Abstract
This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, 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 activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis.—Cristóbal, S., B. Ochoa, and O. Fresnedo. Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes. J. Lipid Res. 1999. 40: 715–725.
Highlights
This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes
The differential CEH purification was assessed by testing cholesteryl esterase, triacylglycerol lipase, and nonspecific esterase activity
A number of properties, including differential purification, N-terminal amino acid sequence, no affinity for heparin and concanavalin A (ConA), kinetic and immunological properties, and sensitivity to metal ions and to serine and sulfhydryl specific reagents, distinguish the enzyme described in this study from the microsome resident proteins triacylglycerol lipase, nonspecific carboxylesterase, and retinyl palmitate hydrolase, from the hepatic lipase that transits the secretory pathway and from the homologous cholesterol ester hydrolases located in cytosol and lysosomes
Summary
This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p -nitrophenyl acetate was higher than with p -nitrophenyl butyrate. Described below is a purification strategy for a cholesteryl ester hydrolase from rat liver microsomes using conventional protein purification procedures, preparation of neutralizing antibodies, and characterization of some properties of this enzyme. On the basis of differential purification, and structural, immunological, kinetic and regulatory features, the purified CEH can be clearly differentiated from the cytosolic CEH, the lysosomal CEH, the hepatic lipase, the retinyl ester hydrolase, the triacylglycerol lipase, and nonspecific microsomal carboxylesterases
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