Incorporation of Na(+),K(+)-ATP-ase into the thiolipid biomimetic assemblies via the fusion of proteoliposomes

The black lipid membranes (BLMs) are artificial membrane systems that have been widely used in the study of different biological processes. In this paper the planar bilayer lipid membranes have been used to study the behavior of thiolipid molecules-dipalmitoyl-phosphatidyl-ethanolamine-mercaptopropionamide (DPPE-MPA) and cholesteryl 3-mercaptopropionate (Chs-MPA)-as compared to classical BLM made of natural lipids. We present our experiments on black thiolipid bilayer (BTM) formation from a thiolipid solution and basic results of pump currents generated by sodium-potassium pump-Na(+),K(+)-ATP-ase-introduced to such bilayer systems via proteoliposome adsorption with subsequent fusion. Our results imply that no substantial difference exists between BLMs formed from classical lipids and those made from thiolipids used in this study. The same thiolipid molecules were subsequently used for the formation of covalently bound, tethered bilayer lipid membranes (t-BLMs) on polycrystalline gold electrodes. Similarly, as in the case of BLMs, we took advantage of proteoliposome adsorption/fusion to obtain a t-BLM system with reconstituted enzyme. The vesicle fusion on hydrophobic or hydrophilic substrates is one of the main ways to obtain a bilayer system with incorporated biological species. In this paper we present also our preliminary results of electrochemical experiments using rapid solution exchange technique on such t-BLMs systems and their comparison with painted solid supported membranes (SSMs) and BLMs. We have also followed the process of vesicles fusion onto thiolipid monolayer by means of in situ atomic force microscopy in tapping mode (TM-AFM). On the basis of these experiments, we conclude that DPPE-MPA and Chs-MPA molecules used in our experiments preserve lipid properties, allowing for at least partial reconstitution of Na(+),K(+)-ATP-ase into such t-BLMs. On the other hand, the relatively compact organization on polycrystalline gold and the hydrophobic nature of the first monolayer of tethered thiolipids slows down the proteoliposome fusion onto such monolayers and consequently hinders the protein insertion. However, this effect can be overcome by mechanical stimulus that facilitates proteoliposome delamination onto the self-assembled monolayer.     

Fluorescence transduction of an enzyme-substrate reaction by modulation of lipid membrane structure

The emission intensity of the fluorophore nitrobenzoxadiazoledipalmitoylphosphatidylethanolamine (NBD-PE) is sensitive to local environmental structure when this species is used as a component of a phospholipid membrane. The physical and electrostatic structure of a membrane may be modulated by selective chemical reactions, and the resulting alteration in fluorescence intensity provides transduction of such selective chemical processes. One example is the reaction between the extrinsic membrane-associated enzyme acetylcholinesterase (AChE) and the substrate acetylcholine (ACh), which produces an increase in hydronium ion activity at the surface of a lipid membrane. A mechanism of transduction of the enzymatic reaction by lipid monolayer membranes was investigated by spectrofluorimetric methods and fluorescence microscopy. Mixed monolayers composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) which contained 30 mol-% or more of DPPA and 1 mol-% of NBD-PE provided transduction of the AChEACh reaction. Reaction of micromolar concentrations of ACh with AChE-monolayer systems induced increases in fluorescence intensity of up to 50%. Direct observation of the microscopic structure of lipid monolayers on a time scale of minutes showed that the reaction did not drastically affect the distribution of coexisting microscopic phase domains that were present in the monolayers The fluorescence imaging and spectroscopic results did indicate that massive structural reorganization at a molecular level probably occurred in a period of seconds. The results are consistent with an electrostatic mechanism of perturbation of the structure of the monolayer in which local pH gradients associated with the reaction of AChE with substrate altered the extent of ionization of DPPA in the headgroup zone of the membrane.     

