Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.     

Continuous fluorometric assay of Ca2+ transport by liposomes with Quin 2 entrapped: effect of phospholipase A2 and unsaturated long-chain fatty acids

The amount of free calcium in the cytoplasm is important in stimulation coupled with a number of cellular functions. The putative ionophoretic action of membrane lipid metabolites on Ca2+ offers convenient explanation of the stimulation-coupled mobilization of cytoplasmic Ca2+. To analyze the ionophoretic action of the lipid metabolites, we devised a sensitive method to study Ca2+ transport that uses liposome-entrapped Quin 2. A calcium ionophore, A23187, increased the fluorescence intensity of the Ca2+-Quin 2 complex as a function of Ca2+ transport into liposomes. A similar Ca2+ flux into the liposomes was induced by phospholipase A2 (PLA2) and by various long-chain fatty acids in liposomes that consist of phospholipids containing unsaturated fatty acids. The potencies of the fatty acids for Ca2+ transport is inversely correlated with their melting points. The oxidized products of the unsaturated fatty acids increased the Ca2+ and nonspecific permeability of the biological membranes. These results suggest that stimulation-coupled PLA2 activation might mediates the mobilization of cytoplasmic Ca2+.     

Formation of giant liposomes promoted by divalent cations: critical role of electrostatic repulsion.

Spontaneous formation of giant unilamellar liposomes in a gentle hydration process, as well as the adhesion energy between liposomal membranes, has been found to be dependent on the concentration of divalent alkali cations, Ca2+ or Mg2+, in the medium. With electrically neutral phosphatidylcholine (PC), Ca2+ or Mg2+ at 1-30 mM greatly promoted liposome formation compared to low yields in nonelectrolyte or potassium chloride solutions. When negatively charged phosphatidylglycerol (PG) was mixed at 10%, the yield was high in nonelectrolytes but liposomes did not form at 3-10 mM CaCl2. In the adhesion test with micropipette manipulation, liposomal membranes adhered to each other only in a certain range of CaCl2 concentrations, which agreed with the range where liposome did not form. The adhesion range shifted to higher Ca2+ concentrations as the amount of PG was increased. These results indicate that the divalent cations bind to and add positive charges to the lipids, and that membranes are separated and stabilized in the form of unilamellar liposomes when net charges on the membranes produce large enough electrostatic repulsion. Under the assumption that the maximum of adhesion energy within an adhesive range corresponds to exact charge neutralization by added Ca2+, association constants of PC and PG for Ca2+ were estimated at 7.3 M(-1) and 86 M(-1), respectively, in good agreement with literature values.     

Spontaneous lipid vesicle fusion with electropermeabilized cells

Fusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process.     

Induction of Ca2+ transport in liposomes by insulin.

The requirement of extracellular Ca2+ for insulin action has been indicated by past studies. With a view to understand the interaction of insulin with Ca2+ in the vicinity of the cell membrane, we have examined the ability of insulin and its constituent polypeptide chains A and B to translocate Ca2+ and Mg2+ across the lipid bilayer in two sets of synthetic liposomes. The first were unilamellar vesicles made of dimyristoylphosphatidylcholine and contained the Ca2+ sensor dye arsenazo III. Peptide-mediated Ca2+ and Mg2+ transport in these vesicles was monitored at 37 degrees C in a neutral buffer containing CaCl2 or MgCl2 using a difference absorbance method. In the second set, multilamellar vesicles of egg lecithin containing trapped fura-2 were employed and the cation transport was followed at 20 degrees C by fluorescence changes in the dye. Control experiments indicated that the hormonal peptides caused no appreciable perturbation of the vesicles leading to leakage of contents or membrane fusion. In both liposome systems, substantial Ca2+ and Mg2+ transport was observed with insulin and the B chain; the A chain was less effective as an ionophore. Quantitative analysis of the transport kinetic data on the B chain showed a 1:1 peptide-Ca2+ complex formed inside the membrane. In light of the available structural data on Ca2+ binding by insulin and insulin receptor, our results suggest the possibility of the hormone interacting with the receptor with the bound Ca2+.     

