Amyloglucosidase enzymatic reactivity inside lipid vesicles

Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG) (EC 3.2.1.3) from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, V _max and K _m , were determined by initial velocity measurements, and K _i was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose) formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.     

Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers

The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.     

Interaction of liposome with immobilized chitosan during main phase transition

It has been recently demonstrated that chitosan in aqueous solution alters the phase behavior and structure of a phospholipid bilayer (Fang, N.; et al. Biomacromolecules 2001, 2, 1161-1168). Until now, the physical driving forces between chitosan and the phospholipid bilayer upon their initial encounter remains unknown. In this study, confocal reflectance interference contrast microscopy (C-RICM), phase contrast microscopy and bioadhesion modeling are concurrently applied to probe the interaction of phospholipid vesicle with immobilized chitosan at various temperatures, pH, and osmotic stress. First, the successful immobilization of chitosan on amino-silanized glass is indicated by the increases in both the degree of vesicle deformation and adhesion energy of vesicles adhering on chitosan modified substrate in comparison with those on amino-silanized glass. Second, the phase transition of a phospholipid bilayer does not modulate the adhesion strength at the chitosan-biomembrane interface at pH 7.4. With increase of the degree of protonation on the chitsoan backbone at pH 4, the adhesion energy is increased by 5-fold for vesicles of all sizes compared to that in pH 7.4. Furthermore, pH reduction amplifies the thermal-induced response of larger vesicles on the immobilized chitosan layer. Interestingly, a moderate increase of osmotic stress maximizes the degree of vesicle deformation and adhesion energy at 23 degrees C and dampens the effect of phase transition on vesicle adhesion. Overall, this study demonstrates the quantitation of chitosan-biomembrane interactions that will be critical for future applications of chitosan in biological systems.     

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T(m). In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T(m) and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 microm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.     

Precision parameters from spin-probe studies of membranes using a partitioning technique. Application to two model membrane vesicles

A new version of the ESR spin probe partitioning method is developed and applied to the study of hydration properties of dimyristoyl-phosphatidylglycerol (DMPG) and dimyristoyl-phosphatidylcholine (DMPC) vesicles as functions of salt concentration and temperature above the lipid phase transition. The small spin probe di-tert-butyl nitroxide (DTBN) is used in order to achieve motionally narrowed Electron Spin Resonance (ESR) spectra which may be analyzed with high precision. The new method relies on the use of the second harmonic display of the ESR spectrum followed by spectral line fitting. Spectral fitting yields precise ESR parameters giving detailed information on the surroundings of the spin probe in both phospholipid and aqueous phases. The nitrogen hyperfine coupling constant of DTBN arising from those probes occupying the vesicles is used to study the hydration of the vesicle surface. The hydration properties of the negatively charged vesicle surface of DMPG vesicles are affected by the addition of salt at all temperatures. In contrast, the hydration of DMPC vesicles does not change with salt concentration at the low temperatures. However, at higher temperatures the hydration properties of DMPC vesicle are affected by salt which is interpreted to be due to the faster motion of the phospholipid molecules. The partitioning of the spin probe increases with salt concentration for both DMPG and DMPC vesicles, while water penetration decreases simultaneously. The spin probe in the phospholipid bilayer exhibits anisotropic motion and the extent of the anisotropy is increased at the higher salt concentrations.     

Change in volume magnetic susceptibility at the phase transition of dipalmitoylphosphatidylcholine.

We have observed a change at 41 degrees C in the relative volume magnetic susceptibility of an aqueous dispersion containing 13 wt% multilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles. The magnitude of the change is consistent with the known density change of the phospholipid bilayer and the assumption that the mass susceptibility of the system is constant through the transition. The superconducting susceptometer used in this study of the sharp transition of DPPC will be able to detect 1% changes in bilayer density for 10 wt% dispersions even when the transition temperature and transition width of phospholipid vesicle under various experimental conditions.     

