Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.     

Determination of lipid asymmetry in human red cells by resonance energy transfer

This report describes the application of a resonance energy transfer assay to determine the transbilayer distribution of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipid analogues. The validity of this technique was established by determining the relationship between the distance of separation of lissamine rhodamine B labeled phosphatidylethanolamine (N-Rho-PE) acceptor lipid and NBD-labeled donor lipid and energy transfer efficiency. By determination of the distance between probes at 50% transfer efficiency (R0), the distance between fluorophores distributed symmetrically (outer leaflet label) and asymmetrically in artificially generated vesicles was determined. Calculation of the average distance between probes revealed a 14-A difference between NBD-lipid and N-Rho-PE localized in the same leaflet and in opposing leaflets, respectively. Application of this technique to the study of the transbilayer distribution of NBD-lipid in human red blood cells (RBC) showed that exogenously supplied NBD-phosphatidylserine (NBD-PS) was selectively transported to the inner leaflet, whereas NBD-phosphatidylcholine remained in the outer leaflet. In contrast, pretreatment of the RBC with diamide (a SH cross-linking reagent) blocked the transport of NBD-PS. The absence or presence of NBD-PS in the outer leaflet was independently verified by employing "back-exchange", trinitrobenzenesulfonic acid derivatization, and decarboxylation with PS decarboxylase experiments. These control experiments yielded results which confirmed the lipid distributions determined by the resonance energy transfer assay.     

Fluorescence assay for phospholipid membrane asymmetry.

Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine lipid transport and membrane structure. We have developed a method for chemically modifying NBD-labeled lipids in both artificial and biological membranes. This was achieved by treating fluorescently labeled membranes with dithionite (S2O4(-2)). When small unilamellar vesicles containing NBD-labeled phospholipids were reacted with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicles' bilayer was reduced. Seven different NBD-lipid analogues, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent 7-amino-2,1,3-benzoxadiazol-4-yl-lipid derivatives. To assess the feasibility of using this reagent in biological systems, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanol ami ne was inserted into the outer leaflet of the plasma membrane of CHO-K1 cells. Subsequent incubation of these cells with a nontoxic concentration of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. These data suggest that dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivatives. We demonstrate how reduction of NBD-lipids with dithionite can be used to prepare asymmetrically labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochemical investigations, including the measurement of phospholipid translocase activity.     

Cholesterol transfer between lipid vesicles. Effect of phospholipids and gangliosides.

The effect of lipid composition on the rate of cholesterol movement between cellular membranes is investigated using lipid vesicles. The separation of donor and acceptor vesicles required for rate measurement is achieved by differential centrifugation so that the lipid effect can be quantified in the absence of a charged lipid generally used for ion-exchange-based separation. The rate of cholesterol transfer from small unilamellar vesicles (SUVs) containing 50 mol% cholesterol to a common large unilamellar vesicle (LUV) acceptor containing 20 mol% cholesterol decreases with increasing mol% of sphingomyelin in the SUVs, while phosphatidylethanolamine and phosphatidylserine have no appreciable effect at physiologically relevant levels. There is a large decrease in rate when phosphatidylethanolamine constitutes 50 mol% of donor phospholipids. Interestingly, gangliosides which have the same hydrocarbon moiety as sphingomyelin exert an opposite effect. The effect of spingomyelin seems to be mediated by its ability to decrease the fluidity of the lipid matrix, while that of gangliosides may arise from a weakening of phosphatidylcholine-cholesterol interactions or from a more favourable (less polar) microenvironment for the desorption of cholesterol provided by the head-group interactions involving sugar residues. If the effect of asymmetric transbilayer distribution of lipids is taken into consideration, the observed composition-dependent rate changes could partly account for the large difference in the rates of cholesterol desorption from the inner and outer layers of plasma membrane. Such rate differences may be responsible for an unequal steady-state distribution of cholesterol among various cellular membranes and lipoproteins.     

