Domain formation in sphingomyelin/cholesterol mixed membranes studied by spin-label electron spin resonance spectroscopy

Interactions of palmitoylsphingomyelin with cholesterol in multilamellar vesicles have been studied over a wide range of compositions and temperatures in excess water by using electron spin resonance (ESR) spectroscopy. Spin labels bearing the nitroxide free radical group on the 5 or 14 C-atom in either the sn-2 stearoyl chain of phosphatidylcholine (predominantly 1-palmitoyl) or the N-stearoyl chain of sphingomyelin were used to determine the mobility and ordering of the lipids in the different phases. Two-component ESR spectra of the 14-position spin labels demonstrate the coexistence first of gel (L(beta)) and liquid-ordered (L(o)) phases and then of liquid-ordered and liquid-disordered (L(alpha)) phases, with progressively increasing temperature. These phase coexistences are detected over a limited range of cholesterol contents. ESR spectra of the 5-position spin labels register an abrupt increase in ordering at the L(alpha)-L(o) transition and a biphasic response at the L(beta)-L(o) transition. Differences in outer splitting between the C14-labeled sphingomyelin and phosphatidylcholine probes are attributed to partial interdigitation of the sphingomyelin N-acyl chains across the bilayer plane in the L(o) state. In the region where the two fluid phases, L(alpha) and L(o), coexist, the rate at which lipids exchange between phases (<7 x 10(7) s(-)(1)) is much slower than translational rates in the L(alpha) phase, which facilitates resolution of two-component spectra.     

Cholesterol modulation of sphingomyelinase activity at physiological temperatures

Bacillus cereus sphingomyelinase activity was assayed on large unilamellar vesicles composed of sphingomyelin (SM)/cholesterol (Ch) mixtures at varying proportions. Natural (egg) SM was used with a gel–fluid transition temperature at ca. 40 °C. When the enzyme was assayed at 37 °C, the activity on pure SM was exceedingly low, but a small increase was observed as soon as some Ch was added, and a large enhancement of activity occurred with Ch proportions above 25 mol%. The data were interpreted in terms of sphingomyelinase activity being higher in the cholesterol-induced liquid-ordered phase than in the gel phase. The abrupt increase in activity above 25 mol% Ch would occur as a result of a change in domain connectivity, when the Ch-rich liquid-ordered domains coalesced. In equimolar SM/Ch mixtures, that were in the liquid-ordered state in a wide range of temperatures, sphingomyelinase activity was virtually constant in the 30–70 °C range. The results demonstrate that at the mammalian and bird physiological temperatures Ch modulates sphingomyelinase activity, and that this can occur precisely because most SM have a gel–fluid transition temperature above the physiological temperature range. In addition, Ch activation of sphingomyelinase and the strong affinity of Ch for SM allow the rapid, localised and self-contained production of the metabolic signal ceramide in specific microdomains (rafts).     

Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol

The structural transitions in aqueous dispersions of egg-sphingomyelin and bovine brain-sphingomyelin and sphingomyelin co-dispersed with different proportions of cholesterol were compared during temperature scans between 20° and 50 °C using small-angle and wide-angle X-ray scattering techniques. The Bragg reflections observed in the small-angle scattering region from pure phospholipids and codispersions of sphingomyelin:cholesterol in molar ratios 80:20 and 50:50 could all be deconvolved using peak fitting methods into two coexisting lamellar structures. Electron density profiles through the unit cell normal to the bilayer plane were calculated to derive bilayer and water layer thicknesses of coexisting structures at 20° and 50 °C. Codispersions of sphingomyelin:cholesterol in a molar ratio 60:40 consisted of an apparently homogeneous bilayer structure designated as liquid-ordered phase. Curve fitting analysis of the wide-angle scattering bands were applied to correlate changes in packing arrangements of hydrocarbon in the hydrophobic domain of the bilayer with changes in enthalpy recorded by differential scanning calorimetry. At 20 °C the wide-angle scattering bands of both pure sphingomyelins and codispersions of sphingomyelin and cholesterol could be deconvolved into two symmetric components. A sharp component located at a d-spacing of 0.42 nm was assigned to a gel phase in which the hydrocarbon chains are oriented perpendicular to the bilayer plane. A broader symmetric band centered at d-spacings in the region of 0.44 nm was assigned as disordered hydrocarbon in dispersions of pure sphingomyelin and as liquid-ordered phase in codispersions of sphingomyelin and cholesterol. It is concluded from the peak fitting analysis that cholesterol is excluded from gel phases of egg and brain sphingomyelins at 20 °C. The gel phases coexist with liquid-ordered phase comprised of egg-sphingomyelin and 27 mol% cholesterol and brain-sphingomyelin and 33 mol% cholesterol, respectively. Correlation of the disappearance of gel phase during heating scans and the enthalpy change recorded by calorimetry in codispersions of sphingomyelin and cholesterol leads to the conclusion that a major contribution to the broadened phase transition endotherm originates from dilution of the cholesterol-rich liquid-ordered phase by mobilization of sphingomyelin from the melting gel phase.     

