ISOLATION OF RAT CARDIAC SARCOPLASMIC-RETICULUM WITH IMPROVED CA-2+ UPTAKE AND RYANODINE BINDING

The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 m KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mm oxalate at 37°C was 0.45 μmol/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 μmol/min-mg in the presence of 10 μm ruthenium red. The Ca2+-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 μmol/min-mg and 0.84 μmol/min-mg in the absence and presence of 10 μm ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nm and a Bmax of 3.7 pmol/mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values of Bmax, EP and Ca2+-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR. © 1991.