Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.     

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

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

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

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

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

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

Measuring peptide translocation into large unilamellar vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.     

Osmotically induced shape changes of large unilamellar vesicles measured by dynamic light scattering

Static and dynamic light scattering measurements have been used to characterize the size, size distribution, and shape of extruded vesicles under isotonic conditions. Dynamic light scattering was then used to characterize osmotically induced shape changes by monitoring changes in the hydrodynamic radius (R(h)) of large unilamellar vesicles (LUVs). These changes are compared to those predicted for several shapes that appear in trajectories through the phase diagram of the area difference elasticity (ADE) model (. Phys. Rev. E. 52:6623-6634). Measurements were performed on dioleoylphosphatidylcholine (DOPC) vesicles using two membrane-impermeant osmolytes (NaCl and sucrose) and a membrane-permeant osmolyte (urea). For all conditions, we were able to produce low-polydispersity, nearly spherical vesicles, which are essential for resolving well-defined volume changes and consequent shape changes. Hyper-osmotic dilutions of DOPC vesicles in urea produced no change in R(h), whereas similar dilutions in NaCl or sucrose caused reductions in vesicle volume resulting in observable changes to R(h). Under conditions similar to those of this study, the ADE model predicts an evolution from spherical to prolate then oblate shapes on increasing volume reduction of LUVs. However, we found that DOPC vesicles became oblate at all applied volume reductions.     

Structural basis of anthracycline selectivity for unilamellar phosphatidylcholine vesicles: an equilibrium binding study

Fluorescence anisotropy titration was used to determine the equilibrium binding affinities of several anthracycline antitumor antibiotics for sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Eight daunomycin analogues, all differing from the parent by one structural change in the aglycon portion of the molecule, as well as four anthracycline congeners modified in the amino sugar were studied. Double-reciprocal plots were used to determine overall binding affinities (K). It was shown that structural changes in both the aglycon and amino sugar portions of the daunomycin molecule strongly modulated K values for DMPC and DPPC bilayers. For modifications in the aglycon portion of an anthracycline, a correlation between drug hydrophobicity and membrane affinity was observed. The number of binding sites per phospholipid molecule (n) and the apparent association constant (Kapp) where K = nKapp, were determined at several temperatures for adriamycin, daunomycin, and carminomycin. The n values were found to be independent of temperature for fluid-phase DMPC or solid-phase DPPC bilayers. The Kapp values (25 degrees C) ranged from (0.82-4.4) X 10(5) M-1 for DMPC vesicles to (4.4-7.3) X 10(5) M-1 for DPPC vesicles. Although the Kapp values for the three drugs were similar for a particular bilayer, major differences were noted in the values of n and, therefore, in the overall vesicle affinities (nKapp). van't Hoff plots showed that anthracycline binding was exothermic; in all cases but one binding was accompanied by a decrease in entropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extrusion of electroformed giant unilamellar vesicles through track-etched membranes

Unilamellar vesicle populations having a narrow size distribution and mean radius below 100 nm are preferred for drug delivery applications. In the present work, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used to prepare giant unilamellar vesicles (GUVs) by electroformation and multilamellar vesicles (MLVs) by thin film hydration. Our experiments show that in contrast to MLVs, a single-pass extrusion of GUVs through track-etched polycarbonate membranes at moderate pressure differences is sufficient to produce small liposomes having low polydispersity index. Moreover, we observe that the drug encapsulating potential of extruded liposomes obtained from GUVs is significantly higher compared to liposomes prepared by extrusion of MLVs. Furthermore, our experiments carried out for varying membrane pore diameters and extrusion pressures suggest that the size of extruded liposomes is a function of the velocity of GUV suspensions in the membrane pore.     

Cholesterol transfer from small and large unilamellar vesicles

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

Protein purification by affinity binding to unilamellar vesicles

A novel purification technique is proposed which employs affinity-ligand-modified liposomes to specifically purify bioactive macromolecules from solution. This process is demonstrated with avidin as the model biomolecule and biotin as the affinity ligand. Biotin is covalently bound to the surface of small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylethanolamine (DMPE). The number of accessible binding sites on the liposomes is determined by titration with avidin, and the kinetics of binding are evaluated by monitoring the concentration of free avidin in solution after the addition of biotinylated liposomes. The specificity of the process is determined by following the affinity binding of avidin to biotinylated liposomes in the presence of model impurities (i.e., lysozyme and cytochrome C). Liposome-bound avidin is separated from the impurities by ultrafiltration through a membrane which retains the liposomes.     

Quantitative analysis of the binding of ezrin to large unilamellar vesicles containing phosphatidylinositol 4,5 bisphosphate

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP₂) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP₂. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ∼5 μM for PIP₂-LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP₂-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ∼30 nm²) binding to a single PIP₂ can block access to neighboring PIP₂ molecules and thus contributes to lower the accessible PIP₂ concentration. In addition, no evidence exists for a clustering of PIP₂ induced by ezrin addition.     

Interaction of wheat alpha-thionin with large unilamellar vesicles.

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

Curvature elasticity and refolding of OmpA in large unilamellar vesicles

The stability of OmpA in large unilamellar vesicles of dilauroyl phosphatidylcholine was studied using different concentrations of urea. The effective energy of unfolding, as determined from refolding experiments, is greater than that for small sonicated unilamellar vesicles by an amount that is compatible with estimates of the elastic energy of highly curved vesicles. The on-rate for refolding and insertion is slower for large unilamellar vesicles than for small unilamellar vesicles, which indicates a contribution of vesicle strain also to the free energy of the transition state.     

