title: Phospholipase C-induced aggregation and fusion of cholesterol-lecithin small unilamellar vesicles.
abstract: We have investigated the effects of the Ca(2+)-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.
authors.affiliation: Department of Chemical Engineering, University of Delaware, Newark 19716.
authors.forename: A S
authors.initials: AS
authors.lastname: Luk
authors.forename: E W
authors.initials: EW
authors.lastname: Kaler
authors.forename: S P
authors.initials: SP
authors.lastname: Lee
mesh_terms: D002784:Cholesterol / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008051:Lipid Bilayers; D008561:Membrane Fusion; D009281:Naphthalenes / Q000737:chemistry; D010713:Phosphatidylcholines / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000737:chemistry; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D009281:Naphthalenes; D010713:Phosphatidylcholines; D011726:Pyridinium Compounds; C046466:N,N'-4-xylylenebis(pyridinium); D002784:Cholesterol; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D010738:Type C Phospholipases