Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver.
The effect of various physicochemical forms of substrate on the activity of acid cholesteryl ester hydrolase isolated from rat liver lysosomes was studied. The amount of sodium taurocholate was varied in the substrate mixture which contained constant amounts of egg phosphatidylcholine (PC) and cholesteryl oleate. The resulting substrate forms produced were PC vesicles, PC vesicles with incorporated sodium taurocholate, mixed micelles, and mixed micelles together with free bile salt micelles. Gradually increasing amounts of sodium taurocholate activated cholesteryl oleate hydrolysis until the molar sodium taurocholate/PC ratio of ca. 0.6; thereafter hydrolytic activity decreased rapidly. The presence of sodium taurocholate micelles clearly inhibits cholesteryl oleate hydrolysis. We therefore propose that the activation observed at low bile salt concentrations depends on bile salt interaction with the substrate vehicle, whereas the inhibition observed at high bile salt concentrations depends on sodium taurocholate interacting with the enzyme. When comparing different phospholipid components in the supersubstrate, the enzyme activity was highest in the presence of dioleoyl PC and decreased when present with dipalmitoyl PC and egg PC. Egg lysoPC completely inhibited the enzyme activity. A net negative charge on the surface of the vesicle substrate increased cholesteryl ester hydrolase activity while a net positive charge on the surface inhibited the enzyme activity. Only part of the product inhibition of cholesteryl oleate hydrolase caused by Na-oleate was reversible when tested with bovine serum albumin present in the incubation mixture.
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D002787:Sterol Esterase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013656:Taurocholic Acid / Q000494:pharmacology
D009829:Oleic Acids; D010743:Phospholipids; D019301:Oleic Acid; D013656:Taurocholic Acid; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase