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In this study the effect of a transmembrane electrical potential on the phospholipid headgroup conformation was investigated using the 2H-NMR technique. Large unilamellar vesicles were prepared of dioleoylphosphatidylcholine, specifically 2H-labeled at the alpha- or beta-position of the choline group. No conformational change of the phosphocholine headgroup could be detected after induction of a valinomycin-induced K(+)-diffusion potential across the bilayer. However, this method could be used to measure the redistribution of tetraphenylphosphonium across the bilayer in response to delta psi, which reorients the phosphocholine headgroups in the opposite bilayer-water interfaces.

In this overview liposomes are described as bilayer-bounded vesicles which under defined conditions act as ideal osmometers according to the Boyle van't Hoff law. Investigations on osmotic volume changes, directly or indirectly by taking advantage of changes in light scattering, are considered and applications in permeability measurements are discussed. Solute-solvent interactions occurring in isotonic swelling experiments are analysed in view of an irreversible thermodynamic description. In a second part liquid crystalline lipid bilayers are characterized as highly selective permeability bilayers and the physical principles underlying this selectivity are considered. Attention is given to special physical and chemical conditions that may cause structural defects in the bilayer organization and can affect the selective permeability properties of the bilayer or completely deteriorate its barrier function. Finally an evaluation is given of intrinsic ionophoretic activity in lipid bilayers containing negatively charged lipids.

A new property of the presequence of the mitochondrial precursor protein cytochrome oxidase subunit IV is presented. This mitochondrial presequence induces interbilayer contacts between large unilamellar vesicles consisting of phosphatidylcholine and cardiolipin. The presequence-vesicle aggregates can be dissociated by applying a membrane potential across the bilayers (negative inside). These effects require the presence of cardiolipin and are not observed for other negatively charged phospholipids. We propose a role for the presequence in the formation and dissociation of mitochondrial contact sites.

The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%.

From data obtained by differential scanning calorimetry phase diagrams were constructed, using a thermodynamically based fitting method. The following binary mixtures of phosphatidylcholines in water were studied: 14:0/14:0-glycerophosphocholine/16:0/16:0-glucerophosphocholine, 14:0/14:0-glycerophosphocholine/18:0/18:0-glycerophosphocholine, 12:0/12:0-glycerophosphocholine/16:0/16:0-glycerophosphocholine, 18:1t/18:1t-glycerophosphocholine/14:0/14:0-glycerophosphocholine and 18:1t/18:1t-glycerophosphocholine/16:0/16:0-glycerophosphocholine. A comparison is made of the present results with those obtained using probe techniques and the differences are discussed.

It is shown by 31P-NMR and small angle X-ray scattering that induction of an hexagonal HII phase in dioleoylphosphatidylcholine model membranes by external addition of gramicidin A' depends on the solvent which is used to solubilize the peptide. Addition of gramicidin from dimethylsulfoxide or trifluoroethanol solution leads to HII phase formation whereas addition of the peptide from ethanol does not. This solvent dependence is shown by circular dichroism to be correlated with the peptide conformation. The channel conformation appears to be responsible for HII phase formation by gramicidin.

(1) A selective increase in permeability is induced in cardiolipin/phosphatidylcholine bilayers at Ca2+ concentrations of 1--3 mM. At higher concentrations of Ca2+ the permeability barrier is completely destroyed. (2) The selective increase in permeability is correlated with the formation of lipid particles visualized by freeze-fracture electron microscopy and an isotropic signal in 31P-NMR spectra. (3) Lowering the Ca2+ concentration shows reduction in permeability but the formation of the lipid particles is a non-reversible process. (4) At higher Ca2+ concentrations, 31P-NMR spectra and freeze-fracture results indicate the formation of the hexagonal phase, explaining the disappearance of the permeability barrier.

