Polyethylene glycol-induced lipid mixing but not fusion between synthetic phosphatidylcholine large unilamellar vesicles

Abstract

We have examined the effect of poly(ethylene glycol) (PEG) on stable large unilamellar vesicles formed by a rapid extrusion technique and composed of pure synthetic phosphatidylcholines. The lipid systems studied were the saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the monounsaturated 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). PEG at all concentrations (3.8-40 wt %) induced lipid mixing between large vesicles composed of these phosphatidylcholines. Extensive leakage of internal contents also occurred at high PEG concentrations. However, in contrast to our previous report [Parente, R. A., & Lentz, B. R. (1986) Biochemistry 25, 6678], we could detect no mixing of internal contents indicative of fusion. This discrepancy is due to environmental factors that affect the behavior of 8-amino-naphthalene-1,3,6-trisulfonic acid (ANTS), the fluorophore used in the assay for contents mixing and leakage [McIntyre, Parks, Massenburg, & Lentz (1991) (submitted)]. In agreement with the results of the fusion assay, quasielastic light-scattering measurements revealed no increase in vesicle size following treatment with PEG. These results emphasize the importance of using assays for both membrane mixing and contents mixing to demonstrate fusion, since significant lipid mixing occurred in the absence of fusion. We conclude that large vesicles composed of pure phosphatidylcholine do not fuse in the presence of even high concentrations of PEG. However, DOPC vesicles containing a small amount of an amphipathic "impurity" have been shown to fuse in the presence of PEG at 23 degrees C. These results are discussed in terms of their implications for the mechanism of PEG-induced membrane fusion.

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

“…If fusion is occurring, this observation indicates that it must be hemi-fusion, in which only the outer monolayer lipids mix at bilayer contact sites. Hemi-fusion can occur when 2 bilayers are brought into close physical contact (Burgess et al, 1991), but does not always result from vesicle aggregation (Fujii et al, 1993). The electrostatic aggregation of POPG vesicles by HNP-2 could lead to such close contact, and the amphipathic nature of the peptide could lead to the sort of bilayer destabilization that promotes hemi-fusion (Siegel, 1993).…”

Spontaneous lipid transfer between organized lipid assemblies

“…Other manipulations of the aqueous mileu known to affect spontaneous interbilayer lipid transfer involve addition of chaotropic salts [69,72] or poly(ethylene)glycol [130][131]. In such cases, it is difficult to distinquish whether the primary effect is restructuring of the bulk aqueous milieu or the highly polarized hydration zone associated with the surface of organized lipid assemblies.…”

“…Based on their results, Lentz and co-workers [130][131] proposed that poly(ethylene glycol) enhances the rate of intervesicular lipid transfer by dehydrating and aggregating the PC vesicles thereby altering the properties of the aqueous gap between the membrane surfaces.…”

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

“…The lipid mix was dissolved in chloroform [33], the bulk of the organic solvent was removed by evaporation under a stream of N 2 gas, and the remaining trace level of chloroform was removed by incubation for 30 min under vacuum. The dried lipid film was re-hydrated with 20 mM HEPES, pH 7.4, containing 100 mM KCl for 30 min, vortexed for 5 min, and subjected to 5 freeze-thaw cycles.…”