An improved method for the isolation of rat cardiac sarcoplasmic reticulum

Abstract

Preparations of cardiac sarcoplasmic reticulum (CSR) isolated from the rat by differential centrifugation have been widely used for measuring alterations in intracellular calcium flux in response to metabolic and pharmacologic disruptions. However, the purity of these SR fractions has not been firmly established. Using a combination of differential and linear sucrose gradient centrifugation, we have isolated rat CSR with high specific activity and purity. By SDS-PAGE analysis, the preparation is enriched in a protein (110 kD) of similar size to the Ca2+-ATPase of SR from other sources. Gels stained with the dye 'Stains-All' reveal a blue colored 55 kD band, confirming the presence of calsequestrin, the intraluminal low-affinity calcium binding protein of SR. The presence of the transmembrane 53 kD glycoprotein of SR was confirmed by endoglycosidase-H treatment followed by SDS-PAGE and also by a modified Western blotting technique. The rate of calcium uptake in this preparation averages 130 nmol/mg over the first minute of accumulation, approximately 4 times that previously reported for rat CSR. Calcium uptake in our preparation was essentially complete within 5 minutes. Preparations isolated by this method should be of value in future studies measuring alterations in rat CSR function.