Effect of substrate physical state on the activity of acid cholesteryl ester hydrolase

The effects of the physicochemical properties of the substrate vehicle on the activity of acid cholesteryl ester hydrolase (ACEH; EC 3.1.1.13) isolated from rat liver lysosomes have been studied. In particular, the influence of the physical state of the neutral lipid core of substrate emulsion particles on the enzymatic activity has been probed in the light of previous studies on the clearance of cholesteryl esters (CE) from lipid-loaded cells which indicated that inclusions that are in the isotropic (liquid) state can be hydrolyzed faster than those in the anisotropic (liquid-crystalline) state. In the present study, such lipid inclusions were isolated from cultured cells and used as substrates for the hydrolase. No appreciable difference between the hydrolysis rates of isotropic and anisotropic inclusions was observed; the Vmax values were 93.0 +/- 6.7 and 84.0 +/- 3.3 nmol CE/mg.h, respectively. To elucidate the factors which affect the activity of ACEH, model inclusions were prepared by sonication and used as substrates. The physical state of these models was varied in a systematic way by changes of droplet composition and incubation temperature. The rate of hydrolysis was found to be insensitive to the physical state of the core of the model inclusions in good agreement with the results obtained with cellular inclusions. However, the activity of ACEH is sensitive to such interfacial properties of the lipid droplets as surface area available to the enzyme, net surface charge and surface solubility of the substrate CE molecules. The enzymatic activity is also sensitive to the amount of free cholesterol present in the emulsion droplets. The interfacial concentration and molecular packing of substrate CE molecules in the droplet surface significantly affect the hydrolytic activity of ACEH.