Synthesis of a new phosphatidylserine spin-label and calcium-induced lateral phase separation in phosphatidylserine-phosphatidylcholine membranes.

A new phosphatidylserine spin label with nitroxide stearate attached at the 2 position has been synthesized by the reaction of spin-labeled CDP-diglyceride with L-serine under the catalytic action of phosphatidylserine synthetase. Some structural properties of pure phosphatidylserine (PS) and binary PS-phosphatidylcholine (PC) membranes were studied with the spin label. PS membrane became solidified on lowering solution pH, 50% solidification being attained at pH 3.5. The membrane was also solidified by addition of Ca-2+. The effect of Ba-2+,Sr-2+, and Mg-2+ was smaller than that of Ca-2+. The calcium-induced lateral phase separation in the binary membrane was studied from the side of the calcium-receiving lipid. The results confirmed and extended our previous conclusion drawn with PC spin label. The phase diagram of the binary membrane in the presence of Ca-2+ was determined. Not all PS molecules were aggregated to form the solid patches but some remained dissolved in the fluid PC matrix. The fluid PS fraction was larger for the membranes containing more PC. The membrane with 10% PS still had a significant fraction of solid phase. The rate of calcium-induced aggregation was greatly dependent on the PS content. The aggregation was almost complete within 5 min in the membrane containing 67% PS, while it was still proceeding after several hours in the membrane with 20% PS. The rate-limiting step was suggested to be in the formation of "stable" nuclei consisting of larger aggregates. The possible biological significance of the ionotropic phase separation was discussed whereby a transient density fluctuation was emphasized.     

Effect of membrane phase transition on long-time calcium-induced fusion of phosphatidylserine vesicles

Dynamic light scattering has been used to study the temperature dependence of the extent of long-time calcium-induced fusion of sonicated vesicles composed of various natural and synthetic phosphatidylserine with different acyl chains. The vesicles of each composition are found to exhibit a peak temperature in the vicinity of which the extent of fusion shows a distinct maximum. The fusion peak temperature increases as the bilayer gel-to-liquid-crystal phase transition temperature increases. The results suggest a role played by membrane fluidity in determining fusion efficiency.     

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca²⁺-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca²⁺ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca²⁺ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca²⁺ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca²⁺ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca²⁺, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca²⁺ concentration, and identical to that with pure phosphatidylserine in excess Ca²⁺. The results imply that Ca²⁺-induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.     

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles. Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca2+-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca2+ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca2+ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca2+ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca2+ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca2+, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca2+ concentration, and identical to that with pure phosphatidylserine in excess Ca2+. The results imply that Ca2+- induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.     

Dynamic morphology of calcium-induced interactions between phosphatidylserine vesicles.

Structural changes in phosphatidylserine vesicles exposed to calcium chloride for various times have been observed by means of video-enhanced light microscopy and freeze-fracture electron microscopy. Large flat double-bilayer diaphragms form at the contacts between aggregated vesicles within milliseconds. Bilayers at and outside of diaphragms rupture and allow vesicles to collapse completely by flattening against each other within seconds. Collapse through intermediate states to a stable multilamellar phase is complete within minutes. The Ca-induced attraction energy and the resultant flattening at contacts between vesicles is far beyond that needed to stress bilayers to the point of rupture. Although the destabilizing response to this stress is preferential to the diaphragm region, 40% of adhering pairs rupture outside of the diaphragm region rather than fuse with each other. In this respect the mechanism of fusion between these vesicles may be fundamentally different from the controlled fusion process in cells.     

Osmotic stress induces a phase transition from interdigitated gel phase to bilayer gel phase in multilamellar vesicles of dihexadecylphosphatidylcholine

We have investigated the effects of poly(ethylene glycol) (PEG) on the structure and phase behavior of multilamellar vesicles of dihexadecylphosphatidylcholine (DHPC-MLVs) using an X-ray diffraction method. At low concentrations of PEG-6K (MW = 7500), DHPC-MLVs were in an interdigitated gel (L(beta)I) phase, a gel phase with interdigitated hydrocarbon chains. At around 24% (w/v) PEG 6K, a phase transition from the L(beta)I phase to a bilayer gel phase occurred in the DHPC-MLVs, and above this concentration, they were in a bilayer gel phase. On the other hand, ethylene glycol (EG), the monomer of PEG, did not induce this phase transition in the DHPC-MLVs. A mechanism of this phase transition is proposed and discussed; a decrease in the repulsive interaction between the head groups of the phospholipids in the bilayer gel phase with an increase in PEG concentration, which is due to a decrease in the cross-sectional area of the head group region by osmotic stress, may be the main reason for this phase transition.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current.

