Oligomerization of Clostridium perfringens epsilon-toxin is dependent upon membrane fluidity in liposomes

Clostridium perfringens epsilon-toxin binds to receptors on MDCK cells and forms a heptamer in membranes. The mechanism behind the oligomerization of epsilon-toxin was studied using carboxyfluorescein (CF)-loaded liposomes composed of various phosphatidylcholines (PCs). The toxin caused CF to leak from liposomes in a dose-dependent manner. The toxin-induced leakage of CF, binding of the toxin to liposomes, and formation of a functional oligomer increased as the phase-transition temperature (Tm) of the PC used in the liposomes decreased. Surface plasmon resonance analysis using an HPA sensorchip (BIAcore) also revealed that the binding of the toxin to liposomes increased with a decrease in the Tm of the PC used in liposomes. The oligomer that was formed in 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID)-treated liposomes was labeled, indicating that it inserts into a hydrophobic region. Furthermore, the rate of epsilon-toxin-induced CF leakage was enhanced by treatment with phosphatidylethanolamine or diacylglycerol, which is known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. We show that membrane fluidity in the liposome plays an important role in the binding of the toxin to liposomes, insertion into the hydrophobic region in the bilayer of liposomes, and the assembly process in the bilayer.     

Effect of N-acyl-phosphatidylethanolamine on the Membrane Fusion Between Sendai Virus and Liposome

We have compared the properties of two N-acyl derivatives of dilauryl phosphatidylethanolamine on lipid polymorphism, vesicle leakage and Sendai virus fusion. The derivatives contained either an N-lauroyl group (NLPE) or an N-acetyl group (NAcPE). Only the NAcPE markedly affected the bilayer to hexagonal transition temperature of dielaidoyl phosphatidylethanolamine, shifting it to higher values. In contrast the NLPE slightly lowered this phase transition temperature. The two lipids also have opposite effects on leakage from small unilamellar vesicles of egg phosphatidylcholine. The NLPE inhibits leakage, while the NAcPE promotes it. This vesicle stabilizing effect of NLPE against leakage is not manifested in alterations of rates or extents of Sendai virus fusion to liposomes of egg phosphatidylethanolamine plus 2% ganglioside G_D1a. The NLPE has no effect, while the NAcPE reduces the observed fusion, at least in part as a consequence of a reduction in the final extent of fusion. These results demonstrate that the bilayer stabilizing effects of NLPE do not result in a lower rate of viral fusion. Furthermore, these bilayer stabilizing effects against leakage are not solely a function of the lipid headgroup but also require a structure with three long acyl chains. The reduced leakage is not related to a loss in monolayer curvature strain.     

Spontaneous vesiculation of uncharged phospholipid dispersions consisting of lecithin and lysolecithin

The work presented here demonstrates that the phenomenon of spontaneous vesiculation is not restricted to charged lipids and lipid mixtures, but occurs also in isoelectric phospholipid mixtures consisting of egg phosphatidylcholine (EPC) and egg lysophosphatidylcholine (lyso-EPC). 1H high-resolution NMR and freeze-fracture electron microscopy have been used to characterize the mixed EPC/lyso EPC dispersions in excess H2O. The predominant phase in these mixed phospholipid dispersions is smectic (lamellar) at least up to approximately 70% lysophosphatidylcholine. The type of phospholipid aggregate formed in excess H2O depends on the mole ratio diacyl to monoacyl phosphatidylcholine. The dispersive (lytic) action of lysophosphatidylcholine on phosphatidylcholine bilayers becomes effective at lysophospholipid contents in excess of approximately 10%. Large multilamellar liposomes are disrupted and replaced by smaller particles, mainly unilamellar vesicles. Between 30 and 70% lysophosphatidylcholine a significant proportion of the total phospholipid is present as small unilamellar vesicles (SUV) of a diameter of 23 nm (range: 20-70 nm). At even higher lysophosphatidylcholine contents the fraction of phospholipid present as small mixed micelles with a diameter smaller than about 14 nm grows at the expense of the vesicular structures. There is a second effect of increasing the quantity of lysophosphatidylcholine in phosphatidylcholine bilayers: the presence of lysophosphatidylcholine in excess of 10% renders the phospholipid bilayer more permeable to ions as compared to pure phosphatidylcholine bilayers. The key factor in inducing spontaneous vesiculation is probably not the charge but the wedge-like shape of the lysophospholipid molecule. The molecular shape may give rise to an asymmetric distribution of lysophosphatidylcholine between the two halves of the bilayer, thus stabilizing highly curved bilayers as present in SUV.     

Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature

We have examined whether there is a relationship between the lamellar-hexagonal phase transition temperature, TH, and the initial kinetics of H+- and Ca2+-induced destabilization of phosphatidylethanolamine (PE) liposomes. The liposomes were composed of dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine (EPE), or phosphatidylethanolamine prepared from egg phosphatidylcholine by transesterification (TPE). These lipids have well-spaced lamellar-hexagonal phase transition temperatures (approximately 12, approximately 45, and approximately 57 degrees C) in a temperature range that allows us to measure the initial kinetics of bilayer destabilization, both below and above TH. The liposomes were prepared at pH 9.5. The TH of EPE and TPE was measured by using differential scanning calorimetry, and it was found that the TH was essentially the same at low pH or at high pH in the presence of 20 mM Ca2+. At temperatures well below TH, either at pH 4.5 or at pH 9.5 in the presence of Ca2+, the liposomes aggregate, leak, and undergo lipid mixing and mixing of contents. We show that liposome/liposome contact is involved in the destabilization of the PE liposomes. The temperature dependence of leakage, lipid mixing, and mixing of contents shows that there is a massive enhancement in the rate of leakage when the temperature approaches the TH of the particular PE and that lipid mixing appears to be enhanced. However, the fusion (mixing of aqueous contents) is diminished or even abolished at temperatures above TH. At and above the TH, a new mechanism of liposome destabilization arises, evidently dependent upon the ability of the PE molecules to adapt new morphological structures at these temperatures. We propose that this destabilization demarks the first step in the pathway to the eventual formation of the HII phase. Thus, the polymorphism accessible to PE is a powerful agent for membrane destabilization, but additional factors are required for fusion.     

Gramicidin conformational studies with mixed-chain unsaturated phospholipid bilayer systems.

The transition of gramicidin from a nonchannel to a channel form was investigated using mixed-chain phosphatidylcholine lipid bilayers. Gramicidin and phospholipids were codispersed, after removal of the solvents chloroform/methanol or trifluoroethanol which resulted in nonchannel and channel conformations, respectively, as confirmed using circular dichroism (CD). The fluorescence emission maxima of the nonchannel form were shifted toward shorter wavelengths by heating at 60 degrees C (for 0-12 h), which converted it to a channel form, again as confirmed by CD. The channel form did not respond to heat treatment. Heat treatment also increased the fluorescence anisotropy of the nonchannel gramicidin tryptophans. The rate of transition from the nonchannel to channel conformation was found to be faster if phosphatidylethanolamine was present in combination with phosphatidylcholine compared to phosphatidylcholine alone. Also, gramicidin in bilayers of the polyunsaturated 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine converted more rapidly compared to 1-palmitoyl-2-oleoylphosphatidylcholine. Using the fluorescence anisotropy of the membrane lipid probe 1,6-diphenyl-1,3,5-hexatriene, it was also shown that the motional properties of the surrounding lipid acyl chains differed for the channel and nonchannel gramicidin conformations. The possibility that lipids tending to favor the hexagonal phase (HII) would enhance the rate of the nonchannel to channel transition was supported by 31P NMR which revealed the presence of some HII lipids in the channel preparations. The results of this study suggest that gramicidin may serve as a useful model for similar conformational transitions in other more complex membrane proteins.     

