Phospholipase C-induced aggregation and fusion of cholesterol-lecithin small unilamellar vesicles.
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.
Luk
A S
AS
Department of Chemical Engineering, University of Delaware, Newark 19716.
Kaler
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EW
Lee
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SP
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*
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