Stability and fusion of lipid vesicles containing headgroup-modified analogues of phosphatidylethanolamine

We have used lipid mixing, contents mixing and contents-leakage assays to characterize the divalent cation-mediated interactions of vesicles composed of various headgroup-modified analogues of phosphatidylethanolamine, PE (N- and C-2-alkylated derivatives, and analogues with increased separations of the phosphoryl and amino groups) together with a low mole percentage of phosphatidylserine (PS). Vesicles containing different structural analogues of PE exhibit marked differences, both in the threshold divalent cation concentrations that are required to initiate vesicle-vesicle interactions and in the rates of contents mixing and leakage observed at suprathresholds divalent cation concentrations. The efficiencies of divalent cation-promoted contents leakage, and to a slightly lesser extent those of contents mixing, for PS/PE (analogue) vesicles show a marked inverse correlation with the lamellar-to-hexagonal II transition temperature (TH) of the PE (analogue) component. However, the destabilization kinetics for such vesicles show no abrupt changes over the temperature range around the equilibrium TH value measured for the vesicle lipids. Vesicles combining PS with different PE analogues exhibit divalent cation thresholds for aggregation that are not correlated with the TH values of the PE (analogue) components but appear instead to be correlated with the equilibrium interbilayer separations measured in multilamellar dispersions of these species. We have identified headgroup-modified analogues of PE that can be used to prepare vesicles that fuse more rapidly under a given set of conditions, or that show a bette ratio of fusion-to-contents-leakage rates, than do PE-containing vesicles. These results may be useful both for understanding better the bases for the high fusion-supporting ability of PE and for the preparation of lipid vesicles 'tailored' for particular practical applications.     

Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles

The interactions of unilamellar vesicles containing phosphatidylcholine (PC) and phosphatidic acid (PA) in the presence of calcium and magnesium were examined by fluorometric assays of vesicle lipid mixing, contents mixing, and contents leakage and by spray-freezing freeze-fracture electron microscopy. These results were correlated with calorimetric and fluorometric measurements of divalent cation induced lateral segregation of lipids in these vesicles under comparable conditions. PA-PC vesicles in the presence of calcium show a rapid but limited intermixing of vesicle lipids and contents, the extent of which increases as the vesicle size decreases or the PA content increases. Calcium produces massive aggregation and efficient mixing of the contents of vesicles containing high proportions of dioleoyl-PA or egg PA, but vesicle coalescence in the latter case is followed rapidly by vesicle collapse and massive leakage of contents. The effects of magnesium are similar for vesicles of very high PA content. However, in the presence of magnesium, vesicles containing lower amounts of PA exhibit "hemifusion", a mode of interaction in which vesicles aggregate and mix approximately 50% of their lipids, apparently representing the lipids of the outer monolayer of each vesicle, without significant mixing of vesicle contents or collapse of the vesicles. Fluorometric measurements of lipid lateral segregation demonstrate that lateral redistribution of lipids in PA-PC vesicles begins at submillimolar concentrations of divalent cations and shows no abrupt change at the "threshold" divalent cation concentration, above which coalescence of vesicles is observed. By correlating calorimetric and fluorometric measurements of lipid lateral segregation and mixing of vesicle components, we can demonstrate that lipid segregation is at least strongly correlated with calcium-promoted coalescence of PA-PC vesicles and is essential to the magnesium-promoted interactions of vesicles of low PA contents.     

Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.     

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR(2), a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

PlcHR(2) is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR(2) when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR(2) cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR(2) on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR(2)-induced liposome aggregation, as long as Ca(2+) is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis相似文献   

Docking and fast fusion of synaptobrevin vesicles depends on the lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane

The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in ∼20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., of 10 and ∼20 ms, respectively.     

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements.

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR2, a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

PlcHR₂ is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR₂ when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR₂ cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR₂ on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR₂-induced liposome aggregation, as long as Ca²⁺ is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis < vesicle aggregation < total lipid mixing < inner lipid mixing < contents mixing. Concomitant measurements of the threshold diacylglyceride + ceramide concentrations in the bilayer show that late events, e.g. lipid mixing, require a higher concentration of PlcHR₂ products than early ones, e.g. aggregation. When the above results are examined in the context of the membrane effects of other phospholipid phosphocholine hydrolases it can be concluded that aggregation is necessary, but not sufficient for membrane fusion to occur, that diacylglycerol is far more fusogenic than ceramide, and that vesicle membrane permeabilization occurs independently from vesicle fusion.     