The influence of proteins and peptides on the phase properties of lipids

This paper reviews model membrane studies on the modulation of the macroscopic structure of lipids by lipid-protein interactions, with particular emphasis on the gramicidin molecule. This hydrophobic peptide has three main effects on lipid polymorphism: (1) in lysophosphatidylcholine it triggers a micellar to bilayer transition, (2) in phosphatidylethanolamine it lowers the bilayer to hexagonal H_II phase transition temperature and (3) in phosphatidylcholine and other bilayer preferring lipids it is able to induce the formation of an H_II phase. From experiments in which the gramicidin molecule was chemically modified it can be concluded that the tryptophan residues play a determining role in the peptide-induced changes in polymorphism. The experimental data lead to the proposal that gramicidin molecules have a tendency to self-associate, possibly mediated by tryptophan-tryptophan interactions and organize into tubular structures such as found in the H_II phase.     

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).     

Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes

Single bilayer vesicles were prepared by sonication of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine. Incubation with lysophospholipase results in a fast hydrolysis of 80–90% of lysophosphatidylcholine. The remaining lysophosphatidylcholine is only very slowly hydrolysed. There results are interpreted as lysophosphatidylcholine being asymmetrically distributed over the two halves of the bilayer. The slow phase of lysophosphatidylcholine hydrolysis sets an upper limit to the rate of transbilayer movement of lysophosphatidylcholine. The half time of this process at 37° C is estimated to be about 100 h. Incorporation of cholesterol in the vesicles reduces the distributional asymmetry of lysophosphatidylcholine to the extent of an outside-inside ratio of 60 : 40. [¹⁴C]Lysophosphatidylcholine introduced into the outer monolayer of such vesicles by intervesicular transfer of lysophosphatidylcholine remains virtually completely available for hydrolysis by lysophospholipases, corroborating the interpretation that transbilayer movement of lysophosphatidylcholine in these vesicles is an extremely slow process.In handshaken liposomes consisting of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine 15–20% of lysophosphatidylcholine is readily available for exogenous lysophospholipase. This pool may represent lysophosphatidylcholine in the outer monolayer of the liposomes.     

Synthesis and physical properties of phosphatidylcholine labelled with 9-(2-anthryl)nonanoic acid,a new fluorescent probe

Synthesis and physical properties of a new anthracene fatty acid, 9-(2-anthryl)nonanoic acid, and the corresponding anthracene-phosphatidylcholines which were obtained by condensing the acid with sn-1-palmitoyl-lysophosphatidylcholine (PAPC) and with egg lysophosphatidylcholine (EAPC) are described. Differential scanning calorimetry experiments show that these lipids can undergo a liquid-crystal to gel phase transition at temperatures of 15°C and 18°C for EAPC and PAPC, respectively. In monolayers, PAPC exhibits a compression curve nearly superimposable to that of dipalmitoylphosphatidylcholine (DPPC), with a molecular area of 0.48 nm² at π = 30 mN m^?1. The data indicate that in these lipids, the anthracene group is only slightly more bulky than a normal acyl chain and that it does not significantly affect the regular phospholipid molecular packing. In ethanol solutions or when incorporated into egg phosphatidylcholine liposomes in a molar ratio of 1%, these lipids display UV absorption spectra and fluorescence emission spectra similar to those of 2-methyl anthracene. For EAPC liposomes, a broad and structureless fluorescence emission spectrum centered at around 450 nm, was recorded, suggesting the occurrence of anthracene excimers. As ascertained by UV spectrophotometry, differential scanning calorimetry, fluorescence polarization and anthracene photodimerization experiments, EAPC displays good miscibility properties with lipids in the liquid state (egg phosphatidylcholine) or in the gel state (distearoylphosphatidylcholine (DSPC)). The potential of these anthracene derivatives for studying the dynamics and the topological distribution of lipids in biomembranes is discussed.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Intramolecular excimer formation of pyrene-labeled lipids in lamellar and inverted hexagonal phases of lipid mixtures containing unsaturated phosphatidylethanolamine

