Mixed monolayers of straight-chain/branched-chain phospholipids. I. Mixed monolayers of distearoyl phosphatidylcholine and diisoeicosanoyl phosphatidylcholine

The temperature dependence of the force/area isotherms of monolayer of distearoyl phosphatidylcholine (DSPC), diisoeicosanoyl phosphatidylcholine (DIEPC) and a complete mixed compositional range of these two lecithins are reported. The isotherms for DSPC closely resemble those previously reported for dipalmitoyl phosphatidylcholine but are shifted to higher temperatures by 16 degrees C. The isotherms of DIEPC, an iso-branched lecithin, show differences from these obtained for similar straight-chain lecithins in that the full condensed isotherms are more expanded, the fully expanded isotherms are more condensed and therefore the liquid expanded (LE)/liquid condensed (LC) intermediate region is significantly reduced. This means that the condensed state is more disordered and the expanded state is less disordered than the corresponding states in straight-chain lecithins. Data for the mixed films are interpreted in terms of surface pressure/mole fraction phase diagrams and both energies and entropies of compression associated with the LE/LC transition. The phase diagrams at 34.1 degrees C, 35.8 degrees C and 38.5 degrees C are all of the negative azeotropic type with the surface pressure minimum point shifting with temperature. The thermodynamic analysis indicates that from 34.1 degrees C to 38.5 degrees C the driving force for mixing changes from the entropy to the energy of the transition. It would seem that at the lower temperature the packing of the distearoyl lecithin is perturbed by the diisoeicosanoyl lecithin, while at higher temperatures the very high entropy of pure or nearly pure diisoeicosanoyl lecithin results in other mixtures having less entropy than would be expected on an ideal mixing basis.     

Peroxidation of phosphatidylcholine and cholesterol in mixed monolayers]

Peroxidation of phosphatidylcholine and cholesterol in mixed monolayers at the air-water surface is studied. It is shown that the rate of phosphatidylcholine peroxidation is abruptly decreased in the presence of cholesterol.     

Imaging of the domain organization in sphingomyelin and phosphatidylcholine monolayers

The lateral organization of biomembranes has gained significant interest when the fluid mosaic model was challenged by the model of "lipid rafts". Several lipid classes like cholesterol and sphingolipids are considered to be essential for their formation. Here we investigate the lateral domain formation in binary mixtures of sphingomyelin and phosphatidylcholine. Both are major lipid components of lipoproteins and mammalian cell membranes at various molar ratios. Surface pressure-area isotherms and surface potential-area isotherms of monolayers composed of these lipids clearly indicated non-ideal mixing. In addition, Brewster angle microscopy provided a well-suited approach to image the formation of lateral domains. These images demonstrated that pure sphingomyelin forms very stable finger-like domains that exhibit a distinct internal organization suggesting an anisotropic orientation of the acyl side chains. Similar behavior was found for mixtures containing more than 60 mol% sphingomyelin. With increasing content of phosphatidylcholine the domain size decreased and the surface pressure, where domain formation occurred, increased. At lower sphingomyelin content (30-60 mol%) rather round-shaped, smaller domains were observed. Thus, the potential of sphingomyelin domains as potentially important building blocks for actual domains that could be building blocks for raft formation is suggested, even without the presence of cholesterol. In addition, these observations may suggest a role for the distinct molar ratio of these key lipids frequently found in physiologically relevant particles such as low and high density lipoproteins or the outer leaflet of the human erythrocyte membrane.     

Proton magnetic relaxation studies of mixed phosphatidylcholine/fatty acid and mixed phosphatidylcholine bimolecular bilayers.

High resolution proton spin-lattice relaxation times (T1), spin-spin relaxation times (T2) and resonance linewidths were measured above the gel-to-liquid crystal transition temperature (Tm), in phosphatidylcholine bilayers possessing various degrees of intramolecular motional anisotrophy at the level of various alkyl chain proton groups. The experiments were designed to test the hypothesis that coupled trans-gauche isomerizations along the chains can be responsible for the anisotropic motion of phosphatidylcholine proton groups in bilayer membranes (Horwitz, A.F., Horsley, W.J. and Klein, M.O. (1972) Proc. Natl. Acad. Sci. U.S. 69,500). Systematic series of structural perturbations of the bilayer were achieved in mixed phosphatidylcholine/fatty acid and in mixed phosphatidylcholine bilayers where the degree of motional anisotrophy of the chains' proton groups was gradually reduced by progressively increasing the chain length disparity of the two components. The systematic T1 and T2 variations observed were interterpreted on the basis of the Woessner's treatment for computing the relaxation times of a spin pair reorienting randomly about an axis which, in turn, tumbles randomly (Woessner, D.E. (1962) J. Chem. Phys. 36, 1). The results confirmed in a qualitative sense the original hypothesis made by Horwitz et al. The time-averaged structural interpretations suggested by the mangetic relaxation studies are in agreement with low-angle X-ray diffraction results obtained below Tm. In addition, the T1 values evaluated at various temperatures in dipalmitoyl phosphatidylcholine vesicles incorporated with either 2H-labeled or unlabeled palmitic acid chains indicated that the average intermolecular contribution to the spin-lattice relaxation rate of the proton groups of the phosphatidylcholine chains appears comparable to the intramolecular term at temperatures moderately higher than Tm, but becomes less and less important as the temperature is further increased above the thermal transition.     