Ca2+-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles

The effect of cholesterol on the Ca2+-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca2+ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c11, and of the fusion reaction itself, f11. In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c11 and f11 increase steeply with the Ca2+ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c11 decreases, while the rate of the actual fusion reaction, f11, increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.     

Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.     

Changes during hibernation in different phospholipid and free and esterified cholesterol serum levels in black bears

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P < 0.05). The percentage increase during hibernation was more in free cholesterol (57%) than in esterified cholesterol (27%). Analysis of subclasses of serum phospholipids showed that choline containing phospholipids, i.e., sphingomyelin (SPG) (14%) and phosphatidylcholine (PC) (76%), are the major phospholipids in the serum of bear. The minor phospholipids included 8% of phosphatidylserine (PS) + phosphatidylinositol (PI), while phosphatidylethanolamine (PE) was only 2% of the total phospholipids. A comparison of phospholipid subclasses showed that PC, PS + PI and SPG were significantly increased, while PE was significantly decreased (P < 0.05) in the hibernating state as compared with the active state in black bears. These results suggest that the catabolism of phospholipids and cholesterol is decreased during hibernation in black bears, leading to their increased levels in the hibernating state as compared with the active state. In summary, our results indicate that serum cholesterol and phospholipid fractions (except PE) are increased during hibernation in bears. It is proposed that the increase of these lipids may be due to the altered metabolism of lipoproteins that are responsible for the clearance of the lipids.     

Interaction of wheat alpha-thionin with large unilamellar vesicles.

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.     

Influence of gp41 fusion peptide on the kinetics of poly(ethylene glycol)-mediated model membrane fusion

The fusion peptide of the HIV fusion protein gp41 is required for viral fusion and entry into a host cell, but it is unclear whether this 23-residue peptide can fuse model membranes. We address this question for model membrane vesicles in the presence and absence of aggregating concentrations of poly(ethylene glycol) (PEG). PEG had no effect on the physical properties of peptide bound to membranes or free in solution. We tested for fusion of both highly curved and uncurved PC/PE/SM/CH (35:30:15:20 mol %) vesicles and highly curved PC/PE/CH (1:1:1) vesicles treated with peptide in the presence and absence of PEG. Fusion was never observed in the absence of PEG, although high peptide concentrations led to aggregation and rupture, especially in unstable PC/PE/CH (1:1:1) vesicles. When 5 wt % PEG was present to aggregate vesicles, peptide enhanced the rate of lipid mixing between curved PC/PE/SM/CH vesicles in proportion to the peptide concentration, with this effect leveling off at peptide/lipid (P/L) ratios approximately 1:200. Peptide produced an even larger effect on the rate of contents mixing but inhibited contents mixing at P/L ratios >1:200. No fusion enhancement was seen with uncurved vesicles. The rate of fusion was also enhanced by the presence of hexadecane, and peptide-induced rate enhancement was not observed in the presence of hexadecane. We conclude that gp41 fusion peptide does not induce vesicle fusion at subrupturing concentrations but can enhance fusion between highly curved vesicles induced to fuse by PEG. The different effects of peptide on the rates of lipid mixing and fusion pore formation suggest that, while gp41 fusion peptide does affect hemifusion, it mainly affects pore formation.     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

Modeling degranulation with liposomes: Effect of lipid composition on membrane fusion