Modeling of matrix vesicle biomineralization using large unilamellar vesicles

Stable, large unilamellar vesicles (LUV) have been constructed that model matrix vesicles (MV) in inducing de novo mineral formation when incubated in synthetic cartilage lymph (SCL). Using a dialysis method for incorporation of predetermined pure lipid, electrolyte and protein constituents, the detergent n-octyl beta-D-glucopyranoside enabled formation of stable, impermeable LUV with a diameter ( approximately 300 nm), lipid composition (phosphatidylcholine-phosphatidylserine-cholesterol, 7:2:2, molar ratio) and enclosed inorganic phosphate level (25-100 mM) similar to that of native MV. Mineral formation by these LUVs was measured by 45Ca(2+) uptake and FTIR analysis following incubation in SCL. Addition of the ionophore A23187 to SCL enabled 45Ca(2+) uptake comparable to that of native MV. FTIR analysis revealed that crystalline mineral formed in the LUV during incubation in SCL, but not in the absence of ionophore. This mineral had an IR absorption spectrum like that of the acid-phosphate-rich, octacalcium phosphate-like mineral formed by native MV. Perturbing the LUV membrane with either detergents or phospholipase A(2) following prior incubation in SCL enabled egress of mineral crystallites from the vesicle lumen, stimulating further mineral formation. Annexin V, a major protein in native MV with known Ca(2+) channel activity, incorporated into the LUV lumen or added to the external medium, induced only limited 45Ca(2+) uptake. This indicates that additional factors are required for annexin V to form Ca(2+) channels. Nevertheless for the first time, stable LUVs have been constructed with MV-like lipid, electrolyte, and protein composition and size that induce formation of mineral like that formed by native MV.     

Uniform preparations of large unilamellar vesicles containing anionic lipids

A general procedure for the preparation of large unilamellar vesicles of selected sizes has been developed. The procedure consists of dissolving the lipid in organic solvent, washing with mild acid, removing the solvent, adding salt (0.15 M KCl) solution, and adjusting the pH (raising it to about pH 10 and lowering it immediately to pH 7.55). The procedure takes less than 30 min. The resulting unilamellar vesicles are of a single size with a rather low standard deviation. The sizes of these preparations range between 150 and 1000 nm in diameter. Sizes and polydispersities were measured to within 1-2% by photon correlation spectroscopy. Vesicle size varies with the phospholipid structure, the composition of the phospholipid mixture, the ionic strength of the medium, the alkyl chain composition, the cholesterol content of the phospholipid mixture, and the timing of the pH adjustment procedure. Uniform preparations of vesicles have been obtained from the dioleoyl esters of phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine, from diphytanyl ethers of glycolipid sulfate, phosphatidylglycerol, phosphatidylglycerol phosphate, and phosphatidylglycerol sulfate, from bovine liver phosphatidylinositol, from Escherichia coli phosphatidylethanolamine, from membrane lipid extracts from E. coli and Holabacterium cutirubrum, and from dodecanesulfonate-alkanol mixtures and free oleic acid. The preparation of unilamellar vesicles from oleic acid is novel, and the size range is 600-3000 nm; the preparations are relatively uniform. Vesicles of phospholipids in which sucrose and trehalose replace salt as the impermeant do not differ significantly from those prepared in pentaerythritol.     

Optical changes in unilamellar vesicles experiencing osmotic stress.

Membrane properties that vary as a result of isotropic and transmembrane osmolality variations (osmotic stress) are of considerable relevance to mechanisms such as osmoregulation, in which a biological system "senses" and responds to changes in the osmotic environment. In this paper the light-scattering behavior of a model system consisting of large unilamellar vesicles of dioleoyl phosphatidyl glycerol (DOPG) is examined as a function of their osmotic environment. Osmotic downshifts lead to marked reductions in the scattered intensity, whereas osmotic upshifts lead to strong intensity increases. It is shown that these changes in the scattering intensity involve changes in the refractive index of the membrane bilayer that result from an alteration in the extent of hydration and/or the phospholipid packing density. By considering the energetics of osmotically stressed vesicles, and from explicit analysis of the Rayleigh-Gans-Debye scattering factors for spherical and ellipsoidal shells, we quantitatively demonstrate that although changes in vesicle volume and shape can arise in response to the imposition of osmotic stress, these factors alone cannot account for the observed changes in scattered intensity.     

Kinetics of melittin binding to phospholipid small unilamellar vesicles

We have used the decrease in the fluorescence intensity of the single tryptophan residue of bee venom melittin at long emission wavelengths that accompanies binding of the peptide to phospholipid small unilamellar vesicles to determine the rate of binding through the use of stopped-flow fluorometry in the millisecond range. We have found the rate to depend on the degree of saturation of the lipid acyl chains as well as on the physical state of the bilayer, the net electric charge of the polar headgroups, and the lipid-to-melittin molar ratio R. For zwitterionic lipids (i) the binding process is found to exhibit negative cooperativity, and (ii) the rate-limiting step appears to be penetration of the protein into the hydrophobic region of the bilayer. For negatively charged lipids the results show that binding is a very fast process that seems to be electrostatic in nature.     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Effect of sodium taurocholate and sodium cholate on short-circuit current on amphibian membranes

The effects of the bile salts, sodium taurocholate (NaTc) and sodium cholate (NaCh), and toad bile gallbladder (bile) on short-circuit current (SCC) across isolated skin, and sodium taurocholate (NaTc) on isolated bladder of Bufo arenarum toads were tested. Sodium taurocholate (NaTc), sodium cholate (NaCh) and toad bile gallbladder (bile) promoted an increase in SCC, when added to the external side. The stimulatory effect was reversible after rinsing the preparation for 60 min. Implications on in vivo renal function of these results are discussed.