Effect of streptolysin S on liposomes Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [¹⁴C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine > C18 : 1 phosphatidylcholine > C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0°C and 4°C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23°C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Effect of streptolysin S on liposomes. Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [14C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine greater than C18: I phosphatidylcholine greater than C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0 degrees C and 4 degrees C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23 degrees C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Partitioning of Tetrachlorophenol into Lipid Bilayers and Sarcoplasmic Reticulum: Effect of Length of Acyl Chains,Carbonyl Group of Lipids and Biomembrane Structure

We report results of a partitioning study of 2,3,4,6-tetrachlorophenol (TeCP). In the study we explored (1) the effect of the length of acyl chains of lipids (C16:1 – C24:1) and alkanes (C6–C16), (2) the role of the carbonyl group of lipids, and (3) the effect of molecular structure of the sarcoplasmic reticulum membrane on TeCP partitioning. Mole fraction partition coefficients have been measured using equilibrium dialysis for un-ionized (HA), and ionized (A) species, Kp_x^HA, Kp_x^A. Their values are concentration-dependent. Partition coefficients were analyzed in terms of a model that accounts for saturation of membrane associated with the finite area of partition site, and electrostatic interactions of (A-) species with charged membrane. Limiting values of partition coefficients, corresponding to infinite dilution of solute, Kp_x0^HA, Kp_x0^A were obtained. Kp_x0^HA and Kp_x0^A measure the strength of solute-membrane interactions. Studies were done with single-layered vesicles of lipids with variable chain length: 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (C16:1), 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1), 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22:1), and 1 ,2-dinervonoyl-sn-glycero-3-phosphocholine (C24:1), and egg-PC. Kp_x0 for transfer of TeCP from water into lipid membranes was found to be independent of the length of acyl chains, whereas Kp_x0 for transfer from water into alkanes increased with the length of alkane. The effect of the carbonyl CO group of lipids on partitioning was measured using 1,2-di-o-octadecenyl-sn-glycero-3-phosphocholine (CO absent) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (CO present) liposomes. Carbonyl groups, known to change dipolar potential, had no effect on partitioning. Partition coefficients of un-ionized and ionized forms of TeCP were invariant to the presence of proteins and other membrane components of sarcoplasmic reticulum (SR) membrane.     

Effects of phosphatidylserine on membrane incorporation and surface protection properties of exchangeable poly(ethylene glycol)-conjugated lipids

Liposomes containing the acidic phospholipid phosphatidylserine (PS) have been shown to avidly interact with proteins involved in blood coagulation and complement activation. Membranes with PS were therefore used to assess the shielding properties of poly(ethylene glycol 2000)-derivatized phosphatidylethanolamine (PE-PEG(2000)) with various acyl chain lengths on membranes containing reactive lipids. The desorption of PE-PEG(2000) from PS containing liposomes was studied using an in vitro assay which involved the transfer of PE-PEG(2000) into multilamellar vesicles, and the reactivity of PS containing liposomes was monitored by quantifying interactions with blood coagulation proteins. The percent inhibition of clotting activity of PS liposomes was dependent on the PE-PEG(2000) content. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG(2000) which transferred out slowly from PS liposomes was able to abolish >80% of clotting activity of PS liposomes at 15 mol%. This level of DSPE-PEG(2000) was also able to extend the mean residence time of PS liposomes from 0.2 h to 14 h. However, PE-PEG(2000) with shorter acyl chains such as 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-PEG(2000) were rapidly transferred out from PS liposomes, which resulted in a 73% decrease in clotting activity inhibition and 45% of administered intravenously liposomes were removed from the blood within 15 min after injection. Thus, PS facilitates the desorption of PE-PEG(2000) from PS containing liposomes, thereby providing additional control of PEG release rates from membrane surfaces. These results suggest that membrane reactivity can be selectively regulated by surface grafted PEGs coupled to phosphatidylethanolamine of an appropriate acyl chain length.     

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG(2000)-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG(2000)), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)). Transfer of DSPE-PEG(2000) to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG(2000) was transferred efficiently even at 4 degrees C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG(2000) to become evenly distributed between the two liposome populations (T(EQ)) at 4 degrees C and 37 degrees C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.     

Liposomes alter thermal phase behavior and composition of red blood cell membranes

Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.     

Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals the stoichiometry at which free cholesterol clusters disappear.