The penetration of local anesthetics into phosphatidylcholine monolayers.

The penetration of tetracaine into monolayers of phosphatidylcholine and trioctanoin at different surface pressures, and the penetration of dibucaine, tetracaine, butacaine, lidocaine, and procaine into monolayers of didecanoylphosphatidylcholine at II = 10 mN/m was determined by the use of a modified Gibbs adsorption equation. These data were shown to fit a geometric model and compared favorably with data determined by a method based on the geometric model. The penetration of tetracaine into phosphatidylcholine monolayers was pressure dependent. At II = 10 mN/m, the local anesthetics penetrate into a phosphatidycholine monolayer in the order: dibucaine greater than tetracaine greater than butacaine greater than lidocaine greater than procaine. This correlates with their potencies in blocking nerve conduction and inhibiting phospholipase A2.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Binding of substance P to monolayers and vesicles made of phosphatidylcholine and/or phosphatidylserine.

Analyses of interactions between substance P (SP) and phospholipids were performed by combined surface pressure and surface potential measurements in monolayers and by 13C-NMR experiments on liposomes. This study was carried out using synthetic SP molecules: [1-13C-Gly9]SP and [1-13C-Gly2]SP. Injection of SP into the aqueous subphase led to an expansion of phosphatidylcholine (PtdCho) or phosphatidylserine (PtdSer) monolayer surface area. An apparent association constant of SP for PtdSer was estimated to be around 10(6)-10(-7) M-1. The surface potential delta V/n varied linearly with the molecular area whereas the variation of surface pressure was biphasic, suggesting that at least two binding states contributed to the monolayer expansion. These two states Si (SP is inserted into the bilayer) and Ss (SP is stuck on the surface) were observed on vesicular membranes by 13C-NMR. The kinetic of interconversion between these two states can be estimated by NMR, the Ss state being the stablest one. No perpendicular insertion of SP into these vesicular preparations seemed to occur, as previously postulated. However, SP might form aggregates in contact with these model systems, leading to a loss of permeability of the lipid vesicles.     

Domain formation in phosphatidylinositol monophosphate/phosphatidylcholine mixed vesicles

Phosphoinositides have been shown to control membrane trafficking events by targeting proteins to specific cellular sites, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. To shed light on the processes that lead to the formation of phosphoinositide-enriched microdomains, phosphatidylcholine/phosphatidylinositol monophosphate (phosphatidylinositol-3-phosphate (PI-3P), -4-phosphate (PI-4P), or -5-phosphate (PI-5P)) mixed vesicles were investigated by calorimetric (DSC) Fourier transform infrared spectroscopic (FTIR), and fluorescence resonance energy transfer (FRET) measurements. The experiments furnished results consistent with a pH-dependent formation of phosphatidylinositol monophosphate-enriched microdomains. The domain formation was most pronounced between pH approximately 7 and approximately 9.5, whereas slightly acidic pH values (pH 4) resulted in the disintegration of the domains. This pH-dependent phosphatidylcholine/phosphatidylinositol monophosphate demixing was observed for the gel phase (FTIR experiments) as well as for the fluid lipid phase (FRET measurements). The observed microdomains are presumably stabilized by hydroxyl/hydroxyl as well as hydroxyl/phosphomonoester and phosphodiester interactions. While the pH dependence of the mutual phosphatidylinositol monophosphate interaction was largely the same for all investigated phosphatidylinositol monophosphates, it turned out that the relative stability of phosphatidylinositol monophosphate-enriched microdomains (pH 7-9.5) was governed by the position of the phosphomonoester group at the inositol ring (PI-4P > PI-5P > PI-3P). Demixing was also observed for phosphatidylcholine/phosphatidylinositol mixed vesicles; however, in this case the microdomain formation was only slightly affected by pH changes.     

