Biochemical Society Transactions (1992) 20 349s Cooperative unit and enthalpy studies of cholesterol and cholesteryl esters in dimyristoylphosphatidylcholine liposomes.
(a)
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RICHARD J. MALCOLMSON*, PAUL H. BESWICK’, JOHN HIGINBOTHAM*, PIERRE 0. PRIVAP and LAURENT SAUNIER+.
Deparmnts of *Applied Chemical and Physical Sciences and +Biological Sciences, Napier Polytechnic. 10 Colinton Road. Edinburgh EHlO SDT, U.K. The reduction of the main chain-melting transition in phospholipid bilayers with increasing cholesterol concentration has been well documented [ 11. The effects of low concentrations of cholesterol, and particularly its esters, have been less weU studied, particularly with regard to the pretransition. Apolar esters are poorly soluble in polar lipids but their concentration in membranes varies from negligible in Schwann cell membranes to 8%in lysosomal membranes [2], with high concentrationsin malignant kidney cells [3] and in the pathogenesis of atherosclerosis [4]. Their presence affects the properties of membranes. Questions arise about the link between changes in lipid concentration and pathogenicity. To answer these one can use Differential Scanning Calorimetry @SC) to study the physical properties of model membranes i.e. Dimyristoylphosphatidylcholine (DMFT) liposomes containing varying amounts of cholesterol or its esters. This technique gives information on the enthalpy (AH) of phase transitions and the size of the cooperative unit (No), which is the average number of phospholipid molecules in microdomains of the non-dominant phase. No is given by dividing the van? Hoff enthalpy by AH. Liposomes were prepared by dissolving DMFT and cholesterol/ ester in chloroform-methanol(2: 1 v/v). The solvent was evaporated off in round-bottomed flasks under vacuum at 35OC. The vacuum was maintained for 3-4 hours after apparent evaporation with repeated flushing with N2 Liposome preparations were produced by mixing the lipid with an equal mass of 25 mM Tris-HCl, pH 7.2, containing 1 mM EDTA, at 35OC for 15 minutes in a rotary evaporator followed by agitation on a vortex mixer. For DSC measurements, a scan rate of 0.5 Wmin and sample mass of 15 mg were employed. At low concentrationsof all four added lipids ( 4 . 5 mol%), the enthalpies of the main and pretransitions were increased while No was decreased. At higher concentrations (0.5-3 mol%), the enthalpies of both transitions were reduced while No showed less of an obvious trend. In the case of the pretransition, the greatest effect was observed with cholesterol while the cholesteryl esters, particularly the unsaturated oleate (18:l) and linoleate (18:2), had a greater effect on the main transition. It was also noted that for the unsaturated esters, concentrationsabove 1.5 mol% resulted in the main transition on the DSC curve being composed of two overlapping components, indicating phase separation and thus placing an upper limit on their solubility in DMPC. For the saturated ester, cholesteryl stearate (18:O). no such effect was observed and the upper limit of solubility is thought to be at least 2 mol%. It is known from NMR studies [ 5 ] that phospholipid head-group disorder increases by ca. 20% during the pretransition while the acyl chains remain in the gel phase. Therefore, it may be that the pretransition is governed mainly by changes in head-group interactions at the bilayer surface which are modified by the presence of the polar -OH group of cholesterol while the less polar ester linkage (COO) will cause a less significant Abbreviations used: DSC, differential scanning calorimetry; AH, phase transition enthalpy; DMPC, dimyristoylphosphatidylcholine;NMR. nuclear magnetic resonance; No, cooperative unit.
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R Fig. 1. Effect of cholesterollcholesterylester concentrntionson AH (a)and No (b)for the pretransition of DMPC liposomes. (A - cholesteryl stearate; 0 - cholesteryl linoleate; W - cholesteryl oleate; 0 - cholesterol). A representation of the error in the data has been shown for 2 mo18 cholesteryl stearate. (Note: 1 cal = 4.184 J).
perturbation. The main transition is the result of formerly all-trans phospholipid acyl chains gaining motional freedom and adopting gauche configurations. The sterically “bulkier” esters (particularly the unsaturated esters) possibly disrupt chain packing in the gel phases to a greater extent than cholesterol, giving a reduction in the number of all-trans chains. The additional acyl chain possessed by the esters may help to order phospholipid chains to a greater extent than cholesterol in the liquid crystalline phase and further reduce the difference between the rippled gel and liquid crystalline phases. Accordingly, the reduction in main transition enthalpy at 1.5 mol% ester is similar to that at 3 mol% cholesterol. This work was supported by a grant from the Association for
International Cancer Research. 1. Mabrey, S., Mateo, P.L. & Sturtevant, J.M.(1990)Biochemistry 1 7 ( 1 2 ) , 2464-2468. 2. Parsons, D.S. (1975) Bio/ogica/Membranes,1st edn., p.24,
Clarendon Press, Oxford. 3. Gebhard, R.L., Clayman, R.V., Prigge, W.F., Figenshau, R., Staley, N.A., Reesey,C. & Bear, A. (1987)J. Lip. Res. 2 8,1177-1184. 4. Gorrissen, H., Tulbch, A.P. & Cushley, R.J. (1980) Biochem. J. 1 9, 3422-3429. 5. Sackmann, E., (1980) in Lquid Crystals of One- and TwoDimensional Order (Helfrich,W. & Heppke, G., eds.)p.310, Springer-Verlag,Berlin. New York.