In this study, we have used cholesterol oxidase as a probe to study cholesterol/phospholipid interactions in mixed monolayers at the air/water interface. Mixed monolayers, containing a single phospholipid class and cholesterol at differing cholesterol/phospholipid molar ratios, were exposed to cholesterol oxidase at a lateral surface pressure of 20 mN/m (at 22 degrees C). At equimolar ratios of cholesterol to phospholipid, the average rate of cholesterol oxidation was fastest in unsaturated phosphatidylcholine mixed monolayers (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and egg yolk phosphatidylcholine), intermediate in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and slowest in sphingomyelin monolayers (egg yolk or bovine brain sphingomyelin). The average oxidation rate in mixed monolayers was not exclusively a function of monolayer packing density, since egg yolk and bovine brain sphingomyelin mixed monolayers occupied similar mean molecular areas even though the measured average oxidation rate was different with these two phospholipids. This suggests that the phospholipid acyl chain composition influenced the oxidation rate. The importance of the phospholipid acyl chain length on influencing the average oxidation rate was further examined in defined phosphatidylcholine mixed monolayers. The average oxidation rate decreased linearly with increasing acyl chain lengths (from di-8:0 to di-18:0). When the average oxidation rate was examined as a function of the cholesterol to phospholipid (C/PL) molar ratio in the monolayer, the otherwise linear function displayed a clear break at a 1:1 stoichiometry with phosphatidylcholine mixed monolayers, and at a 2:1 C/PL stoichiometry with sphingomyelin mixed monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. II. Effect of membrane lipid composition in a liposome system

In the first paper of this series, it was shown that a toxin from the sea anemone Stoichactis helianthus increased the permeability of black lipid membranes due to transmembrane channel formation. In the present study, we have used liposomes to examine the reactivity of the toxin with different phospholipids. Membrane damage was assessed by measuring the release of ⁸⁶Rb⁺ and ¹⁴C-labeled membrane lipid. For the different lipids, the rank order of marker release was: sphingomyelin > C18: 2 phosphatidylcholine > C18: 1 phosphatidylcholine > C18: 0 phosphatidylcholine > C16: 0 phosphatidylcholine = C14: 0 phosphatidylcholine. In C14: 0 and C16: 0 phosphatidylcholine liposomes there was no ¹⁴C-labeled lipid release and only 13 to 16% ⁸⁶Rb⁺ release which corresponds to the ⁸⁶Rb⁺ content in the outermost aqueous shell of multilamellar liposomes. This indicates that membrane damage was limited to the outermost bilayer. In liposomes prepared with the other lipids, the extent of release of both markers increased proportionately with the length and the degree of unsaturation of the lipids' acyl side chains. Sphingomyelin liposomes were the most susceptible with 47% of the ¹⁴C-labeled lipid marker and 90% of the ⁸⁶Rb⁺ marker being released. The large extent of ¹⁴C-labeled lipid release is attributed to a detergent-like activity of the toxin which presumably is due to the amphipathic nature of the protein. Thus, the toxin can inflict membrane damage in two ways: (1) channel formation, and (2) detergent action. The importance of one mechanism or the other apparently varies depending on membrane structure and lipid composition.     

Amphotericin B-induced ion flux is markedly attenuated in phosphatidylglycerol membrane as evidenced by a newly devised fluorometric method

The effect of phospholipid head group on the membrane-permeabilizing activity of amphotericin B (AmB) was examined using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) liposomes. The activity of AmB was evaluated as K⁺ influx measured as pH change inside liposomes by fluorescent measurements of 2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein (BCECF). AmB showed prominent permeability in POPC liposomes, whereas hardly inducing ion flux in POPG membrane. POPC added to POPG liposomes as a minor constituent markedly enhanced membrane permeability, indicating the importance of a phosphonocholine group of PC for the drug’s activity.     

Effect of fatty acyl domain of phospholipids on the membrane-channel formation of Staphylococcus aureus alpha-toxin in liposome membrane.