Degranulation involves the regulated fusion of granule membrane with plasma membrane. To study the role of lipid composition in degranulation, large unilamellar vesicles (LUVs) of increasing complexity in lipid compositions were constructed and tested for Ca²⁺-mediated lipid and contents mixing. Lipid-mixing rates of LUVs composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were strongly decreased by the addition of either phosphatidylcholine (PC) or sphingomyelin (SM), while phosphatidylinositol (PI) had little effect. Complex LUVs of PCPESMPIPS (2427201613, designed to emulate neutrophil plasma membranes) also showed very low rates of both lipid mixing and contents mixing. The addition of cholesterol significantly lowered the Ca²⁺ threshold for contents mixing and increased the maximum rates of both lipid and contents mixing in a dose-dependent manner. Membrane remodeling, which occurs in neutrophil plasma membranes upon stimulation, was simulated by incorporating low levels of phosphatidic acid (PA) or a diacylglycerol (DAG) into complex LUVs containing 50% cholesterol. The addition of PA both lowered the Ca²⁺ threshold and increased the rate of contents mixing in a dose-dependent manner, while the DAG had no significant effect. The interaction of dissimilar LUVs was also examined. Contents-mixing rates of LUVs of two different cholesterol contents were intermediate between the rates observed for the LUVs of identical composition. Thus, cholesterol needed to be present in only one fusing partner to enhance fusion. However, for PA to stimulate fusion, it had to be present in both sets of LUVs. These results suggest that the rate of degranulation may be increased by a rise in the cholesterol level of either the inner face of the plasma membrane or the outer face of the granule membrane. Further, the production of PA can promote fusion, and hence degranulation, whereas the subsequent conversion of PA to DAG may reverse this promotional effect.Abbreviations ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - DiC₈ 1,2-dioctanoyl-sn-glycerol - DPX p-xylene-bis-pyridinium bromide - LUV large unilamellar vesicle - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - R18 octadecyl rhodamine - SM sphingomyelin     

Changes in phospholipids and free fatty acids in the brains of mice preconditioned by hypoxia.

Changes in the composition and contents of phospholipids and free fatty acids were observed and compared in three groups: (A) unpreconditoned normal controls, (B) exposure to 1 run of hypoxia and (C) exposure to 4 runs of hypoxia. In group B, the content of phosphatidyl ethanolamine (PE), phosphatidyl serine (PS) and free fatty acids (FFAs) increased significantly and the content of phosphatidyl choline (PC) and sphingomyelin (SM) decreased significantly. While in group C the content of PE, PS, PC and FFAs changed significantly when compared with that of group B, all phospholipid (except SM) and FFA contents tended to decrease to the level of group A. No new FFA was seen in the brain homogenates in any of the three groups. These results suggest that the changes in the content of mouse brain phospholipids and FFAs may be adaptive and involved in the animals' tolerance to hypoxia.     

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.     

The rate of fusion of phospholipid vesicles and the role of bilayer curvature

Kinetics of Ca²⁺-induced fusion of phosphatidylserine vesicles is studiied for lipid concentrations varying from 1 μM to 100 μM. Fusion is monitored by mixing of aqueous vesicle contents and by explicitly accounting for leakage. The analysis provides separately rates of aggregation and fusion. The rate of fusion per se decreases steeply with vesicle size.     

Phosphatidylserine Inhibits and Calcium Promotes Model Membrane Fusion

PEG-mediated fusion of SUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, cholesterol, and dioleoylphosphatidylserine was examined to investigate the effects of PS on the fusion mechanism. Lipid mixing, content mixing, and content leakage measurements were carried out with vesicles containing from 0 to 8 mol % PS and similar amounts of phosphatidylglycerol. Fitting these time courses globally to a 3-state (aggregate, intermediate, pore) sequential model established rate constants for each step and probabilities of lipid mixing, content mixing, and leakage in each state. Charged lipids inhibited both the rates of intermediate and pore formation as well as the extents of lipid and contents mixing, although electrostatics were not solely responsible for inhibition. Ca²⁺ counteracted this inhibition and increased the extent of fusion in the presence of PS to well beyond that seen in the absence of charged lipids. The effects of both PS and Ca²⁺ could be interpreted in terms of a previous proposal for the nature of lipid fluctuations that account for transition states for the two steps of the fusion process examined. The results suggest a more significant role for Ca²⁺-lipid interactions than is acknowledged in current thinking about cell membrane fusion.Abbreviations used: SUVs, small unilamellar vesicles; DOPC, 1,2-dioleoyl-3-sn-phosphatidylcholine; DOPE, 1,2-dioleoyl-3-sn-phosphatidylethanolamine; SM, sphingomyelin (bovine brain); CH, Cholesterol; DOPS, 1,2-dioleoyl-3-sn-phosphatidylserine; PS, phosphatidylserine; DOPG, 1,2-dioleoyl-3-sn-phosphatidylglycerol; PG, phosphatidylglycerol; TES, N- tris(hydroxymethyl)methyl}2-2-aminoethane sulfonic acid; PEG, poly(ethylene glycol); CM, contents mixing; LM, lipid mixing