Proton- and calcium-induced fusion and destabilization of liposomes

Abstract

A new liposome fusion assay has been developed that monitors the mixing of aqueous contents at neutral and low pH. With this assay we have investigated the ability of H+ to induce membrane destabilization and fusion. The assay involves the fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and its quencher N,N'-p-xylylenebis(pyridinium bromide) (DPX). ANTS is encapsulated in one population of liposomes and DPX in another, and fusion results in the quenching of ANTS fluorescence. The results obtained with the ANTS/DPX assay at neutral pH give kinetics for the Ca2+-induced fusion of phosphatidylserine large unilamellar vesicles (PS LUV) that are very similar to those obtained with the Tb3+/dipicolinic acid (DPA) assay [Wilschut, J., & Papahadjopoulos, D. (1979) Nature (London) 281, 690-692]. ANTS fluorescence is relatively insensitive to pH between 7.5 and 4.0. Below pH 4.0 the assay can be used semiquantitatively by correcting for quenching of ANTS due to protonation. For PS LUV it was found that, at pH 2.0, H+ by itself causes mixing of aqueous contents, which makes H+ unique among the monovalent cations. We have shown previously that H+ causes a contact-induced leakage from liposomes composed of phosphatidylethanolamine and the charged cholesteryl ester cholesteryl hemisuccinate (CHEMS) at pH 5.0 or below, where CHEMS becomes protonated. Here we show that H+ causes lipid mixing in this pH range but not mixing of aqueous contents. This result affirms the necessity of using both aqueous space and lipid bilayer assays to comprehend the fusion event between two liposomes.

Fungal extracellular ribotoxins as insecticidal agents

“…The breakdown of the permeability barrier of lipid bilayers can be analyzed using an assay employing the fluorescence probe 8-aminonaphtalene1,3,6-trisulfonic acid (ANTS) and its colisional quencher N,Np-xilene-bispyridinium bromide (DPX) (Ellens et al, 1985(Ellens et al, , 1986. When they are encapsulated into lipid vesicles, the release of the intravesicular content to the external medium results in dilution of probe and quencher, with the concomitant increase in ANTS fluorescence.…”

The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner

“…The phospholipid concentration was measured using the method of Bartlett (33). The leakage of encapsulated solutes was assayed as described by Ellens et al (34). The ANTS/ DPX-loaded lipid vesicles (final lipid concentration of 0.1 mM) were treated with the appropriate amounts of Bcl-x L ΔTM or its mutants in a fluorimeter cuvette at 37 °C with constant stirring.…”

Interaction of wheat α‐thionin with large unilamellar vesicles

“…For assays of vesicle leakage, the buffer contained in addition 25 mM ANTS and 90 mM DPX. For the assays of fusion, the buffer contained either 50 mM of ANTS or 180 mM of DPX (Ellens et al, 1985). Nonencapsulated fluorescent probes were separated from the vesicle suspension through a Sephadex G-75 gel filtration column (Pharmacia, Uppsala, Sweden) eluted with buffer.…”

“…Fusion of the vesicles (mixing of aqueous contents) was assayed as described by Ellens et al (1985). Briefly, liposomes containing 50 mM ANTS were mixed with liposomes containing 180 mM DPX in a 1 : 1 proportion.…”