Ca 2+-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles

Abstract

The effect of cholesterol on the Ca 2+-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca 2+ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c 11 , and of the fusion reaction itself, f 11 . In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c 11 and f 11 increase steeply with the Ca 2+ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c 11 decreases, while the rate of the actual fusion reaction, f 11 , increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.