Assay, kinetics, and lysosomal localization of an acid cholesteryl esterase in rabbit aortic smooth muscle cells.

A sensitive radioisotope microassay has been developed for the estimation of cholesteryl ester hydrolase (EC 3.1.1.13) in preparations of isolated rabbit aortic smooth muscle cells. Kinetic studies served to establish optimal assay conditions, which involve incubation in a total volume of 0.2 ml containing 5 pM cholesteryl oleate tritiated in the cholesterol moiety, 0.4 mM egg yolk lecithin, 0.3 mu taurocholate, 25 pg of bovine serum albumin (defatted) per ml, and 0.05 M sodium acetate bufIer, pH 4.25. The labeled cholesterol was separated from the ester by thin layer chromatography with double development. Hydrolysis of as little as 1 pmole of substrate could be detected by this method. Under these conditions no measurable activity was found at neutral or alkaline pH in either phosphate or Tris buffer. The enzyme is several times more active on cholesteryl esters of unsaturated fatty acids than on those of saturated fatty acids in the Cl8 and CZO series. Among esters of even numbered saturated fatty acids (Cl2 to &Jr cholesteryl myristate (C,,) was hydrolyzed fastest. Fractionation studies indicate that cholesteryl esterase is associated with lysosomes in aortic smooth muscle cells.