Promotion of hexagonal phase formation and lipid mixing by fatty acids with varying degrees of unsaturation

A series of C18 and C22 fatty acids, with varying degrees of unsaturation, were tested for their ability to alter the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. Lowering the pH from 7.4 to 6.4 greatly decreased the bilayer to hexagonal phase transition temperature of fatty acid-phosphatidylethanolamine mixtures. At pH 7.4, increasing unsaturation of the fatty acid generally increased their hexagonal phase-forming ability. However, oleic acid had somewhat greater hexagonal phase-forming capacity and docosahexaenoic acid somewhat less than would be expected for their degree of unsaturation. At pH 6.4 the difference among fatty acids was small and except for stearic acid, all had similar hexagonal phase forming tendencies. The fatty acids cause acid-induced fusion. There is little effect of fatty acid structure on membrane fusion.     

Fusion of phosphatidylethanolamine-containing liposomes and mechanism of the L alpha-HII phase transition

The initial kinetics of fusion and leakage of liposomes composed of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) have been correlated with the phase behavior of this lipid. Gagné et al. [Gagné, J., Stamatatos, L., Diacovo, T., Hui, S. W., Yeagle, P., & Silvius, J. (1985) Biochemistry 24, 4400-4408] have shown that this lipid is lamellar (L alpha) below 20 degrees C, is hexagonal (HII) above 70 degrees C, and shows isotropic 31P NMR resonances at intermediate temperatures. This isotropic state is also characterized by complex morphological structures. We have prepared DOPE-Me liposomes at pH 9.5 and monitored the temperature dependence of the mixing of aqueous contents, leakage, and changes in light scattering upon reduction of the pH to 4.5. At and below 20 degrees C, where the lipid is in the L alpha phase, there is very little aggregation or destabilization of the liposomes. Between 30 and 60 degrees C, i.e., where the lipid is in the isotropic state, the initial rates of liposome fusion (mixing of aqueous contents) and leakage increase. At temperatures approaching that where the hexagonal HII phase transition occurs, the initial rates and extents of fusion decrease, whereas leakage is enhanced. Similar results were found for dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine (2:1) liposomes. These results clearly establish a common mechanism between the appearance of the isotropic state (between the L alpha and HII phases) and the promotion of liposome fusion. We propose a simple model to explain both the observed behavior of phosphatidylethanolamine-containing membranes with respect to liposome fusion and/or lysis and the beginning of the L alpha-HII phase transition.     

Inverted hexagonal and cubic phases induced by alpha-tocopherol in fully hydrated dispersions of dilauroylphosphatidylethanolamine

The effect of alpha-tocopherol on the thermotropic phase behaviour and structure of aqueous dispersions of 1,2-di-lauryl-sn-glycero-3-phosphoethanolamine was examined by synchrotron X-ray diffraction. The pure phospholipid exhibited a lamellar gel to liquid-crystal phase transition at 30 degrees C on heating at 3 degrees C min(-1) between 10 degrees C and 90 degrees C. The transition was reversible with a temperature hysteresis of 0.3 degrees C on cooling. At temperatures less than 10 degrees C only lamellar gel phase of the pure phospholipid was seen in co-dispersions of up to 20 mol % alpha-tocopherol. The presence of 2.5 mol % alpha-tocopherol caused the appearance of inverted hexagonal phase at temperatures just below the main phase transition temperature that co-existed with the lamellar gel phase. The intensity of scattering from the hexagonal-II phase increased with increasing proportion of alpha-tocopherol in the mixture and in proportions greater than 10 mol % it persisted at temperatures above the main transition and co-existed with the lamellar liquid-crystal phase of the pure phospholipid. At higher temperatures all co-dispersions containing up to 15 mol % alpha-tocopherol showed the presence of cubic phases. These phases indexed a Pn3m or Pn3 space grouping. When the proportion of alpha-tocopherol was increased to 20 mol % the only non-lamellar phase observed was inverted hexagonal phase. This phase co-existed with lamellar gel and liquid-crystal phases of the pure phospholipid, but was the only phase present at temperatures >60 degrees C. The X-ray diffraction data were used to construct a partial phase diagram of the lipid mixture in excess water between 10 degrees and 90 degrees C and up to 20 mol % alpha-tocopherol in phospholipid.     

Role of the position of unsaturation on the phase behavior and intrinsic curvature of phosphatidylethanolamines.