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.     

Phase transitions in sphingomyelin thin filsm. A spin label study

3-Spiro-(2′-(N-oxyl-4′,4′-dimethyloxazolidine)) — cholestane, (I) and 12-spiro-(2′-(N-oxyl-4′,4′-dimethyloxazolidine))-stearic acid (II) have been used as molecular probes to study the interaction of sphingomyelin and cholesterol in both dry and hydrated oriented films at different temperatures. The presence of 50 mole percent cholesterol causes a gel to liquid crystalline phase transition of bovine brain sphingomyelin at 20°C. A temperature induced phase transition involving the phospholipid polar groups has been detected. The mean transition temperature from a rigid to a fluid bilayer lattice structure is 32°C ±0.5°C in hydrated equimolar sphingomyelin — cholesterol films.     

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C₁₂-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L_o domains but induces the formation of a gel-like phase. The activation of phospholipase A₂ by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.     

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).     

Lamellar gel-lamellar liquid crystal phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from aqueous trehalose solutions. A real-time X-ray diffraction study

The mechanism of the phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from fully hydrated gel phase (L beta') in the presence of trehalose has been investigated by real-time X-ray diffraction methods. Sequential diffraction patterns were recorded with an accumulation time of 3 s during heating and 1.2 s during cooling between about 20 and 80 degrees C. A transition is observed in the range 47-53 degrees C that involves structural events typical of a lamellar gel-lamellar liquid-crystal (L beta--L alpha) transformation. This transition is completely reversible with a temperature hysteresis of 2-3 degrees C and thereby resembles the main phase transition of fully hydrated dipalmitoylphosphatidylcholine multilayers. The mechanism of the transition from L beta to L alpha as seen in the wide-angle scattering profiles show that the sharp peak at about 0.41 nm, characteristic of the gel phase, broadens and shifts progressively to about 0.44 nm towards the end of the transition. A temperature jump of 6C degrees/s through the phase transition region of a freeze-dried dipalmitoylphosphatidylcholine: trehalose mixture (molar ratio 1:1) showed that the phase transition had a relaxation time of about 2 s which is similar to that of the main transition in the fully hydrated lipid. X-ray diffraction studies of the melting of dipalmitoylphosphatidylcholine freeze-dried from the lamellar-gel phase in the absence of trehalose showed a transition at above 70 degrees C. The low-angle diffraction data of phospholipid/trehalose mixtures are consistent with an arrangement of trehalose molecules in a loosely packed 'monolayer' separating bilayers of phospholipid. Trehalose appears to reduce the direct interbilayer hydrogen bond coupling thereby modifying the thermal stability and the phase transition mechanism of the bilayers.     

Structure and interactions of ether- and ester-linked phosphatidylethanolamines

The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientational order in the choline headgroup of sphingomyelin: A 14N-NMR study

An aqueous dispersion of fully hydrated bovine sphingomyelin was studied using ¹⁴N-NMR spectroscopy. Spectra were obtained as a function of temperature over the range 15–80°C, in both the liquid crystal and gel phases. In the liquid crystal phase, powder pattern lineshapes were obtained, whose quadrupolar splitting slowly decreases with increasing temperature. The spectra are increasingly broadened as the temperature is lowered through the phase transition into the gel phase. The linewidths and the second moments of these spectra indicate that the onset of a broad phase transition occurs at approx. 35°C, in agreement with previous calorimetric and ³¹P-NMR measurements. There is no evidence from the lineshapes for an hexagonal phase in this system, and this conclusion is supported by X-ray diffraction measurements carried out on aqueous dispersions of sphingomyelin in both phases. Assuming that the static nitrogen quadrupole coupling constant is the same for both sphingomyelin and dipalmitoyl-l-α-phosphatidylcholine (DPPC), the decrease observed in the quadrupolar splitting of sphingomyelin compared to that of DPPC indicates that the orientational order of the choline headgroup in liquid crystalline sphingomyelin is not the same as that of its counterpart in DPPC. Preliminary relaxation time measurements of $\text{T}{}_1$ and $\text{T}{}_2$ are presented which suggest that there are also dynamic differences between sphingomyelin and DPPC in the choline headgroup.     

