The effects of various peptides on the thermotropic properties of phosphatidylcholine bilayers

The effects of an amino acid derivative (N-benzoyl-l-argininamide), four small peptides (Phe-Gly-Phe-Gly, gastrin-related peptide (Trp-Met-Arg-Phe-NH₂), tetragastrin (Trp-Met-Asp-Phe-NH₂), pentagastrin (Boc-βAla-Trp-Met-Asp-Phe-NH₂)) and one medium-sized peptide. glucagon (29 residues), on the gel-to-liquid crystalline transition of a multilamellar suspension of dimyristoylphosphatidylcholine have been studied by means of high-sensitivity differential scanning calorimetry. At low concentrations of added solutes, the temperature at which the excess apparent specific heat in the gel-to-liquid crystalline phase transition of the lipid is maximal is lowered by an amount proportional to the total concentration of the peptide, with proportionality constants ranging from ?0.018 K mM^?1 for Phe-Gly-Phe-Gly to ?3.1 K mM^?1 for the gastrin-related peptide. The lipid mixtures involving the first two solutes listed above exhibited approximately symmetrical curves of excess apparent specific heat vs. temperature. The curves for the other solutes were asymmetric, and could be well represented as the sum of either two or three two-state curves. The asymmetry, which was especially pronounced in the cases of pentagastrin and glucagon, thus appeared to be due to the presence of components having lower and/or higher transition temperatures than that of the lipid. Pentagastrin and glucagon (R.M. Epand and J.M. Sturtevant, Biochemistry 20 (1981) 4603) have much smaller effects on the gel-to-liquid crystalline phase transition of dipalmitoylphosphatidylcholine than on that of the dimyristoyl analog.     

The effect of dolichol on the permeability properties of phosphatidylcholine bilayers

The permeability of liposomes to water, glucose, Ca2+ and alkaline cations was monitored by recording the change in absorbance at 450 nm using a rapid reaction stopped-flow spectrophotometer. Liposomes were prepared with egg phosphatidylcholine and concentrations of dolichol ranging from 0.1% to 9% (w/w). Net permeability of phosphatidylcholine bilayers to alkaline cations was induced by the incorporation of dolichol. This effect was not observed in the case of non-charged solutes like glucose or in that of alkaline earth cations such as calcium. Permeation of K+ was significantly increased above the phase transition temperature. These results suggest that dolichols may play a role in biological membranes, besides the well-known glycosyl carrier function in the biosynthesis of glycoproteins.     

Structure and properties of mixed-chain phosphatidylcholine bilayers

The structural and thermotropic properties of the hydrated mixed-chain phosphatidylcholines (PCs), C(8):C(18)-PC and C(10):C(18)-PC, have been studied by X-ray diffraction and differential scanning calorimetry. For fully hydrated C(8):C(18)-PC, the reversible chain melting transition is observed at 9.9 degrees C (delta H = 7.3 kcal/mol). X-ray diffraction at 0 degrees C (below the chain melting transition) shows a small bilayer repeat distance, d = 51.0 A, and a sharp, symmetric wide-angle reflection at 4.1 A, characteristic of a mixed interdigitated bilayer gel phase [see McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038-4044; Hui, S. W., Mason, J. T., & Huang, C. (1984) Biochemistry 23, 5570-5577]. At 30 degrees C (above the chain melting transition), a diffuse band is observed at 4.5 A characteristic of an L alpha phase but with an increased bilayer periodicity, d = 61 A. Both the calculated lipid bilayer thickness (d1) and that determined directly from electron density profiles (dp-p) show unusual increases as a consequence of chain melting. In contrast, fully hydrated C(10):C(18)-PC shows an asymmetric endothermic transition at 11.8 degrees C. Below the chain melting transition, two lamellar phases are present, corresponding to coexisting interdigitated (d = 52.3 A) and noninterdigitated (d = 62.5 A) bilayer gel phases. The relative amounts of these phases depend upon the low-temperature incubation and/or hydration conditions, suggesting conversions, albeit kinetically complex, between metastable, and stable phases. The different behavior of C(8):C(18)-PC and C(10):C(18)-PC, as well as their positional isomers, is rationalized in terms of the molecular conformation of PC.     

