The sarcoplasmic reticulum is a lipid-protein membrane system that regulates the intracellular concentration of free Ca 2 ions in skeletal and cardiac muscle. The Ca 2 ions ATPase enzyme, which is the main protein component of isolated sarcoplasmic reticulum, has been identified as the Ca 2 ion transporting protein or Ca 2 ion pump. The mode of involvement of phospholipids in Ca 2 ions transport will be studied with purified preparations of sarcoplasmic reticulum from skeletal and cardiac muscle. The role of phospholipids on the enzymatic and physico-chemical properties of the Ca 2 ion ATPase enzyme will be investigated by replacing the lipids of the purified ATPase with chemically well-defined species. The investigations will include detailed kinetic analysis and EPR studies with paramagnetic spin labels incorporated into either the lipid or the protein moiety. The role of phospholipids in Ca 2 ion transport will be analyzed by modifying the polar head group of phosphatidyl ethanolamine and studying the effect on the coupling between ATP hydrolysis and Ca 2 ion transport. Reconstitution of membrane vesicles from the purified Ca 2 ion ATPase, calsequestrin and phospholipids with different polar head groups and fatty acid chains will be attempted. The information obtained in all these studies will be used to reconstitute a system that can carry out Ca 2 ion transport coupled to the hydrolysis of ATP as efficiently as the native sarcoplasmic membranes do. The lipid content and composition of a purified cardiac sarcoplasmic reticulum fraction will be analyzed. The requirement for phospholipids in cardiac Ca 2 ion transport and Ca2 ion-ATPase activity will be investigated by delipidation and phospholipid replacement experiments.
