine Triphosphate, Animals, Biological Transport, Calcium, Calcium Radioisotopes, Cations, Divalent, Cells, Cultured, Gene Expression, Humans, Hydrolysis, Isotope Labeling, Mammals, Myocardium, Sarcoplasmic Reticulum Calcium-Transporting ATPases, 0306 Physical Chemistry (incl. Structural), 0601 Biochemistry and Cell Biology, 1103 Clinical Sciences, 3101 Biochemistry and cell biology

Abstract:

Sarco-/endoplasmic reticulum (SR/ER) Ca(2+) pumps (SERCAs) build up vital Ca(2+) gradients across the intracellular SR/ER membrane, helping to control cell function, proliferation, growth, differentiation, and death. We describe two techniques to measure the SERCA activity either in mammalian culture cells overexpressing SERCAs or in muscle tissue containing high levels of endogenous SERCAs. As Ca(2+) transport is tightly coupled to ATP hydrolysis, it is possible to determine the rate of Ca(2+)-dependent ATP hydrolysis and use it as a measure for SERCA activity or, in a second approach, to quantify ATP-stimulated uptake of radioactive (45)Ca(2+). Here, we first provide an overview of the mechanism of Ca(2+)-transport ATPases and show how this can be taken advantage of in protocols for measuring Ca(2+) pump activity.