Binding Sites for Cholesterol on Ca2+-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)

Lipid-protein interactions and Ca2+-ATPase function

“…2 of Meissner et al [6]. Reconstitution of Ca'+-ATPase with fluorescent phospholipids was carried out by the method of detergent dilution as described by Johannson et al [7).…”

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: Solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine

“…DOPC was chosen because it has been previously shown to maximize the recovery of activity when reconstituted [13,33]. To maximize the replacement of endogenous lipid, a high lipid-to-Ca# + -ATPase ratio of 1500 : 1 was used.…”

Ca2+-ATPase Activity and Lens Lipid Composition in Reconstituted Systems

“…Studies indicate that cholesterol may bind to non annular site(s) (Simmonds et al, 1982 ;Ding et al, 1994). When lipids are in the liquid crystalline phase, cholesterol has been shown to cause only a slight inhibition or no effect at all on Ca# + -ATPase activity (Johannsson et al, 1981 ;Madden, King and Quinn, 1981 ;Cheng et al, 1986).…”

“…Because lens lipid composition and structure change with age, cataract and region in human lenses (references above) and since Ca# + -ATPase activity has been shown to be sensitive to cholesterol content (Warren et al, 1975 ;Ding et al, 1994 ;Lee et al, 1994 ;Quin, Gomez and Madden, 1980), and to the crystalline phase of the lipid hydrocarbon chains (Squier, Bigelow and Thomas, 1988 ;Starling, East and Lee, 1995 ;Lee et al, 1995), the effect of these lipids on SERCA1 Ca# + -ATPase activity was investigated using SERCA1 type Ca# + -ATPase from rabbit muscle reconstituted into lens lipid and semi synthetic dihydrosphingomyelin vesicles enriched with cholesterol and phosphatidylcholine.…”