INTERACTION OF PHOSPHATIDIC-ACID AND PHOSPHATIDYLSERINE WITH THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM AND THE MECHANISM OF INHIBITION()

The sarcoplasmic reticulum of skeletal muscle contains anionic phospho lipids as well as the zwitterionic phosphatidylcholine and phosphatidy lethanolamine. Here we study the effects of anionic phospholipids on t he activity of the Ca2+-ATPase purified from the membrane. Reconstitut ion of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C-18:1)PS] or dioleoylphosphatidic acid [di(C-18:1)PA] leads to a decrease in ATP ase activity. Measurements of the quenching of the tryptophan fluoresc ence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcho line close to 1 and suggest that phosphatidic acid only binds to the A TPase at the bulk lipid sites around the ATPase. Addition of di(C-18:1 )PS or di(C-18:1)PA to the ATPase in the short-chain dimyristoleoylpho sphatidylcholine [di(C-14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the resp onse of tryptophan fluorescence intensity to Ca2+ binding. It is concl uded that the lipid headgroup and lipid fatty acyl chains have separat e effects on the function of the ATPase. The anionic phospholipids hav e no significant effect on Ca2+ binding to the ATPase; the level of Ca 2+ binding to the ATPase, the affinity of binding and the rate of diss ociation of Ca2+ are unchanged by reconstitution into di(C-18:1)PA. Th e major effect of the anionic lipids is a reduction in the maximal lev el of binding of MgATP. This is attributed to the formation of oligome rs of the Ca2+-ATPase, in which only one molecule of the ATPase can bi nd MgATP dimers in di(C-18:1)PS and trimers or tetramers in di(C-18:1) PA. The rates of phosphorylation and dephosphorylation for the proport ion of the ATPase still able to bind ATP are unaffected by reconstitut ion. Larger changes were observed in the level of phosphorylation of t he ATPase by P-1, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase labelled with 4-(brom omethyl)-6,7-dimethoxycoumarin was also changed in di(C-18:1)PS and di (C-18:1)PA, so that effects of Mg2+ became comparable with those seen on phosphorylation for the unreconstituted ATPase. The anionic phospho lipids could induce a conformational change in the ATPase on binding M g2+ equivalent to that normally induced by phosphorylation or by bindi ng inhibitors such as thapsigargin.