Synergistic effects of micromolar concentrations of Zn2+ and Ca2+ on membrane fusion

Resonance Energy Transfer between N-(7-nitro-2,1,3 benzoxadiazol -4 yl) phosphatidyl ethanolamine and N-Lissamine-Rhodamine B sulfonyl) phosphatidyl ethanolamine embedded in two different populations of small unilamellar vesicles made of phosphatidyl serine has been used to study the fusion process induced by Zn2+ and Ca2+. Lipid intermixing demonstrating fusion of liposome membranes can already be observed at 125 and 250 mumol/l of Zn2+. After short time pre-incubations with micromolar concentrations of Zn2+ as low as 150 mumol/l, Ca2+ induces an instantaneous increase of vesicle fusion. The lipid intermixing induced by micromolar concentrations of Ca2+ (250-500 mumol/l) could be increased up to 4 times when pre-incubated with 150 or 200 mumol/l of Zn2+. The effect of 1 mM of Ca2+ alone on lipid intermixing can be mimicked by 150 mumol/l of Zn2+ followed by 500 mumol/l of Ca2+. Our data demonstrate that Zn2+ and Ca2+ act synergistically to affect cation-induced membrane fusion. We suggest that Zn2+ specifically alters the physical state of phospholipid membranes making them more prone to calcium-triggered fusion.     

H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes.

The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.     

Demonstration of Na+-dependent Ca2+ efflux using low concentrations of fluorometric Ca2+ probes

The effects of different concentrations of the fluorometric Ca2+ probes, fura-2 and indo-1, on Ca2+ transients in cultured rat aortic smooth muscle cells were examined. When stimulated with the agonists, angiotensin II and arginine vasopressin, cells incubated with low concentrations of fura-2 or indo-1 (less than 1 microM) produced Ca2+ transients characterized by a small increase followed by a dramatic decrease in fluorescence below the original baseline. This effect of agonists was concentration-dependent, reversible, and blocked by receptor antagonists. In contrast to the agonists, stimulation of Ca2+ transients with depolarizing concentrations of K+ or with caffeine did not produce decreases in fluorescence and Ca2+ levels at any loading concentration of probe. The decrease in Ca2+ observed with agonists was dependent on the presence of extracellular Na+. These data suggest that under certain loading conditions, fluorescent Ca2+ indicators measure agonist-stimulated Ca2+ efflux mediated by a Na+/Ca2+ exchange mechanism.     

Rate of solute incorporation to liposomes evaluated from encapsulated enzymes activities

There are numerous studies on systems comprising an enzyme encapsulated in unilamellar liposomes and its substrate initially present in the external aqueous media. Most of these studies are focused on enzyme stability and activity in a restricted media. However, the rate of the process is also determined by the capacity of the substrate to permeate towards the liposome inner pool. In spite of this, there are few studies aimed at a quantitative evaluation of the substrate permeation rate and its lifetime inside the liposome pool. In the present work, we describe, in terms of a very simple mechanism, the permeation of glucose and hydrogen peroxide in DPPC unilamellar liposomes. To this aim, we evaluated the rate of the process employing encapsulated glucose oxidase and catalase in the kinetic diffusion controlled limit. Under this condition, the rate of the process becomes zero order in the enzyme and allows a direct evaluation of the rate constant for the permeation process and the lifetime of a substrate molecule incorporated into the liposome inner pool.     

Effect of divalent metal ions on annexin-mediated aggregation of asolectin liposomes

Annexins belong to a family of Ca2+- and phospholipid-binding proteins that can mediate the aggregation of granules and vesicles in the presence of Ca2+. We have studied the effects of different divalent metal ions on annexin-mediated aggregation of liposomes using annexins isolated from rabbit liver and large unilamellar vesicles prepared from soybean asolectin II-S. In the course of these studies, we have found that annexin-mediated aggregation of liposomes can be driven by various earth and transition metal ions other than Ca2+. The ability of metal ions to induce annexin-mediated aggregation decreases in the order: Cd2+ > Ba2+, Sr2+ > Ca2+ > Mn2+ > Ni2+ > Co2+. Annexin-mediated aggregation of vesicles is more selective to metal ions than the binding of annexins to membranes. We speculate that not every type of divalent metal ion can induce conformational change sufficient to promote the interaction of annexins either with two opposing membranes or with opposing protein molecules. Relative concentration ratios of metal ions in the intimate environment may be crucial for the functioning of annexins within specialized tissues and after treatment with toxic metal ions.     

