Purification of rat liver lysosomal cholesteryl ester hydrolase.
An acidic cholesteryl ester hydrolase (EC 3.1.1.13) from rat liver lysosomes was purified approximately 120-fold with 5% recovery of the original homogenate activity. The sequential steps were: digitonin solubilization, agarose gel filtration, DEAE-agarose and CM-agarose column chromatography. The enzyme was at least 90%  pure as judged by polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. It exhibited a molecular weight of about 60 000 as determined by sodium dodecyl sulfate polyacrylamide electrophoresis and gel filtration. The soluble enzyme required substrate which was incorporated into phospholipid vehicles for optimal activity. On the contrary, aggregates of the enzyme required a substrate preparation that involved the direct addition of cholesteryl ester in acetone. The enzyme also catalyzed the hydrolysis of emulsions of triacylglycerol. The ratio of the two activities remained almost constant during purification suggesting that the two activities (EC 3.1.1.13 and EC  3.1.1.3, respectively) may be the result of the broad specificity of one enzyme. The effects of some inhibitors and some properties of the enzyme have been studied and discussed.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D002847:Chromatography, Agarose; D004072:Digitonin; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D011092:Polyethylene Glycols; D051381:Rats; D002787:Sterol Esterase / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000201:enzymology
D011092:Polyethylene Glycols; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase; D004072:Digitonin; D002118:Calcium