A fluorescence assay to monitor vesicle fusion and lysis

An assay based on the fluorescent compound 2',7'-([bis(carboxymethyl)amino]methyl) fluorescein (calcein) has been developed to investigate vesicle fusion and lysis. The assay involves encapsulating the nonfluorescent Co2+ complex of calcein in one set of vesicles and EDTA in a second set. If fusion occurs, EDTA chelates Co2+, releasing calcein which may be assayed by means of its intense fluorescence. Leakage of either component of the reaction can be directly quantitated by titrating the external medium with the other component. This assay was used to investigate the effects of calcium ion on small phosphatidylserine vesicles, a system reported to undergo fusion. It revealed immediate and extensive leakage of the encapsulated contents; membrane fusion must, therefore, be simultaneously with or subsequent to, loss of vesicle integrity. This assay is convenient and flexible; it should be useful in a variety of investigations of membrane-bounded compartments.     

A fluorescence quenching technique for the measurement of paramagnetic ion concentrations at the membrane/water interface. Intrinsic and X537A-mediated cobalt fluxes across lipid bilayer membranes

We have characterized the quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine by Co2+ in egg phosphatidylcholine (PC) lipid bilayer vesicles. The quenching constant obtained is 59 M-1. We demonstrate one use of this fluorescence quenching technique by measuring intrinsic and X537A-mediated transmembrane Co2+ fluxes in large unilamellar PC vesicles. The intrinsic rate constant for Co2+ flux we measure is 3 X 10(-6) S-1. We confirm that the neutral Co approximately (X537A)2 complex is the main component of the X537A-mediated cobalt flux. Since this method measures the concentration of Co2+ at the site of the fluorophore, it is generally applicable to the measurement of paramagnetic ion concentrations in the region of the membrane/water interface.     

Fusion of synaptic vesicle membranes with planar bilayer membranes.

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.     

The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles

Co2+ quenched the fluorescence of the lipid probes NBD-phosphatidylethanolamine (NBD-PE) and lissamine-rhodamine phosphatidylethanolamine (N-Rh-PE) incorporated into lipid vesicles, according to a collisional quenching mechanism in agreement with the Stern-Vollmer law. The quenching coefficient (Q) for NBD-PE, incorporated into uncharged phosphatidylcholine (PC) vesicles was 13.8 M-1. This value was equal to the quenching coefficient of water-soluble NBD-taurine in aqueous solution, indicating that Co2+ was readily accessible to the outer surface of PC vesicles. In phosphatidylserine-phosphatidylethanolamine (PS-PE) (1:1) vesicles, quenching was also proportional to Co2+ concentration but Q was 114 mM-1, some 8000-fold smaller. Using the Gouy-Chapman-Stern model we demonstrated that the surface density of Co2+ bound to lipid was linear with Co2+ concentration in the medium up to 7%. Co2+-associated phospholipid would in turn quench NBD-PE or N-Rh-PE by collisional quenching with lateral diffusion. We investigated the ability of Co2+ to permeate PS-PE (1:1) vesicles. Co2+ quenched fluorophores on the outer surface of large unilamellar vesicles, formed by reverse-phase evaporation. In small unilamellar vesicles Co2+ quenched probes on both outer and inner surfaces, indicating rapid permeation of the ions into the vesicles. Using stopped-flow rapid mixing, we measured the rate of influx of Co2+, and correcting for surface potential using the Gouy-Chapman-Stern model, we calculated a permeability coefficient of 10(-12) cm/s for Co2+ concentrations below 300 microM. Above this concentration, there was a very steep rise in the permeability coefficient, indicating that binding of Co2+ induces defects in the bilayer of these vesicles. This may be related to the ability of the vesicles to undergo membrane fusion. A method for calculating the membrane surface potential from Co2+ quenching data is presented.     

ATP-driven copper transport across the intestinal brush border membrane

The divalent metal ion transporter DMT1 is localized in the brush border membrane (BBM) of the upper small intestine and has been shown to be able to transport Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. Belgrade rats have a glycine-to-arginine (G185R) mutation in DMT1, which affects its function. We investigated copper transport with BBM vesicles of Belgrade rats loaded with calcein, which exhibits fluorescence quenching by various metal ions. Transport of copper was disrupted in unenergized BBM vesicle of b/b Belgrade rats, as had been described for iron transport, while +/b vesicles exhibited normal transport by DMT1. When either b/b or +/b vesicles were loaded with ATP and magnesium, similar high-affinity accumulation of copper was observed in both types of vesicles. Thus, brush border membranes possess an ATP-driven, high-affinity copper transport system which could serve as the primary route for copper uptake by the intestine.     

Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels

A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.     

Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.     