By use of carboxyfluorescein-loaded multilamellar liposomes prepared from synthetic phosphatidylcholine (PC) or sphingomyelin and cholesterol in a molar ratio of 1:1, we studied whether or not fatty acyl domain of the phospholipids affects the membrane-damaging action (or channel formation) of Staphylococcus aureus alpha-toxin on the phospholipid-cholesterol membranes. Our data indicated: (1) that toxin-induced carboxyfluorescein-leakage from the liposomes composed of saturated fatty acyl residue-carrying PC and cholesterol was decreased with increasing chain length of the acyl residues between 12 and 18 carbon atoms, although toxin-binding to the liposomes was not significantly affected by the length of fatty acyl residue; (2) that unsaturated fatty acyl residue in PC or sphingomyelin molecule conferred higher sensitivity to alpha-toxin on the phospholipid-cholesterol liposomes, compared with saturated fatty acyl residues; and (3) that hexamerization of alpha-toxin, estimated by SDS-polyacrylamide gel electrophoresis, occurred more efficiently on the liposomes composed of PC with shorter fatty acyl chain or unsaturated fatty acyl chain. Thus, hydrophobic domain of the phospholipids influences membrane-channel formation of alpha-toxin in the phospholipid-cholesterol membrane, perhaps by modulating packing of phospholipid, cholesterol and the toxin in membrane.     

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks

Apolipoprotein mediated formation of nanodisks was studied in detail using apolipophorin III (apoLp-III), thereby providing insight in apolipoprotein-lipid binding interactions. The spontaneous solubilization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles occured only in a very narrow temperature range at the gel-liquid-crystalline phase transition temperature, exhibiting a net exothermic interaction based on isothermal titration calorimetry analysis. The resulting nanodisks were protected from proteolysis by trypsin, endoproteinase Glu-C, chymotrypsin and elastase. DMPC solubilization and the simultaneous formation of nanodisks were promoted by increasing the vesicle diameter, protein to lipid ratio and concentration. Inclusion of cholesterol in DMPC dramatically enhanced the rate of nanodisk formation, presumably by stabilization of lattice defects which form the main insertion sites for apolipoprotein α-helices. The presence of fully saturated acyl chains with a length of 13 or 14 carbons in phosphatidylcholine allowed the spontaneous vesicle solubilization upon apolipoprotein addition. Nanodisks with C13:0-phosphatidylcholine were significantly smaller with a diameter of 11.7 ± 3.1nm compared to 18.5 ± 5.6 nm for DMPC nanodisks determined by transmission electron microscopy. Nanodisk formation was not observed when the phosphatidylcholine vesicles contained acyl chains of 15 or 16 carbons. However, using very high concentrations of lipid and protein (>10mg/ml), 1,2,-dipalmitoyl-sn-glycero-3-phosphocholine nanodisks could be produced spontaneously although the efficiency remained low.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Influence of phospholipid chain length on verotoxin/globotriaosyl ceramide binding in model membranes: comparison of a supported bilayer film and liposomes

The importance of the surrounding lipid environment on the availability of glycolipid carbohydrate for ligand binding was demonstrated by studying the influence of phosphatidylcholine fatty acid chain length on binding of verotoxins (VT1 and VT2c) to their specific cell surface receptor, globotriaosylceramide (Gb₃) in the presence of auxiliary lipids both in a microtitre plate surface bilayer film and in a liposome membrane model system. In the microtitre assay, both VT1 and VT2c binding to Gb₃ was increased as a function of decreasing PC acyl chain length likely resulting in increased Gb₃ exposure. In the liposome assay VT1 binding was similarly modulated, however the effect on VT2c binding was more complex and did not follow a simple function of increased carbohydrate exposure. Earlier work established that C22:1 and C18:1Gb₃ fatty acid homologues were the preferred Gb₃ receptor isoforms in the microtitre assay for VT1 and VT2c respectively. This selectivity was maintained in C16PC containing liposomes, but in C14PC liposomes, binding to C22:1Gb₃ (but not C18:1Gb₃) was elevated such that this Gb₃ species now became the preferred receptor for both toxins. This change in verotoxin/Gb₃ homologue binding selectivity in the presence of C14PC did not occur in the microtitre bilayer format. These results are consistent with our proposal that these toxins recognize different epitopes on the Gb₃ oligosaccharide. We infer that relative availability of these epitopes for toxin binding in an artificial bilayer is influenced not only by the exposure due to the discrepancy between the fatty acyl chain lengths of Gb₃ and PC, but by the physical mode of presentation of the bilayer structure. Such acyl chain length differences have a more marked effect in a supported bilayer film whereas only the largest discrepancies affect Gb₃ receptor function in liposomes. The basis of phospholipid modulation of glycolipid carbohydrate accessibility for receptor function is likely complex and will involve phase separation, gel/liquid crystalline transition, packing and lateral mobility within the bilayer, suggesting that such parameters should be considered in the assessment of glycolipid receptor function in cells.     