The bilayer-to-hexagonal phase transition temperatures (T(H)) of di-18:1(C) phosphatidylethanolamine with double bonds at positions 6, 9, and 11 are 37 degrees C, 8 degrees C, and 28 degrees C, respectively, as measured by differential scanning calorimetry and x-ray diffraction. Thus T(H) exhibits a minimum when the C=C is around position 9, similar to what has been found for the gel-to-liquid crystalline phase transition temperature in other lipids. Factors that may contribute to the dependence of T(H) on double bond position were studied by x-ray diffraction of the hexagonal phases in the presence and absence of added alkane, with or without the osmotic stress of polyethylene glycol, and over a wide temperature range. The lattice dimensions show that the intrinsic radius of lipid monolayer curvature increases as the double bond is moved toward the tail ends. A measure of the bending moduli of these lipid monolayers shows a higher value for the 9 position, and lower values for the other two. Consideration of the bilayer-to-hexagonal transition in terms of bending and interstitial energies provides a rationale for the relative values of T(H).     

Diacylglycerols, lysolecithin, or hydrocarbons markedly alter the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines

The bilayer to hexagonal phase transition temperatures of dielaidoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylethanolamine are 65.6 and 71.4 degrees C, respectively. Using high-sensitivity differential scanning calorimetry, I have shown that these transition temperatures are extremely sensitive to the presence of small amounts of other lipid components. For example, at a mole fraction of only 0.01, dilinolenin lowers the bilayer to hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine by 8.5 degrees C. Other diacylglycerols have similar effects on this transition temperature, although the degree of unsaturation of the acyl chains has some effect, with distearin being less potent. In comparison, the 20-carbon alkane eicosane lowers this transition temperature by 5 degrees C, while palmitoyl-lysolecithin raises it by 2.5 degrees C. Similar effects of these additives on the bilayer to to hexagonal phase transition temperature are observed with dielaidoylphosphatidylethanolamine. At these concentrations of additive, there is no effect on the gel-state to liquid-crystalline-state transition temperature. The observed shifts in the temperature of the bilayer to the hexagonal phase transition can be qualitatively interpreted in terms of the effects of these additives on the hydrophilic surface area and on the hydrophobic volume. Substances expanding the hydrophobic domain promote hexagonal phase formation and lower the bilayer to hexagonal phase transition temperature. The sensitivity of the bilayer to hexagonal phase transition temperature to the presence of additives is at least as great as that which has been observed for any other lipid phase transition.     

The effect of protons or calcium ions on the phase behavior of phosphatidylserine-cholesterol mixtures.

The influence of protons or calcium ions on the miscibility of cholesterol in phosphatidylserine has been examined using differential scanning calorimetry and X-ray diffraction. At pH 2.6, where the carboxyl group of the serine moiety is protonated, two endothermic transitions are observed in cholesterol-phosphatidylserine mixtures. The midpoint of the first is at 35 degrees C in the absence of cholesterol and decreases to approx. 15 degrees C for molar fraction of cholesterol 0.5. The second transition is centered at approx. 44 degrees C, almost independent of cholesterol content. The two lower temperature phases are lamellar and the high temperature phase has hexagonal symmetry. Cholesterol is more miscible in protonated phosphatidylserine than in the sodium form: cholesterol crystals are detected at a molar ratio of phosphatidylserine to cholesterol of about 1.7:1 as compared to about 2.3:1 at neutral pH. In the presence of calcium ions (1.3 Ca2+ per phosphatidylserine), a lamellar phase is observed with layer spacing 53 A which is independent of temperature (25 degrees C-65 degrees C) and of cholesterol content. Calcium ions cause reduced cholesterol solubility: crystallites are detected already at a molar ratio of 4:1.     

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

The structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine codispersed with a branched-chain phosphatidylcholine

