Journal of Lipid Research, Vol 34, 211-217, Copyright © 1993 by Lipid Research, Inc.
Lipid vesicle fusion induced by phospholipase C activity in model bile
TE Little, H Madani, SP Lee and EW Kaler
Department of Medicine, University of Washington, Seattle 98195.
Using a system of phosphatidylcholine-cholesterol vesicles to model the
vesicle phase of mammalian bile (1:1 molar ratio) we evaluated whether very
small amounts of C. perfringens phospholipase C activity (0.5-6.5 nmol/min
per ml) could lead to vesicle fusion, a precursor step for cholesterol
precipitation in gallbladder bile. Quasielastic light scattering
spectroscopy (QLS) was used to monitor vesicle growth and aggregation in
model bile (0.89 mM total lipid) in the presence of phospholipase C.
Vesicle growth over 2 h could be detected with phospholipase activity as
little as 0.5 nmol/min per ml. Vesicle growth was sustainable over days in
the absence of Ca2+ once as little as 3-7 mol% diacylglycerol had been
generated as a result of the initial phospholipase C treatment. The
presence of fusion intermediates was confirmed using transmission electron
microscopy. In addition, kinetically slow vesicle fusion with intravesicle
content mixing and minimal leakage was also confirmed by fluorescence
spectroscopy using two populations of vesicles containing 5 mM TbCl3 or 50
mM dipicolinic acid. Efficient fusion (40% maximum fluorescence) was
obtained at 30 min at 25 degrees C with phospholipase C activity. This
level of enzyme activity approximates that found in human gallbladder bile
(1.2 nmol/min per ml). We conclude that the hydrolysis products of
phospholipase C activity can, in very small amounts (3-7 mol%
diacylglycerol), lead to destabilization and fusion of cholesterol-
saturated biliary vesicles. A reappraisal of the importance of
phospholipase C hydrolysis products in the pathogenesis of cholesterol
gallstones is warranted based on these observations.
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