Comparative study of lipolysis by PLA2 of DOPC substrates organized as monolayers, bilayer vesicles and nanocapsules

The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. One biologically relevant example is the enzymatic hydrolysis of DOPC by PLA(2), which results in cleavage of phospholipids molecules into water insoluble lipolytic products, namely oleic acid and lysophospholipid. In general, the enzymatic activity depends on the substrate organization and molecular environment of the catalytic reaction. The lipolysis by phospholipase A(2) of dioleoylphosphatidylcholine substrates organized as monolayer, bilayers vesicles and lipid nanocapsules was studied by measuring the decrease of the surface area at constant surface pressure or increase of the surface pressure at constant area at air-water interface. A kinetic model describing the coupling of the catalytic act with corresponding interfacial phenomena was developed. By using the kinetic model the values for the global hydrolytic kinetic constants were obtained. The obtained value for the monolayer is five orders of magnitude higher than this obtained with small unilamellar vesicles and six orders of magnitude higher then those obtained with lipid nanocapsules. The comparison shows that the enzymatic catalytic act occurring in the lipid environment of the monolayer is more efficacious than at the vesicle and nanocapsules interfaces.     

Conformational analyses of bacillomycin D, a natural antimicrobial lipopeptide, alone or in interaction with lipid monolayers at the air-water interface

Bacillomycin D is a natural antimicrobial lipopeptide belonging to the iturin family. It is produced by Bacillus subtilis strains. Bacillomycin D is characterized by its strong antifungal and hemolytic properties, due to its interaction with the plasma membrane of sensitive cells. Until now, only few limited analyses were conducted to understand the biological activities of bacillomycin D at the molecular level. Our purpose was to analyze the conformation of bacillomycin D using IR spectroscopy and to model its interactions with cytoplasmic membranes using Langmuir interfacial monolayers. Our findings indicate that bacillomycin D contains turns and allow to model its three-dimensional structure. Bacillomycin D formed a monolayer film at the air-water interface and kept its turn conformation, as shown by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). To identify the membrane lipid target of bacillomycin D, its interactions with pure lipid monolayers were analyzed and an original behavior of the lipopeptide toward cholesterol-containing monolayers was shown. This original behavior was lost when bacillomycin D was interacting with pure cholesteryl acetate monolayers, suggesting the involvement of the alcohol group of cholesterol in the lipopeptide-cholesterol interaction.     

Interactions of a fungistatic antibiotic, griseofulvin, with phospholipid monolayers used as models of biological membranes

Griseofulvin (GF) is an oral antibiotic for widely occurring superficial mycosis in man and animals caused by dermaphyte fungi; it is also used in agriculture as a fungicide. The mechanism of the biological activity of GF is poorly understood. Here, the interactions of griseofulvin with lipid membranes were studied using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE) monolayers spread at the air/water interface. Surface pressure (Pi), electric surface potential (Delta V), grazing incidence X-ray diffraction (GIXD), and Brewster angle microscopy (BAM) were used for studying pure phospholipid monolayers spread on GF aqueous solutions, as well as mixed phospholipid/GF monolayers spread on pure water subphase. Moreover, phospholipase A2 (PLA2) activity toward DLPC monolayers and molecular modeling of the GF surface and lipophilic properties were used to get more insight into the mechanisms of GF-membrane interactions. The results obtained show that GF has a meaningful impact on the film properties; we propose that nonpolar interactions are by and large responsible for GF retention in the monolayers. The modification of membrane properties can be detected using both physicochemical and enzymatic methods. The results obtained may be relevant for elaborating GF preparations with increased bioavailability.     

Hyaluronidase binds differently DPPC,DPPS or GlcNAc-bearing glycolipid biomimetic monolayers

Bovine testis hyaluronidase (btHyal) had been shown to have direct effects on cancer cells and to be a useful adjuvant in several medicines. Furthermore this enzyme had been found to be membrane-associated. Thus, in this work, the interactions between btHyal and membranes were analyzed by using lipid monolayers at the air–water interface as a biomimetic membrane system. This allowed us to define the btHyal interactions with two residues of hyaluronic acid (a btHyal substrate), GlcNAc and carboxylic group, which are present in cholesteryl-triethoxy-N-acetylglucosamine (Chol-E₃-GlcNAc) and in DPPS, respectively. btHyal bound preferentially Chol-E₃-GlcNAc monolayers and showed a decreasing affinity for Chol-E₃-GlcNAc-DPPC monolayers containing decreasing amount of glycolipid, suggesting a crucial role of the glycolipid GlcNAc. Furthermore the significant btHyal binding to DPPS was not affected by the presence of free GlcNAc in the subphase. These results and the absence of significant binding of btHyal to pure DPPC monolayer suggest that the protein interacts with the lipid monolayer by mimicking the enzyme–substrate interactions or by electrostatic interactions.     

