An assay for sarcoplasmic reticulum Ca 2+-ATPase activity in muscle homogenates

Abstract

A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca 2+-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca 2+-ATPase activity, while eliminating Ca 2+-dependent myofibrillar ATPase activity and reducing Ca 2+-independent or background ATPase activity. High [Ca 2+] (20 m m) could be used to selectively inhibit the SR Ca 2+ ATPase. Identification of the Ca 2+-dependent ATPase activity in muscle homogenates as being SR Ca 2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 m m Ca 2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca 2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca 2+-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 μg of tissue (wet weight) in a volume of 25 μl.