EVIDENCE THAT THE EFFECTS OF PHOSPHOLIPIDS ON THE ACTIVITY OF THE CA2-ATPASE DO NOT INVOLVE AGGREGATION()

The Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum, solubilized in monomeric form in C(12)E(8), has been reconstituted by dialysis in to sealed vesicles of dioleoyl phosphatidylcholine [di(C-18:1)PC], dim yristoleoyl phosphatidylcholine [di(C-14:1)PC], dinervonyl phosphatidy lcholine [di(C-24:1)PC] or dipalmitoyl phosphatidylcholine [di(C-16:0) PC] in the gel phase, at a phospholipid/ATPase molar ratio of 10 000:1 . Cross-linking experiments show that ATPase molecules are present in these reconstituted vesicles as isolated monomeric species. ATPase act ivities for the reconstituted vesicles are about half of those for the ATPase reconstituted with the same lipid in unsealed membrane fragmen ts, attributed to a close to random orientation for the ATPase molecul es in the reconstituted vesicles. ATPase activities for the ATPase in reconstituted vesicles of di(C-14:1)PC or di(C-24:1)PC are less than i n vesicles of di(C-18:1)PC, and no activity could be detected for the ATPase in di(C-16:0)PC in the gel phase. It is concluded that effects of lipids on the activity of the ATPase are independent of any changes in the state of aggregation of the ATPase. Inhibition of ATPase activ ity by spermine and by the hydrophilic domain of phospholamban are obs erved both for the unreconstituted ATPase and for the ATPase in recons tituted vesicles, so that inhibition is independent of any aggregation caused by these polycationic species. Stimulation of ATPase activity by jasmone is also observed both for the unreconstituted ATPase and fo r the ATPase in reconstituted vesicles, so that stimulation of the ATP ase also does not follow from any change in the state of aggregation o f the ATPase.