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.     

Differential ability of cholesterol-enriched and gel phase domains to resist benzyl alcohol-induced fluidization in multilamellar lipid vesicles

Benzyl alcohol (BA) has a well-known fluidizing effect on both artificial and cellular membranes. BA is also likely to modulate the activities of certain membrane proteins by decreasing the membrane order. This phenomenon is presumably related to the ability of BA to interrupt interactions between membrane proteins and the surrounding lipids by fluidizing the lipid bilayer. The components of biological membranes are laterally diversified into transient assemblies of varying content and order, and many proteins are suggested to be activated or inactivated by their localization in or out of membrane domains displaying different physical phases. We studied the ability of BA to fluidize artificial bilayer membranes representing liquid-disordered, cholesterol-enriched and gel phases. Multilamellar vesicles were studied by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid, which display different phase partitioning. Domains of different degree of order and thermal stability showed varying abilities to resist fluidization by BA. In bilayers composed of mixtures of an unsaturated phosphatidylcholine, a saturated high melting temperature lipid (sphingomyelin or phosphatidylcholine) and cholesterol, BA fluidized and lowered the melting temperature of the ordered and gel phase domains. In general, cholesterol-enriched domains were more resistant to BA than pure gel phase domains. In contrast, bilayers containing high melting temperature gel phase domains containing a ceramide or a galactosylceramide proved to be the most effective in resisting fluidization. The results of our study suggest that the ability of BA to affect the fluidity and lateral organization of the membranes was dependent on the characteristic features of the membrane compositions studied and related to the intermolecular cohesion in the domains.     

Kinetics of calcium-induced mixing of lipids and aqueous contents of large unilamellar phosphatidylserine vesicles

Calcium ion-induced fusion events in suspensions of large unilamellar phosphatidylserine (PS) liposomes were monitored by fluorescence methods. Mixing of vesicle contents was studied by measuring the increase in terbium emission intensity due to formation of a complex between Tb³⁺ ions and dipicolinic acid trapped in the liposomes. Lipid redistribution was determined with the aid of the resonance transfer of excitaton energy using dipalmitoylphosphatidylethanolamine labelled with the donor N-(7-nitro-2,1,3-benzoxadiazol-4-yl) or the acceptor tetramethylrhodamine at the free amino group. The two methods yielded significantly different results. While recombination of contents could not be detected at Ca²⁺ concentrations below 2.5 mM the threshold concentration for lipid mixing was 1 mM. For saturating Ca²⁺ concentrations (>5 mM Ca²⁺) initial rates were higher by almost an order of magnitude for lipid mixing than for recombination of liposome contents. These observations indicate that the observation of rapid lipid mixing phenomena does not allow one to draw conclusions as to the fate of the enclosed volumes.     

Thermal acoustic radiation from multilamellar vesicles in lipid phase transition

A new acoustical method for the investigation of lipid phase transition is introduced based on the measurement of the thermal acoustic radiation (TAR) inherent in lipids. The TAR of multilamellar vesicles from dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) was measured in the megahertz range and the variations in the radiation intensity during the lipid phase transition were recorded. Two types of variations are possible: if the temperature of the vesicles decreases (in the process of transition from the liquid crystalline state to the gel state) then the TAR intensity increases, and if the temperature increases (in the reverse transition) then the TAR intensity decreases. These effects are connected with an increase in the ultrasonic absorption in the vesicles under lipid phase transition. Basing on the results of the TAR investigation, a new theoretical estimate has been developed of the variation in the absorption coefficient during the lipid phase transition. In this estimate, the variation is equated to the ratio of the phase transition entropy to the gas constant.     

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.     

