Fusion Of Phospholipid Vesicles Research Articles

PH- and ionic strength-dependent fusion of phospholipid vesicles induced by pardaxin analogues or by mixtures of charge-reversed peptides.

The fusogenic properties of the neurotoxin paradaxin and eight of its analogues with small unilamellar vesicles (SUV), composed of egg phosphatidylcholine and phosphatidylserine (PC/PS), were investigated. Fusion was demonstrated by a lipid-mixing assay and by an increase in vesicle size as revealed by electron microscopy. The lipid-mixing assay was performed at either neutral (pH 6.8) or acidic (pH 4.5) conditions, in solutions containing either high or low salt concentrations. A low level of fusion could be induced at neutral pH only by pardaxin derivatives with amino groups at both the peptide's backbone and N-terminus. However, a marked enhancement in the fusogenic activity occurred when amino groups were present also in the C-terminus. Pardaxin analogues in which amino groups were substituted by carboxylic groups induced elevated levels of fusion only at high salt concentrations where enhancement of aggregation occurs, and acidic pH, which increased alpha-helicity. The influence of mutual interactions between pardaxin's analogues possessing complementary charges on the lipid-mixing process was also studied. At neutral pH and high salt, an inactive acidic analogue increased the fusogenic activity of a complementary-charged basic peptide. However, such mutual interactions at low salt concentrations reduced the fusogenic activity of the pardaxin analogues. Analogues containing D-amino acids were not fusogenic, thus demonstrating the structural specificity of these observations. The results indicate that the charge, alpha-helical structure, and aggregation of peptide monomers play an important role in the fusogenic ability of polypeptides.

Annexin I interactions with human neutrophil specific granules: fusogenicity and coaggregation with plasma membrane vesicles

The interactions of annexin I with specific granules isolated from human neutrophils were investigated. Unfractionated cytosol induced Ca 2+-dependent granule self-aggregation and fusion of granules with model phospholipid vesicles. High Ca 2+ concentrations were required for these processes (500–600 μM for the half-maximal rate of granule self-aggregation; 100–200 μM for the half-maximal rate of fusion with phospholipid vesicles). These activities were inhibited by a monoclonal antibody specific for annexin I and immunodepletion of cytosol by this antibody greatly reduced activity, implicating annexin I as the major mediator of these processes in neutrophil cytosol. The fact that the Ca 2+ concentration dependences differed for different membranes suggests that specificity may be contolled by the type of intracellular membrane involved and the local Ca 2+ concentration. Trypsin treatment of granules enhanced the rate of fusion of phospholipid vesicles with granules, suggesting that access to phospholipids in the granule membrane may be modulated by granule proteins or that a fusogenic protein factor in the granule membrane is activated by trypsin treatment. Coaggregation of specific granules with plasma membrane vesicles mediated by Ca 2+ and annexin I was suggested by the fact that granules preincubated with Ca 2+, cytosol and plasma membrane vesicles blocked the fusion of subsequently added phospholipid vesicles with the plasma membrane vesicles. These data suggest a role for annexin I as part of a multiprotein system involved in membrane—membrane contact necessary for exocytosis of specific granules in human neutrophils.

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.

1] Fusion assays monitoring intermixing of aqueous contents

This chapter discusses fusion assays monitoring intermixing of aqueous contents. The fusion of phospholipid vesicles has been used as a model system for understanding the molecular mechanisms of membrane fusion in biological systems. Fusion is monitored by a variety of techniques, including differential scanning calorimetry, dynamic light scattering, and gel filtration. These techniques essentially measure changes in the system under study before and after the induction of fusion. Methods to study the kinetics of the fusion process include measuring the dilution of spin-labeled phospholipids from labeled to unlabeled vesicles as an indicator of lipid mixing, monitoring the intermixing or dilution of fluorescent phospholipids, and following the intermixing of aqueous contents of vesicles. The processes taking place during membrane fusion are (1) adhesion or aggregation of two membranes or vesicles, (2) establishment of molecular contact between the membranes, (3) membrane destabilization at the area of intermembrane contact, (4) merging of membrane components, and (5) coalescence of the internal aqueous contents of the vesicles or compartments bounded by the membranes.

Computer detection of the rapid diffusion of fluorescent membrane fusion markers in images observed with video microscopy.