Changes in the lipid dynamics of liposomal membranes induced by poly(ethylene glycol): free volume alterations revealed by inter- and intramolecular excimer-forming phospholipid analogs.

Influence of osmotic shrinkage, swelling, and dehydration on large unilamellar liposomes (LUVs) of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) was investigated using the fluorescent lipid probes 1-palmitoyl-2-[10-(pyren-1-yl)]-decanoyl-sn-glycero-3-phosphocholi ne (PPDPC) and 1,2-bis[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC). Increasing concentrations of poly(ethylene glycol) (PEG, average molecular weight of 6000) producing osmotic gradients delta omega up to 250 mOsm/kg were first added to the outside of LUV labeled with 0.1 mol% of either of the above fluorescent phospholipids. The resulting osmotic shrinkage was accompanied by a progressive reduction in the lateral diffusion of the membrane-incorporated PPDPC, evident as a decrease in the rate of its intermolecular excimer formation. In contrast, under the same conditions the rate of intramolecular excimer formation by bisPDPC increased. Notably, signals opposite to those described above were observed for both of the fluorescent probes upon osmotic swelling of DOPC liposomes with encapsulated PEG. The lateral diffusion of PPDPC became progressively reduced upon membrane dehydration due to increasing concentrations of symmetrically distributed PEG (with equal polymer concentrations inside and outside of the liposomes) when neither shrinkage nor swelling occurs while enhanced excimer formation by bisPDPC was evident. The later results were interpreted in terms of osmotically induced changes in the hydration of lipids. In brief, the removal of water from the phospholipid hydration shell diminishes the effective size of the polar headgroup, which subsequently allows for an enhanced lateral packing of the phospholipid acyl chains. Our findings are readily compatible with membrane free volume Vf changes due to osmotic forces under three different kinds of stress (shrinkage, swelling, and dehydration) applied on the lipid bilayers.     

Structural aspects of pressure effects on infrared spectra of mixed-chain phosphatidylcholine assemblies in D2O

The barotropic behavior of D2O dispersions of 1-stearoyl-2-caproyl-sn-glycero-3-phosphocholine, C(18):C(10)PC, a highly asymmetric phospholipid in which the length of the fully extended acyl chain at the sn-1 position of the glycerol backbone is twice as long as that at the sn-2 position, has been investigated by high-pressure Fourier transform infrared spectroscopy. This asymmetric phosphatidylcholine bilayer at room temperature displays a pressure-induced phase transition corresponding to the liquid-crystalline----gel phase transition at 1.4 kbar. A conformational ordering of the lipid acyl chains is observed to take place abruptly at the transition pressure of 1.4 kbar. However, the lamellar lipid molecules and their acyl chains remain to be orientationally disordered in the gel phase until the applied pressure reaches 5.5 kbar. In the gel phase of fully hydrated C(18):C(10)PC, the asymmetric lipid molecules assemble into mixed interdigitated bilayers with perpendicular orientation of the zigzag planes among neighboring acyl chains. The role of excess water played in the interchain structure and the behavior of excess water and bound water under high pressure are also discussed.     

The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity

Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.     

Structure of polymerizable lipid bilayers. V. Synthesis, bilayer structure and properties of diacetylenic ether and ester lipids.