Effects of lipid chain length on the surface properties of alkylaminomethyl rutin and of its mixture with model lecithin membrane

Three model flavonoid-based bioactive molecules with different lipid chain lengths (RuCn: n=8, 12, 18) were newly synthesized. The surface properties [surface pressure (π)-area (A), surface potential (ΔV)-surface pressure (π) and dipole moment (u(⊥))-surface pressure (π)] of pure RuCn and the lecithin membrane compounds had been investigated by using the Langmuir monolayer technology. The results suggested that the distinctive monolayer behavior of RuCn is strongly dependent on the lipid chain length. The great differences in the monolayer properties brought by the lipid chain length could be attributed to two major factors: (i) the ionization degree of the bulky hydrophilic head group (including hydroxyl and NH groups) alters its local field solely via the surface potential; (ii) tring molecular (or dipole) packing density within monolayers. The excess Gibbs energy (ΔG((ex))) calculated for the RuCn-lecithin mixed monolayers infers that higher stability of the mixed monolayer can be strengthened as the lipid chain length decreases. And the addition of RuCn into lecithin membrane may increase the total u(⊥) of the binary mixed monolayers, which could inhibit the hydration of the lecithin's hydrophilic head groups. The shorter the lipid chain length of RuCn (e.g., RuC8) is, the higher the surface activity can be. Our findings provide a molecular basis for the application of such class of biomolecules in the functional food, cosmetics and medicine.     

Assembly of antibodies in lipid membranes for biosensor development

An investigation of the incorporation of antibody in lipid films of a composition that has been used for biosensor preparation is reported. IgG that is incorporated into lipid monolayers prepared from 7:3 mixtures of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic acid is edge-active, and enters and penetrates the fluid region of the mixed-phase system when monolayers are held at low pressure (< 20 mN/m). It was found that there is an “exclusion pressure” observed in pressure-area (π-A) curves that are collected for monolayers that contain antibody. This term refers to a specific threshold of lateral pressure (which is reached by monolayer compression) that can cause explusion of antibody from the interior of a membrane. Microscopic images of monolayers containing the fluorescent phospholipid nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE), or antibody labeled with tetramethylrhodamine isothiocyanate (TRITC), were used to determine the structure of membranes, and the location of effects on structure caused by IgG. Ellipsometric measurements of lipid monolayers that were cast onto silicon wafers by the LangmuirBlodgett method were used to study the thickness of monolayers and to investigate the structural changes that occurred at the “exclusion pressure.” Both the use of fluorescent antigen and ellipsometry indicated that antibody binding activity was present and was dependent on compression pressure. The effects of pH and ionic strength of subphase, antibody concentration, incubation time, and lateral pressure have been examined. The results may indicate the conditions that can be used to improve the incorporation of active IgG for preparation of biosensors that are based on lipid membranes.     

Properties of mixed lipid monolayers assembled on hydrophobic surfaces through vesicle adsorption

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.     

Negatively charged phospholipids trigger the interaction of a bacterial Tat substrate precursor protein with lipid monolayers

Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection-absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron-sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate-membrane interactions.     

Ceramide-enriched microdomains in planar membranes

Ceramide has a large effect on the properties of biological membranes, increasing lipid order and promoting lateral phase separation, and plays an important role in cell signaling. This review provides an overview of recent studies of the effects of direct ceramide incorporation and enzymatic ceramide generation on planar supported membranes, including lipid monolayers and supported lipid bilayers. Recent studies have focused on understanding the nucleation, growth and morphology of ceramide gel domains, characterizing the properties of ceramide-rich membrane phases and investigating the effects of ceramide on phase-separated membranes with co-existing liquid-ordered and fluid phases, as models for cellular membranes.     