Calcium binds to dioleoylphosphatidate/dioleoylphosphatidylcholine (DOPA/DOPC) (20:80, mol%) multilamellar vesicles in the presence of a calcium ionophore with stoichiometry of about 0.6 nmol calcium per nmol phosphatidate and an apparent dissociation constant of about 1.7 mM. Experiments on the behaviour of monomolecular films at an air/water interface show that calcium-phosphatidate binding results in a decrease in the area of the polar region of the phosphatidate molecule, probably caused by headgroup dehydration and partial charge neutralization. At calcium concentration higher than about 3 mM calcium neutralizes the negatively charged membrane surface of DOPA/DOPC (20:80, mol%) large unilamellar vesicles, and vesicle aggregation is observed. At 10 mM of calcium this results in a low level of vesicle fusion. These observed processes are not attended with calcium-induced phosphatidylcholine transbilayer movement in the membranes of DOPA/DOPC (20:80, mol%) large unilamellar vesicles. When these findings are compared with the results of a previous study on the permeability behaviour of large unilamellar vesicles of the same phospholipid composition under comparable conditions (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108) the following conclusions can be drawn. At low millimolar calcium concentrations (less than 2.5 mM) calcium does not occupy all the binding sites of the membrane, no membrane-membrane interactions are observed and a selective translocation of calcium and calcium-chelating anions is appearing. The mechanism of this translocation may be explained by the formation of uncharged dehydrated complexes of calcium, phosphatidate and calcium chelator, which can pass the membrane via transient occurring non-bilayer structures. Between 3 and 10 mM of calcium an a selective permeability increase of the vesicular membrane is found, which is not a consequence of vesicle fusion but apparently of vesicle aggregation, possibly causing packing defects in the membrane.

The permeability behaviour of dioleoylphosphatidate/dioleoylphosphatidylcholine (20:80, mol%) large unilamellar vesicles at low millimolar calcium concentrations is different for various solutes. Between 0.5 mM and 2.5 mM of calcium a selective influx of calcium and efflux of enclosed calcium chelating anions is observed. At higher calcium concentrations the membrane loses its barrier function for a large variety of solutes. These permeability increases are a specific consequence of calcium phosphatidate interactions, because control experiments in which calcium was replaced by magnesium or in which dioleoylphosphatidate was replaced by dioleoylphosphatidylglycerol showed under the same conditions no permeability changes. These results are discussed on the basis of various putative mechanistic models for phosphatidate-mediated calcium translocation across membranes. Furthermore a kinetical model is presented by which the observed selective calcium and calcium-chelator translocation can be explained.

Aspects of osmotic properties of liposomes, prepared from synthetic lecithin, above, at and below the gel to liquid crystalline phase transition temperature are described. The experiments show that liposomal membranes with their lipids in the gel state are still permeable to water. The rate of water permeation changes drastically on passing the transition temperature. The water permeation has activation energies of 9.5 +/- 1.28 and 26.4 +/- 0.9 kcal/mol above and below the transition temperature, respectively, indicating that the diffusion processes take place by different mechanisms. With respect to the barrier properties of the liposomes in the vicinity of the transition temperature, the following conclusions can be made. (1) Studying the osmotic shrinkage of liposomes at a fixed temperature near the transition point, the experiments indicate that dimyristoyl phosphatidylcholine membranes are highly permeable to glucose under these conditions, where liquid and solid domains co-exist. Under the same conditions the osmotic experiments did not indicate a strong increase in glucose permeability of dipalmitoyl phosphatidylcholine membranes as compared to the situation above and below the transition temperature. (2) On the other hand, perturbations of the phase equilibrium by temperature varations resulted in a marked increase of the glucose permeation through dipalmitoyl phosphatidylcholine bilayers. Once a new phase equilibrium of liquid and solid regions is established the permeation rate of glucose is much less.