Micromolar concentrations of Zn2+ potentiates Ca2+-induced phase separation of phosphatidyl serine containing liposomes

Fluorescence quenching of 1-acyl-2-[6[(7 nitro-2,1,3-benzoxadiazol-4yl) amino]caproyl] phosphatidyl choline in small unilamellar vesicles consisting of phosphatidyl serine has been used to monitor the lipid phase separation induced by Zn2+ and Ca2+. Phase separation of vesicle membranes was observed with Zn2+ at concentrations as low as 125 microM. Low concentrations of Zn2+ required long incubation times to reach maximal quenching (120 minutes at 375 microM). When low concentrations of Ca2+ were added to the preparation during the developing phase of Zn2+-induced quenching, an explosive increase in fluorescence quenching was instantenously observed. Phase separation induced by sub-millimolar concentrations of Ca2+ could be increased at least 4 times when vesicles were pre-incubated with 250 microM of Zn2+.     

Aluminum-induced lipid phase separation and membrane fusion does not require the presence of negatively charged phospholipids

The interaction of Aluminum with phosphatidyl serine lipid vesicles containing variable amounts of phosphatidyl ethanolamine, phosphatidyl choline and cholesterol has been studied by lipid phase separation monitored by fluorescence quenching. The interaction of Al3+ with neutral phospholipid membranes has also been investigated. Maximal lipid phase separation can be demonstrated in mixed phosphatidyl ethanolamine-cholesterol vesicles when using concentrations of aluminum between 87.5 and 125 microM. Millimolar concentrations of Ca2+, Mn2+, Cd2+ and Zn2+ were without any effect. Aluminum also induced fusion of phospholipid membranes monitored by resonance energy transfer between N-(7-nitro-2,1,3, benzoxadiazol-4 yl) phosphatidyl ethanolamine and N-(lissamine Rhodamine B-sulfonyl) phosphatidyl ethanolamine, either when containing low amounts of phosphatidyl serine (12.5%) or without any negatively charged phospholipid. Aluminum-induced fusion of liposomes was also monitored by the fluorescence of the terbium-dipicolinic acid complex (Tb-DPA3-) formed during fusion of vesicles containing either Tb-(citrate)6- complex or sodium salt of dipicolinic acid.     

Stable adhesion of phospholipid vesicles to modified gold surfaces

Phospholipid vesicles are well-studied biomembrane mimics that are of increasing interest in drug delivery, immunoassays, and sensor chips. In a number of biosensor applications it is desirable to be able to adhere vesicles to a surface in a manner which does not result in their rupture or fusion. Such behavior should, in principle, be achievable by controlling the vesicle-surface and vesicle-vesicle interactions. We have varied vesicle composition and charge (phosphatidylcholine, phosphatidylcholine-phosphatidic acid 18 mol%) and solution ionic strength, to study the adhesion of fluorescent vesicles to glass, gold, and gold modified with chemisorbed acetyl-cysteine. The extent of chemisorption was characterized with angle-resolved X-ray photoelectron spectroscopy (ARXPS), and vesicle integrity and behavior was studied using entrapped and lipophilic fluorescent markers, together and in separate measurements. Vesicle fusion (by energy transfer), adhesion of intact vesicles (with entrapped calcein) and diffusion coefficients (by photobleaching recovery) were monitored using confocal fluorescence microscopy. Acetyl-cysteine modified gold surfaces were shown to be appropriate substrates for adhesion of intact vesicles. Finally, as a 'proof of principle' for fluorescence amplification, release of a self-quenching entrapped reporter dye (calcein) by the detergent Triton X-100 was followed in real time.     

Optical measurement of aqueous potassium concentration by a hydrophobic indicator in lipid vesicles

An assay was developed for K+ in aqueous solution at neutral pH. The method was based on the change in optical absorbance of the hydrophobic indicator 7-(n-decyl)-2-methyl-4-(3',5'-dichlorophen-4'-one)indonaphthl++ +-1-ol (MEDPIN) in phospholipid vesicles. Formation of a ternary complex between a valinomycin-K+ pair and the anionic form of MEDPIN in the bilayer resulted in an absorption band at 584 nm. K+ concentration was determined by monitoring the MEDPIN absorbance at 584 nm and MEDPIN quenching of lissamine rhodamine B sulfonylphosphatidylethanolamine (L-RhB-PE) fluorescence by an energy-transfer mechanism. Both the fluorescence intensity and lifetime of L-RhB-PE decreased by more than 25% upon addition of 50 mM K+. Kinetic studies using stopped-flow photometry showed a single-exponential reaction of MEDPIN and valinomycin in vesicles with aqueous K+ (maximum rate 1.7 s-1) that was dependent upon [valinomycin] and [K+]. The lipid surface charge was shown to influence the ratio of anionic to neutral MEDPIN at constant pH, and to alter the sensitivity of MEDPIN absorbance to aqueous [K+]. A 1:20 neutral/negative lipid mole ratio was optimal for K+ detection at pH 7.4. Spectroscopic and kinetic data suggest that the optical response of MEDPIN to K+ involves the formation of a ternary complex between K+, valinomycin and MEDPIN.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

