Application of 2-hydroxypropyl-beta-cyclodextrin in the assay of acyl-CoA:cholesterol acyltransferase and neutral and acid cholesterol ester hydrolases

Abstract

The utility of 2-hydroxypropyl-beta-cyclodextrin for increasing the sensitivity of assays for the microsomal acylCoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-beta-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-beta-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.