Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes

This report describes a purification procedure for a cholesteryl ester hydr olase (CEH) from female rat liver microsomes, and some structural, immunolo gical, kinetic, and regulatory properties of the enzyme that distinguish th e microsomal CEH from other hepatic cholesteryl ester-splitting enzymes, CE H was purified 12.4-fold from reisolated microsomes using sequential solubi lization by sonication, polyethylene glycol precipitation, fractionation wi th hydroxyapatite, anion exchange chromatography, and chromatography on hyd roxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-f old over nonspecific esterase activity and 56-fold over triacylglycerol lip ase activity, In sharp contrast with most esterases and lipases, CEH did no t bind to concanavalin A-Sepharose and heparin-Sepharose, After polyacrylam ide gel electrophoresis, the purified enzyme exhibited two silver-stained b ands, but only the protein electroeluted from the low mobility band had CEH activity, Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence, The purified enzyme had optimal ac tivity at pH 6 and no taurocholate requirements, and was inhibited by the s erine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfh ydryl specific reagents, It hydrolyzed cholesteryl oleate much more efficie ntly than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat l iver microsomes contain a bile salt-independent catalytic protein that is r elatively specific for cholesteryl ester hydrolysis.