We have developed an algorithm for automated detection of the dynamic pattern characterizing flashes of fluorescence in video images of membrane fusion. The algorithm detects the spatially localized, transient increases and decreases in brightness that result from the dequenching of fluorescent dye in phospholipid vesicles or lipid-enveloped virions fusing with a planar membrane. The flash is identified in video images by its nonzero time derivative and the symmetry of its spatial profile. Differentiation is implemented by forward and backward subtractions of video frames. The algorithm groups spatially connected pixels brighter than a user-specified threshold into distinct objects in forward- and backward-differentiated images. Objects are classified as either flashes or noise particles by comparing the symmetries of matched forward and backward difference profiles and then by tracking each profile in successive difference images. The number of flashes identified depends on the brightness threshold, the size of the convolution kernel used to filter the image, and the time difference between the subtracted video frames. When these parameters are changed so that the algorithm identifies an increasing percentage of the flashes recognized by eye, an increasing number of noise objects are mistakenly identified as flashes. These mistaken flashes can be eliminated by a human observer. The algorithm considerably shortens the time needed to analyze video data. Tested extensively with phospholipid vesicle and virion fusion with planar membranes, our implementation of the algorithm accurately determined the rate of fusion of influenza virions labeled with the lipophilic dye octadecylrhodamine (R18).

Annexin I-mediated vesicular aggregation: mechanism and role in human neutrophils.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.

Fusion of phospholipid vesicles induced by an amphiphilic model peptide: close correlation between fusogenicity and hydrophobicity of the peptide in an alpha-helix.

A model peptide with 51 amino acid residues consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and a turn sequence of Asn-Pro-Gly at the center of the molecule has a random conformation at neutral pH but adopts an amphiphilic alpha-helical form in the presence of various salts or nucleotides [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396; Goto, Y., Okamura, N., & Aimoto, S. (1991) J. Biochem. (Tokyo) 109, 746-750]. The interaction of this model peptide with liposome membranes and the resulting alpha-helical conformational transition and membrane fusion as well as the effect of the nucleotide ATP on these events were examined at neutral pH. The peptide associated stoichiometrically with liposome membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) in a molar ratio of 2:1, resulting in formation of an amphiphilic alpha-helix and induction of fusion of the liposomes. However, the final fusion level was not correlated with the amount of binding or the helix content and was found to increase on an increase in hydrophobicity of the peptide in the alpha-helical form by neutralization of its positive charges by the negative charges of PS. In contrast, in the presence of ATP, the peptide bound completely to the PS/PC membranes at a lower concentration of liposome and concomitantly induced membrane fusion, indicating that ATP cooperates with PS to neutralize the charges of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane destabilization by N-terminal peptides of viral envelope proteins.

The fusion of lipid enveloped viruses with cellular membranes is thought to be mediated by the insertion into the target membrane of the N-terminal polypeptides of viral spike glycoproteins. Since membrane destabilization is a necessary step in membrane fusion, we investigated whether synthetic peptides with amino acid sequences corresponding to the N-termini of influenza virus hemagglutinin (HA2), vesicular stomatitis virus G-protein and Sendai virus F-protein, induce the destabilization and fusion of phospholipid vesicles. Membrane destabilization by the peptides was monitored by the release of aqueous contents of large unilamellar phospholipid vesicles. Aggregation was detected by a resonance energy transfer assay. Membrane fusion was followed by means of assays for the intermixing of phospholipids and of aqueous contents. The 17-amino acid HA2 peptide (HA2.17) destabilized phosphatidylcholine (PC) vesicles even at neutral pH, but the rate and extent of destabilization increased at lower pH. This peptide did not mediate appreciable release of contents from phosphatidylserine (PS) vesicles. HA2.17 induced neither aggregation nor fusion of PC or PS vesicles. In contrast, the 7-amino acid N-terminal peptide of G-protein (G.7) destabilized PS-containing membranes and not pure PC vesicles. Although G.7 caused aggregation of and lipid mixing between PS vesicles, it did not mediate any detectable intermixing of aqueous contents. The presence of cholesterol in PC membranes did not affect the destabilization caused by the N-terminal peptide of Sendai virus F-protein (F1.7), suggesting that cholesterol is not necessary for the effective interaction of this peptide with membranes, contrary to earlier proposals. Our results support the hypothesis that the hydrophobic N-terminal region of certain viral envelope proteins insert into and destabilize target membranes.

