APPLICATION OF 2-HYDROXYPROPYL-BETA-CYCLODEXTRIN IN THE ASSAY OF ACYL-COA-CHOLESTEROL ACYLTRANSFERASE AND NEUTRAL AND ACID CHOLESTEROL ESTER HYDROLASES

The utility of 2-hydroxypropyl-beta-cyclodextrin for increasing the se nsitivity of assays for the microsomal acyl CoA:cholesterol acyltransf erase, 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 validat ed by assessing: (i) linearity of product formation with incubation ti me and protein amount, and saturation with substrate, and (ii) the eff ect of treatments of cells or of subcellular fractions on enzyme activ ities. Delivery of cholesterol dissolved in 2-hydroxypropyl-beta-cyclo dextrin to the acyl-CoA:cholesterol acyltransferase assay mixture rais ed the enzyme activity more than 8-fold and was twice that measured wh en cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-beta-cyclo dextrin itself was partially effective, apparently by making endogenou s cholesterol more accesible to the enzyme. Inclusion of 2-hydroxyprop yl-beta-cyclodextrin in cholesterol ester hydrolase assays using stand ard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esteras e activity by about 50%. Differences in the catalytic activity oi acyl -CoA:cholesterol acyltransferase and cholesterol ester hydrolases caus ed by treatment of hepatocytes with compound 58-035 or 25-hydroxychole sterol, or of subcellular fractions with NaF, were maintained when enz ymes were assayed with cyclodextrin. The results indicate that 2-hydro xypropyl-beta-cyclodextrin is a suitable vehicle for delivering choles terol to acyl-CoA:cholesterol acyltransferase and enhances the sensiti vity of standard assays of the enzymes governing the intrahepatic hydr olysis of cholesteryl esters.