Assay for the transbilayer distribution of glycolipids. Selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine- egg phosphatidylethanolamine;-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A(2) confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing <1 mol% radiolabeled glucosylceramide or short-chain C(6)-glucosylceramide, carboxy-TEMPO oxidized half the glucosylceramide. However, if surface C(6)-glucosylceramide was first depleted by bovine serum albumin (BSA) (extracting 49 +/- 1%), 94% of the remaining C(6)-glucosylceramide was resistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surface of LUVs without affecting inner leaflet glucosylceramide. At pH 9.5 and at 0 degrees C, the reaction reached completion by 20 min.     

Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of Deltadnf1Deltadnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by Deltadnf1Deltadnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.     

Absence of transbilayer diffusion of spin-labeled sphingomyelin on human erythrocytes. Comparison with the diffusion of several spin-labeled glycerophospholipids

We have measured the transbilayer diffusion at 4 degrees C of spin labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid in the human erythrocyte membrane. Measurements were also carried out in ghosts, released without ATP, and on large unilamellar vesicles made with total lipid extract. As reported previously (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755), the amino phospholipids are rapidly transported from the outer to the inner leaflet on fresh erythrocytes, whereas phosphatidylcholine diffuses slowly. We now show that phosphatidic acid behaves like phosphatidylcholine: approximately 10% is internalized in 5 h at 4 degrees C. Under the same experimental conditions, no inward transport of sphingomyelin can be detected. In ghosts resealed without ATP, all glycerophospholipids tested diffuse slowly from the outer to the inner leaflet (approx. 10% in 5 h) while no transport of sphingomyelin is seen. Finally in lipid vesicles, the inward diffusion of all glycerophospholipids is less than 2% in 5 h and a very small transport of sphingomyelin can be measured. These results confirm the existence of a selective inward aminophospholipid transport of fresh erythrocytes and suggest a slow and passive diffusion of all phospholipids on ghosts, resealed without ATP, as well as on lipid vesicles.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]     

Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles

The thermodynamics of binding of the antibacterial peptide magainin 2 amide (M2a) to negatively charged small (SUVs) and large (LUVs) unilamellar vesicles has been studied with isothermal titration calorimetry (ITC) and CD spectroscopy at 45 degrees C. The binding isotherms as well as the ability of the peptide to permeabilize membranes were found to be qualitatively and quantitatively similar for both model membranes. The binding isotherms could be described with a surface partition equilibrium where the surface concentration of the peptide immediately above the plane of binding was calculated with the Gouy-Chapman theory. The standard free energy of binding was deltaG0 approximately -22 kJ/mol and was almost identical for LUVs and SUVs. However, the standard enthalpy and entropy of binding were distinctly higher for LUVs (deltaH0 = -15.1 kJ/mol, deltaS0 = 24.7 J/molK) than for SUVs (deltaH0 = -38.5 kJ/mol, deltaS0 = -55.3 J/molK). This enthalpy-entropy compensation mechanism is explained by differences in the lipid packing. The cohesive forces between lipid molecules are larger in well-packed LUVs and incorporation of M2a leads to a stronger disruption of cohesive forces and to a larger increase in the lipid flexibility than peptide incorporation into the more disordered SUVs. At 45 degrees C the peptide easily translocates from the outer to the inner monolayer as judged from the simulation of the ITC curves.     

The Curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content,and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm)radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, ~40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained ~20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets ofthe bilayer. The proportion oftotal PE residing in the outer leaflet was unaffected by changes in either the cholesterol orPE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions ofpalmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

The curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, approximately 40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained approximately 20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets of the bilayer. The proportion of total PE residing in the outer leaflet was unaffected by changes in either the cholesterol or PE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate- and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions of palmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

Phosphatidylserine decarboxylase: generation of asymmetric vesicles and determination of the transbilayer distribution of fluorescent phosphatidylserine in model membrane systems