Fusion of phospholipid vesicles with a Langmuir lipid monolayer

The interactions of phospholipid vesicles with monolayers were investigated by film balance. The results revealed that the molecular transfer from vesicle to monolayer was strongly influenced by vesicle dimensions, vesicle phases and surface charge. Using this method, we obtained quantum information about molecule transfer which may furnish a model system to study intermembrane interactions.

Ca(2+)-induced fusion of phospholipid vesicles containing free fatty acids: modulation by transmembrane pH gradients.

The influence of a transmembrane pH gradient on the Ca(2+)-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol % oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets.

Sequence of critical events involved in fusion of phospholipid vesicles induced by clathrin

Membrane fusion induced by clathrin is accompanied by several events such as conformational change, membrane binding and association of clathrin, and membrane aggregation (Maezawa et al. (1989) Biochemistry 28, 1422–1428; Maezawa and Yoshimura (1990) Biochem. Biophys. Res. Commun. 173, 134–140). To clarify the sequence of these events, we examined their time-courses by reducing the pH of the medium from 7.4 to a given pH in the range of 3.5–5.0 at 25°C or 10°C. Large unilamellar vesicles composed of phospatidylserine and phosphatidylcholine were used in most experiments. The half-time for conformational change of clathrin was less than those for membrane binding and association of clathrin. The half-times and the initial rates of membrane binding and association of clathrin were similar order of magnitude, although the pH-profiles of the initial rates of the two events were somewhat different. Membrane aggregation started after membrane binding of clathrin. A lag phase was observed in the time-course of membrane fusion, whereas there was no lag phase in membrane binding and association of clathrin and membrane aggregation. Moreover, the lag time before fusion was independent of the clathrin concentration, although the initial rates of these three events were dependent on it, suggesting that the three reactions are not responsible for the lag phase before fusion, and that there is some other event(s) in the lag time. On the other hand, there was a threshould-pH in the pH profile of the lag-time and the threshold-pH coincided with the critical pH at which the final associated state of clathrin was apparently reversed in the presence and absence of liposomes, suggesting that the event(s) in the lag phase may be related to this final associated state of clathrin molecules on the liposome membranes. These results indicate that clathrin-induced fusion of liposomes is initiated through the following sequential events: conformational change of clathrin, membrane binding and association of clathrin, which occur simultaneously but independently, membrane aggregation, an event(s) in the lag phase, and actual fusion.

Fusion and Fragmentation of Phospholipid Vesicles by Apohemoglobin at Low pH1

Human apohemoglobin in acidic media was found to induce fusion of phosphatidylcholine/phosphatidylserine (1:1) vesicles at low protein concentration but to fragment the same vesicles to form micellar complex at high protein concentration. The fusion was demonstrated by size increase, vesicle content mixing, lipid mixing, and electron microscopy. The micellization of phospholipid vesicles was observed by light scattering, gel filtration, and electron microscopy. The hydrophobic labeling of the apohemoglobin/vesicle complex followed by CNBr cleavage of apohemoglobin showed that an N-terminal segment of the beta subunit with a molecular weight of approximately 6,000 seems to be mainly involved in the fusion process, but the whole sequences of both alpha and beta chains participate in the micellization process.

Annexin-mediated membrane fusion of human neutrophil plasma membranes and phospholipid vesicles

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 μm in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca 2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1–9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1–9)annexin I was observed at Ca 2+ concentrations as low as aproximately 5 μM, suggesting that the truncated form of this protein may be active at physiologically low Ca 2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca 2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca 2+-dependent degranulation of human neutrophils.

Metaphase chromosomes from mammalian cells stimulate fusion of artificial phospholipid vesicles

Isolated mitotic chromosomes are able to form complexes with phosphatidylcholine liposomes in the presence and absence of Ca 2+ ions, in the latter case in the presence of polyamines. Interactions with chromosomes stimulates liposome fusion. The fusion is promoted by condensed and EDTA-decondensed chromosomes.