The structure and thermotropic phase behaviour of a fully hydrated binary mixture of dipalmitoylphosphatidylcholine and a branched-chain phosphatidylcholine, 1, 2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine, were examined using differential scanning calorimetry, synchrotron X-ray diffraction and freeze-fracture electron microscopy. The branched-chain lipid forms a nonlamellar phase when dispersed alone in aqueous medium. Mixed aqueous dispersions of the two phospholipids containing less than 33 mol% of the branched-chain lipid form lamellar phases over the whole temperature range were studied (4 degrees C to 60 degrees C). When present in proportions greater than 33 mol% it induces a hexagonal phase in mixed aqueous dispersions with dipalmitoylphosphatidylcholine at temperatures above the fluid phase transition. At temperatures below 35 degrees C a hexagonal phase coexists with a gel bilayer phase. The lamellar<-->nonlamellar transition can be explained satisfactorily on the basis of the shape of the molecule expressed in terms of headgroup and chain cross-sectional areas. At temperatures below 35 degrees C macroscopic phase separation of two gel phases takes place. Freeze-fracture electron microscopy revealed that one gel phase consists of bilayers with a highly regular, periodic superstructure (macro-ripples) whereas the other phase forms flat, planar bilayers. The macro-ripple phase appears to represent a relaxation structure required to adapt to the packing constraints imposed by the incorporation of the branched-chain lipid into the dipalmitoylphosphatidylcholine host bilayer. The data suggest that structural changes that take place on cooling the mixed dispersion below the lamellar<-->nonlamellar phase transition temperature cannot be adequately described using the molecular form concept. Instead it is necessary to take into account the detailed molecular form of the guest lipid as well as its physical properties.     

Cholesterol favors phase separation of sphingomyelin

The phase behavior of mixed lipid dispersions representing the inner leaflet of the cell membrane has been characterized by X-ray diffraction. Aqueous dispersions of phosphatidylethanolamine:phosphatidylserine (4:1 mole/mole) have a heterogeneous structure comprising an inverted hexagonal phase H(II) and a lamellar phase. Both phases coexist in the temperature range 20-45 degrees C. The fluid-to-gel mid-transition temperature of the lamellar phase assigned to phosphatidylserine is decreased from 27 to 24 degrees C in the presence of calcium. Addition of sphingomyelin to phosphatidylethanolamine/phosphatidylserine prevents phase separation of the hexagonal H(II) phase of phosphatidylethanolamine but the ternary mixture phase separates into two lamellar phases of periodcity 6.2 and 5.6 nm, respectively. The 6.2-nm periodicity is assigned to the gel phase enriched in sphingomyelin of molecular species comprising predominantly long saturated hydrocarbon chains because it undergoes a gel-to-fluid phase transition above 40 degrees C. The coexisting fluid phase we assign to phosphatidylethanolamine and phosphatidylserine and low melting point molecular species of sphingomyelin which suppresses the tendency of phosphatidylethanolamine to phase-separate into hexagonal H(II) structure. There is evidence for considerable hysteresis in the separation of lamellar fluid and gel phases during cooling. The addition of cholesterol prevents phase separation of the gel phase of high melting point sphingomyelin in mixtures with phosphatidylserine and phosphatidylethanolamine. In the quaternary mixture the lamellar fluid phase, however, is phase separated into two lamellar phases of periodicities of 6.3 and 5.6 nm (20 degrees C), respectively. The lamellar phase of periodicity 5.6 nm is assigned to a phase enriched in aminoglycerophospholipids and the periodicity 6.3 nm to a liquid-ordered phase formed from cholesterol and high melting point molecular species of sphingomyelin characterized previously by ESR. Substituting 7-dehydrocholesterol for cholesterol did not result in evidence for lamellar phase separation in the mixture within the temperature range 20-40 degrees C. The specificity of cholesterol in creation of liquid-ordered lamellar phase is inferred.     

Effects of temperature on the yeast cell cycle analyzed by flow cytometry

The effects of temperature (in the range 15-36 degrees C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30 degrees C, and a transition point is apparent at about 20 degrees C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21-36 degrees C, while duration of G2+ M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell-cycle model for budding yeast.     

The effect of pH and temperature on the reaction of fully reduced and mixed-valence cytochrome c oxidase with dioxygen

The reaction of fully reduced and mixed-valence cytochrome oxidase with O2 has been followed in flow-flash experiments, starting from the CO complexes, at 428, 445, 605 and 830 nm between pH 5.8b and 9.0 in the temperature range of 2-40 degrees C. With the fully reduced enzyme, four kinetic phase with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 2.5 x 10(4), 1.0 x 10(4) and 800 s(-1), respectively, are observed. The rates of the three last phases display a very small temperature dependence, corresponding to activation energies in the range 13-54 kJ x mol(-1). The rates of the third and fourth phases decrease at high pH due to the deprotonation of groups with pKa values of 8.3 and 8.8, respectively, but also the second phase appears to have a small pH dependence. In the reaction of the mixed-valence enzyme, three kinetic phases with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 6000 and 150 s(-1), respectively, are observed. The third phase only has a small temperature dependence, corresponding to an activation energy of 20 kJ x mol(-1). No pH dependence could be detected for any phase. Reaction schemes consistent with the experimental observations are presented. The pH dependencies of the rates of the two final phase in the reaction of the fully reduced enzyme are proposed to be related to the involvement of protons in the reduction of a peroxide intermediate. The temperature dependence data suggest that the reorganization energies and driving forces are closely matched in all electron transfer steps with both enzyme forms. It is suggested that the slowest step in the reaction of the mixed-valence enzyme is a conformation change involved in the reaction cycle of cytochrome oxidase as a proton pump.     