Alterations in the organization of phosphatidylcholine/cholesterol bilayers by tetrahydrocannabinol

The interactions of delta 9-tetrahydrocannabinol (THC) with various phosphatidylcholines (PCs) was studied in model membranes by differential scanning calorimetry. THC present in PC bilayers above a certain concentration complexed stoichiometrically with phospholipids containing both saturated and unsaturated fatty acids. When the bilayer PCs were sufficiently dissimilar for phase separation to occur, THC preferentially associated with the lower melting point lipid. The presence of cholesterol below 20 mol% in dipalmitoylphosphatidylcholine bilayers enhanced THC X PC complex formation. Above 20 mol% cholesterol, there was no indication of THC X dipalmitoylphosphatidylcholine complex formation. This is in agreement with a phase rearrangement occurring in PC bilayers at concentrations of cholesterol of approximately 20 mol%. These studies suggest several possible mechanisms for the modulation of membrane activities by hydrophobic drugs such as THC.     

Lateral interactions of pig apolipoprotein A-1 with egg yolk phosphatidylcholine and with cholesterol in mixed monolayers at the triolein-saline interface.

Interfacial tensions of egg yolk phosphatidylcholine (PC) and cholesterol monolayers adsorbed at the triolein-saline interface were measured in the presence and absence of pig apolipoprotein A-1 (apoA-1) in the saline phase. In the absence of apoA-1, the adsorptions of PC and cholesterol at the interface from the triolein phase are cooperative, showing large lateral attractive interactions between the PC molecules and the cholesterol molecules in the monolayer. In the presence of apoA-1, the PC adsorption is anti-cooperative, indicating strong lateral attractive interactions between the PC and the apoA-1 molecules, i.e., apparently, repulsive lateral interactions between the PC molecules. On the other hand, lateral interactions of very low magnitude are observed between the cholesterol and apoA-1 molecules in the monolayer. Values of the lateral interaction energy are evaluated from the adsorption data by the Defay-Prigogine-Flory theory of monolayers. The large difference in lateral interaction energy with apoA-1 between PC and cholesterol in a mixed monolayer is discussed in connection with current problems in lipoprotein catabolism: reverse cholesterol transport, alterations in affinity of lipid particles to apoA-1, and formation of high-density lipoproteins and abnormal lipoproteins.     

pH-dependent domain formation in phosphatidylinositol polyphosphate/phosphatidylcholine mixed vesicles

Phosphatidylinositol polyphosphates (PI-PPs) have been shown to mediate a large variety of physiological processes by attracting proteins to specific cellular sites. Such site-specific signaling requires local accumulation of PI-PPs, and in light of the rich headgroup functionality, it is conceivable that hydrogen bond formation between adjacent headgroups is a contributing factor to the formation of PI-PP-enriched domains. To explore the significance of hydrogen bond formation for the mutual interaction of PI-PPs, this study aims to characterize the pH-dependent phase behavior of phosphatidylcholine/phosphatidylinositol bisphosphate and trisphosphate mixed vesicles by differential scanning calorimetry, infrared transmission spectroscopy, and fluorescence resonance energy transfer measurements. For pH values >7-7.5, the experiments yielded results consistent with dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylinositol polyphosphate gel phase demixing, whereas for moderately acidic conditions, an enhanced mixing was observed. Similarly, this pH-dependent formation of PI-PP-enriched domains was also found for the physiologically important fluid phase. The stability of PI-PP-enriched domains and to some extent the pH dependence of the domain formation was governed by the number as well as the position of the phosphomonoester groups at the inositol ring.     

Sphingomyelin content conditions insertion of daunorubicin within phosphatidylcholine monolayers

Cell death induced by the chemotherapeutic drug daunorubicin (DNR) implicates an apoptotic pathway originating at the plasma membrane and characterized by sphingomyelin (SM) hydrolysis and ceramide generation. The mechanisms by which such a drug (hypothetically passively diffusing across a structural membrane) can trigger SM hydrolysis is unknown, but raises the question of the precise interaction between DNR and membrane lipid constituents. In this initial study, using alternative current polarography together with voltammetry, we report that after a first step of adsorption, insertion of DNR within a condensed phosphatidylcholine monolayer was significantly facilitated by SM content.     