A calorimetric study of the thermotropic behaviour of mixtures of brain cerebrosides with other brain lipids

We have used a computer-controlled differential scanning calorimeter to determine the phases present in mixtures of the brain galactocerebrosides with other representative brain lipids. There are two types of brain galactocerebroside, those which possess an alpha-hydroxy substituent on the acyl chain (HFA) and those that do not (NFA). In the liquid crystalline state both cerebrosides were miscible with all the lipids studied, but in the gel state they were immiscible with cholesterol and the brain phosphatidylcholines. However, cholesterol mixtures in which the cholesterol mole fraction exceeded one third formed homogeneous metastable gel states on cooling from above the melting point of the cerebroside. Relaxation to the stable two phase state took place slowly over several hours. The solubilities of the galactocerebrosides in the other main brain sphingolipid, sphingomyelin, were much higher. Only in the case of the NFA galactocerebroside and at low mole fractions of sphingomyelin was immiscibility detected. Ternary mixtures of the two cerebrosides with sphingomyelin/cholesterol and phosphatidylcholine/cholesterol (PC/Chol) showed different miscibility characteristics. On cooling from 80 degrees C all mixtures formed homogeneous gel states. However, on standing the cerebrosides separated into discrete gel phases in all mixtures but one, that in which HFA galactocerebrosides were mixed with sphingomyelin and cholesterol. The cerebroside in the mixture with the composition closest to that of myelin, HFA/PC/Chol, melted at 38 degrees C. On scanning guinea pig CNS myelin which had been equilibrated at 5 degrees C a transition was detected with Tmax 33 degrees C. On the basis of comparison with the HFA/PC/Chol mixture we propose that the transition in myelin at this temperature is due to the melting of a galactocerebroside gel phase.     

Interaction of cholesterol with various glycerophospholipids and sphingomyelin

The influence of cholesterol on the phase behavior of glycerophospholipids and sphingomyelins was investigated by spin-label electron spin resonance (ESR) spectroscopy. 4-(4,4-Dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoic acid (5-SASL) and 1-stearoyl-2-[4-(4,4-dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoy l]-sn- glycero-3-phosphocholine (5-PCSL) spin-labels were employed for this purpose. The outer hyperfine splitting constants, Amax, measured from the spin-label ESR spectra as a function of temperature were taken as empirical indicators of cholesterol-induced changes in the acyl chain motions in the fluid state. The Amax values of 5-PCSL exhibit a triphasic dependence on the concentration of cholesterol for phosphatidylcholines and bovine brain sphingomyelin. We interpret this dependence as reflecting the existence of liquid-disordered, ld, liquid-ordered, lo, and coexistence regions, ld + lo. The phase boundary between the ld and the two-phase region and the boundary between the lo and the two-phase region in the phosphatidylcholine-cholesterol systems coalesce at temperatures 25-33 degrees C above the main-chain melting transition temperature of the cholesterol-free phosphatidylcholine bilayers. In the case of bovine brain sphingomyelin, the ld-lo phase coalescence occurs about 47 degrees C above the melting temperature of the pure sphingomyelin. The selectivity of interaction of cholesterol with glycerophospholipids of varying headgroup charge was studied by comparing the cholesterol-induced changes in the Amax values of derivatives of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine spin-labeled at the fifth position of the sn-2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (L_o) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38°C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20°–50°C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 2. Infrared and 31P NMR spectroscopic studies

