Effect of unesterified cholesterol on the activity of cholesteryl ester transfer protein.
Cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from high-density lipoprotein to triacylglycerol-rich lipoproteins and the transfer of triacylglycerols in the reverse direction. The activity of CETP has been studied using a continuous fluorescence assay which measures the excimer fluorescence of cholesteryl 1-pyrene decanoate in a synthetic donor microemulsion as the indicator of cholesteryl ester transfer. Emulsions were composed of cholesteryl oleate and egg phosphatidylcholine and had an average particle size of 14 +/- 1 nm as calculated from the molar volume of the components. The effect of changing the physical state of the emulsion surface was examined by including unesterified cholesterol in the donor and acceptor particles. The rate of CETP-induced transfer of the fluorescent cholesteryl ester between microemulsion particles increased when unesterified cholesterol was present at concentrations up to 17 mol% relative to phospholipid. The presence of cholesterol also changed the exchange kinetics from an apparent single-exponential to a double-exponential phenomenon. Binding of CETP to the emulsion surface was accompanied by an enhancement of fluorescence which was used to measure the binding equilibria. The enhancement of exchange due to the presence of cholesterol did not correlate with any increased binding of CETP to the emulsion surface. 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 acceptor. The studies demonstrate the use of microemulsions of defined size and composition for the study of the mechanism of action of CETP.
Rajaram
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OV
Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Chan
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RY
Sawyer
W H
WH
D002352:Carrier Proteins / Q000378:metabolism*; D002784:Cholesterol / Q000494:pharmacology*; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism; D004655:Emulsions; D006023:Glycoproteins; D006801:Humans; D007700:Kinetics; D008297:Male; D010713:Phosphatidylcholines / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014316:Tritium
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D004655:Emulsions; D006023:Glycoproteins; D010713:Phosphatidylcholines; C090923:cholesteryl 1-pyrene decanoate; D014316:Tritium; C014409:cholesteryl oleate; D002784:Cholesterol