EFFECT OF UNESTERIFIED CHOLESTEROL ON THE ACTIVITY OF CHOLESTERYL ESTER TRANSFER PROTEIN

Cholesteryl ester transfer protein (CETP) catalyses the transfer of ch olesteryl ester from high-density lipoprotein to triacylglycerol-rich lipoproteins and the transfer of triacylglycerols in the reverse direc tion. The activity of CETP has been studied using a continuous fluores cence assay which measures the excimer fluorescence of cholesteryl 1-p yrene decanoate in a synthetic donor microemulsion as the indicator of cholesteryl ester transfer. Emulsions were composed of cholesteryl ol eate and egg phosphatidylcholine and had an average particle size of 1 4+/-1 nm as calculated from the molar volume of the components. The ef fect of changing the physical state of the emulsion surface was examin ed by including unesterified cholesterol in the donor and acceptor par ticles. The rate of CETP-induced transfer of the fluorescent cholester yl ester between microemulsion particles increased when unesterified c holesterol was present at concentrations up to 17 mol % relative to ph ospholipid. The presence of cholesterol also changed the exchange kine tics from an apparent single-exponential to a double-exponential pheno menon. Binding of CETP to the emulsion surface was accompanied by an e nhancement of fluorescence which was used to measure the binding equil ibria. The enhancement of exchange due to the presence of cholesterol did not correlate with any increased binding of CETP to the emulsion s urface. The presence of unesterified cholesterol in the donor did not affect the rate of transfer of the fluorescent cholesteryl ester when unlabelled emulsion was replaced by high-density lipoprotein as the ac ceptor. The studies demonstrate the use of microemulsions of defined s ize and composition for the study of the mechanism of action of CETP.