The permeation of water through liposomal membranes composed of various saturated phosphatidylcholine plus gramicidin A was studied as a function of temperature. 1. The presence of gramicidin in the liposomal bilayers caused an increase in water permeability. Below the phase transition temperature this effect could be measured quite clearly in all the systems we tested, but the extent of the increase was largely dependent on the length of the hydrocarbon chains. 2. Increasing amounts of gramicidin caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel-liquid crystalline phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. Differential scanning calorimetry analysis of the system containing these relatively small amounts of gramicidin still showed a clear transition from the liquid crystalline to the gel state with only a slight reduction in the enthalpy change. 3. In liposomes composed of dimyristoyl, dipalmitoyl and saturated egg phosphatidylcholine there was a concomitant decrease in the activation energy of water permeation in the presence of gramicidin below and above the phase transition temperature. The activation energy for water permeation through longer chained distearoyl phosphatidylcholine liposomal bilayers was the same with or without gramicidin in the bilayer. 4. It is concluded that the ability of gramicidin to form conducting channels in a gel state bilayer depends on the thickness of the paraffin core.

Light scattering, birefringence and X-ray studies showed that liposomes, with lipid molecules orientated in bilayers, are formed from egg licithin/lysolecithin mixtures up to 50 mol percent of lysolecithin; above this concentration much smaller mixed micelles are formed. Permeability studies demonstrated a dramatic increase in the permeability of the liposomes when the lyso concentration exceeds 22.5 mol percent. X-ray studies indicated a significant decrease in bilayer thickness with increasing lysolecithin concentration. It is suggested that decreased interaction energy between the lipid molecules in the bilayer is responsible for the inability of the thin bilayers to act as an effective permeability barrier.

Ca2+ induces a structural change in phosphatidylcholine-cardiolipin bilayers, which is visualised by freeze-fracturing as lipidic particles associated with the bilayer and is detected by 31P-NMR as isotropic motion of the phospholipids. In this structure a rapid transbilayer movement of phosphatidylcholine and a highly increased permeability towards Mn2+ are observed.

Calorimetric experiments showed a marked effect of Ca2+ and Mg2+ on the thermotropic behaviour of dimyristoyl phosphatidylglycerol. 2. Concentrations of Ca2+ and Mg2+ lower than 1 ion to 2 molecules of phosphatidylglycerol produced a shift of the phase transition to higher temperatures and an increase in the enthalpy change which is consistent with a closer packing of the lipid molecules in the liposomes. 3. Above the 1:2 ratio, freeze-fracture electron microscopy demonstrated typical "crystal" structures both in the presence of Ca2+ and Mg2+. In the presence of Mg2+ a metastable behaviour was noticed in the calorimetric experiments. 4. A Ca2+- and Mg2+-induced shift in the transition temperature and an increase in the enthalpy change was also observed in a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine. However, these mixed samples remained liposomal in structure at any concentration of the divalent ions. 5. Liposomes prepared from a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the absence of divalent cations are permeable in the range 10-50 degrees C. Bilayers of mixtures neutralized by Ca2+ or Mg2+ were demonstrated to be completely impermeable to K+, except in the vicinity of the phase transition. 6. The leak of ions from liposomes of a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the vicinity of the phase transition temperature was considerably less in the presence of Ca2+ than in the presence of Mg2+. 7. It is concluded that there is a correlation between the calorimetric data and the permeability properties of dimyristoyl phosphatidylglycerol-containing bilayers with respect to the influence of Ca2+ and Mg2+.

(1) The thermotropic behaviour of dimyristoyl phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylcholine was investigated by differential scanning calorimetry and freeze-fracture electron microscopy as a function of pH and of Ca2+ concentration. (2) From the thermotropic behaviour as a function of pH, profiles could be constructed from which apparent pK values of the charged groups of the lipids could be determined. (3) Excess Ca2+ induced a shift of the total phase transition in 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-glycerophosphoglycerol mixtures. In 14 : 0/14 : 0-glycerophosphocholine bilayers containing 16 : 0/16 : 0-glycerophosphoglycerol lateral phase separation was induced by Ca2+. (4) Up to molar ratios of 1 : 2 of 14 : 0/14 : 0-glycerophosphoserine to 14 : 0/14: 0-glycerophosphocholine, excess Ca2+ induced lateral phase separation. Addition to mixtures of higher molar ratios caused segregation into different structures: the liposome organization and the stacked lamellae/cylindrical organization. (5) Addition of excess Ca2+ to mixtures of 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-phosphatidic acid caused, independent of the molar ratio, separation into two structural different organizations. (6) The nature of Ca2+-induced changes in bilayers containing negatively charged phospholipids is strongly dependent on the character of the polar headgroup of the negatively charged phospholipid involved.