Interaction of the herbicide atrazine with model membranes. II: Effect of atrazine on fusion of phospholipid vesicles

The effect of atrazine on Ca 2+ induced fusion of cardiolipin(CL) and phosphatidylserine (PS) vesicles is studied by Tb 3+/dipicolinic acid fluorescence and turbidity measurements. The interaction of herbicide with CL and PS membranes is studied by DPH fluorescence polarization. At low concentrations the pesticide partially inhibits fusion, especially in CL vesicles. Higher concentrations of atrazine decrease inhibition of fusion in CL, while fusion is slightly increased in PS. The Ca 2+-induced increase of turbidity is not affected by atrazine in both PS and CL aggregation experiments. DPH polarization measurements show a perturbation only of the membrane hydrophobic core of PS, in presence of Ca 2+. It is hypothesized that this biphasic effect shown by low and high atrazine concentrations on Ca 2+-induced fusion of vesicles is due to a different localization of the pesticide in the membrane.

Peptide inhibitors of enveloped virus infection inhibit phospholipid vesicle fusion and Sendai virus fusion with phospholipid vesicles.

Small hydrophobic peptides that are capable of inhibiting Sendai virus infection of cells (Richardson, C. D., Scheid, A., and Choppin, P. W. (1980) Virology 105, 205-222) are also capable of inhibiting membrane fusion in a pure lipid vesicle system. Large unilamellar vesicles of N-methyl dioleoylphosphatidylethanolamine containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid and/or p-xylene bis (pyridinium bromide) were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) fluorescence assay. Sendai virus fusion with lipid vesicles was measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride, a lipid mixing assay for fusion. The efficiency with which the peptides carbobenzoxy-D-Phe-L-PheGly, carbobenzoxy-L-Phe-L-Tyr, and carbobenz-oxy-Gly-L-Phe inhibit fusion of N-methyl dioleoyl-phosphatidylethanolamine large unilamellar vesicles directly paralleled their previously known effectiveness in blocking virus infectivity of cultured cells. In addition, above a certain concentration threshold, the inhibitory peptides decreased the initial rate of leakage from lipid vesicles. The inhibition by these peptides of virus-vesicle fusion followed the same order of potency as for vesicle-vesicle fusion. The observation of the same relative potency of these peptides toward inhibition of virus-cell infection, and virus-vesicle and vesicle-vesicle membrane fusion suggested that these peptides inhibited virus-cell infection by inhibiting the ability of the virus to fuse with the cell. Furthermore, these results suggest that the mechanism of inhibition of all three fusion events may have steps in common.

Characterization of Ca2+-dependent phospholipid binding, vesicle aggregation and membrane fusion by annexins

The annexins are a family of structurally similar, Ca2(+)-dependent, phospholipid-binding proteins. We compared six members of this family (calpactin I heavy chain, lipocortins I and III, endonexin II, p68 and protein II) to determine their phospholipid-binding specificities, as well as their ability to promote aggregation and fusion of phospholipid vesicles. The Ca2+ requirement for all of the proteins was lowest for binding to vesicles composed of phosphatidic acid, followed by phosphatidylserine and then phosphatidylinositol. Only protein II, p68, lipocortin III and endonexin II bound to vesicles composed of phosphatidylethanolamine, and none bound to phosphatidylcholine. Both calpactin I heavy chain and lipocortin I promoted aggregation of phosphatidylserine- or phosphatidylinositol-containing vesicles in the presence of less than 10 microM-Ca2+. Lipocortin I promoted fusion of liposome membranes by lowering threshold Ca2+ concentrations. Although calpactin I heavy chain did not affect threshold Ca2+ concentrations, it did increase the rate and extent of spontaneous fusion. In contrast, p68 inhibited fusion at threshold Ca2+ concentrations. Whereas previous reports have emphasized properties that the annexins have in common, these findings reveal quantitative and qualitative differences among the annexins which may relate to distinct intracellular functions.

