Study of the lecithin:Cholesterol acyltransferase reaction with liposome and high density lipoprotein substrates

Abstract

The activity of highly purified preparations of human plasma lecithin:cholesterol acyltransferase were stabilized by precipitating the enzyme with ammonium sulfate and using the dilutions of the particulate lecithin:cholesterol acyltransferase suspension for enzyme assays. Ammonium sulfate concentrations in the assay mix up to 0.1 M had no significant effect on lecithin:cholesterol acyltransferase activity. The basic enzymatic properties of lecithin : cholesterol acyltransferase were investigated using liposomes and high density lipoprotein (HDL) substrates. pH optima for both substrates was approximately 8.0. The temperature dependence of lecithin : cholesterol acyltransferase activity resulted in non-linear Arrhenius plots with both substrates. The activity vs. temperature (°C) curves showed slight inflections at 30°C, which may have been due to the relatively rapid inactivation of the enzyme above this temperature. HDL 3 was found to be a better substrate than HDL or HDL 2. HDL 3 was also considerably better than egg phosphatidylcholine/cholesterol liposomes as an lecithin:cholesterol acyltransferase substrate. Addition of HDL 2 to a reaction mix of enzyme and HDL, indicated that HDL 2 acts as an inhibitor of cholesterol esterification in this system.