Lipid-protein surface films generated from membrane vesicles: selfassembly,composition, and film structure

Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 l vesicle suspensions containing 25 g protein yield at least 50 cm² film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure ( 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (±0.2) nm². Offprint requests to: H. Schindler     

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.     

Lipid mobility in the assembly and expression of the activity of the prothrombinase complex

A phospholipid or membrane surface is a required component of the prothrombinase complex, yet little is known about the influence of the lipid on the assembly and expression of this complex. Vesicles composed of synthetic phospholipids were used to investigate the effects of membrane "fluidity" on the prothrombinase complex. All vesicle types studied were capable of supporting the prothrombinase reaction which in each case was characterized by a similar apparent Km. The binding constants for the interaction of Factor Va and prothrombin with synthetic phospholipid vesicles were not significantly affected by temperature. The rate of thrombin production, however, increased with increasing temperature. The fluidity of the vesicles was assessed by measuring the fluorescence lifetimes, steady state anisotropies, and differential phase fluorometry of diphenylhexatriene embedded in the vesicles. No correlation was observed between the fluidity of the vesicles and the steady-state rate of thrombin production, even when the enzymatic activity was monitored below and above the phase transition temperature of the lipid vesicles. A distinct correlation, however, was found between the fluidity of the vesicle and the time required to reach the maximum rate of thrombin production (pre-steady-state interval). We believe that this "lag" time corresponds to the time required for the assembly of the prothrombinase complex. Thus, although lipid fluidity does affect the assembly of the prothrombinase complex, after the complex is assembled, this property has little effect on the catalytic process itself.     

Lipid vesicle adsorption versus formation of planar bilayers on solid surfaces.

The absorption and spreading behavior of lipid vesicles composed of either palmitoyloleoylphosphatidylcholine (POPC) or Escherichia coli lipid upon contact with a glass surface was examined by fluorescence measurements. Fluorescently labeled lipids were used to determine 1) the amount of lipid adsorbed at the surface, 2) the extent of fusion of the vesicles upon contact with the surface, 3) the ability of the adsorbed lipids to undergo lateral diffusion, and 4) the accessibility of the adsorbed lipids by external water soluble molecules. The results of these measurements indicate that POPC vesicles spread on the surface and form a supported planar bilayer, whereas E. coli lipid vesicles adsorb to the surface and form a supported vesicle layer. Supported planar bilayers were found to be permeable for small molecules, whereas supported vesicles were impermeable and thus represented immobilized, topologically separate compartments.     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

Exchange and interactions between lipid layers at the surface of a liposome solution

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. p] Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

STUDIES ON SEEDS : III. Isolation and Structure of Lipid-Containing Vesicles

Two structurally distinct lipid vesicles are present in pea and bean cotyledons during the first few days of germination. Both were isolated by sucrose density gradient centrifugation without significant morphological changes. Lipid vesicles of one type were elongated into a sausage-like or flattened-saccular shape, and were interassociated into sheets which were usually one vesicle thick. These sheets remained intact during homogenization and centrifugation, because some of the lipid vesicles in the sheet were interconnected through their bounding membranes, and because there seemed to be a bonding substance between adjacent vesicles. These vesicles were called "composite" lipid vesicles to distinguish them from the more usual, or "simple," lipid vesicles of other plant and animal tissues. Lipid vesicles of the other type were usually larger than the composite lipid vesicles and were always spherical in form. These vesicles remained single and did not interassociate into sheets. They were probably equivalent to the simple lipid vesicles of other tissues.     

Exchange and interactions between lipid layers at the surface of a liposome solution.

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.Download video file.^{(48M, mov)}     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

Phospholipid vesicles promote human hemoglobin oxidation.

We have studied the heme oxidation kinetics of purified human hemoglobin (Hb) in the presence of lipid vesicles of dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine that exhibited minimal lipid peroxidation. We showed that the lipid vesicles enhanced Hb oxidation and that small unilamellar vesicles (SUVs) exerted a larger effect than large unilamellar vesicles (LUVs). We have determined pseudo first-order rate constants for the initial disappearance of oxygenated ferrous Hb (k0) and for the initial formation of several ferric Hb species (methemoglobin, hemichrome, and choleglobin) in the presence of SUVs and LUVs. k0 and other rate constants depended linearly on lipid-to-hemoglobin molar ratio (lipid/Hb), with k0SUV (h-1) = k0auto (h-1) + 3.7 x 10(-3) x lipid/Hb, and k0LUV (h-1) = k0auto (h-1) + 0.2 x 10(-3) x lipid/hb, where k0auto is the rate constant for Hb autoxidation in the absence of vesicles. Thus, in the absence of lipid peroxidation products, lipid vesicles themselves promote Hb oxidation by enhancing the rate of Hb oxidation. The enhanced oxidation was inhibited by catalase, but not by butylated hydroxytoluene. The rate constants were independent of Hb concentration, in the range of about 3.1 to 100 microM. We suggest that the lipid surface properties, including surface curvature, surface energy, and hydrophobicity, promote hemoglobin oxidation.     

