STABILIZATION OF RAT CARDIAC-SACROPLASMIC-RETICULUM CA-2+ UPTAKE ACTIVITY AND ISOLATION OF VESICLES WITH IMPROVED CALCIUM-UPTAKE ACTIVITY

The Ca2+ uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca2+ uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0° or 37°, but the Ca2+ uptake activity was relatively stable at 25°. The decay of Ca2+ uptake activity at 0° could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca2+ uptake activity of homogenates kept on ice but not of homogenates kept at 37°. We also found that release of the CSR from the cellular debris required homogenization in high KCI. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 μmol/min-mg in the absence of CSR calcium channel blockers and 0.67 μmol/min/mg in the presence of 10 μM ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca2+-ATPase rates averaged 1.07 μmol/min/mg and 0.88 μmol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a 'crude' preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca2+-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function. © 1990 Kluwer Academic Publishers.