The rates of intramolecular excimer formation of di(1'-pyrenemyristoyl)phosphatidylcholine (dipyPC) in dioleoylphosphatidylethanolamine (DOPE), egg PE/diolein (DG) and dilinoleoyl-PE (DLPE)/1-palmitoyl-2-oleoyl-PC (POPC) were studied at different temperatures and lipid compositions. Both the excimer-to-monomer intensity ratio and the excimer association rate constant were employed to quantify the rate of excimer formation. The latter was calculated from the measured monomer fluorescence lifetime of dipyPC. We observed that the rate of excimer formation was sensitive to either the temperature-induced or lipid composition-induced lamellar-to-inverted hexagonal phase transition of the above lipid systems. As the lipids entered the inverted hexagonal phase, the rate of excimer formation increased at the temperature-induced phase transition for DOPE, but decreased at the composition-induced phase transition for both TPE/DG and DLPE/POPC systems by increasing the DG% and decreasing the PC%, respectively. We conclude that the rate of intramolecular excimer formation of dipyPC in the non-lamellar phase is influenced both by the intra-lipid free volume of the hydrocarbon region and the intra-rotational dynamics of the two lipid acyl chains.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.     

Asymmetric addition of ceramides but not dihydroceramides promotes transbilayer (flip-flop) lipid motion in membranes

Transbilayer lipid motion in membranes may be important in certain physiological events, such as ceramide signaling. In this study, the transbilayer redistribution of lipids induced either by ceramide addition or by enzymatic ceramide generation at one side of the membrane has been monitored using pyrene-labeled phospholipid analogs. When added in organic solution to preformed liposomes, egg ceramide induced transbilayer lipid motion in a dose-dependent way. Short-chain (C6 and C2) ceramides were less active than egg ceramide, whereas dihydroceramides or dioleoylglycerol were virtually inactive in promoting flip-flop. The same results (either positive or negative) were obtained when ceramides, dihydroceramides, or diacylglycerols were generated in situ through the action of a sphingomyelinase or of a phospholipase C. The phenomenon was dependent on the bilayer lipid composition, being faster in the presence of lipids that promote inverted phase formation, e.g., phosphatidylethanolamine and cholesterol; and, conversely, slower in the presence of lysophosphatidylcholine, which inhibits inverted phase formation. Transbilayer motion was almost undetectable in bilayers composed of pure phosphatidylcholine or pure sphingomyelin. The use of pyrene-phosphatidylserine allowed detection of flip-flop movement induced by egg ceramide in human red blood cell membranes at a rate comparable to that observed in model membranes. The data suggest that when one membrane leaflet becomes enriched in ceramides, they diffuse toward the other leaflet. This is counterbalanced by lipid movement in the opposite direction, so that net mass transfer between monolayers is avoided. These observations may be relevant to the physiological mechanism of transmembrane signaling via ceramides.     

Diacylglycerol and the promotion of lamellar-hexagonal and lamellar-isotropic phase transitions in lipids: implications for membrane fusion.

Changes in steady-state fluorescence anisotropy of 1 -(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene TMA-DPH) are applied to the detection of lamellar-hexagonal transitions in egg phosphatidylethanolamine. Even low (2 mole%) proportions of diacylglycerol decrease the hexagonal transition temperature considerably, as confirmed by differential scanning calorimetry. Diacylglycerol is also found to promote a lamellar to "isotropic" (Q(224) cubic) transition in mixtures of phosphatidylcholine: phosphatidylethanolamine:cholesterol. This nonreversible transition is also observed by (31)P nuclear magnetic resonance and detected as a large increase in TMA-DPH steady-state anisotropy. The same technique reveals as well that lysophosphatidylcholine counteracts the effect of diacylglycerol and stabilizes the lamellar phase in both transitions. Diacylglycerol and lysophosphatidylcholine are known to respectively promote and inhibit membrane fusion in a variety of systems. These data are interpreted in support of the hypothesis of a highly bent structural fusion intermediate ("stalk"). They also show the interest of lipid-phase studies in predicting and rationalizing membrane fusion mechanisms.     