The polymorphic phase behavior of aqueous dispersions of a number of representative phosphatidylcholines with methyl iso-branched fatty acyl chains was investigated by Fourier transform infrared (FT-IR) and phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy. For the longer chain phosphatidylcholines, where two transitions are resolved on the temperature scale, the higher temperature event can unequivocally be assigned to the melting of the acyl chains (i.e., a gel/liquid-crystalline phase transition), whereas the lower temperature event is shown to involve a change in the packing mode of the methylene and carbonyl groups of the hydrocarbon chains in the gel state (i.e., a gel/gel transition). The infrared spectroscopic data suggest that the methyl iso-branched phosphatidylcholines assume a partially dehydrated, highly ordered state at low temperatures, resembling the Lc phase recently described for the long-chain n-saturated phosphatidylcholines. At higher temperatures, some branched-chain phosphatidylcholines appear to assume a fully hydrated, loosely packed gel phase similar to but not identical with the P beta, phase of their linear saturated analogues. Thus, the iso-branched phosphatidylcholine gel/gel transition corresponds, at least approximately, to a summation of the structural changes accompanying both the subtransition and the pretransition characteristic of the longer chain n-saturated phosphatidylcholines. The infrared spectroscopic data also show that, in the low-temperature gel state, there are significant differences between the odd- and even-numbered isoacylphosphatidylcholines with respect to their hydrocarbon chain packing modes as well as to their head group and interfacial hydration states.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two types of hydrocarbon chain interdigitation in sphingomyelin bilayers

Vibrational Raman spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of synthetic DL-erythro-N-lignoceroylsphingosylphosphocholine [C(24):SPM], a racemic mixture of two highly asymmetric hydrocarbon chain length sphingomyelins. Raman spectral peak-height intensity ratios of vibrational transitions in the C-H stretching-mode region show that the C(24):SPM-H2O system undergoes two thermal phase transitions centered at 48.5 and 54.5 degrees C. Vibrational data for fully hydrated C(24):SPM are compared to those of highly asymmetric phosphatidylcholine dispersions. The Raman data are consistent with the plausible model that the lower temperature transition can be ascribed to the conversion of a mixed interdigitated gel state (gel II) to a partially interdigitated gel state (gel I) and that the higher temperature transition corresponds to a gel I----liquid-crystalline phase transition. The observation of a mixed interdigitated gel state (gel II) at temperatures below 48.5 degrees C implies that biological membranes may have lipid domains in which some of the lipid hydrocarbon chains penetrate completely across the entire hydrocarbon width of the lipid bilayer.     

Hydration properties of N-(alpha-hydroxyacyl)-sphingosine: X-ray powder diffraction and FT-Raman spectroscopic studies

The thermotropic properties of N-(alpha-hydroxyacyl)-sphingosine (CER[AS]) in dry and hydrated state were studied by means of X-ray powder diffraction and FT-Raman spectroscopy. The polymorphic states of the CER[AS]/water mixture (lamellar crystalline, lamellar hexagonal gel, liquid crystalline) depend on the thermal pre-treatment of the sample. Only by heating the CER[AS]/water mixture above the melting chain transition can the system be hydrated. At room temperature, both dry and hydrated states form lamellar structures, which differ in their repeat distance and packing of hydrocarbon chains. Above the melting chain transition, hydrated CER[AS] forms a liquid crystalline hexagonal phase, whereas anhydrous CER[AS] forms an isotropic liquid phase. The various phases of hydrated CER[AS] are distinguished on the basis of the corresponding Raman spectra.     

The effect of the phase transition on the hydration and electrical conductivity of phospholipids.

Adsorption isotherms for various saturated phosphatidylcholines have been obtained. Lipids above and below their phase transition temperature differ only in the amount of water adsorbed and not in the nature of their adsorption isotherms. Cholesterol has an effect similar to that of increasing unsaturation in the hydrocarbon chains. Decreasing the length of the hydrocarbon chains for lipids below their phase transition temperature has no effect on the isotherms. If the chain length is short enough so that the lipids are above their transition temperature, however, a large increase in water adsorption occurs. All of the phospholipids exhibit a rapid increase of electrical conductivity for a few water molecules adsorbed per lipid molecule. All of the phospholipids show a saturation in conductivity at greater amounts of adsorbed water; the shape of the saturation region depends on whether the lipids are above or below their phase transition temperature. The activation energy for the electrical conductivity process depends on whether the hydrated lipids are in the "liquid-like" of the crystalline state, being lower for phospholipids in the liquid-like state. If the lipids are hydrated above their phase transition temperatures, their activation energies are lower than if they are hydrated below the transition temperature. Cholesterol lowers the activation energy. The phosphatidylcholines can be characterized by different activation energies, depending both upon their physical state and the presence of unsaturation in their hydrocarbon chains.     

Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol

The effect of temperature on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC), with and without addition of cholesterol, were studied using differential scanning calorimetry, Fourier transformed infrared spectroscopy, atomic force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temperature regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was observed for different POPC/ceramide mixtures (up to approximately 25 mol % ceramide). This observed phase coexistence scenario is in contrast to that reported previously for this mixture, where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addition of cholesterol to the POPC/ceramide mixtures shows a gradual transition from a gel/fluid to gel/liquid-ordered phase coexistence scenario as indicated by the different experimental techniques used in our experiments. This last result suggests the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixtures studied in contrast to that observed at similar molar concentrations with other ceramide-base-containing lipid mixtures (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Additionally, we observe a critical cholesterol concentration in the ternary mixtures that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.     

A synchrotron X-ray diffraction characterization of the structure of complexes formed between sphingomyelin and cerebroside

Specific lipid-lipid interactions are believed to be responsible for lateral domain formation in the lipid bilayer matrix of cell membranes. The miscibility of glucocerebroside and sphingomyelin extracted from biological tissues has been examined by synchrotron X-ray powder diffraction methods. Fully hydrated binary mixtures of egg-sphingomyelin codispersed with glucosylceramide rich in saturated C22 and C24 N-acyl fatty acids were subjected to heating scans between 20 and 90 °C at 2 °C·min(-1). X-ray scattering intensity profiles were recorded at 1 °C intervals simultaneously in both small-angle and wide-angle scattering regions. A gel phase characterized by a single symmetric peak in the wide-angle scattering region was transformed in all mixtures examined to a fluid phase at about 40 °C, similar to dispersions of pure egg-sphingomyelin. A coexisting lamellar structure was identified at temperatures up to about 75 °C which was characterized by a broad Bragg reflection. The scattering intensity of this structure increased relative to the structure assigned as bilayers of pure sphingomyelin with increasing proportions of glucosylceramide in the mixture. The relationship between the scattering intensities of the two peaks and the relative mass fractions of the two lipids showed that the bilayers assigned to a glucosylceramide-rich structure were composed of sphingomyelin and glucosylceramide in molar ratios of 1 : 1 and 2 : 1, respectively, at temperatures below and above the order-disorder phase transition temperature of the sphingomyelin (40 °C).     

Calorimetric and spectroscopic studies of the thermotropic phase behavior of lipid bilayer model membranes composed of a homologous series of linear saturated phosphatidylserines

The thermotropic phase behavior of lipid bilayer model membranes composed of the even-numbered, N-saturated 1,2-diacyl phosphatidylserines was studied by differential scanning calorimetry and by Fourier-transform infrared and (31)P-nuclear magnetic resonance spectroscopy. At pH 7.0, 0.1 M NaCl and in the absence of divalent cations, aqueous dispersions of these lipids, which have not been incubated at low temperature, exhibit a single calorimetrically detectable phase transition that is fully reversible, highly cooperative, and relatively energetic, and the transition temperatures and enthalpies increase progressively with increases in hydrocarbon chain length. Our spectroscopic observations confirm that this thermal event is a lamellar gel (L(beta))-to-lamellar liquid crystalline (L(alpha)) phase transition. However, after low temperature incubation, the L(beta)/L(alpha) phase transition of dilauroyl phosphatidylserine is replaced by a higher temperature, more enthalpic, and less cooperative phase transition, and an additional lower temperature, less enthalpic, and less cooperative phase transition appears in the longer chain phosphatidylserines. Our spectroscopic results indicate that this change in thermotropic phase behavior when incubated at low temperatures results from the conversion of the L(beta) phase to a highly ordered lamellar crystalline (L(c)) phase. Upon heating, the L(c) phase of dilauroyl phosphatidylserine converts directly to the L(alpha) phase at a temperature slightly higher than that of its original L(beta)/L(alpha) phase transition. Calorimetrically, this process is manifested by a less cooperative but considerably more energetic, higher-temperature phase transition, which replaces the weaker L(beta)/L(alpha) phase transition alluded to above. However, with the longer chain compounds, the L(c) phase first converts to the L(beta) phase at temperatures some 10-25 degrees C below that at which the L(beta) phase converts to the L(alpha) phase. Our results also suggest that shorter chain homologues form L(c) phases that are structurally related to, but more ordered than, those formed by the longer chain homologues, but that these L(c) phases are less ordered than those formed by other phospholipids. These studies also suggest that polar/apolar interfaces of the phosphatidylserine bilayers are more hydrated than those of other glycerolipid bilayers, possibly because of interactions between the polar headgroup and carbonyl groups of the fatty acyl chains.