Effects of fatty acid alpha-hydroxylation on glycosphingolipid properties in phosphatidylcholine bilayers.

The role of glycosphingolipid fatty acid alpha-hydroxylation as a modulator of glycolipid organization and dynamics was considered by 2H-NMR in bilayer membranes. For these experiments, galactosylceramides were prepared in which the natural fatty acid mixture was replaced with perdeuterated 18-carbon hydroxylated or non-hydroxylated stearic acid. The L-stereoisomer of N-(alpha-OH-stearoyl-d34)galactosylceramide and its naturally-occurring D-alpha-OH analogue, were isolated for independent study. Bilayers were formed using 10 mol% galactosylceramide in a shorter chain phospholipid, dimyristoylphosphatidylcholine, in an attempt to reproduce several features of glycolipid-phospholipid interactions typical of cell membranes. Spectra of deuterated galactosylceramide in gel phase phospholipid membranes indicated that alpha-hydroxylation led to greater motional freedom and/or conformational disorder, with no measurable difference between D- and L-alpha-OH fatty acid derivatives. In fluid phosphatidylcholine bilayers the effects were modest. Glycolipid fatty acid hydroxylation led to broadening of the range of order parameters associated with methylene groups near the membrane surface (frequently referred to as the 'plateau region') - this effect being more marked for the naturally-occurring (D) stereoisomer. The degree of overall molecular order sensed by the glycolipid fatty acid chain in a fluid host matrix was minimally affected by alpha-hydroxylation; although the plateau region of the D isomer was slightly more ordered than that of the L isomer and the non-hydroxylated species. These results suggest that a significant aspect of the alpha-hydroxy group effect on glycosphingolipid behaviour in bilayer membranes with low glycolipid content was interference with glycolipid packing amongst host phospholipids in the upper portion of the acyl chains. For the D stereoisomer, there was some evidence that the hydroxy group led to strengthening of interlipid interaction near the membrane surface.     

Mixed-chain phosphatidylcholine bilayers: structure and properties

Calorimetric and X-ray diffraction data are reported for two series of saturated mixed-chain phosphatidylcholines (PCs), 18:0/n:0-PC and n:0/18:0-PC, where the sn-1 and sn-2 fatty acyl chains on the glycerol backbone are systematically varied by two methylene groups from 18:0 to 10:0 (n = 18, 16, 14, 12, or 10). Fully hydrated PCs were annealed at -4 degrees C and their multilamellar dispersions characterized by differential scanning calorimetry and X-ray diffraction. All mixed-chain PCs form low-temperature "crystalline" bilayer phases following low-temperature incubation, except 18:0/10:0-PC. The subtransition temperature (Ts) shifts toward the main (chain melting) transition temperature (Tm) as the sn-1 or sn-2 fatty acyl chain is reduced in length; for the shorter chain PCs (18:0/12:0-PC, 12:0/18:0-PC, and 10:0/18:0-PC), Ts is 1-2 degrees C greater than Tm, and the subtransition enthalpy (delta Hs) is much greater than for the longer acyl chain PCs. Tm decreases with acyl chain length for both series of PCs except 18:0/10:0-PC, while for the positional isomers, n:0/18:0-PC and 18:0/n:0-PC, Tm is higher for the isomer with the longer acyl chain in the sn-2 position of the glycerol backbone. The conversion from the crystalline bilayer Lc phase to the liquid-crystalline L alpha phase with melted hydrocarbon chains occurs through a series of phase changes which are chain length dependent. For example, 18:0/18:0-PC undergoes the phase changes Lc----L beta'----P beta'----L alpha, while the shorter chain PC, 10:0/18:0-PC, is directly transformed from the Lc phase to the L alpha phase. However, normalized enthalpy and entropy data suggest that the overall thermodynamic change, Lc----L alpha, is essentially chain length independent. On cooling, the conversion to the Lc phases occurs via bilayer gel phases, L beta', for the longer chain PCs or through triple-chain interdigitated bilayer gel phases, L beta, for the shorter chain PC 18:0/12:0-PC and possibly 10:0/18:0-PC. Molecular models indicate that the bilayer gel phases for the more asymmetric PC series, 18:0/n:0-PC, must undergo progressive interdigitation with chain length reduction to maintain maximum chain-chain interaction. The L beta phase of 18:0/10:0-PC is the most stable structure for this PC below Tm. The formation and stability of the triple-chain structures can be rationalized from molecular models.     

Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives

alpha-Tocopherol and alpha-tocopheryl succinate are biologically active lipids. The activity of these lipids may be related to how they affect membrane physical-chemical properties. Utilizing fluorescence methods, we have investigated the effect of alpha-tocopherol, alpha-tocopheryl succinate, and alpha-tocopheryl acetate on the properties of model membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In liquid-crystalline phase phospholipid bilayers, alpha-tocopherol decreased acyl chain mobility and decreased the interfacial polarity, but had no effect on the interfacial surface charge. In contrast, alpha-tocopheryl succinate had little effect on acyl chain motion or interfacial hydration, but increased the interfacial surface charge. alpha-Tocopheryl acetate had very little effect on any of the measurements of these bilayer properties. In a gel phase bilayer, alpha-tocopherol decreased acyl chain order, whereas alpha-tocopheryl succinate and alpha-tocopheryl acetate did not. Each alpha-tocopheryl derivative had a different effect on interfacial polarity, however, only alpha-tocopheryl succinate increased the interfacial surface charge. The acylation of alpha-tocopherol abolishes its antioxidant activity and generates molecules with different membrane physical properties. The non-polar acetate group of alpha-tocopheryl acetate locates this compound in a region of the bilayer where it has little effect on bilayer interfacial properties. The free carboxyl group of alpha-tocopheryl succinate is located in the interfacial region of the bilayer where it increases the membrane surface charge.     

The effect of cholesterol on the structure of phosphatidylcholine bilayers

The effect of cholesterol on the structure of phosphatidylcholine bilayers was investigated by X-ray diffraction methods. Electron density profiles at 5 Å resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayer was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12–16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.     

The interaction of ions with phosphatidylcholine bilayers

The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in ²H₂O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant $\text{K}{}_{\mathrm{<mtext>b</mtext>}}$ varies as a function of the quantity of metal ion bound and as a function of the Cl^? concentration. The apparent binding constant at “zero loading” is $\text{K}{}_0\text{= 1.25 · 10}{}^4\text{L}{}^2\text{·}\text{M}{}^{\mathrm{?}}\text{at}\text{0.15}\text{M KCl}$. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl^? concentration.The interaction of lanthanides and divalent cations such as Ca²⁺ and Mg²⁺ is independent of pH in the pH range 3–7⁺ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is $\text{Cl}{}^{\mathrm{?}}\text{< Br}{}^{\mathrm{?}}\text{< NO}{}_3{}^{\mathrm{?}}\text{< SCN}{}^{\mathrm{?}}\text{< I}{}^{\mathrm{?}}\text{< ClO}{}_4{}^{\mathrm{?}}$. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca²⁺ and Mg²⁺ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO₂²⁺ showing the highest affinity.     

Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.     

Effect of encapsulated Ca2+ on the surface properties of curved phosphatidylcholine bilayers

In this paper, the influence of Ca2+ on the adsorption properties of 1,8-anilinonaphthalenesulfonate (ANS) and analogous probes to sonicated vesicles of phosphatidylcholine was studied by means of spectrofluorometry. The fluorescence of ANS added to the vesicle dispersion increases with the Ca2+ concentration in the inner media but remains constant when Ca2+ concentration is changed in the outside solution. However, the fluorescence decreases when large anions such as ClO4- are present in the external solution. Ca2+ inside large liposomes promotes a similar behaviour to that found in sonicated vesicles when they are osmotically contracted in hypertonic media. The results can be interpreted in terms of Ca2+ adsorption on the inner interface and a cooperative interaction between the monolayers.     

Topology of gel-phase domains and lipid mixing properties in phase-separated two-component phosphatidylcholine bilayers.