The presence of lysophosphatidylcholine in chromaffin granules

Lysophosphatidylcholine is thought to be a characteristic component of the chromaffin granules in adrenal glands. By the use of a t.l.c. system that resolves minor phospholipids satisfactorily, this subcellular location was confirmed in the present study in bovine glands. However, phospholipid degradation was demonstrated in homogenates of the adrenal medulla and cortex under conditions similar to those of subcellular fractionation (incubation at 4°C for 90min). Phosphatidylethanolamine and cardiolipin were hydrolysed, but the concentration of lysophosphatidylcholine did not change, indicating that the latter was present in the medulla before this treatment. Attempts were made to decrease the time between death of the animal and the extraction of lipids. Lysophosphatidylcholine was easily demonstrable in lipid extracts of the dissected medulla and even in those of the whole bovine gland. For practical reasons it is not possible to decrease further the time lapse before extraction in the case of this animal. Adrenal glands were obtained from anaesthetized and untreated rabbits. These were frozen immediately in liquid N₂ and the lipids were extracted. In a control experiment, the glands from rabbit were dissected and treated in the same manner as with those of ox, and then the lipids were extracted. No lysophosphatidylcholine was detected in the extracts from glands frozen in liquid N₂ but lysophosphatidylcholine was observed in the controls. These results suggest that lysophosphatidylcholine is not a component of chromaffin granules, but is produced if the period between death of the animal and lipid extraction is unduly prolonged. To discover whether lysophosphatidylcholine affected the permeability barrier properties of chromaffin granules, sonicated liposomes of egg phosphatidylcholine alone or with lysophosphatidylcholine (15mol/100mol) were prepared. Both types were shown by electron microscopy to be largely made up of single bilayer vesicles. The exchange diffusion of [¹⁴C]dopamine was measured across their membranes. Both types of liposomes had similar capture volumes (0.5μl/μmol of phospholipid), and the activation energies of the exchange diffusion of dopamine were also similar (31kJ/mol). These results indicate that the presence of this proportion of lysophosphatidylcholine in chromaffin-granule membranes is not likely to influence their barrier properties towards catecholamines.     

