title: Binding sites for cholesterol on Ca(2+)-ATPase studied by using a cholesterol-containing phospholipid.
abstract: Phosphatidylcholines have been synthesized containing a cholesterol moiety at the 2-position of the glycerol backbone. Fluorescence quenching studies show that cholesterol-containing phosphatidylcholines can bind at the lipid-protein interface of the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum, with an affinity half that of dioleoylphosphatidylcholine. The ATPase activity measured for the ATPase reconstituted with the cholesterol-containing phosphatidylcholine containing an oleoyl fatty acyl chain, (C18:1, CHS)PC, is less than that measured for the ATPase reconstituted with dioleoylphosphatidylcholine. The activity measured for the ATPase reconstituted with the cholesterol-containing phosphatidylcholine containing a myristoleoyl fatty acyl chain, (C14:1, CHS)PC, is less than that measured in (C18:1,CHS)PC and is comparable to that measured in dimyristoleoylphosphatidylcholine (di(C14: 1)PC. The stoichiometry of Ca2+ binding to the ATPase is two Ca2+ ions bound per ATPase molecule in the native membrane or in (C18:1,CHS)PC, but one bound per ATPase molecule in di(C14:1)PC or (C14: 1,CHS)PC. Addition of cholesterol to the ATPase in di(C14:1)PC or (C14:1,CHS)PC increases the Ca2+ binding stoichiometry to the usual 2:1, but the binding stoichiometry remains 1:1 in mixtures of di(C14: 1)PC and (C14:1,CHS)PC. Removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity for the ATPase in the native membrane, but an increase in fluorescence intensity for the ATPase in di(C14:1)PC or (C14:1,CHS)PC. Addition of cholesterol to the ATPase in di(C14:1)PC or (C14:1,CHS)PC reverses this change. It is concluded that cholesterol linked to a phospholipid molecule can interact with the ATPase only at the lipid-protein interface.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Southampton, U.K.
authors.forename: J
authors.initials: J
authors.lastname: Ding
authors.forename: A P
authors.initials: AP
authors.lastname: Starling
authors.forename: J M
authors.initials: JM
authors.lastname: East
authors.forename: A G
authors.initials: AG
authors.lastname: Lee
mesh_terms: D001665:Binding Sites; D001966:Bromine; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D010713:Phosphatidylcholines / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D010713:Phosphatidylcholines; D002784:Cholesterol; D000252:Calcium-Transporting ATPases; D001966:Bromine