APPLICATION OF 2-HYDROXYPROPYL-BETA-CYCLODEXTRIN IN THE ASSAY OF ACYL-COA-CHOLESTEROL ACYLTRANSFERASE AND NEUTRAL AND ACID CHOLESTEROL ESTER HYDROLASES
M. Liza et al., APPLICATION OF 2-HYDROXYPROPYL-BETA-CYCLODEXTRIN IN THE ASSAY OF ACYL-COA-CHOLESTEROL ACYLTRANSFERASE AND NEUTRAL AND ACID CHOLESTEROL ESTER HYDROLASES, Lipids, 31(3), 1996, pp. 323-329
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.