Electrical Properties of Phospholipid Vesicles

The capacitance of the membrane of phospholipid vesicles and the electrical properties of the vesicle interior have been determined. To this end the electrical properties of phospholipid vesicles have been investigated over a frequency range extending from 1 kHz to 100 MHz. The dielectric behavior is characterized by two dispersions, one placed between 1 kHz and 1 MHz and the other between 1 and 100 MHz. The relaxational behavior at low frequencies is explained by counterion movement tangential to the vesicle surface and a reasonable value for the fixed charge of the vesicles is calculated from the dispersion magnitude. The relaxation at high frequencies is of the Maxwell-Wagner type and appears caused by the phospholipid bilayer bounding the interior phase of the vesicles. It is consistent with the existence of a closed bilayer with a capacitance of about 2 μF/cm² and an internal phase similar to the vesicle suspending medium. There is no indication of other than normally structured water inside the small vesicles.     

Asymmetric fusion between phospholipid vesicles and vesicles formed from synthetic di(n-alkyl)phosphates

We have investigated the fusion behavior of a mixed vesicle system consisting of vesicles prepared from the simple synthetic surfactants di(n-dodecyl)phosphate (DDP) or di(n-tetradecyl)phosphate (DTP) and vesicles prepared from the phospholipids phosphatidylserine (PS) or dioleoylphosphatidylcholine (DOPC). Fusion between the vesicles, induced by Ca2+, was determined by a resonance energy transfer assay for lipid mixing, sucrose density gradient analysis, and electron microscopy. We demonstrate that synthetic surfactant vesicles can specifically engage in asymmetric fusion events, provided that the incubation temperature is kept below the gel-liquid crystalline phase-transition temperature (Tc) of the synthetic amphiphile (29 and 48 degrees C for DDP and DTP, respectively) and that the physical state of the target membrane is fluid. Asymmetric fusion of DDP or DTP vesicles was most efficient with PS vesicles, but it also occurred with zwitterionic PC vesicles. In the latter case, fusion proceeded spontaneously, but the process was markedly accelerated upon addition of Ca2+. Furthermore, in contrast to a massive transformation of bilayer into nonbilayer hexagonal HII tubular structures, as occurs upon symmetric Ca(2+)-induced fusion of DDP vesicles, asymmetric fusion with phospholipid bilayers predominantly leads to the formation of larger vesicles. This indicates that both PS and DOPC stabilize the DDP bilayer structure in the fusion product.     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Frequency-dependent electrodeformation of giant phospholipid vesicles in AC electric field

A model of vesicle electrodeformation is described which obtains a parametrized vesicle shape by minimizing the sum of the membrane bending energy and the energy due to the electric field. Both the vesicle membrane and the aqueous media inside and outside the vesicle are treated as leaky dielectrics, and the vesicle itself is modeled as a nearly spherical shape enclosed within a thin membrane. It is demonstrated (a) that the model achieves a good quantitative agreement with the experimentally determined prolate-to-oblate transition frequencies in the kilohertz range and (b) that the model can explain a phase diagram of shapes of giant phospholipid vesicles with respect to two parameters: the frequency of the applied alternating current electric field and the ratio of the electrical conductivities of the aqueous media inside and outside the vesicle, explored in a recent paper (S. Aranda et al., Biophys J 95:L19–L21, 2008). A possible use of the frequency-dependent shape transitions of phospholipid vesicles in conductometry of microliter samples is discussed.     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Effect of gangliosides on membrane permeability studied by enzymic and fluorescence-spectroscopy techniques.

