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.
Highlights
Ryanodine-sensitive vesicles accumulated negligible Ca" during the preload, and were recovered from the lower density regions of the sucrose gradient
The explanation for the inability of these additional sarcoplasmic reticulum subfractions to accumulate Ca2+ions was unclear. It waspossible that the vesicles had become leaky or weredamaged during their preparation and were unable to retain any Ca2+ions that they might have transported [6, 14], or that their membranes were inverted such that any active Ca2+transport by them would have been directed outward into the incubation medium [11, 13, 16]. We suggest that these cardiac sarcoplasmic reticulum vesicles thought previously to be deficient in Ca" uptake are capable of transporting substantial amounts of Caz+,and that they constitute a distinct subpopulation of sarcoplasmic reticulum vesicles in cardiac microsomes
In agreement with the results of Levitsky et al [7] and Misselwitz et al [13],we found that only a fraction of the total sarcoplasmic reticulum vesicles accumulated Ca2+oxalate during the loading step, and that these vesicles continued to transport considerable Ca2+after they were isolated
Summary
Subfractionation of Crude Cardiac Sarcoplasmic Reticulum Vesicles-Crude cardiac sarcoplasmic reticulum vesicles were isolated as described previously [12]. The crude vesicle pellets (75 to 100 mg ofprotein) were resuspended in 40 mlof an ice-coldmedium containing 50 mM histidine, 100 m~ KCI, 65 mM MgCh, 60 mM NazATP, 25 m~ Tris/EGTA,' 20 m~ CaCh, and 5 mM Tridoxalate (pH 7.1). Basal ATPase activities were determined in the same media, which had no added Ca2+and 1 m~ Tris/EGTA. In experiments in which Ca2+uptake and Ca2+dependent ATPaseactivities were measured simultaneously, aliquots were taken from the same suspension of vesicles to determine the amount of Ca2+uptake and the total ATPase activity. An additional sample was incubated with EGTA and no added Ca2+to determine the basal ATPase activity. Membrane protein (200 pg) was incubated in 1 ml of ice-cold medium containing 50 m~ histidine, 3 mM MgClz, 100 mM KCl, and 50 p~ CaC12 with or without 2 m~ Tris/EGTA. All other reagents were purchased from the Sigma Chemical Co
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