The influence of the lipid mixing properties on the lateral organization in a two-component, two-phase phosphatidylcholine bilayer was investigated using both an experimental (fluorescence recovery after photobleaching (FRAP)) and a simulated (Monte Carlo) approach. With the FRAP technique, we have examined binary mixtures of 1-stearoyl-2-capryl-phosphatidylcholine/1,2-distearoyl-phosphat idylcholine (C18C10PC/DSPC), and 1-stearoyl-2-capryl-phosphatidylcholine/1,2-dipalmitoyl-phospha tid ylcholine (C18C10PC/DPPC). Comparison with the 1,2-dimyristoyl-phosphatidylcholine/1,2-distearoyl-phosphatidylcholine (DMPC/DSPC) previously investigated by FRAP by Vaz and co-workers (Biophys. J., 1989, 56:869-876) shows that the gel phase domains become more effective in restricting the diffusion coefficient when the ideality of the mixture increases (i.e., in the order C18C10PC/DSPC-->C18C10PC/DPPC-->DMPC/DSPC). However, an increased lipid miscibility is accompanied by an increasing compositional dependence: the higher the proportion of the high-temperature melting component, the less efficient the gel phase is in compartmentalizing the diffusion plane, a trend that is best accounted for by a variation of the gel phase domain shape rather than size. Computer-simulated fluorescence recoveries obtained in a matrix obstructed with obstacle aggregates of various fractal dimension demonstrate that: 1) for a given obstacle size and area fraction, the relative diffusion coefficient increases linearly with the obstacle fractal dimension and 2) aggregates with a lower fractal dimension are more efficient in compartmentalizing the diffusion plane. Comparison of the simulated with the experimental mobile fractions strongly suggests that the fractal dimension of the gel phase domains increases with the proportion of high-temperature melting component in DMPC/DSPC and (slightly) in C18C10PC/DPPC.     

Insights into the dynamics of DMSO in phosphatidylcholine bilayers.

The solvation effects of dimethyl sulfoxide (DMSO) on the phase stability of dimyristoylphosphatidylcholine (DMPC) have been fully characterized using differential scanning calorimetry (DSC) and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of the sub-, pre-, and main transitions of DMPC were found to increase linearly with increasing mole fraction of DMSO up to mole fraction X=0.13 DMSO/H(2)O. Beyond X=0.13, the pre-transition peak started to merge with the peak representing the main transition. Simultaneously, the subtransition peak began to disappear as its transition temperature also decreased. At X=0.18, with both the subtransition and pre-transition absent, the main transition between the planar gel and the liquid-crystalline phase was observed at 30.3 degrees C. Transition enthalpy values indicated that the subgel, planar gel and rippled gel phases are most stable at X=0.11, 0.16 and 0.20 DMSO/H(2)O, respectively. This demonstrates that DMSO exerts distinct effects on each respective phase and corresponding transition. Temperature-dependent fluorescence emission scans show an increase in hydration as the system proceeds from the subgel phase all the way to the liquid-crystalline phase and correlated well with the effects of DMSO on the transition temperatures of DMPC observed in our calorimetry data. Initial observations for the sub- and main transition are further confirmed by fluorescence anisotropy using DPH as a probe. The results illustrate the differences in the microviscosity of each phase and how DMSO affects the phase transitions. Ultimately, our results suggest the most likely mechanism governing the biological actions of DMSO may involve the regulation of the solvation effects of water on the phospholipid bilayer.     

Temperature dependence of the repulsive pressure between phosphatidylcholine bilayers.

Bilayer structure and interbilayer repulsive pressure were measured from 5 to 50 degrees C by the osmotic stress/x-ray diffraction method for both gel and liquid crystalline phase lipid bilayers. For gel phase dibehenoylphosphatidylcholine (DBPC) the bilayer thickness and pressure-distance relations were nearly temperature-independent, and at full hydration the equilibrium fluid spacing increased approximately 1 A, from 10 A at 5 degrees C to 11 A at 50 degrees C. In contrast, for liquid crystalline phase egg phosphatidylcholine (EPC), the bilayer thickness, equilibrium fluid spacing, and pressure-distance relation were all markedly temperature-dependent. As the temperature was increased from 5 to 50 degrees C the EPC bilayer thickness decreased approximately 4 A, and the equilibrium fluid spacing increased from 14 to 21 A. Over this temperature range there was little change in the pressure-distance relation for fluid spacings less than approximately 10 A, but a substantial increase in the total pressure for fluid spacings greater than 10 A. These data show that for both gel and liquid crystalline bilayers there is a short-range repulsive pressure that is nearly temperature-independent, whereas for liquid crystalline bilayers there is also a longer-range pressure that increases with temperature. From analysis of the energetics of dehydration we argue that the temperature-independent short-range pressure is consistent with a hydration pressure due to polarization or electrostriction of water molecules by the phosphorylcholine moiety. For the liquid crystalline phase, the 7 A increase in equilibrium fluid spacing with increasing temperature can be predicted by an increase in the undulation pressure as a consequence of a temperature-dependent decrease in bilayer bending modulus.     