Four diacetylenic phosphatidylcholines (PC's) have been synthesized and the structures of bilayers of these lipids have been determined at low resolution by low-angle X-ray diffraction. The PC's all have 18-carbon chains but differ with respect to the ether/ester linkage at the sn-1 and sn-2 positions and the relative position of the diacetylene moiety: diester-PC (1): 1,2-bis(octadeca-4',6'-diynoyl)-sn-glycero-3-phosphocholine diester-PC (2): 1-(octadeca-4',6'-diynoyl)-2-(octadeca-5',7'-diynoy l)-sn- glycero-3-phosphocholine diester-PC (3): 1,2-bis(octadeca-8',10'-diynoyl)-sn-glycerol-3-phosphocholin e diether-PC (4): 1-O-(octadeca-4',6'-diynyl)-2-O-(octadeca-5",7"-din yl)-sn- glycero-3-phosphocholine Only (1) exhibits the typical bilayer profile, whereas (2), (3) and (4) show evidence of interdigitation and/or significant disorder. Only (1) polymerized effectively upon illumination with 254 nm light, turning deep blue in seconds, indicating the formation of long, well-ordered polydiacetylenic structures. Liposomes of these derivatives were tested for permeability by osmotic swelling. Polymerized liposomes of (1) underwent osmotic swelling with urea, glycerol, and acetamide more rapidly than did liposomes of stearoyl-oleoyl-PC, but the initial rates of osmotic swelling of polymerized liposomes of (1) were 3-10-times lower than those of unpolymerized liposomes of (1). Blue polymerized multilayer samples of (1) exhibited an irreversible thermochromic transition to red at approx. 40 degrees C. Differential scanning calorimetry with liposome suspensions of (1) revealed an endotherm at 28.3 degrees C with a transition enthalpy of 40 J/g. PC (1) is a potentially useful diacetylenic lipid which exhibits facile, complete polymerization and a bilayer thickness comparable to that of biomembrane lipids.     

Occurrence of glyceryl ethers in the phosphatidylcholine fraction of surfactant from dog lungs

The molecular species of ether-linked lipids in the phosphatidylcholine (PC) fraction of the pulmonary surfactant obtained from the lavage fluid of dog were characterized. A combination of base-catalyzed methanolysis, phospholipase C treatment, gas-liquid chromatography, and mass spectrometry procedures were applied. The phospholipid composition of the surfactant, obtained by phosphorus assay of lipids separated by silica gel G thin-layer chromatography (TLC), was: PC (75%), phosphatidylglycerol (10%), phosphatidylethanolamine (7%), plus small amounts of sphingomyelin, phosphatidylinositol, and phosphatidylserine. The major components of the PC were 1,2-diacylPC (95%), and 1-O-alkyl-2-acylPC (5%). No detectable amounts of 1-O-alkyl-1'-enyl-2-acylPC or di-alkyl-1-enylPC were observed. The acyl groups present in the diacylPC were 14:0 (5%), 16:0 (68%), 16:1 (12%), 18:0 (6%), 18:1 (7%) and 18:2 (2%). The predominant alkyl ether chains located at the carbon 1 position of the 1-O-alkyl-2-acylPC were 16:0 (84%), 18:0 (5%) and 18:1 (14%). At the carbon 2 position only a 16:0 fatty acyl residue was detected. In three out of seven animals platelet-activating factor-like activity, as determined by a platelet aggregation assay, was isolated by TLC. This aggregating activity was lost upon base-catalyzed methanolysis, but was restored by functional levels after acetylation.     

Structural stability of the erythrocyte anion transporter, band 3, in different lipid environments. A differential scanning calorimetric study

In order to understand how subtle variations in lipid structure can influence the stability of an integral membrane protein, the purified, delipidated anion transport domain of human erythrocyte band 3 was reconstituted into a series of well-defined lipids and examined by differential scanning calorimetry. From the calorimetric scans, plots of denaturation temperature (Tm), enthalpy (delta Hd), and heat capacity (delta Cdp) as a function of phospholipid chain length, degree of unsaturation, headgroup type, and cholesterol content were constructed. The data show that the stability of the 55,000-dalton membrane-spanning domain of band 3 is exquisitely sensitive to the acyl chain length of its phospholipid environment, increasing almost linearly from a Tm of 47 degrees C in dimyristoleylphosphatidylcholine (C14:1) to 66 degrees C in dinervonylphosphatidylcholine (C24:1). The integral domain was also found to be significantly stabilized by increasing the degree of saturation of the fatty acyl chains and by elevating the cholesterol content of the membrane. Although band 3 was native in all reconstituted lipid systems, the transport protein's stability was clearly much greater in zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) than anionic lipids (phosphatidylserine and phosphatidylglycerol). Enthalpy and delta Cdp values were generally within the ranges expected of globular proteins in the various reconstituted systems, except the values for the anionic and polyunsaturated phospholipids were anomalously low. Much of the data can be accounted for by the hypothesis that band 3 has a long hydrophobic cross-section and that a close match between the hydrophobic zone of the membrane-spanning protein and the nonpolar region of the bilayer is necessary for maximum protein stability. Because the integral domain of band 3 may be structurally representative of a larger group of transport proteins, the data should be useful in interpreting structural observations on protein-lipid interactions in other membrane systems.