Interaction of a peripheral protein of the erythrocyte membrane, band 4.1, with phosphatidylserine-containing liposomes and erythrocyte inside-out vesicles

Interactions of band 4.1 with mixed phospholipid membranes [phosphatidylserine (PtdSer), phosphatidylethanolamine, phosphatidylcholine, etc.] and erythrocyte inside-out vesicles were studied. Band 4.1 showed a higher affinity to PtdSer-containing membranes. The amount of binding to PtdSer-containing liposomes was larger than that to PtdSer-lacking liposomes. The amount of binding to inside-out vesicles did not change significantly on a protease treatment of the vesicles. The amount of band 4.1 bound on inside-out vesicles decreased on PtdSer-decarboxylase treatment of the vesicles. Ca2+ acted inhibitory to the binding of band 4.1. Band 4.1 together with PtdSer-containing vesicles but not with PtdSer-lacking vesicles induced gelation of spectrin-actin copolymer solution. Ca2+ inhibited the gelation. Fluorescence energy transfer from PtdSer-containing vesicles to band 4.1 was larger than that from PtdSer-lacking vesicles. Band 4.1 caused a marked release of tempocholine from preloaded PtdSer-containing liposomes but not from PtdSer-lacking liposomes. The release was larger from liposomes containing more PtdSer. Ca2+ was inhibitory to the tempocholine release. We suggest from these results that band 4.1 provides another anchoring site for the cytoskeletal spectrin-actin network to PtdSer domains in the inner layer of erythrocyte membrane. This anchoring may be involved in functional regulation since the interaction causes the membrane permeability change that is dependent on Ca2+.     

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.     

Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.

Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.     

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.     

Demonstration of a calcium influx in cytolytic T lymphocytes in response to target cell binding

By using the Ca2+-sensitive dye indo-1, an antigen-specific increase in intracellular Ca2+ in cloned cytolytic T lymphocytes (CTL) was measured under conditions that were permissive for T cell-mediated cytolysis. To synchronize lethal hit delivery in a suspension of effector and target cells, a modification of the cation pulse method in which Ca2+ is added to preformed conjugates of CTL and target cells was used. Conjugate formation was unaffected by the absence of extracellular Ca2+ under these conditions. Lytic activity of these cloned CTL was markedly reduced in the absence of extracellular Ca2+ and was restored upon Ca2+ repletion. When indo-1-loaded CTL were preincubated with target cells in the absence of extracellular Ca2+, a marked antigen-specific increase in indo-1 fluorescence, indicative of an increase in intracellular Ca2+, was observed after repletion of extracellular Ca2+. This increase in intracellular Ca2+ was shown to be due solely to changes in the CTL and not the target cell within the time course of the experiment, and results from the influx of extracellular Ca2+. Antibody to the T cell receptor for antigen also evokes a similar increase in intracellular Ca2+ in CTL under these conditions. This method provides a means for the direct examination of the response of CTL to cellular antigen as well as soluble antibody and is a versatile and valuable tool for the study of CTL function.     

Transmission electron microscopy observations of sonication-induced changes in liposome structure.

Freeze-fracture Transmission Electron Microscopy (TEM) was used to show that sonication does not homogeneously disrupt liposome dispersions to form vesicles. Many large multilamellar particles remain intact after sonication and small, unilamellar vesicles are present after just 10 s of exposure. Small vesicles appear to coexist with large liposomes even before sonication. The mechanical and thermal stresses induced by sonication nucleate liquid crystalline defects in the liposomes, including edge and screw dislocations and +1 disclinations, but the Dupin cyclide structure of unsonicated liposomes is still recognizable in the larger particles after sonication. Defects in the bilayer organization may provide pathways for enhanced transport within the liposome, as well as from the liposome interior to exterior. A screw dislocation-catalyzed mechanism of liposome-to-vesicle conversion is proposed that accounts for the TEM observations.     

Lanthanum as a calcium-substituting ion for binding to sarcoplasmic reticulum ATPase

Ca2+ binding and internalization in sarcoplasmic reticulum ATPase can be investigated by the use of La3+ as a Ca2+ analog. Displacement kinetics of Ca2+ bound by La3+ in native vesicles is a slow biphasic process (k1 = 0.55 s-1 and k2 = 0.05 s-1) that is consistent with the existence of two Ca2+ binding populations whereas in leaky vesicles there appears to be a single population (k = 0.57 s-1). Rapid quench experiments demonstrate that Ca2+ internalization occurs with an initial burst (approximately 8 nmol/mg protein) associated with the presence of a phosphate-donor substrate in the reaction medium. While acid quenching for measurements of phosphoenzyme is instantaneous, La3+ quenching allows completion of one catalytic and transport cycle due to the slow La3+ exchange with Ca2+. This explains the apparent inconsistencies in the kinetics and stoichiometry of phosphoenzyme formation and Ca2+ internalization that are observed under certain experimental conditions.     

Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle.

Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase.     

Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.     

H+- and Ca2+-induced fusion and destabilization of liposomes

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.