Effect of ions on the organization of phosphatidylcholine/phosphatidic acid bilayers

Lipid bilayers are two-dimensional fluids. Here, the effect of monovalent ion concentration on the mixing, and consequently the organization, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) bilayers has been examined. Epifluorescence microscopy was used to visualize the organization. Fluorescence recovery after photobleaching and attenuated total reflection-Fourier transform infrared spectroscopy were used to assess the fluidity of the lipids. At high ionic strength the DOPC and DOPA lipids appear uniformly mixed. Upon lowering the ionic strength, rapid separation is observed. The DOPA-rich regions appear fractal-like and exhibit hysteresis in their properties. The lipids freely exchange between the two regions. These experiments clearly demonstrate the significant effect that electrostatics can have on membrane organization.     

Effect of calcium and temperature on mixed lipid-valinomycin monolayers. A comparison of glycosphingolipids (ganglioside GT1b, sulphatides) and phosphatidylcholine

The influence of calcium and temperature on pure lipid (bovine brain PC, sulphatides, ganglioside GT1b), valinomycin and mixed lipid-valinomycin monolayers at the air/water interface was studied. In mixed films, evidence was found that the two components were miscible. On the other hand, at higher surface pressures, phase separation occurs in the cases of PC and sulphatides. Measuring the area requirement and the collapse pressure the stability of both lipid and the peptide was increased in particular due to ganglioside-valinomycin interaction. The addition of 10(-5) M calcium into the subphase at 20 and 37 degrees C and surface pressures of 10 and 20 mN/m led to a condensing effect in ganglioside mixtures, with formation of aggregates as indicated also by the nearly ideal behaviour of two component monolayers.     

Physicochemical studies of the interaction of the lipoheptapeptide surfactin with lipid bilayers of L-alpha-dimyristoyl phosphatidylcholine

To understand the biological action of surfactin from Bacillus subtilis we investigated its effects on the phase transition of L-alpha-dimyristoyl phosphatidylcholine (DMPC)-vesicles from the crystalline to the fluid state using differential scanning calorimetry; light scattering; small angle neutron scattering and cryo-electron microscopy. DSC-thermograms revealed two phase transition peaks. Light scattering profiles showed two branches with characteristic hysteresis phenomena. With both techniques the same values of the phase transition temperatures T(m1) and T(m2) of 23.5 and 23 degrees C were obtained indicating two forms of DMPC-surfactin aggregates which could be visualized by cryo-electron microscopy. Until 4 mol% surfactin the vesicular form predominated, but was accompanied by bilayered membrane fragments by increasing the biosurfactant concentrations. At surfactin concentrations higher than 15 mol% smaller DMPC-surfactin micelles of ellipsoidal conformation were formed, as demonstrated by small angle neutron scattering. In addition, by "Poor Man's" temperature-jump-relaxation spectroscopy slow transients in the phase transition of vesicular DMPC-surfactin aggregates with relaxation times of 20-30 s were detected which presumably indicate the slow dissipation of intermediate lipid-and surfactin domains formed after the main phase transition on the way to the fluid state. This process is accelerated by surfactin.     

Deuterium nuclear magnetic resonance studies of bile salt/phosphatidylcholine mixed micelles

Mixed micelles of deoxycholate (DOC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) have been prepared in which the POPC was specifically deuterated in the 2-, 6-, 10-, or 16-position of the palmitoyl chain or in the N-methyl position of the choline head group. The deuterium nuclear magnetic resonance (2H NMR) spectrum of each of these specifically deuterated mixed micelles consists of a singlet whose line width depends upon the position of deuteration. Spin-spin relaxation times indicate a gradient of mobility along the POPC palmitoyl chain in the mixed micelle, with a large increase in mobility on going from the 10- to the 16-position. Spin-lattice relaxation times (T1's) demonstrate a similar gradient of mobility. Both trends in NMR relaxation behavior are consistent with a bilayer arrangement for the solubilized POPC. 2H T1 times for DOC/POPC micelles are significantly shorter than those measured in other bilayer systems, indicating unusually tight phospholipid acyl chain packing in the mixed micelle.     

Studies on mixed monolayers of phospholipids and fusogenic lipids.

1. The behaviour of mixed monolayers of 14 different lipids with preparations of erythrocyte lipids, purified natural and synthetic phospholipids, cholesterol and galactosylceramide was investigated. 2. The mean areas occupied per molecule in mixed films containing lipids that are fusogenic for hen erythrocytes were compared with those for corresponding films containing lipids that are inactive as fusogens. 3. Fusogenic lipids were found to exhibit interactions, which were not shown by non-fusogenic lipids, in mixed monolayers with several species of phospholipid, particularly those containing a choline head group. 4. Heterogeneity in the hydrophobic chains of phosphatidylcholine, their degree of unsaturation and the presence of cholesterol had little effect on the interaction of phosphatidylcholine with fusogenic lipids. 5. Fusogenic lipids showed little specific interaction with natural or synthetic preparations of phosphatidylethanolamine. 6. The possible significance of these observations in relation to the action of fusogenic lipids on biological membranes is discussed in the light of the asymmetrical distribution of phospholipids in erythrocyte membranes.     

