THE RELATIONSHIP BETWEEN PHOSPHOLIPID CONTENT AND CA2-ATPASE ACTIVITYIN THE SARCOPLASMIC-RETICULUM()

The relationship between the phospholipid composition of sarcoplasmic reticulum and the activity of the Ca2+, Mg2+-stimulated ATPase was ana lyzed by digestion of membrane phospholipids with phospholipase C and A(2) enzymes of diverse specificity and by detergent extraction. Phosp holipase C of Clostridium perfringens and Clostridium welchii, that hy drolyze preferentially phosphatidylcholine (PC), inhibited the Ca2+-AT Pase activity parallel with the depletion of phosphatidylcholine from the membrane. Phospholipase C of Bacillus cereus hydrolyzed in additio n to PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), ca using complete inhibition of Ca2+-stimulated ATPase activity. Digestio n of sarcoplasmic reticulum with the phospholipase A(2) of snake or be e venom produced similar effects. The phosphatidylinositol (PI)-specif ic 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 re versed by octaethyleneglycol dodecyl ether (C(12)E(8)). These experime nts suggest that the full phospholipid content of native sarcoplasmic reticulum (similar or equal to 100 mol phospholipid per mol Ca2+-ATPas e), is required for ATPase activity and there is no indication that PE , PS, and PI play a specific role in ATP hydrolysis. Extraction of sar coplasmic reticulum phospholipids by detergents such as deoxycholate, cholate and C(12)E(8) also caused proportional inhibition of ATPase ac tivity with the decrease in phospholipid content; the parallel extract ion of PC, PE and PI left the phospholipid composition largely unchang ed during delipidation. These observations do not support the requirem ent for a 'lipid annulus' of similar or equal to 30 phospholipid molec ules/Ca2+-ATPase as proposed by Hesketh et al. ((1976) Biochemistry 15 , 4145-4151) or the specific interaction of phosphatidylethanolamine w ith the ATPase molecule proposed by Bick et al. ((1991) Arch. Biochem. Biophys. 286, 346-352).