Fluorescence quenching in model membranes An analysis of the local phospholipid environments of diphenylhexatriene and gramicidin A′

Interactions between the fluorophors diphenylhexatriene or gramicidin A′ and lipids are examined using a spin-labeled phosphatidylcholine as a fluorescence quenching probe. It is found that in phospholipid vesicles of mixed lipid composition at temperatures where phospholipids are completely in the liquid crystal phase, several different species of phosphatidylcholines are randomly distributed around the fluorophors. In vesicles of mixed lipid composition which can undergo thermally induced phase separations, the fluorescence quenching observed at lower temperatures reflects a non-random distribution of lipids around each fluorophor. This observation is explained in terms of the partition of fluorophor between a spin-labeled lipid-rich liquid crystal phase, and a spin-labeled lipiddepleted gel phase. Gramicidin A′ strongly favors partition into the liquid crystal phase, whereas diphenylhexatriene partitions about equally between the two lipid phases. A method is described utilizing fluorescence quenching for the calculation of the partition coefficient for a fluorophor. The partition coefficients so calculated are shown to be in good agreement with previously reported values derived from other methods. It is also shown that Ca²⁺-induced lipid phase separations can be monitored by fluorescence quenching.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.     

Effects of dolichol on membrane permeability

Small vesicles containing the tetra-anionic fluorescent probe calcein were prepared by sonication of mixtures of plant phosphatidylethanolamine, plant phosphatidylcholine, and dolichol. Following chromatography, the isolated vesicles were found to retain entrapped calcein over the temperature range of 15 to 40 degrees C. Utilizing an assay measuring the fluorescence quenching of entrapped calcein by cobalt ions, the presence of dolichol in the membranes was found to promote the permeability of the phospholipid bilayers to the divalent cation. The permeability was shown to be dependent on temperature with an increase in rate of 17-fold between 15 and 35 degrees C although the plant phospholipids used in these experiments have no known phase transition within this temperature range. The incorporated dolichol was distributed uniformly throughout the vesicle population. Similar vesicles prepared from phosphatidylethanolamine and phosphatidylcholine without added dolichol, from phosphatidylcholine alone, or with phosphatidylcholine and dolichol were far less permeable to the divalent cation under the same assay conditions. These results demonstrate that dolichols have significant effects on the permeability properties of phospholipid bilayers that contain phosphatidylethanolamine.     

Distances between functional sites of the Ca2+ + Mg2(+)-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler

Cobalt ion inhibits the Ca2+ + Mg2(+)-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2(+)-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies. 1. The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluorescein-labeled sarcoplasmic reticulum membranes due to Co2+ binding to the Ca2+ + Mg2(+)-ATPase. Therefore, we conclude that Co2+ interacts with Ca2+ binding sites. 2. Co2+.ATP can be used as a substrate by this enzyme with Vmax of 2.4 +/- 0.2 mumol ATP hydrolyzed min-1 (mg protein)-1 at 20-22 degrees C and pH 8.0, and with a K0.5 of 0.4-0.5 mM. 3. Co2+ partially quenches, about 10 +/- 2%, the fluorescence of fluorescein-labeled sarcoplasmic reticulum Ca2+ + Mg2(+)-ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1-1.8 nm or 1.3-2.1 nm for one or two equidistant Co2+ binding sites, respectively. 4. Co2+.ATP quenches about 20-25% of the fluorescence of fluorescein-labeled Ca2+ + Mg2(+)-ATPase, from which we obtain a distance of 1.1-1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.     

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.     

Phospholipid-binding properties of bovine factor V and factor Va.

Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.     

Fusogenic activity of delta-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin delta-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of delta-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Localization of adriamycin in model and natural membranes. Influence of lipid molecular packing

The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster ovary cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2-APam, 7-ASte, 12-ASte and anthracene-phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Egg-yolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems. Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0-100 microM, the efficiency of energy transfer was 12-ASte greater than 7-ASte greater than 2-APam, with 100% energy transfer for 12-ASte above a drug concentration of 30 microM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7-ASTe greater than 12-ASte = 2-APam was observed. Measurements of fluorescence quenching using the non-permeant Cu2+ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer. Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains. In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of these cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments. These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.