Resistance of Ca2+-ATPase to dilution by excess phospholipid in reconstituted vesicles

Ca2+-ATPase and other membrane proteins of the sarcoplasmic reticulum membrane from rabbit skeletal muscle have been reconstituted into lipid vesicles with increasing amounts of phosphatidylcholine. The protein composition and phospholipid concentration of these vesicles were analyzed by determining the density of the reconstituted membrane vesicles on linear H2O-2H2O gradients, in a constant concentration of sucrose. In all combinations of the Ca2+-ATPase with a weight excess of phosphatidylcholine, the reconstitute vesicles had a phospholipid-to-protein ratio similar to that of the native sarcoplasmic reticulum membrane, even though both solubilization and mixing had occurred. These vesicles of low phospholipid and high protein content exhibited all the original Ca2+-ATPase activity and ATP-stimulated calcium transport. The Ca2+-ATPase, and the calcium-binding to a lesser extent, may order the lipid in such a manner so as to maintain the initial stoichiometry of lipid to protein observed in the native sarcoplasmic reticulum membrane.