| Abstract: | Two subpopulations of cardiac sarcoplasmic reticulum vesicles were resolved functionally, based on their sensitivities to the drug ryanodine. These two subpopulations of sarcoplasmic reticulum vesicles, termed ryanodine-sensitive and ryanodine-insensitive, were separated by preloading crude cardiac microsomes with Ca2+ oxalate in the presence of ATP, followed by sucrose density gradient centrifugation. Ryanodine-insensitive vesicles accumulated most of the Ca2+ oxalate during the preload, and constituted the densest subfraction recovered from the sucrose gradient. These ryanodine-insensitive vesicles exhibited the highest density of Ca2+ pumps, and accounted for 10 to 15% of the total protein in crude cardiac microsomes. Ryanodine-insensitive vesicles continued to transport substantial amounts of Ca2+ after isolation. Ryanodine-sensitive vesicles accumulated negligible Ca2+ during the preload, and were recovered from the lower density regions of the sucrose gradient. On a milligrams of protein basis, these vesicles were present in 7-fold excess over ryanodine-insensitive vesicles. Ryanodine-sensitive vesicles transported low amounts of Ca2+ under normal incubation conditions, but 3 X 10(-4) M ryanodine strikingly increased their Ca2+ uptake 5- to 10-fold. Ca2+ uptake by ryanodine-sensitive vesicles was uniquely regulated by Ca2+ ion concentration. Elevation of the ionized Ca2+ concentration from 2 to 4 microM increased Ca2+ uptake by these vesicles greater than 5-fold, but had no effect on their Ca2+-dependent ATPase activity. These ryanodine- and Ca2+ concentration-dependent effects were apparent for only ryanodine-sensitive vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed distinct differences in polypeptide staining between ryanodine-sensitive and ryanodine-insensitive vesicles, confirming by an independent method that the two populations of vesicles were different. These data provide the first biochemical evidence for functional and structural heterogeneity of cardiac sarcoplasmic reticulum vesicles. |
