BINDING-SITES FOR CHOLESTEROL ON CA2-ATPASE STUDIED BY USING A CHOLESTEROL-CONTAINING PHOSPHOLIPID()

Phosphatidylcholines have been synthesized containing a cholesterol mo iety at the 2-position of the glycerol backbone. Fluorescence quenchin g studies show that cholesterol-containing phosphatidylcholines can bi nd at the lipid-protein interface of the Ca2+-ATPase from skeletal mus cle sarcoplasmic reticulum, with an affinity half that of dioleoylphos phatidylcholine. The ATPase activity measured for the ATPase reconstit uted with the cholesterol-containing phosphatidylcholine containing an oleoyl fatty acyl chain, (C18:1,CHS)PC, is less than that measured fo r the ATPase reconstituted with dioleoylphosphatidylcholine. The activ ity measured for the ATPase reconstituted with the cholesterol-contain ing phosphatidylcholine containing a myristoleoyl fatty acyl chain, (C 14:1,CHS)PC, is less than that measured in (C18:1,CHS)PC and is compar able to that measured in dimyristoleoylphosphatidylcholine (di(C14:1)P C. The stoichiometry of Ca2+ binding to the ATPase is two Ca2+ ions bo und per ATPase molecule in the native membrane or in (C18:1,CHS)PC, bu t one bound per ATPase molecule in di(C14:1)PC or (C14:1,CHS)PC. Addit ion of cholesterol to the ATPase in di(C14:1)PC or (C14:1,CHS)PC incre ases 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 Ca2+-bound ATPase results in a decrease in tryptophan fluorescence intensity for the ATPase in the native membran e, 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 chol esterol linked to a phospholipid molecule can interact with the ATPase only at the lipid-protein interface. Free cholesterol, although large ly excluded from the lipid-protein interface, can bind at other hydrop hobic sites on the ATPase. It is suggested that these sites could be l ocated between transmembrane alpha-helices.