Structural differences among phosphatidylcholine, phosphatidylethanolamine, and mixed phosphatidylcholine/phosphatidylethanolamine multilayers: an infrared absorption study

We have examined the infrared absorption spectra from 4000 to 250 cm^?1 of multilayers of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylcholine/phosphatidylethanolamine (1:1 m/m) as a function of hydration, pH, and fatty acid composition. Characteristic splittings of the CH₂ bending and rocking modes and the position of the phosphoryl absorption at ca. 1240 cm^?1 reveal differences in acyl chain packing and head group conformation in the various films. Spectra demonstrate the importance of NH → O hydrogen bonding of the ethanolamine head group and the prerequisite head group conformation (tangent to the multilayer plane) in establishing these structural differences. The general appearance of the P-O-C stretching region (~1050 cm^?1) in the pure and mixed films further supports these conclusions and shows that the spectra clearly distinguish among the different head group orientations. Self-association of phosphatidylethanolamine is sometimes sufficient to prevent formation of mixed phases with phosphatidylcholine at neutral pH. The amount of fine structure, particularly in the low-frequency (800?200 cm^?1) region, in spectra of films of anhydrous, saturated-chain phospholipids decreases considerably when the films are monohydrated, when mixed phases exist, or when there are unsaturations in the acyl chains. These changes likely result from decreased crystal field effects in the spectra as the phosphatide packing density is decreased by any of the above procedures. Furthermore, the absence of other changes upon complete hydration of phosphatidylcholine films suggests that only the initial water is tightly bound to the lipid.     

Characterization of mixed monolayers of phosphatidylcholine and a dicationic gemini surfactant SS-1 with a langmuir balance: effects of DNA

Monolayers of a cationic gemini surfactant, 2,3-dimethoxy-1,4-bis(N-hexadecyl-N;N-dimethyl-ammonium)butane dibromide (abbreviated as SS-1) and its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (pi-A) curves and determined the elastic area compressibility modulus (C) as a function of lateral packing pressure and the mole fraction of the cationic lipid (X(SS-1)), with and without DNA in the subphase. Both SS-1 and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (X(SS-1) < 0.05) of SS-1 in a POPC monolayer condensed the film dramatically, up to 20% at 30 mN/m. This effect is suggested to reflect reorientation of the P(-)-N(+) dipole of the POPC headgroup. Accordingly, the magnitude of the condensing effect diminishes with X(SS-1) and is not observed for mixed films of dioleoylglycerol and SS-1. Reorientation of the P(-)-N(+) dipole is further supported by the pronounced increase in monolayer dipole potential psi due to SS-1. The presence of DNA in the subphase affected the mixed POPC/SS-1 monolayers differently depending on the constituent lipid stoichiometry as well as on the DNA/SS-1 charge ratio. At a DNA concentration of 0.63 microM (in base pairs) condensation of neat POPC monolayers was evident, and this effect remained up to X(SS-1) < 0.5, corresponding to DNA/SS-1 charge ratio of 1.25. An expansion due to DNA, evident as an increase in DeltaA/molecule, was observed at X(SS-1) > 0.5. At a higher concentration of DNA (1.88 microM base pairs) in the subphase corresponding to DNA/SS-1 charge ratio of 3.75 at X(SS-1) = 0.5, condensation was observed at all values of X(SS-1).     

Calcium association with phosphatidylserine. Modification by cholesterol and phosphatidylcholine in monolayers and bilayers

We have examined the association of Ca2+ with phosphatidylserine/cholesterol and phosphatidylserine/dimyristoylphosphatidylcholine mixed monolayers using a surface radiocounting technique. No Ca2+ association with pure monolayers of the uncharged molecules was observed. The Ca2+/phosphatidylserine surface ratio was approximately 1:2 in expanded monolayers of the pure anionic lipid and in phosphatidylserine/phosphatidylcholine mixtures. An increase in surface-associated Ca2+ to a number ratio of 1:1 was observed in phosphatidylserine/cholesterol films when the mole fraction of cholesterol was raised to 0.5 and above and the phospholipid number density held constant. We interpret these findings as a prevention of intermolecular salt formation by the sterol. Further support is provided by particle electrophoresis.