The effect of gangliosides on membrane permeability was investigated by studying the kinetic properties of cytochrome c oxidase, the activity of which, when the enzyme is reconstituted in phospholipid vesicles, is dependent on membrane permeability to H+ and K+. The experiments indicate that three different gangliosides (GM1, DD1a, GT1b) incorporated into cytochrome c oxidase-containing phospholipid vesicles stimulate enzymic activity, in the absence of ionophores, most probably by disorganizing the bilayer lipid assembly and increasing its permeability to ions. This interpretation was confirmed by fluorescence-spectroscopy experiments in which the rate of passive leakage of carboxyfluorescein entrapped in the vesicles was measured. Cholera toxin, or its isolated B-subunit, added to GM1-containing proteoliposomes inhibited cytochrome c oxidase activity, indicating the lack of formation, under these experimental conditions, of channels freely permeable to H+ or K+.     

Physiochemical Modeling of Vesicle Dynamics upon Osmotic Upshift

We modeled the relaxation dynamics of (lipid) vesicles upon osmotic upshift, taking into account volume variation, chemical reaction kinetics, and passive transport across the membrane. We focused on the relaxation kinetics upon addition of impermeable osmolytes such as KCl and membrane-permeable solutes such as weak acids. We studied the effect of the most relevant physical parameters on the dynamic behavior of the system, as well as on the relaxation rates. We observe that 1) the dynamic complexity of the relaxation kinetics depends on the number of permeable species; 2) the permeability coefficients (P) and the weak acid strength (pK_a-values) determine the dynamic behavior of the system; 3) the vesicle size does not affect the dynamics, but only the relaxation rates of the system; and 4) heterogeneities in the vesicle size provoke stretching of the relaxation curves. The model was successfully benchmarked for determining permeability coefficients by fitting of our experimental relaxation curves and by comparison of the data with literature values (in this issue of Biophysical Journal). To describe the dynamics of yeast cells upon osmotic upshift, we extended the model to account for turgor pressure and nonosmotic volume.     

Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants

The three-dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes were analyzed by imaging negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer-aided three-dimensional reconstruction. The information on electron micrographs obtained at 6 degrees angular increments from +60 degrees to -60 degrees was digitized by densitometric scanning, Fourier-transformed, corrected for imaging errors, cross-correlated, and synthesized to the three-dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle, indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16-nm height, 2.0-nm wall thickness, and a 3.0-nm torus at the free end with an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane-binding site, and only small pores of up to 2.0-nm in diameter penetrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of approximately 4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest the MAC may mediate increased membrane permeability by protein channel formation in addition to lipid reorientation.     

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.     

Active generation and propagation of Ca2+ signals within tunneling membrane nanotubes

Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch.     

Modeling leakage kinetics from multilamellar vesicles for membrane permeability determination: application to glucose

The glucose permeability of bilayers formed from phosphatidylcholine, Brij30, and sodium octadecyl sulfate has been determined via an enzymatic reaction. Glucose is encapsulated in either uni- or multilamellar vesicles (MLV) and its concentration in the dispersion medium is monitored by spectrophotometry analysis through the rate of glucose oxidase-catalyzed reaction of glucose oxidation. A kinetic model of leakage, taking explicitly into account one, two, or n(w)-walls (n(w) > 1) for the vesicles and assuming an enzymatic Michaelis-Menten behavior, is proposed and used to fit the experimental data. The two-wall model was chosen to fit experimental data obtained on MLV since an average value of 1.7 bilayers was estimated for MLV by cryo-TEM imaging. A permeability value of 5.8 +/- 4.4 10(-9) cm/s was found. The proposed model is validated by the measurement of the bilayer permeability deduced from the modeling of glucose leakage from unilamellar vesicles with the same composition. In this latter case, a value of 8.3 +/- 0.7 10(-9) cm/s is found for the permeability, which is within the error bar of the value found with MLV.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A2: a 13C NMR study

A new approach to study phospholipase A2 mediated hydrolysis of phospholipid vesicles, using 13C NMR spectroscopy, is described. [13C]Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A2 (Crotalus adamanteus). The 13C-labeled carboxyl/carbonyl peaks from the products [lyso-1-palmitoylphosphatidylcholine (LPPC) and palmitic acid (PA)] were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was approximately 50% ionized at pH approximately 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of 13C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.     

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane

Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.