The study of the interaction of a model alpha-helical peptide with lipid bilayers and monolayers

We studied the interaction of the alpha-helical peptide acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)) with tethered bilayer lipid membranes (tBLM) and lipid monolayers formed at an air-water interface. The interaction of L(24) with tBLM resulted in adsorption of the peptide to the surface of the bilayer, characterized by a binding constant K(c)=2.4+/-0.6 microM(-1). The peptide L(24) an induced decrease of the elasticity modulus of the tBLM in a direction perpendicular to the membrane surface, E(radial). The decrease of E(radial) with increasing peptide concentration can be connected with a disordering effect of the peptide to the tBLM structure. The pure peptide formed a stable monolayer at the air/water interface. The pressure-area isotherms were characterized by a transition of the peptide monolayer, which probably corresponds of the partial intercalation of the alpha-helixes at higher surface pressure. Interaction of the peptide molecules with lipid monolayers resulted in an increase of the mean molecular area of phospholipids both in the gel and liquid crystalline states. With increasing peptide concentration, the temperature of the phase transition of the monolayer shifted toward lower temperatures. The analysis showed that the peptide-lipid monolayer is not an ideally miscible system and that the peptide molecules form aggregates in the monolayer.     

A thermodynamic study of GPI-anchored and soluble form of alkaline phosphatase films at the air-water interface

Glycosylphosphatidyl inositol (GPI) anchored proteins are localized and clustered on the outer layer of the plasma membranes forming microdomains. Among them, mammalian alkaline phosphatases (AP-GPI) are widely distributed enzymes. They can also exist as soluble proteins without anchor (APs). Using the Langmuir film technique, we study the thermodynamic properties of monolayers for both protein forms at the air-buffer interface. The enzymatic activity is maintained at the interface but the adsorption of the two forms of AP is very different. AP-GPI presents a higher surface activity and a larger molecular area than the soluble form. The molecular area deduced for high surface pressures suggests a different organization of the monolayers for these two forms. APs molecules seem to adsorb as a multilayer at the interface while AP-GPI appear to be orientated with the major axis parallel to the interface. This orientation allows the accessibility of AP-GPI enzymatic sites that are turned in direction of the subphase as in vivo where the active sites must be turned outside of the membrane.     

Interactions of lipases with lipid monolayers. Facts and questions

Among the proteins, lipolytic enzymes provide a valuable model for studying protein-lipid interactions. Lipases having a catalytic action which is strictly dependent upon the presence of a lipid interface were used in the present study in order to gain better insight into protein-lipid interactions. Most of the data presented here were obtained using the monolayer technique, by recording (either independently or simultaneously) the lipolytic activity, the amount of protein adsorbed to the lipid monolayer, and the surface pressure variations following protein adsorption. Several non-enzymatic proteins were used as controls in order to determine how lipase behaviour differs from that of other proteins. At all initial surface pressures tested, with zwitterionic monolayers, a good correlation was observed between the amount of lipase bound to the monolayer and the surface pressure increase, in agreement with previous studies. Conversely, with neutral lipid monolayers the amount of lipase bound to the monolayer was not found to be surface pressure dependent. This latter behaviour observed with lipases on neutral films is not specific to lipases, since it was also observed with bovine serum albumin and beta-lactoglobulin A. Lipase activity in the presence of various proteins was investigated with monomolecular films of glycerol didecanoate, either at constant surface area or at constant surface pressure. Depending upon the nature of the lipase and the protein, inhibition of lipase activity was either observed or not. Inhibition was correlated with a decrease in lipase surface concentration. The ability of the various proteins to inhibit lipolysis is: (i) a function of their excess versus lipase in the bulk phase, and: (ii) correlated with their penetration capacity (i.e., the initial rate of surface pressure increase of a glycerol didecanoate monolayer having an initial surface pressure of 20 dyn/cm, after the injection-of the protein). Since lipase inhibition was observed with low surface densities of inhibitory proteins, a long-range effect is probably involved in the mechanism of interfacial lipase inhibition. The nature of the ionic charge added to the monolayer by the protein is not critical for determining lipase adsorption or desorption. It is hypothesized that the lack of lipase adsorption to, or desorption from, the lipid monolayer results from a change in the organization of the hydrocarbon moiety of the lipid.     

