Journal of Lipid Research, Vol 17, 239-247, Copyright © 1976 by Lipid Research, Inc.
The use of phospholipid vesicles for in vitro studies on cholesteryl ester hydrolysis
P Brecher, J Chobanian, DM Small and AV Chobanian
Radiolabeled cholesteryl oleate was incorporated into vesicles prepared
from egg yolk lecithin and utilized as a substrate for studies of sterol
ester hydrolases present in rat liver homogenates. The cholesteryl oleate
was shown to be associated with vesicles (unilamellar liposomes) using
Sepharose 4B chromatography. With this substrate, two different cholesteryl
ester hydrolytic enzymes were demonstrated in subcellular fractions from
the liver homogenates. In the lysosome-rich fraction an acid hydrolase was
present, while in the cytosol fraction (150,000 g supernatant), hydrolytic
activity was shown to occur with an optimum pH between 8 and 8.5. The
substrate was characterized by Sepharose chromatography both before and
after incubation with the liver fraction and was not dramatically altered
even by rigorous incubation conditions. The lysosomal enzyme preparation
was capable of hydrolyzing almost all the cholesteryl oleate in the
vesicles. Hydrolysis of the phospholipid was proportionately much less than
that of the cholesteryl oleate. Comparisons were performed between the
vesicle preparation and an alternate substrate preparation involving the
direct addition of cholesteryl oleate in acetone solution. The vesicles
appeared to be a better substrate for the lysosomal enzyme whereas the
activity in the cytosol fraction did not distinguish between the two
substrate preparations. Unsonicated suspensions of cholesteryl oleate and
lecithin did not serve as suitable substrates for the enzymes. These
studies demonstrate the applicability of cholesteryl ester-containing
vesicles as a useful substrate for studying cholesteryl ester hydrolysis in
vitro.
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