Phospholipase C-Induced Aggregation and Fusion of Cholesterol—Lecithin Small Unilamellar Vesicles

Andrew S. Luk, Eric W. Kaler, Sum P. Lee

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35 Scopus citations

Abstract

We have investigated the effects of the Ca2+-requiring enzyme phospholipase C on the stability of sonicated vesicles made with different molar ratios of cholesterol to lecithin. Vesicle aggregation is detected by following turbidity with time. Upon the addition of phospholipase C and after a short lag period, the turbidity of a vesicle dispersion increases continuously with time. The rate of increase of turbidity increases with both the enzyme-to-vesicle ratio and the cholesterol content of the vesicles. Vesicle fusion and leakage of contents are monitored by a contents-mixing fusion assay using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX) as the fluorescence probes [Ellens, H., Bentz, J., & Szoka, F. C. (1985) Biochemistry 24, 3099–3106]. The results clearly show that phospholipase C induces vesicle fusion. The rate of vesicle fusion correlates with the enzyme-to-vesicle ratio but not with the cholesterol content of the membrane. Negligible aggregation and fusion of vesicles occurs when the experiment is repeated with buffer free of Ca2+. The membrane-destabilizing diacylglycerol, a product of lecithin hydrolysis by phospholipase C, is speculated to play a major role in driving the observed vesicle aggregation and fusion. The kinetics of vesicle aggregation and vesicle fusion can be predicted by linking Michaelis–Menten enzyme kinetics to a mass-action model.

Original languageEnglish (US)
Pages (from-to)6965-6973
Number of pages9
JournalBiochemistry
Volume32
Issue number27
DOIs
StatePublished - 1993

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