Effect of average phospholipid curvature on supported bilayer formation on glass by vesicle fusion

The adsorption of large unilamellar vesicles composed of various combinations of phosphatidylcholine, phosphatidylethanolamine (PE), monomethyl PE, and dimethyl PE (PE-Me2) onto a glass surface was studied using fluorescence microscopy. The average lipid geometry within the vesicles, described mathematically by the average intrinsic curvature, C(0,ave), was methodically altered by changing the lipid ratios to determine the effect of intrinsic curvature on the ability of vesicles to rupture and form a supported lipid bilayer. We show that the ability of vesicles to create fluid planar bilayers is dependent on C(0,ave) and independent of the identity of the component lipids. When the C(0,ave) was approximately -0.1 nm(-1), the vesicles readily formed supported lipid bilayers with almost full mobility. In contrast, when the C(0,ave) ranged from approximately -0.2 to approximately -0.3 nm(-1), the adsorbed vesicles remained intact upon the surface. The results indicate that the average shape of lipid molecules within a vesicle (C(0,ave)) is essential for determining kinetically viable reactions that are responsible for global geometric changes.     

Targeted liposomes: convenient coupling of ligands to preformed vesicles using "click chemistry"

An efficient and convenient chemoselective conjugation method based on "click chemistry" was developed for coupling ligands to the surface of preformed liposomes. It can be performed under mild conditions in aqueous buffers; the use of a water soluble Cu(I) chelator, such as bathophenanthrolinedisulfonate, was essential to obtain good yields in reasonable reaction times. A model reaction was achieved in which, in a single step, an unprotected alpha-D-mannosyl derivative carrying a spacer arm functionalized with an azide group was conjugated to the surface of vesicles presenting a synthetic lipid carrying a terminal alkyne function. When liposomes composed of saturated phospholipids were used, the reaction conditions developed in the present work did not damage the membranes as measured by the absence of leakage of entrapped 5,6-carboxyfluorescein. Moreover, as assessed by agglutination experiments using concanavalin A, the mannose residues were perfectly accessible on the surface of the targeted vesicles.     

Enhanced binding of phosphatidylserine-containing lipid vesicle targets to RAW264 macrophages

Summary Phosphatidylserine was found to significantly enchance the binding of phospholipid vesicles to RAW264 macrophages. We have measured the kinetics of non-specific uptake of unilamellar vesicles as a function of phosphatidylserine concentration in these model target membranes. Dimyristoylphosphatidylcholine was the principle component of these phospholipid vesicles. In most experiments, radiolabeled phospholipid and 1 mol % each of both a fluorescent phospholipid and a hapten-containing lipid headgroup were utilized. In the presence of specific anti-hapten antibody phosphatidylserine-containing vesicles are rapidly taken up via phagocytosis. The antibody-independent non-specific uptake of phosphatidylserine-free vesicles was low, as previously reported. However, the presence of 5 mol % phosphatidylserine dramatically enhanced the uptake of phospholipid vesicles by macrophages. This uptake was shown to be principally due to binding to the macrophage surface. Incubation of macrophages in the presence of sodium azide or at 4°C, conditions which are known to inhibit phagocytosis, do not influence the uptake of the lipid vesicles. Fluorescence video-intensification microscopy was used to observe the interaction of carboxyfluorescein-loaded vesicles with macrophages. Fluorescence could not be observed when using phosphatidylserine-free vesicles. However, phosphatidylserine-containing vesicles can be observed bound to the cell periphery. Intracellular fluorescence could not be observed. The binding of phosphatidylserine-containing vesicles was enhanced roughly four-fold over phosphatidylserine because the effect could not be observed with membranes containing 1 mol % or 2.5 mol% phosphatidylserine. In addition, the binding enhancement required the presence of divalent cations in the incubation medium.Abbreviations DMPC dimyristoylphosphatidylcholine - PS phosphatidylserine - DNP-PE dinitrophenyl---minocaproyl-phosphatidylethanolamime - NBDPE N-4-nitrobenzo-2-oxa-1, 3-diazole phosphatidylethanolamine - EDTA ethylenediaminetetraacetic acid     

Simultaneous monitoring of electroformation of phospholipid vesicles by quartz crystal microbalance and optical microscopy