Interactions of the local anesthetic tetracaine with glyceroglycolipid bilayers: a 2H-NMR study

We have examined the effects of the local anesthetic tetracaine on the orientational and dynamic properties of glycolipid model membranes. We elected to study the interactions of tetracaine with the pure glycolipid 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) and a mixture of beta-DTGL (20 mol%) in dimyristoylphosphatidylcholine (DMPC) by deuterium NMR (2H-NMR) spectroscopy. 2H-NMR spectra of beta-DTGL have been measured as a function of temperature in the presence of both the charged (pH 5.5) and uncharged forms (pH 9.5) of tetracaine. The results indicate that the anesthetic induces the formation of non-lamellar phases. Specifically, the incorporation of uncharged tetracaine results in the formation of a hexagonal phase which is stable from 52 to 60 degrees C. At lower pH, the spectrum at 52 degrees C is very reminescent of that of the beta-glucolipid alone in a bilayer environment, while as the temperature is elevated to 60 degrees C, a transition from a spectrum indicative of axial symmetry to one due to nearly isotropic motion or symmetry occurs, which may result from the formation of a cubic phase. Although it leads to an alteration in the phase behavior, the presence of tetracaine does not induce large changes in the headgroup orientation of beta-DTGL. In contrast to the pure glycolipid situation, the interaction of tetracaine with beta-DTGL (20 mol%) in DMPC does not trigger the formation of non-lamellar phases, but leads to a slight reduction in molecular ordering. The presence of the charged form of the local anesthetic near the aqueous interface of the bilayer appears to induce a small change in the conformation about the C2-C3 bond of the glycerol backbone of beta-DTGL in the mixed lipid system. Thus, the major influence of the local anesthetic on glycolipids is a change in the stability of the lamellar phase, facilitating conversion to phases with hexagonal or isotropic environments for the lipid molecules.     

Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.     

Labelling of colloidal gold with IgE. A quantitative study using monoclonal IgE anti-beta-lactoglobulin and evaluation of the biological activity of the gold complex with RBL-1 cells

Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal 125I-IgE antibovine milk beta-lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1-8.8 (28 nm in particle diameter). At low coverage of the particles (less that or equal to 5 molecules per particle), the isotherm was independent of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2-5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mapping of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4 degrees C. Surface clusters developed when the cells were incubated at 37 degrees C.     

The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study.

1. The polymorphic phase behaviour of aqueous dispersions of phosphatidylethanolamines isolated from human erythrocytes, hen egg yolk and Escherichia coli have been investigated employing 31P NMR techniques. All species exhibit well defined, reversible bilayer to hexagonal (H11) phase transitions as the temperature is increased. The temperatures at which these transition take place (10, 25--30 and 55--60 degrees C for erythrocyte, egg yolk and E. coli phosphatidylethanolamine, respectively) are sensitive to the fatty acid composition, occurring at a temperature up to 10 degrees C above the high temperature end of the hydrocarbon phase transition as detected by differential scanning calorimetry. In some cases the bilayer to hexagonal (H11) transitions may also be detected employing calorimetric techniques. 2. The addition of equimolar concentrations of cholesterol to these naturally occurring phosphatidylethanolamines does not dramatically affect the bilayer-hexagonal (H11) transition temperature, producing changes of up to 10 degrees C. 3. 18 : 1t/18 : 1t phosphatidylethanolamine undergoes the bilayer to hexagonal (H11) phase transition as the temperature is increased through the interval 50--55 degrees C. Alternatively, hydrated 12 : 0/12 : 0 phosphatidylethanolamine remains in the bilayer phase at temperatures up to 90 degrees C (50 degrees C above the hydrocarbon phase transition temperature). 4. The presence of 100 mM NaCl or 10 mM CaCl2 in aqueous dispersions of egg yolk phosphatidylethanolamine does not alter the temperature-dependent polymorphic phase behaviour significantly. However, at 40 degrees C, increasing the p2H above 8.0 results in progressive inhibition of the hexagonal (H11) phase and the appearance of a phase possibly of cubic structure at p2H 9.0. At p2H 10.0 the bilayer phase is preferred. 5. It is suggested that in biomembranes containing phosphatidylethanolamine as a majority species (such as that of E. coli) the fatty acid composition may primarily reflect the need to maintain bilayer structure. Alternatively, it is pointed out that in mammalian membranes such as that of the erythrocyte, phosphatidylethanolamine tends to destabilize bilayer structure. The resulting possibility that transitory non-bilayer lipid configurations may occur may be directly related to many important properties of biological membranes.     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