1. The role of length and unsaturation of phospholipid acyl chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467--2474). 2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low. 3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chain-length was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length. 4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers. 5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidylglycerol was independent on acyl-chain composition and occurred at very low amounts of phospholipid. 6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gel-to-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids. 7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interfacial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.

(1) The effect of incorporation of different lysophosphatidylcholine species on the structure, barrier properties and dynamics of bilayers made of various phosphatidylcholines both the presence and absence of cholesterol have been investigated by 31P-NMR, freeze-fracture electron microscopy and K+-permeability measurements. (2) In a dispersion of lysophosphatidylcholine : cholesterol (1 : 1) the lipids are organized in extended bilayers. Upon cooling a micellar solution of 1-palmitoyllysophosphatidylcholine below the chain-melting temperature a transition to a lamellar, most likely interdigitating organization is observed. 31P-NMR shows in both situations a marked decrease in effective chemical shift anisotropy. (3) 1-Palmitoyllysophosphatidylcholine can be incorporated up to 30 mol% into liquid crystalline bilayers of dipalmitoylphosphatidylcholine and up to 35 mol% into dioleoylphosphatidylcholine bilayers. Above this concentration micellization of the bilayers occurs. In the gel state, bilayer structure is maintained up to 60 mol% of the lysocompound. (4) 1-Oleoyllysophosphatidylcholine can be incorporated to higher concentrations into liquid crystalline phosphatidylcholine bilayers than the palmitoyl analogue, which can be explained by the more cylindrical shape of the 1-oleoyllysophosphatidylcholine. (5) In marked contrast, incorporation of only 1 mol% of 1-oleoyllysophosphatidylcholine into gel state dipalmitoylphosphatidylcholine already destabilizes bilayer structure and makes the membranes completely permeable for K+. These results are discussed with respect to the mixing properties of the various lysophosphatidylcholines. (6) In general these effects are accompanied by a loss of K+-permeability barrier, which however occurs at lower lysophosphatidylcholine concentrations than needed for the start of micellization. (7) Cholesterol incorporation counteracts the bilayer destabilizing role of lysophosphatidylcholines. (8) 31P-NMR demonstrates with increasing lysophosphatidylcholine concentrations in the bilayers of phosphatidylcholines a decrease in the effective chemical shift anisotropy. As the rigid lattice spectra of lysophosphatidylcholine and phosphatidylcholine are identical, this reflects a change in the conformational and/or motional properties of the phospholipid head groups. This phenomenon might play a role in the observed permeability changes.

comparative studies on bilayer systems of saturated phosphatidylcholines and phosphatidylethanolamines revealed a maximum in ionic permeability in phosphatidylcholine bilayers at the temperature of the gel to liquid-crystalline phase transition but such an increase in permeability was not detectable in bilayers of phosphatidylethanolamine. Furthermore, it was found that at the phase transition temperature the phosphatidylcholine bilayers are subject to rapid hydrolysis by pancreatic phospholipase A2 whereas phosphatidylethanolamine bilayers are not. These differences are discussed in view of detailed information on the molecular organization in the gel and liquid crystalline phases of the two phospholipid classes.