Incorporation of blood clotting factor X into phospholipid model membranes: fluorescence microscopy imaging and subphase effects surrounding the lipid phase transition region

Factor X is a blood clotting protein that associates at membrane surfaces to become activated during the coagulation cascade. A molecular level understanding of the protein-membrane phospholipid interactions has not been reached, although it is thought that the protein binds to phospholipids in the presence of calcium through a bridge with the Gla (gamma-carboxyglutamic acid) domain on the protein. In this work, phospholipid Langmuir monolayers have been utilized as model membranes to study factor X association with phospholipid membrane components. Surface pressure measurements indicate that subphase addition of sodium, magnesium, and calcium ions enhances protein penetration of the lipid monolayer, with the largest association found with calcium ions in the subphase. Fluorescence microscopy images collected after protein penetration of lipid monolayers indicate monolayer condensation in the presence of sodium and magnesium ions. Aggregation of lipid domains is induced when calcium is in the subphase, indicating binding-induced flocculation of surface lipid aggregates. Calcium binding to factor X likely causes a conformational change which allows protein-membrane interaction via hydrophobic association with lipid molecules.     

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.     

Effect of the molecular structure of more common amphiphilic molecules on their interactions with dioleoyl-phosphatidylcholine monolayers

A large number of surface-active or amphiphilic compounds interact with biological membranes and their various models. The surface-active properties of these compounds have been cited as a fundamental factor which determines the tendency of these molecules to bind to, to cross and to perturb the lipid structure of biomembranes and biomembranes models. As well as surface activity, the extent of interaction of a compound with a membrane has been correlated with its lipophilicity. This study shows that the surface activity and lipophilicity of a compound are not the only factors which determine its interaction with a lipid membrane structure. Experiments have been carried out looking at the effect of compounds of different molecular structure on their type of association with self-assembled phospholipid monolayers on mercury electrodes. The paper concludes by demonstrating the properties of different compounds which facilitate their interaction with the supported lipid monolayer.     

Organization, structure and activity of proteins in monolayers

Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.     

Effect of gramicidin on phospholipid-modified monolayers and on ion transfer at a liquid-liquid interface.

The interaction of hybrid lipid/gramicidin A (gA) monolayers with dextran sulfate (DS) and the effect of this interaction on ion transfer at a liquid-liquid interface is reported. The interfacial and physicochemical properties are studied with Langmuir-Blodgett (LB) and electrochemical techniques. The results obtained from compression isotherms demonstrate that the interactions between the different species in the hybrid monolayer vary according to the chemical nature of the lipid (hydrocarbon region and charge of the head group). Interfacial capacitance measured with AC voltammetry indicates that the DS chains form a rather flat and compact layer when adsorbed to either zwitterionic or negatively charged phospholipid monolayers, and that calcium, even at low concentrations, interacts with the monolayers. These results are successfully described by a model based on the solution of the Poisson-Boltzmann equation in the interfacial region. Ion transfer and interactions with the lipid/gA/DS-modified monolayers were also studied with electrochemical techniques. Admittance data show that although the studied ions are not using gA channels for the transfer through the lipid membranes, the incorporation of gA in the lipid domain and the adsorption of DS at the interface have a significant effect on ion transfer across the monolayers. This effect can be explained as a consequence of the modified surface charge and of the compactness of the lipid domain due to its interaction with gA and to calcium and DS adsorption at the interface. The ion-transfer rate, therefore, depends on the composition of the monolayer and the chemical nature of the ion.     

Chemical transduction with fluorescent lipid membranes using selective interactions of acetylcholine receptor with agonist/antagonist and acetylcholine with substrate

Alterations in the physical structure of vesicles and monolayers of phospholipids and soybean lecithin were monitored by measurement on the average fluorescence intensity changes from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-a-phosphatidyl ethanolamine (NBD-PE) located in the lipid matrices. This probe was intimately dispersed at a concentration of 1-2 mol-% in lipid membranes and had an emission sensitive to local environmental structure. Alterations in the structure of soybean lecithin vesicles were induced by the selective interaction of acetylcholine receptor with the agonist carbamylcholine and the antagonist alpha-bungarotoxin. Structural changes in vesicles with a 7:3 mole ratio of dipalmitoylphosphatidyl choline to dipalmitoylphosphatidic acid were observed for selective interactions between acetylcholinesterase and acetylcholine. Enhancement of fluorescence emission from the lipid membranes provided transduction of the selective binding events of the receptor and enzyme. A maximum sensitivity of about a 30% enhancement per micromole of carbamylcholine and a detection limit for the toxin of 10 nM were observed for the receptor. Fluorescence microscopy was used to establish that protein could be incorporated in monolayer lipid membranes and to provide information about potential mechanisms of fluorescence enhancement. These studies show that lipid membranes containing NBD-PE can be used as generic transducers of protein-ligand interactions.