Author:
Holemans Tine,Vandecaetsbeek Ilse,Wuytack Frank,Vangheluwe Peter
Abstract
Sarco-/endoplasmic reticulum (SR/ER) Ca2+ pumps (SERCAs) build up vital Ca2+ 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 Ca2+ transport is tightly coupled to ATP hydrolysis, it is possible to determine the rate of Ca2+-dependent ATP hydrolysis and use it as a measure for SERCA activity or, in a second approach, to quantify ATP-stimulated uptake of radioactive 45Ca2+. Here, we first provide an overview of the mechanism of Ca2+-transport ATPases and show how this can be taken advantage of in protocols for measuring Ca2+ pump activity.
Publisher
Cold Spring Harbor Laboratory
Subject
General Biochemistry, Genetics and Molecular Biology
Cited by
4 articles.
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