Glycophorin was incorporated into large unilamellar vesicles and the bilayer permeability was measured as a function of the lipid composition. In agreement with previous data (Van der Steen, A.T.M., De Kruijff, B. and De Gier, J. (1982) Biochim. Biophys. Acta 691, 13-23) it was found that glycophorin greatly increased the bilayer permeability of DOPC vesicles. This effect was observed for a large variety of phosphatidylcholines, differing in their fatty acid composition and homogeneity. In sharp contrast, it was observed that variations in the polar headgroups by incorporation of DOPE, DOPS and, to a lesser extent, cholesterol, into the DOPC/glycophorin vesicles restored the barrier function. These results are compared to the size of the particles, revealed by freeze-fracture electron microscopy on the glycophorin-containing bilayer and are discussed in the light of various types of lipid-protein interactions and protein aggregation state.

Using enthalphy data from differential scanning calorimetry experiments and 13C-NMR linewidths of specifically (N-Me-13C)-labelled lipids, the miscibility properties of phosphatidylcholines and lysophosphatidylcholines in liposomal dispersons have been investigated. It was found that 16 : 0 lysophosphatidylcholine mixes homogeneously in 16 : 0/16 : 0 phosphatidylcholine bilayers. Mixtures of 16 : 0 lysophosphatidylcholine with 18 : 1c/18 : 1c phosphatidylcholine, of 18 : 1c lysophosphatidylcholine with 16 : 0/16 : 0 phosphatidylcholine and of 18 : 1c lysophosphatidylcholine with 18 : 1c/18 : 1c phosphatidylcholine of exhibited immiscibility in the phosphatidylcholine gel state.

The permeation of water through liposomal membranes composed of phosphatidylcholine plus varying amounts of cholesterol was studied as a function of temperature. 1. Increasing amounts of cholesterol caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel to liquid-crystalline phase transition temperature of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine liposomes. At cholesterol concentrations above about 30 mol % there was no longer a discontinuity in the rate of water permeation. 2. The incorporation of cholesterol produces a steep change in the activation energy of the water permeation above the transition temperature of the saturated lecithin occurring at about 15 mol % of cholesterol. Below the transition temperature there was a gradual decrease in the activation energy of the water permeation in the region of 0 to 33 mol % of cholesterol. 3. In systems containing unsaturated phosphatidylcholines cholesterol also enhanced the activation energy of the water permeation although to a lesser extent. The results indicate that the position of the cis-double bond in the fatty acid chain is very important in this respect. 4. In systems in which cholesterol increased the temperature dependence of the water permeation there is also an enhancement of the temperature dependence of the isotonic glycerol and erythritol swelling by the same number of kcal/mol.

This paper describes experiments showing the importance of the fatty acid chain length on the barrier properties of liposomal bilayers, prepared from saturated lecithins, under conditions of lateral phase separation. 1. Above the gel to liquid crystalline phase transition temperature, liposomes prepared from saturated lecithins with 14 or more carbon atoms per acyl chain exist as stable bilayers, which are practically impermeable to ions. 2. At temperatures well above the transition temperature dilauroyl phosphatidylcholine liposomes exhibited osmotic shrinkage, which was dependent on the ionic size of the solute used to bring about the osmotic gradient, indicating that the permeation through these less stable bilayers takes place mainly via individual diffusion of the permeating ions. 3. An enhanced release of trapped potassium from liposomes was demonstrated in the vicinity of the transition temperature. The extent of the increase, however, depended strongly on the length of the paraffin chain. 4. From measurements of the shrinkage behaviour of liposomes in the vicinity of the transition temperature it is concluded that the increased permeability decreases with increasing diameter of the permeating ion. This finding implies that the increased permeability at the transition temperature cannot be ascribed to "macroscopic" rupture of the liposomal membrane. The maximum permeability in the vicinity of the Tc is discussed in terms of probability and size distribution of statistical pore formation at the boundaries of liquid and solid domains.