Mechanism of liposome destabilization by polycationic amino acids

Polycationic amino acids induce the leakage and fusion of liposomes containing anionic lipids. We have investigated the nature and extent of the changes in membrane physical properties caused by these polypeptides which could result in the observed membrane destabilization. We found that in the range of pH 5 to pH 7 both poly-l-histidine and poly-l-lysine were ineffective in shifting the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine, either in the presence of absence of 1-palmitoyl-2-oleoylphosphatidylserine. We also studied the gel to liquid crystalline phase transition properties of 11 mixtures of phosphatidylserine and phosphatidylethanolamine, both in dimyristoyl forms as well as the 1-palmitoyl-2-oleoyl forms, as a function of pH and in the presence and absence of polycationic amino acids. We observed that these two lipids were largely miscible at all pH values and in the presence and absence of the polypeptides. However, there was some increased tendency for phase separation at higher pH and in the absence of polypeptide. Thus neither changes in curvature strain nor lateral phase separation induced by the polycationic amino acids could account for their marked ability to induce leakage and fusion.Phosphatidylethanolamine labelled with pyrene on one of the acyl chains gives rise to fluorescent emission from both monomer and excimer forms. The ratio of emission intensity from these two forms is indicative of lateral phase separation and the degree of lateral mobility of this probe. In equimolar mixtures of the 1-palmitoyl-2-oleoyl forms of phosphatidylserine and phosphatidylethanolamine in the liquid crystalline phase at 30 °C we find little effect of pH on the ratio of excimer to monomer emission intensity. However poly-l-lysine markedly lowers the fraction of excimer emission from these liposomes through the pH range from 5 to 7. Poly-l-histidine lowers the excimer to monomer emission ratio at pH 5 but not at pH 7. This is opposite to what one would expect for lateral phase separation and is interpreted at being the consequence of the polypeptide lowering the rate of lateral diffusion of the lipids. This effect of poly-l-histidine is observed over a range of temperatures from 0 to 40°C in both gel and liquid crystalline phases. There is no evidence from the behaviour of the pyrene fluorescent probe for lipid interdigitation. We conclude that the promotion of leakage and fusion in anionic liposomes by polycationic amino acids is not a result of large changes in the physical properties or arrangements of the lipids but rather to a surface binding of the polyamino acids.Abbreviations DSC differential scanning calorimetry - DEPE dielaidoylphosphatidylethanolamine - POPS 1-palmitoyl-2-oleoylphosphatidylethanolamine - DMPS dimyristoylphosphatidylserine - DMPE dimyristoylphosphatidylethanolamine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine - T_H bilayer to hexagonal phase transition temperature - pyr-PE 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolaline - E/M ratio of intensities of excimer to monomer emission     

Hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, induced permeability change of phosphatidylcholine bilayers

Interactions of hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, with phosphatidylcholine vesicles were investigated to obtain information on its bioactive mechanism. The peptide induced the leakage of a fluorescent dye, calcein, entrapped in sonicated vesicles. The leakage rate depended on both the peptide and the lipid concentrations. Analysis of this dependency indicated that the leakage was due to the monomeric peptide and that the membrane-perturbing activity of the monomer was higher for solid distearoylphosphatidylcholine vesicles than for fluid egg yolk phosphatidylcholine vesicles. Hypelcin A also affected the gel to liquid-crystalline phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles. The transition was broadened with a reduced transition enthalpy, suggesting the peptide strongly binds the surrounding lipids to perturb the bilayer lipid packing. A circular dichroism study revealed that the helical content of hypelcin A increases upon membrane binding. We concluded that the monomeric peptide with an increased helical content, complexed with the lipids, perturbs the lipid organization and induces the increased permeability.     

The apoptotic protein tBid promotes leakage by altering membrane curvature

The apoptotic protein tBid is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylcholine at a molar ratio of 1:2 in the presence of calcium. When half of the phosphatidylcholine component of these liposomes is replaced with phosphatidylethanolamine, a lipid that promotes negative membrane curvature, the rates of both leakage and lipid mixing caused by tBid are substantially increased. Replacement of cardiolipin with phosphatidylglycerol, a lipid that is structurally similar to cardiolipin but does not promote negative membrane curvature in the presence of calcium, prevents the tBid from promoting leakage. The promotion of leakage by tBid is also inhibited by several substances that promote positive membrane curvature, including lysophosphatidylcholine, tritrpticin, a potent antimicrobial peptide, and cyclosporin A, a known inhibitor of cytochrome c release from mitochondria. We directly measured the effect of tBid on membrane curvature by (31)P NMR. We found that tBid promotes the formation of highly curved non-lamellar phases. All of these data are consistent with the hypothesis that tBid promotes negative curvature, and as a result it destabilizes bilayer membranes. Bcl-X(L) inhibits leakage and lipid mixing induced by tBid. Bcl-X(L) is anti-apoptotic. It reduces the promotion of non-bilayer phases by tBid, although by itself Bcl-X(L) is capable of promoting their formation. Bcl-X(L) has little effect on liposomal integrity. Our results suggest that the anti-apoptotic activity of Bcl-X(L) is not a consequence of its interaction with membranes, but rather with other proteins, such as tBid.