Studies on the phase transitions of lysoderivatives of ethanolamine glycerophospholipids from human brain

The phase transition temperature of 1,2-distearoylglycerophosphocholine is reduced in presence of equimolar amounts of 1-O-(1'-alkenyl)-glycerophosphoethanolamine (ethanolamine lysoplasmalogen) from 53.3 degrees C-54.1 degrees C to 44.0 degrees C-44.9 degrees C at different pH (4.0; 7.2; 9.0; 10.5). 1-Acyl-glycerophosphoethanolamine leads to a smaller reduction of the 1,2-distearoyl-glycerophosphocholine transition temperature: 45.0 degrees C-46.2 degrees C at the same pH-values. 1-Alkyl-glycerophosphoethanolamine (hydrogenated ethanolamine lysoplasmalogen) possesses a transition temperature, which is 3.3 degrees C-4.9 degrees C higher than the hydrogenated 1-acyl-glycerophosphoethanolamine at each pH investigated. At pH 9.0 and, more pronounced, at pH 10.5 we find a reduction of the transition temperature for both these substances, whereas their transition temperature is nearly unchanged at pH 4.0 and 7.2. Our results clearly show that the ether-bonding in the lysoderivative of plasmalogen is responsible for the closer packing compared to the 1-acyl-glycerophosphoethanolamine.     

Accelerated formation of cubic phases in phosphatidylethanolamine dispersions.

By means of x-ray diffraction we show that several sodium salts and the disaccharides sucrose and trehalose strongly accelerate the formation of cubic phases in phosphatidylethanolamine (PE) dispersions upon temperature cycling through the lamellar liquid crystalline-inverted hexagonal (Lalpha-HII) phase transition. Ethylene glycol does not have such an effect. The degree of acceleration increases with the solute concentration. Such an acceleration has been observed for dielaidoyl PE (DEPE), dihexadecyl PE, and dipalmitoyl PE. It was investigated in detail for DEPE dispersions. For DEPE (10 wt% of lipid) aqueous dispersions at 1 M solute concentration, 10-50 temperature cycles typically result in complete conversion of the Lalpha phase into cubic phase. Most efficient is temperature cycling executed by laser flash T-jumps. In that case the conversion completes within 10-15 cycles. However, the cubic phases produced by laser T-jumps are less ordered in comparison to the rather regular cubic structures produced by linear, uniform temperature cycling at 10 degrees C/min. Temperature cycles at scan rates of 1-3 degrees C/min also induce the rapid formation of cubic phases. All solutes used induce the formation of Im3m (Q229) cubic phase in 10 wt% DEPE dispersions. The initial Im3m phases appearing during the first temperature cycles have larger lattice parameters that relax to smaller values with continuation of the cycling after the disappearance of the Lalpha phase. A cooperative Im3m --> Pn3m transition takes place at approximately 85 degrees C and transforms the Im3m phase into a mixture of coexisting Pn3m (Q224) and Im3m phases. The Im3m/Pn3m lattice parameter ratio is 1. 28, as could be expected from a representation of the Im3m and Pn3m phases with the primitive and diamond infinite periodic minimal surfaces, respectively. At higher DEPE contents ( approximately 30 wt%), cubic phase formation is hindered after 20-30 temperature cycles. The conversion does not go through, but reaches a stage with coexisting Ia3d (Q230) and Lalpha phases. Upon heating, the Ia3d phase cooperatively transforms into a mixture of, presumably, Im3m and Pn3m phases at about the temperature of the Lalpha-HII transition. This transformation is readily reversible with the temperature. The lattice parameters of the DEPE cubic phases are temperature-insensitive in the Lalpha temperature range and decrease with the temperature in the range of the HII phase.