Crystallization of water in multilamellar vesicles

The two-step crystallization of water in multilamellar vesicles (MLVs) of phosphatidylcholines has been investigated. The main crystallization occurs near -15 degrees C and involves bulk water. Contrary to unilamellar vesicles, a sub-zero phase transition is observed for MLVs at -40 degrees C that corresponds to the crystallization of interstitial water, as proved by Fourier transform infrared absorption and differential scanning calorimetry (DSC) experiments. Furthermore, by means of the DSC method and, more specifically, using the enthalpy change values Delta H(sub) at the sub-zero transition, the number of water molecules per 1,2-dipalmitoylphosphatidylcholine (DPPC) molecule giving rise to this transition has been estimated for different H(2)O/DPPC molar ratios. The curve of the molecular fraction of water molecules involved in the sub-zero transition versus the H(2)O/DPPC molar ratio exhibits a maximum for H(2)O/DPPC equal to 27 (40% in mass of water) and tends towards zero for H(2)O/DPPC ratio values approaching that of the swelling limit of the membrane. A smaller enthalpy value of the sub-zero transition is found for 1-oleoyl-2-palmitoyl-3-phosphatidylcholine (OPPC) than for DPPC. This may be explained by the decrease of interstitial water's quantity when the lipid contains an unsaturated chain. When troxerutin, a hydrophilic drug, is added to the DPPC multilayers, the decrease of Delta H(sub) and melting enthalpy of bulk water is attributed to a decrease of the entropy of the liquid phase owing to the network of water molecules surrounding troxerutin molecules. In all cases, the experiments revealed that the sub-zero transition occurs only in the presence of excess water with respect to the swelling limit of membranes. This evidence could be, at least qualitatively, related to an increase of membrane pressure on interstitial water subsequent to bulk water crystallization.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).     

Generation of multilamellar and unilamellar phospholipid vesicles

Multilamellar and unilamellar vesicles can be generated by a variety of techniques which lead to systems with differing lamellarity, size, trapped volume and solute distribution. The straight-forward hydration of lipid to produce multilamellar vesicles (MLVs) results in systems which exhibit low trapped volumes and where solutes contained in the aqueous buffer are partially excluded from the MLV interior. Large trapped volumes and equilibrium solute distributions can be achieved by freeze-thawing or by ‘reverse phase’ procedures where the lipid is hydrated after being solubilized in organic solvent. Unilamellar vesicles can be produced directly from MLVs by extrusion or sonication or, alternatively, can be obtained by reverse phase or detergent removal procedures. The advantages and limitations of these techniques are discussed.     

Effect of oligomers of ethylene glycol on thermotropic phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles.

The effect of oligomers of ethylene glycol (EG) on thermotropic phase transitions of dipalmitoylglycerophosphatidylcholine multilamellar vesicles (DPPC-MLV) were investigated. Diethylene glycol (di-EG) had a biphasic effect on transition temperature, reducing pre-transition temperature (Tp) at low concentrations but increasing main transition temperature (Tm) and extinguishing pre-transition at high concentration. Results of the X-ray diffraction method and the excimer method indicated that di-EG induced interdigitated gel phase (L beta 1 phase) in the DPPC membranes at high concentration. Phase diagram of temperature-di-EG concentration for DPPC-MLV was determined by use of X-ray diffraction and differential scanning calorimetry, which was similar to that of temperature-EG concentration. The minimum concentration of di-EG where L beta 1 phase was induced was 42%(w/v), which was larger than that of EG (30%(w/v)). On the other hand, in the presence of triethylene glycol (tri-EG), Tm and Tp increased with an increased in tri-EG concentration, as well as poly(ethylene glycol). These differences, between the effects of di-EG and those of tri-EG, might be due to the differences of their sizes.     

Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.     

Fluorescence depolarization studies of the phase transition in multilamellar phospholipid vesicles exposed to 1.0-GHz microwave radiation

The phase transition in multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles was studied during exposure to continuous wave 1.0-GHz microwave radiation. Fluorescence depolarization measurements using a lipid-seeking molecular probe, diphenylhexatriene (DPH). were performed as a function of temperature. Semilog plots of microviscosity versus temperature illustrate the phase transition which shows a 5°C shift when the vesicles are treated with chloroform as a positive control. No shift of the phase transition was found during exposure to microwave radiation at specific absorption rates between 1 and 30 W/kg. Samples were exposed in a rectangular transmission line (TEM cell), and specific absorption rates were calculated from electrical measurements of incident, reflected, and transmitted power. Samples were exposed to increasing intensities of radiation, while the temperature was maintained at either 23.5 or 25.5 °C; these temperatures represented the two ends of the phase transition region for these vesicles. No statistically significant difference was found between exposed and control samples. These results are in contrast to those of others using laser Raman spectroscopy to measure the phase transition in similar multilamellar vesicles exposed to microwave radiation.