Large unilamellar vesicles (LUV) that contained a fluorescent analog of phosphatidylserine (NBD-PS) were used in model systems to determine the feasibility of employing phosphatidylserine decarboxylase (PS-decarboxylase) to generate asymmetric vesicles and to determine the transbilayer distribution of PS. PS-decarboxylase prepared by sonication of Escherichia coli JA 200 pLC 8-47 was found to be stable in detergent-free buffers and catalyzed the conversion of NBD-PS to NBD-phosphatidylethanolamine (NBD-PE). PS-decarboxylase was capable of decarboxylating virtually all of the NBD-PS present in the outer leaflet of LUV containing a symmetric or asymmetric (outside only) distribution of NBD-PS, but not NBD-PS present in the inner leaflet of the vesicles. The ability of PS-decarboxylase to decarboxylate only NBD-PS located in the outer leaflet of the vesicles was independently verified by resonance energy transfer (between NBD-PS and (lissamine) rhodamine B-labeled phosphatidylethanolamine) and by derivatization with trinitrobenzenesulfonic acid (TNBS). These techniques revealed that the exchangeable pool (the fraction of NBD-PS on the outer leaflet) and the respective fraction of Tnp-(NBD-PS) formed were equivalent to the extent of PS-decarboxylase-mediated decarboxylation of NBD-PS to NBD-PE. These results show that PS-decarboxylase can be used to generate asymmetric vesicles (i.e., PS inside, PE outside) and determine the intrabilayer distribution of PS in model membranes.     

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.     

Sphingomyelin modulates the transbilayer distribution of galactosylceramide in phospholipid membranes

The interrelationships among sphingolipid structure, membrane curvature, and glycosphingolipid transmembrane distribution remain poorly defined despite the emerging importance of sphingolipids in curved regions and vesicle buds of biomembranes. Here, we describe a novel approach to investigate the transmembrane distribution of galactosylceramide in phospholipid small unilamellar vesicles by (13)C NMR spectroscopy. Quantitation of the transbilayer distribution of [6-(13)C]galactosylceramide (99.8% isotopic enrichment) was achieved by exposure of vesicles to the paramagnetic ion, Mn(2+). The data show that [6-(13)C]galactosylceramide prefers (70%) the inner leaflet of phosphatidylcholine vesicles. Increasing the sphingomyelin content of the 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles shifted galactosylceramide from the inner to the outer leaflet. The amount of galactosylceramide localized in the inner leaflet decreased from 70% in pure 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles to only 40% in 1-palmitoyl-2-oleoyl-phosphatidylcholine/sphingomyelin (1:2) vesicles. The present study demonstrates that sphingomyelin can dramatically alter the transbilayer distribution of a monohexosylceramide, such as galactosylceramide, in 1-palmitoyl-2-oleoyl-phosphatidylcholine/sphingomyelin vesicles. The results suggest that sphingolipid-sphingolipid interactions that occur even in the absence of cholesterol play a role in controlling the transmembrane distributions of cerebrosides.     

Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol

The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents.     

CD Spectroscopy of Peptides and Proteins Bound to Large Unilamellar Vesicles

Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.     

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.     

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.     

Spontaneous vesicularization of myelin lipids is counteracted by myelin basic protein

Hand-vortexed dispersions of several lipids (cerebrosides, sulfatides, PC, PE, PS and sphingomyelin), mixed in the ratios found for these categories of lipids in myelin, exhibit 31P-NMR spectra which have contributions from both isotropic and lamellar resonances. Investigation of this system by freeze-fracture electron microscopy and X-ray diffraction revealed that this lipid mixture has spontaneously formed small unilamellar vesicles (SUVs) (diam. approximately 400 A) and large highly convoluted unilamellar vesicles (LUVs) (diam. approximately 1000 A), the latter possibly resulting from aggregation and fusion of the SUV structures. This vesicularization of the myelin lipids was reversed by the addition of myelin basic protein: only large multilamellar aggregates were formed in the presence of protein, as shown by all three experimental methods. Although no rigorous physical-chemical explanation for these phenomena is yet available, the possibility is suggested that the high concentration of cerebrosides and/or phosphatidylethanolamine in this particular mixture of myelin lipids play pivotal roles in the formation of these unusual vesicles. Spontaneous vesicularization of myelin lipids is discussed as a potential pathway toward destabilization of the myelin sheath.