An assay for sarcoplasmic reticulum Ca2(+)-ATPase activity in muscle homogenates.
A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High [Ca2+] (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ 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 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ 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 Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.
Simonides
W S
WS
Laboratory for Physiology, Faculty of Medicine, Free University, Amsterdam, The Netherlands.
van Hardeveld
C
C
D000818:Animals; D000918:Antibody Specificity; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D003720:Densitometry; D003902:Detergents; D005470:Fluorometry; D009132:Muscles / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D000252:Calcium-Transporting ATPases