The relationship between phospholipid content and Ca2+-ATPase activity in the sarcoplasmic reticulum

The relationship between the phospholipid composition of sarcoplasmic reticulum and the activity of the Ca2+, Mg2+-stimulated ATPase was analyzed by digestion of membrane phospholipids with phospholipase C and A2 enzymes of diverse specificity and by detergent extraction. Phospholipase C of Clostridium perfringens and Clostridium welchii, that hydrolyze preferentially phosphatidylcholine (PC), inhibited the Ca2+-ATPase activity parallel with the depletion of phosphatidylcholine from the membrane. Phospholipase C of Bacillus cereus hydrolyzed in addition to PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), causing complete inhibition of Ca2+-stimulated ATPase activity. Digestion of sarcoplasmic reticulum with the phospholipase A2 of snake or bee venom produced similar effects. The phosphatidylinositol (PI)-specific phospholipases of B. cereus and Bacillus thuringiensis caused less than 10% inhibition of the Ca2+-ATPase, accompanied by the hydrolysis of more than 70% of the phosphatidylinositol content of the membrane, without significant change in PC, PE and PS content. The inhibition of ATPase activity by the C type phospholipases was nearly completely reversed by octaethyleneglycol dodecyl ether (C12E8). These experiments suggest that the full phospholipid content of native sarcoplasmic reticulum (congruent to 100 mol phospholipid per mol Ca2+-ATPase), is required for ATPase activity and there is no indication that PE, PS, and PI play a specific role in ATP hydrolysis. Extraction of sarcoplasmic reticulum phospholipids by detergents such as deoxycholate, cholate and C12E8 also caused proportional inhibition of ATPase activity with the decrease in phospholipid content; the parallel extraction of PC, PE and PI left the phospholipid composition largely unchanged during delipidation. These observations do not support the requirement for a 'lipid annulus' of congruent to 30 phospholipid molecules/Ca2+-ATPase as proposed by Hesketh et al. ((1976) Biochemistry 15, 4145-4151) or the specific interaction of phosphatidylethanolamine with the ATPase molecule proposed by Bick et al. ((1991) Arch. Biochem. Biophys. 286, 346-352).