title: Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.
abstract: The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin was also changed in di(C18:1)PS and di(C18:1)PA, so that effects of Mg2+ became comparable with those seen on phosphorylation for the unreconstituted ATPase. The anionic phospholipids could induce a conformational change in the ATPase on binding Mg2+ equivalent to that normally induced by phosphorylation or by binding inhibitors such as thapsigargin.
authors.affiliation: Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, U.K.
authors.forename: K A
authors.initials: KA
authors.lastname: Dalton
authors.forename: J M
authors.initials: JM
authors.lastname: East
authors.forename: S
authors.initials: S
authors.lastname: Mall
authors.forename: S
authors.initials: S
authors.lastname: Oliver
authors.forename: A P
authors.initials: AP
authors.lastname: Starling
authors.forename: A G
authors.initials: AG
authors.lastname: Lee
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002413:Cations, Divalent; D003374:Coumarins / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D010712:Phosphatidic Acids / Q000378:metabolism* / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000838:Anions; D002413:Cations, Divalent; D003374:Coumarins; D005456:Fluorescent Dyes; D010712:Phosphatidic Acids; D010718:Phosphatidylserines; C053769:4-bromomethyl-6,7-dimethoxycoumarin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine