Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement

The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg-yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios (R) were necessary (R greater than 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R greater than 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R greater than 0.1 for EPC vesicles, R greater than 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations (R less than 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel.     

Interaction of the polyene antibiotic amphotericin B with phospholipid bilayer membranes: A circular dichroism study

The circular dichroism of amphotericin B “Fungizone” has been used to study its interaction with lecithin, dimyristoyl and dipalmitoyl phosphadidylcholine vesicles with or without cholesterol. It appears that circular dichroism monitors species other than those monitored by electronic absorption. As a result of association, new spectra appear which are of opposite signs according to wether the vesicles are in the gel state or in the liquid crystalline state; the cholesterol dependance of the rate of these changes is also different according to the state of the vesicles. It is concluded that the model proposed by Hsu Chen and Feingold for the gel state should not be rejected on the basis of data obtained in the liquid crystalline state.     

Interaction between phospholipid bilayer membranes and the polyene antibiotic amphotericin B: Lipid state and cholesterol content dependence

The interaction between amphotericin B and egg yolk phosphatidylcholine, dimyristoyl (DMPC) and dipalmitoyl phosphatidylcholine (DPPC) phospholipid bilayer vesicles has been monitored by the circular dichroism (CD) spectra of amphotericin B at a 1 · 10^?5 M concentration. This method has revealed that amphotericin B may be present in a number of different forms depending on the time elapsed after the mixing, the cholesterol content of the vesicles and the vesicles' physical state. Some striking features of these CD detected species are the following: with egg yolk phosphatidylcholine and a molar cholesterol percentage lower than 25, at 25°C several forms are coexistent, their amount is time-dependent; with dipalmitoyl or dimyristoyl phosphatidylcholines without cholesterol or with a cholesterol molar percentage lower than 25, in the gel state, a form different from the former appears very rapidly; with egg yolk phosphatidylcholine, DMPC and DPPC at a molar cholesterol percentage between 25 and 50 a new form is monitored, identical in the three cases and observed in the liquid crystalline state as well as in the gel state. In the case of the three phospholipids without cholesterol a definite interaction with the antibiotic is observed but with different characteristics according to the nature of lipid.With amphotericin B ‘Fungizone’ the same species are monitored but their appearance is much slower.Two explanations are proposed for the origin of the discrepancies between CD and electronic absorption.     

Transfer of the polyene antibiotic amphotericin B between single-walled vesicles of dipalmitoylphosphatidylcholine and egg-yolk phosphatidylcholine

Amphotericin B transfer between single-walled vesicles of dipalmitoylphosphatidylcholine (DPPC) and of egg phosphatidylcholine, both containing 10 mol% cholesterol, has been studied concurrently by circular dichroism spectroscopy and permeability measurements. At 22°C amphotericin B is rapidly transferred from DPPC to DPPC vesicles as well as from egg phosphatidylcholine to egg phosphatidylcholine vesicles. On the other hand, although amphotericin B is rapidly transferred from egg phosphatidylcholine to DPPC vesicles, it is not transferred from DPPC to egg phosphatidylcholine vesicles. At 48°C, above the transition temperature of DPPC, transfer occurs rapidly both ways. These results are interpreted in terms of difference of association constant of amphotericin B with vesicle membranes in the gel and liquid-crystalline state.     

Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol

The spontaneous incorporation of the polyene antibiotic amphotericin B from a micellar solution into phospholipid vesicles was examined as a function of the lipid composition of the vesicles and their physical state. Virtually no insertion of the antibiotic into egg phosphatidylcholine vesicles was observed even when cholesterol was also present in the bilayer. In contrast, rapid incorporation occurred into systems containing an anionic phospholipid such as phosphatidylglycerol or phosphatidylserine with the fastest rates observed for lipids containing the saturated dimyristoyl fatty acyl species. Insertion of amphotericin B into vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3 mole ratio) was rapid either above, below or within the gel-to-liquid-crystalline phase transition temperature (23 degrees C). The ability of amphotericin B to intercalate into lipid vesicles is discussed in relation to their relative bilayer stabilities.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

Interaction of the polyene antibiotic filipin with model and natural membranes containing plant sterols

The interaction of the polyene antibiotic, filipin, with individual or mixed plant sterols (stigmasterol, sitosterol, campesterol and 24-methylpollinastanol) incorporated into large unilamellar vesicles (LUV) of soybean phosphatidylcholine (PC) as well as the filipin interaction with purified membrane fractions from maize roots containing these sterols was investigated by ultraviolet (UV) absorption and and circular dichroism (CD) spectroscopy. With both types of membrane preparation, dramatic changes in the UV absorption and CD spectra of the antibiotic were evidenced. When LUV containing stigmasterol, sitosterol and/or campesterol were incubated with low filipin concentrations (i.e., for filipin/sterol molar ratios (rst) lower than 1), CD signal characteristic of the formation of filipin-sterol complexes were observed. At higher rst values, the filipin-sterol interaction was shown to be in competition with a filipin-phospholipid interaction. With 24-methylpollinastanol-containing LUV, the filipin-phospholipid interaction was detected even at rst values lower than 1, which suggests a lower affinity of filipin for this sterol and emphasizes the structural differences between delta 5-sterols and 9 beta,19-cyclopropylsterols. With sterol-free soybean PC LUV, a filipin-phospholipid interaction could also be evidenced. With maize root cell membranes containing either delta 5-sterols or 9 beta,19-cyclopropylsterols, CD spectra similar to those obtained in the presence of LUV having these sterols as components were observed. Thus, the protein component of the membranes does not appear to be an important feature.     

Interaction of wheat alpha-thionin with large unilamellar vesicles.

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.     

Interaction of bilirubin with small unilamellar vesicles of dipalmitoylphosphatidylcholine

Interaction of bilirubin with phospholipid bilayers was studied at physiological pH above and below the gel-liquid crystalline phase transition of small unilamellar vesicles of dipalmitoylphosphatidylcholine. Chromatographic, calorimetric and 1H-NMR evidences strongly suggest that dianion form of bilirubin binds to the polar heads of the phosphatidylcholines protruding from the outer leaflet of the vesicles, whilst acid bilirubin, which is insoluble in water, is hydrophobically inserted into the lipophilic region of the bilayers. The surface-bound bilirubin is promptly removed from vesicles, whilst the acid form hydrophobically inserted into the vesicles is firmly bound to the membrane in the gel state. This pool of bilirubin could perturb the chemico-physical properties of the membrane (i.e., fluidity, phase transition, etc. ...) thus contributing to perturbation of the biological properties of living cells.     

Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B

The polyene antibiotic amphotericin B (AMB) is an effective antifungal agent whose therapeutic potential is limited by poor aqueous solubility and toxicity toward host tissues. Addition of apolipoprotein A-I to a multilamellar phospholipid vesicle dispersion containing 20% (w/w) AMB induces the formation of reconstituted high density lipoprotein (rHDL), with solubilization of the antibiotic. Density gradient ultracentrifugation resulted in flotation of the complexes to a density of 1.16 g/ml, and negative stain electron microscopy revealed a population of disk-shaped particles. Native gradient polyacrylamide gel electrophoresis indicated a particle diameter of approximately 8.5 nm. Absorbance spectroscopy provided evidence for AMB integration into the lipid milieu. AMB-rHDLs were potent inhibitors of Saccharomyces cerevisiae growth, yielding 90% growth inhibition at <1 microg/ml yeast culture. In studies with pathogenic fungal species, similar growth inhibition characteristics were observed. Compared with AMB-deoxycholate micelles, AMB-rHDL displayed greatly attenuated red blood cell hemolytic activity and decreased toxicity toward cultured hepatoma cells. In in vivo studies in immunocompetent mice, AMB-rHDLs were nontoxic at 10 mg/kg, and they showed efficacy in a mouse model of candidiasis at concentrations as low as 0.25 mg/kg. These results indicate that AMB-rHDLs constitute a novel formulation that effectively solubilizes the antibiotic and elicits strong in vitro and in vivo antifungal activity with no observed toxicity at therapeutic doses.     

Cholesterol transfer from small and large unilamellar vesicles

The rates of transfer of [14C]cholesterol from small and large unilamellar cholesterol/egg yolk phosphatidylcholine vesicles to a common vesicle acceptor were compared at 37 degrees C. The rate of exchange of cholesterol between vesicles of identical cholesterol concentrations (20 mol%) did not differ from the rate of transfer from donor vesicles containing 20 mol% cholesterol to egg yolk PC vesicles. Further, the rate of transfer of [14C]cholesterol from vesicles containing 15 mol% dicetyl phosphate (to confer a negative charge) was not different from the rate of transfer from neutral vesicles. However, the half-time for transfer of [14C]cholesterol from large unilamellar donor vesicles was about 5-times greater (10.2 h, 80 nm diameter) than from small unilamellar vesicles (2.3 h, 23 nm diameter). These data suggest that increased curvature in small unilamellar vesicles reduces cholesterol-nearest neighbor interactions to allow a more rapid transfer of cholesterol into the aqueous phase.     

Organization of polyene antibiotic amphotericin B at the argon-water interface

Amphotericin B (AmB) is a life-saving antibiotic, used to treat deep-seated mycotic infections. Both the therapeutic and toxic side effects of AmB are directly dependent on its molecular organization. Organization of AmB was studied in monocomponent monomolecular layers formed at the argon-water interface, by means of polarized and non-polarized electronic absorption spectroscopy and analyzed in terms of the exciton splitting theory. The results provide direct indication that AmB forms spontaneously dimers that can be assembled into molecular structures characterized by homogeneous orientational distribution in the monolayer, interpreted as cylindrical pores. The structures are not stable at surface pressures higher than 20 mN/m and therefore dimers are concluded as abundant molecular organization forms of AmB in biomembranes. Possibility of stabilization of the cylindrical structures, at higher surface pressures, by other molecules, e.g. sterols, is also discussed.     

Distribution of cytochrome b5 between small and large unilamellar phospholipid vesicles

Cytochrome b5 is an amphipathic integral membrane protein that spontaneously inserts, post-translationally, into intracellular membranes. When added to preformed phospholipid vesicles, it binds in a so-called "loose" or transferable configuration, characterized by the ability of the protein to rapidly equilibrate between vesicles. In a preliminary report we showed that the distribution of cytochrome b5 among a heterogeneous population of small sonicated phosphatidylcholine vesicles (212 to about 350 A in diameter) lies in favor of the smallest vesicles by a factor of at least 20 (Greenhut, S.F. and Roseman, M.A. (1985) J. Biol. Chem. 260, 5883-5886). In the present studies we have attempted to determine the maximal extent to which bilayer curvature can influence the intervesicle distribution of cytochrome b5, by measuring the distribution of the protein between a population of limit-size vesicles 212 A in diameter and a population of large unilamellar vesicles approximately 1000 A in diameter. (The effect of bilayer curvature on the physical properties of the lipids in the large vesicles is considered to be negligible.) The results show that cytochrome b5 favors the small vesicle population by a factor of about 200. This observation suggests that the formation of highly curved regions in biological membranes (or the formation of regions in which the physical state of the lipids is similar to that in small vesicles) may cause the accumulation of certain membrane proteins at those sites. We also observed that a significant fraction (11-20%) of the cytochrome b5, when added directly to the large vesicles, spontaneously inserts into the "tight," physiologically proper configuration. A possible mechanism is discussed.     

Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.     

Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B. II: study of the interactions of the oligonucleotide/cationic vector complexes with lipid monolayers and lipid unilamellar vesicles

We report a study of the behavior of oligodeoxyribonucleotide (ODN)/amphotericin B3-(N'-dimethylamino)propylamide (AMA) complexes, in the presence of lipid monolayers and large unilamellar vesicles. This study follows the recent discovery of the capacity of AMA, as a new cationic vector, to enhance ODN cellular uptake and efficacy. It aims at investigating the internalization mode of a nucleic acid by AMA. A first study at the air-water interface of AMA and AMA/ODN by surface pressure measurement shows that only free AMA would adsorb at the air-water interface. Second, in the presence of zwitterionic phospholipid- and sterol-containing mixture, ODN-AMA interactions in solution would be higher than lipid-AMA interactions at the interface. In monolayer or with large unilamellar vesicles, AMA monomers adsorb mainly at the phospholipid interface. These results favor a crossing mechanism through AMA channel formation, despite the size of ODN.     

Comparison of the lipid acyl chain dynamics between small and large unilamellar vesicles.

13C NMR spin-lattice relaxation (T1) rates and 13C-1H nuclear Overhauser effects (NOEs) were measured in an identical fashion in two lipid preparations having dramatically different curvatures. The T1 times that were obtained at four magnetic field strengths were fit along with the NOEs to simple models for lipid molecular dynamics. The results indicate that phospholipid chain ordering and dynamics are virtually identical in small and large unilamellar vesicles at the time scales sampled by these 13C-NMR studies. The order parameters and reorientational correlation times that characterize the amplitudes and rates of internal acyl chain motions were equal within experimental error for the methylene segments in the middle of the chains. The only significant differences in order parameters and correlation times between the two vesicle types were small and appeared at the ends of the acyl chains. At the carbonyl end the order was slightly higher in small vesicles than large vesicles, and at the methyl end the order was slightly lower for small vesicles. This indicates that in the more planar systems the acyl chains exhibit a slightly flatter order profile than in more highly curved membranes. The use of the same experimental approach in both small and large vesicle systems provided a more reliable and accurate assessment of the effect of curvature on molecular order than has been previously obtained.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

Association of a protein with membrane vesicles at the collisional limit: studies with blood coagulation factor Va light chain also suggest major differences between small and large unilamellar vesicles

Vesicle size can be a very sensitive modulator of protein-membrane association. In addition, reactions at the collisional limit may be characteristic of many types of protein-membrane or protein-receptor interactions. To probe these effects quantitatively, we analyzed the association of blood clotting factor Va light chain (Va-LC) with phospholipid vesicles of 15-150-nm radius. The number of protein binding sites per vesicle was approximately proportional to vesicle surface area. Association rates approached the collisional limit, and the activation energy for the association reaction was 4.5 +/- 0.5 kcal/mol. In agreement with diffusional theory for this type of interaction at the collisional limit, the observed association rate constant for filling all sites was approximately proportional to the inverse of vesicle radius. This general property has important implications for many systems such as blood coagulation including possible slower association rates and higher Km values for reactions involving whole cells relative to those obtained for phospholipid vesicles. Dissociation rate constants for reactions that are near the collisional limit should also be proportional to the inverse of vesicle size if diffusional parameters are the only factors influencing dissociation. However, Va-LC bound to small unilamellar vesicles (SUVs, less than or equal to 15-nm radius) gave slower dissociation rates than Va-LC bound to large unilamellar vesicles (LUVs, greater than or equal to 35-nm radius). This indicated a change in KI, the intrinsic protein-phospholipid affinity constant for LUVs vs SUVs. The cumulative effect of association and dissociation rates resulted in higher affinity of Va-LC for SUVs than LUVs under equilibrium conditions. The latter was corroborated by competition binding studies. Furthermore, the temperature dependence of both rate constants indicated an entirely entropy-driven binding to LUVs but a largely enthalpy-driven binding to SUVs. Interactions which are largely entropic are thought to be ionic in nature. The differences observed between binding to LUVs and SUVs may reflect thermodynamic differences between these types of phospholipid structures.     

Spectrophotometric analysis of organisation of dipalmitoylphosphatidylcholine bilayers containing the polyene antibiotic amphotericin B

Amphotericin B (AmB) is a polyene antibiotic widely used in the treatment of deep-seated fungal infections. The mode of action of AmB is directly related to the effect of the drug on the lipid phase of biomembranes. In the present work the effect of AmB on the properties of lipid bilayers formed with dipalmitoylphosphatidylcholine (DPPC) and the effect of the lipid phase on the molecular organisation of AmB were studied with application of spectrophotometry in the UV-Vis region. The absorption spectra of AmB in lipid membranes display a complex structure with hypsochromically and bathochromically shifted bands indicative of formation of molecular aggregates of the drug. Formation of molecular aggregates was analysed at different concentrations of the drug in the lipid phase in the range 0.05--5 mol% and at different temperatures in the range 5--55 degrees C. The aggregation level of AmB in the ordered phase of DPPC displayed a minimum corresponding to a concentration of 1 mol% with respect to the lipid. An increase in the aggregation level was observed in the temperature region corresponding to the main phase transition. The structure of molecular aggregates of AmB is analysed on the basis of spectroscopic effects in terms of the exciton splitting model. Analysis of the position of the absorption maximum of AmB in the lipid phase of DPPC in terms of the theory of solvatochromc effects makes it possible to ascribe the refractive indices n=1.40 and n=1.49 to the hydrophobic core of the membrane in the L(alpha) and the P(beta)' phase respectively. Analysis of the aggregation of AmB in the lipid phase in relation to the physical state of the membrane reveals that the temperature range of the main phase transition of a lipid cluster in the immediate vicinity of AmB depends on its concentration. The termination of the phase transition temperature, as read from the AmB aggregation, varies between 42 degrees C at 1 mol% AmB in DPPC and 49 degrees C at 5 mol% AmB in DPPC. The exciton splitting theory applied to the analysis of the spectroscopic data makes it possible to calculate the diameter of the AmB pore as 2.8 A in the gel phase and 3.6 A in the fluid phase of the DPPC membrane, on the assumption that the pore is formed by nine AmB molecules.