A new monofluorinated phosphatidylcholine forms interdigitated bilayers.

16-Fluoropalmitic acid was synthesized from 16-hydroxypalmitic acid using diethylaminosulfur trifluoride. This monofluorinated fatty acid then was used to make 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) as a fluorinated analog of dipalmitoylphosphatidylcholine (DPPC). Surprisingly, we found that the phase transition temperature (Tm) of F-DPPC occurs near 50 degrees C, approximately 10 degrees C higher than its nonfluorinated counterpart, DPPC, as judged by both differential scanning calorimetry and infrared spectroscopy. The pretransition observed for DPPC is absent in F-DPPC. A combination of REDOR, rotational-echo double-resonance, and conventional solid-state NMR experiments demonstrates that F-DPPC forms a fully interdigitated bilayer in the gel phase. Electron paramagnetic resonance experiments show that below Tm, the hydrocarbon chains of F-DPPC are more motionally restricted than those of DPPC. X-ray scattering experiments confirm that the thickness and packing of gel phase F-DPPC is similar to that of heptanetriol-induced interdigitated DPPC. F-DPPC is the first phosphoglyceride containing sn-1 and sn-2 ester-linked fatty acyl chains of equal length that spontaneously forms interdigitated bilayers in the gel state in the absence of inducing agents such as alcohols.     

Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (greater than 10(4) s-1) exchange with all the lipid molecules in the system.     

The lateral distribution of pyrene-labeled sphingomyelin and glucosylceramide in phosphatidylcholine bilayers.

The lateral distribution of N-[10(1-pyrenyl)decanoyl]-sphingomyelin (PyrSPM) and N-[10(1-pyrenyl)decanoyl]-glucocerebroside (PyrGlcCer) was studied in multilamellar vesicles of 1,2-dipalmitoyl-, 1,2-dimyristoyl-, and 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC, DMPC, and POPC, respectively) under anaerobic conditions by determining the excimer-to-monomer fluorescence intensity ratio (E/M) as a function of temperature. The E/M(T) curves for PyrSPM and PyrGlcCer in the three phosphatidylcholine matrices are qualitatively similar to the curves reported for 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]-phosphatidylcholine (PyrPC) in the same three matrix phospholipids (Hresko, R. C., I. P. Sugár, Y. Barenholz, and T. E. Thompson, 1986, Biochemistry, 25:3813-3823). However, there is independent evidence to suggest that sphingomyelin and glucocerebroside are organized in POPC, DPPC, and DMPC in a more complex manner than is PyrPC. In an effort to examine further the relationship between the lateral distribution of the labeled lipid and the shape of an E/M(T) curve, E/M vs. temperature simulations were carried out together with an analysis of the equation that relates E/M to the system parameters. The results indicate that information about the lateral distribution of the pyrene-labeled lipid can be obtained from an E/M(T) curve only for those systems in which the gel to liquid crystalline phase transition temperature of the matrix lipid is higher than that of the pyrene-labeled lipid. However, very little can be known about the system from an E/M(T) curve if the matrix lipid has the lower phase transition temperature.     

Permeability of phosphatidylcholine and phosphatidylethanolamine bilayers

Sodium and glucose effluxes were measured in liposomes formed from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Vesicles composed of a saturated PC display a local permeability maximum in the region of the lipid transition temperature. The height of this maximum is predominantly a function of the thickness of the hydrocarbon chain region. Liposomes formed from a saturated PE do not display such a permeability maximum and in these vesicles the permeability process appears to be controlled by the head group region. It is postulated that the control exerted by the ethanolamine group is due to the reorganization of water structure it induces at the bilayer surface.     

The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide.

A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state 13C MAS NMR spectroscopy.