1. Nuclear magnetic resonance, light scattering and freeze fracturing electron microscopic techniques were used to characterize the size of unilamellar phospholipid vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. 2. Differential scanning calorimetric and light scattering analyses showed that very small unilamellar vesicles obtained by the sonication method exhibit a downward shifted, largely broadened phase transition with a slightly decreased enthalpy change when compared with multilayered liposomes. 3. The phase transition of vesicles with variable diameter as obtained by injection methods resembled the pattern of multilayered liposomes the more the diameter was increased. 4. Repeated cycling through the lipid phase transition was shown to have a progressive effect on a fusion process. This effect was strongly increased when the osmolarity of the medium was enhanced (e.g. by the addition of cryoprotectors). Furthermore it was shown that ice-water of the systems caused abrupt fusion of the lipid structures. 5. Controversial results in the literature on the thermotropic behavior of vesicles could be explained in terms of these fusion processes.

At calcium concentrations up to about 4 mM a selective permeability increase of cardiolipin/dioleoylphosphatidylcholine (50:50, mol%) membranes for calcium and its chelator arsenazo III is observed. Under these conditions calcium does not occupy all the binding sites of cardiolipin at the membrane interface and no vesicle-vesicle interactions are found. Lowering of the cardiolipin content of the vesicles to 20 mol% extends the calcium concentration range in which a selective permeability for calcium and arsenazo III is appearing up to about 12 mM. We suggest that the observed selective permeability increase is caused by transient formation of inverted micellar structures in the membrane with cardiolipin as translocating membrane component for calcium and arsenazo III. At calcium concentrations of 4 mM and higher for 50 mol% cardiolipin-containing vesicles a general permeability increase is found together with calcium-cardiolipin binding in a 1:1 stoichiometry, vesicles aggregation and, above 8 mM of calcium, vesicle fusion. The loss of barrier function of the membrane under these conditions is correlated with vesicle aggregation and may be explained by a transition from a bilayer into a hexagonal HII organization of the phospholipids.

The following phosphatidylethanolamines were studied by differential scanning calorimetry: 1,2-dipalmitoleoyl-, 1,2-dioleoyl-, 1,2-dilauroyl-, 1,2-dielaidyl-, 1,2-dimyristoyl- and 1,2-dipalmitoyl-sn-glycero-3-phosphoryl-ethanolamine. The saturated and trans-unsaturated species underwent thermotropic phase transitions at temperatures about 20-30 degrees C higher than the corresponding phosphatidylcholines but the enthalpy changes were nearly identical. The transition temperatures for the cis-unsaturated species were about the same as those of the corresponding phosphatidylcholines but here the enthalpy change was markedly decreased as compared with the phosphatidylcholines. Freeze-fracture electron microscopy revealed phase changes from a lamellar to a hexagonal phase for 1,2-dipalmitoleoyl- and 1,2-dioleoyl-sn-glycero-phosphorylethanolamine at 20 and 0 degrees C respectively. At these temperatures no transitions were apparent in the calorimeter scan. Incorporation of increasing amounts of cholesterol into phosphatidylethanol-amine bilayers gradually decreased the enthalpy changes of the phase transition in the same manner as was demonstrated before for phosphatidylcholine/cholesterol mixtures. This was studied both for 1,2-dipalmitoleoyl- and 1,2-dimyristoyl-sn-glycerophosphorylethanolamine. In an equimolar mixture of 1,2-dioleoyl- and 1,2-dipalmitoylphosphoryl-ethanolamine, which showed phase separation, cholesterol preferentially decreased the transition of the lowest melting component. In equimolar mixtures of phosphatidylethanolamines and phosphatidylcholines, which showed phase separation, cholesterol preferentially abolished the transition of the phosphatidylcholine component present. This occurred both in experiments where the phosphatidylcholine was the lowest melting and where it was the highest melting component present in the mixture. These experiments strongly suggest that in phosphatidylcholine-phosphatidylethanolamine mixtures at temperatures where both components are in the liquid-crystalline state cholesterol is preferently associated with the phosphatidylcholine component in the mixture.