Monovalent cation-induced fusion of acidic phospholipid vesicles

Fusion of small unilamellar vesicles (SUV) consisting of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and phosphatidylglycerol (PG) from egg yolk, dipalmitoylphosphatidylserine (DPPS) and phosphatidylserine (PS) from bovine brain was studied as a function of monovalent cation concentration. Fusion was detected by measuring the changes in the excimer to monomer fluorescence intensity ratio ( I E/ M ) of pyrene-labeled phospholipid analogues upon fusion of the pyrene-labeled and unlabeled vesicles. No fusion was observed for vesicles consisting of DMPC. PS from bovine brain or PG from egg yolk upon addition of NaCl (up to 1M). However, considerable fusion was evident for vesicles consisting of DMPG or DPPS upon addition of monovalent cations (300 mM to 1 M). Fusion kinetics were fast reaching a plateau after 5 min of addition of cations. The order of efficiency of different monovalent cations to induce the fusion of DMPG vesicles as judged by the changes of the I E/ M ratio was Li + > Na + > K + > Cs +. DSC-scan of sonicated DMPG vesicles showed, in the absence of salt, a phase transition at 19.2°C with enthalpy of 1.1 kcal · mol −1. After incubation in the presence of 600 mM NaCl the DSC scan showed a narrow phase transition at 24.1°C with enthalpy of 6.9 kcal · mole −1 and a pronounced pretransition, both supporting that the fusion of the vesicles had occurred in the presence of NaCl. The results indicate that sonicated vesicles consisting of acidic phospholipids with fully saturated fatty acids fuse in the presence of monovalent cations, whereas those containing unsaturated fatty acids do not.

Deformation and instability of membrane structure of phospholipid vesicles caused by osmophobic association: mechanical stress model for the mechanism of poly(ethylene glycol)-induced membrane fusion

The mechanism of poly(ethylene glycol)-induced fusion of phospholipid vesicles was studied based on the "osmophobic association" theory which was recently proposed both theoretically [Ito, T., Yamazaki, M., & Ohnishi, S. (1989) Biochemistry 28, 5626-5630] and also experimentally [Yamazaki, M., Ohnishi, S., & Ito, T. (1989) Biochemistry 28, 3710-3715]. Osmophobic association and fusion were detected by measuring the light scattering of the vesicle suspension; the former was detected from the increase in light scattering induced by the addition of PEG, and the latter was from the irreversibility of the increase in light scattering. Threshold concentrations of PEG were required not only for osmophobic association but also for fusion. The threshold concentration for fusion depended on the molecular weight of PEG and also on the electrostatic repulsive interaction between phospholipid vesicles, which was manipulated by the use of vesicles with negative surface charge; increasing the molecular weight of PEG lowered the threshold concentration, and increasing the electrostatic repulsive interaction raised it. In addition, a transient leakage of internal contents from the vesicles was observed at the concentration that caused fusion. When the surface charge of the vesicle was varied, the threshold for fusion coincided with that for osmophobic association, provided that the latter exceeded 22 wt % of PEG 6000. However, when the threshold for osmophobic association was less than 22 wt %, the threshold for fusion remained approximately 22 wt %, irrespective of the difference in the threshold for osmophobic association.(ABSTRACT TRUNCATED AT 250 WORDS)

Fusion of phospholipid vesicles produced by the anti-tumour protein α-sarcin

The anti-tumour protein alpha-sarcin causes fusion of bilayers of phospholipid vesicles at neutral pH. This is demonstrated by measuring the decrease in the efficiency of the fluorescence energy transfer between N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidylethano lamine (NDB-PE) (donor) and N-(lissamine rhodamine B sulphonyl)-diacylphosphatidylethanolamine (Rh-PE) (acceptor) incorporated in dimyristoylphosphatidylcholine (DMPG) vesicles. The effect of alpha-sarcin is a maximum at 0.15 M ionic strength and is abolished at basic pH. alpha-Sarcin promotes fusion between 1,6-diphenylhexa-1,3,5-triene (DPH)-labelled DMPG and dipalmitoyl-PG (DPPG) vesicles, resulting in a single thermotropic transition for the population of fused phospholipid vesicles. Bilayers composed of DMPC and DMPG, at different molar ratios in the range 1:1 to 1:10 PC/PG, are also fused by alpha-sarcin. Freeze-fracture electron micrographs corroborate the occurrence of fusion induced by the protein. alpha-Sarcin also modifies the permeability of the bilayers, causing the leakage of calcein in dye-trapped PG vesicles. All of the observed effects reach saturation at a 50:1 phospholipid/protein molar ratio, which is coincident with the binding stoichiometry previously described.