The electroformation of giant vesicles from 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) and optical microscopy, simultaneously using a novel sample cell design. A gold-coated QCM crystal was used as one of the electrodes and an Indium–tin-oxide (ITO)-coated glass slide was used as the second electrode for electroformation. Increases in the frequency and decreases in the dissipation were observed immediately upon voltage application between the two electrodes, indicating the loss of lipid from the QCM surface. Concurrently, we observed vesicles on the QCM electrode surface by differential interference contrast (DIC)-optical microscopy. The lipid-coated substrates were measured with AFM at various stages in the electroformation, and a significant change in the morphology of the lipid film was observed. Ellipsometry was used to find the average thickness of lipid film. The QCM data were fitted to a viscoelastic model to determine the viscoelastic properties and time dependence of the film thickness. All methods used to determine film thickness give values in reasonable quantitative agreement. Differences between the methods are consistent with what one might expect due to what is actually measured in the individual techniques. The comparison between mass loss and observed vesicles suggest that the vesicles formed are first localized to the substrate and then slowly released into the solution. By comparing the mass lost from the lipid film, to the total surface area of lipid vesicles observed, it is apparent that only a relatively small fraction of the lipid goes into the production of unilamellar vesicles with sizes detectable with optical microscopy.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Cell membrane hybrid bilayers containing the G-protein-coupled receptor CCR5

A hybrid bilayer membrane is a planar model membrane that is formed at an alkanethiol monolayer-coated gold surface by the spontaneous reorganization of phospholipid vesicles. Membrane vesicles from monkey kidney COS-1 cells also reorganize at an alkanethiol/lipid monolayer-coated surface resulting in the formation of a cell membrane hybrid bilayer. Atomic force microscopy and spectroscopic ellipsometry indicate that the cell membrane layer is equivalent to the thickness of one leaflet of the membrane and is continuous over large areas. Cell membrane hybrid bilayers were formed from membrane vesicles from COS-1 cells that were transiently transfected with a synthetic human CCR5 chemokine receptor gene. Preparations that contained "inside out" and "right side out" membrane vesicles were used. Binding of monoclonal antibodies to either the amino- or carboxyl-terminus of CCR5 was observed by surface plasmon resonance and confirmed the presence and the random orientation of these integral membrane receptors. Specific and concentration-dependent binding of the beta-chemokine RANTES to the cell membrane hybrid confirmed that CCR5 retained ligand-binding activity. The ability to form cell membrane hybrid bilayers that contain functional G-protein-coupled or other multispanning receptors without requiring protein isolation, purification, and reconstitution offers a promising method for the rapid screening of potential ligands.     

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.     

On the interaction of hemocyanin with lipid bilayer membranes

Osmotic jump experiments were used to measure the ionic permeability induced in lipid vesicles by Megathura crenulata hemocyanin. It was found that this protein strongly increases the conductance of K⁺ and Cl^- through these membranes but not that of SO ₄ ⁼ . These effects were attributed to the formation of ionic channels in the vesicles. We have found that a simple first-order binding model can explain the dependence of the number of pore-containing vesicles both on the time after exposure to hemocyanin and on the protein concentration. Milder effects were attributed to a non-specific adhesion of the protein to the membrane surface. Consistent with the hypothesis of reversible association, vesicles which retained hemocyanin after step sucrose density gradient centrifugation at low ionic strength, lost most of the protein upon recentrifugation at high ionic strength. Consistent with the hypothesis of channel formation bot the above vesicle preparations transferred voltage-dependent hemocyanin channels into planar bilayers when they were made to fuse with them. It is concluded that hemocyanin can interact both specifically, by forming pores within the hydrophobic core of lipid membranes, and non-specifically, probably by means of electrostatic interaction with the surface of the same membrane.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - DOC sodium deoxycholate     

Electrostatic properties of membranes containing acidic lipids and adsorbed basic peptides: theory and experiment

The interaction of heptalysine with vesicles formed from mixtures of the acidic lipid phosphatidylserine (PS) and the zwitterionic lipid phosphatidylcholine (PC) was examined experimentally and theoretically. Three types of experiments showed that smeared charge theories (e.g., Gouy-Chapman-Stern) underestimate the membrane association when the peptide concentration is high. First, the zeta potential of PC/PS vesicles in 100 mM KCl solution increased more rapidly with heptalysine concentration (14.5 mV per decade) than predicted by a smeared charge theory (6.0 mV per decade). Second, changing the net surface charge density of vesicles by the same amount in two distinct ways produced dramatically different effects: the molar partition coefficient decreased 1000-fold when the mole percentage of PS was decreased from 17% to 4%, but decreased only 10-fold when the peptide concentration was increased to 1 microM. Third, high concentrations of basic peptides reversed the charge on PS and PC/PS vesicles. Calculations based on finite difference solutions to the Poisson-Boltzmann equation applied to atomic models of heptalysine and PC/PS membranes provide a molecular explanation for the observations: a peptide adsorbing to the membrane in the presence of other surface-adsorbed peptides senses a local potential more negative than the average potential. The biological implications of these "discreteness-of-charge" effects are discussed.