Submitted on December 22, 2004
Revised on January 14, 2005
Accepted on February 7, 2005
Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases
Yassine Ben Ali, Frederic Carriere, Robert Verger, Stefan Petry, Gunter Muller, and Abdelkarim Abousalham
EIPL-UPR9025, CNRS, Marseille 13009
Corresponding Author: abousal{at}ibsm.cnrs-mrs.fr
Hormone-sensitive lipase (HSL) is thought to contribute importantly to the hydrolysis of cholesterol ester in steroidogenic tissues releasing the cholesterol required for adrenal steroidogenesis. HSL has a broad substrate specificity, since it hydrolyzes triacylglycerols (TAG), diacylglycerols, monoacylglycerols, as well as cholesteryl- and retinyl-esters. In this study, we developed a specific cholesterol esterase assay using CO dispersed in phosphatidylcholine (PC) and gum arabic (GA) by sonication. To continuously monitor the hydrolysis of cholesterol oleate (CO) by pure recombinant HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other purified cholesterol ester-hydrolyzing enzymes. The specific activities measured on CO dispersed in PC and GA were found to be 18, 100, 27 and 3 µmol/min/mg in the case of HSL, cholesterol esterase from Pseudemonas species, Candida rugosa lipase-3 and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is about 4- to 5-fold higher than on long-chain TAG. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were found to be higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro add further molecular insight on the physiological importance of HSL in cholesterol ester catabolism in vivo. Thus HSL could be considered more as a cholesterol ester hydrolase than TAG lipase.
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