Structural changes induced by Triton X-100 on sonicated phosphatidylcholine liposomes

Solubilization of sonicated unilamellar vesicles by Triton X-100 is a complex process. Solubilization starts at low detergent concentrations, as compared to the case of large vesicles, and is accompanied by the simultaneous rapid formation of large multilamellar liposomes. Measurements of lipid and detergent distribution indicate that, at a 1:1 lipid:detergent mole ratio, about one-third of the lipid, with most of the detergent, is solubilized in the form of mixed micelles. The remaining two-thirds are in the form of multilamellar liposomes, virtually free of detergent. Higher detergent concentrations also bring about the solubilization of these liposomes.     

Increased affinity of liposomes derived from total spleen and liver lipids to spleen cells

The interaction of liposomes derived from total lipids of mouse spleen and liver with mouse spleen cells was studied. It was shown that the binding of these liposomes is much higher than the binding of liposomes obtained from a model lipid mixture--phosphatidylcholine--phosphatidylethanolamine--cholesterol (2:1:1). Adherent and nonadherent spleen cells were found to have affinity for liposomes derived from total lipids of spleen or liver. Removal of gangliosides and protein contaminants from the liposomes derived from total spleen lipids caused an increased binding of liposomes to spleen cells. Multilamellar liposomes bound more effectively to ultrasonicated vesicles having a homologous lipid composition than the liposomes with a different lipid composition. The increased affinity of liposomes derived from total lipids of spleen or liver for spleen cells may account for the identical fluidity of the lipid bilayer of liposomes and plasma membranes of spleen cells.     

Phospholipid vesicle formation using nonionic detergents with low monomer solubility. Kinetic factors determine vesicle size and permeability

The method developed previously for formation of unilamellar vesicles from mixed micelles of egg lecithin and octyl glucoside [Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., & Reynolds, J. A. (1981) Biochemistry 20, 833-840] has been extended to allow for (1) use of nonionic detergents with much lower critical micelle concentrations and (2) variation in the time course of detergent removal. The results demonstrate the importance of kinetic factors, especially in the determination of vesicle size: initially formed vesicles are small, but the size increases slowly thereafter if detergent is not removed too quickly. Vesicle size remains fixed when the molar detergent/lipid ratio falls below about 1/1, and detergent removal becomes increasingly difficult thereafter, presumably because flip-flop of detergent from the inner to the outer leaflet of the bilayer membrane is very slow. Residual detergent (to about 25 mol %) has surprisingly little effect on anion permeability but increases cation permeability to the point where the normal discrimination between anions and cations (in pure lipid vesicles) is lost. Detergent added to initially detergent-free vesicles readily partitions into vesicular membranes (presumably only into the outer leaflet) and has a qualitatively similar effect on permeability. Vesicles produced by this method, regardless of residual detergent level, were found to be predominantly unilamellar: no multilamellar liposomes or other lipid aggregates could be detected within the accuracy of the methods employed.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(Na+ + K+)-ATPase from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether ( C12E8 ) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of 'non-oriented' (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the different orientations is the same. The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ratio of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. The ATP-dependent, K+-stimulated Na+-influx is found to be about 35 mumol Na+ per mg (i/o)-protein per min at 22 degrees C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na+ transported per ATP molecule hydrolyzed.     

Barrier properties of glycophorin-phospholipid systems prepared by different methods

Glycophorin was incorporated into large unilamellar dioleoylphosphatidylcholine vesicles by either a detergent dialysis method using octylglucoside or a method avoiding the use of detergents. The vesicles were characterized and the permeability properties and transbilayer movement of lipids in both vesicles were investigated as a function of the protein concentration and were compared to protein-free vesicles. An insight in the permeability properties of the vesicles was obtained by monitoring the ratio potassium (permeant): dextran (impermeant) trap immediately after separation of the vesicles from the external medium. Glycophorin incorporated without the use of detergents in 1:300 protein:lipid molar ratio induces a high potassium permeability for the majority of the vesicles as judged from the low potassium trap (K⁺:dextran trap = 0.21). In contrast, the vesicles in which glycophorin is incorporated via the octylglucoside method (1:500 protein:lipid molar ratio) are much less permeable to potassium (K⁺:dextran trap = 0.67 and $\text{t}\text{1}\text{2}$ of potassium efflux at 22°C is 7.5 h.). The relationship between protein-induced bilayer permeability and lipid transbilayer movement in both vesicle preparations is discussed. Addition of wheat-germ agglutinin to glycophorin-containing vesicles comprised of dioleoylphosphatidylcholine and total erythrocyte lipids caused no or just a small effect (less than 20% release of potassium) on the potassium permeability of these vesicles. Also, addition of lectin to dioleoylphosphatidylethanolamine-glycophorin bilayer vesicles in a 25:1 lipid:glycophorin molar ratio had no effect on the permeability characteristics of the vesicles. In contrast, addition of wheat-germ agglutinin to bilayer vesicles made of dioleoylphosphatidylethanolamine and glycophorin in a 200:1 molar ratio resulted in a release of 74% of the enclosed potassium by triggering a bilayer to hexagonal (H_II) phase transition. The role of protein aggregation and the formation of defects in the lipid bilayer on membrane permeability and lipid transbilayer movement is discussed.     

The effects of charge and size on the interaction of unilamellar liposomes with macrophages

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of unilamellar liposomes from total polar lipid extracts of methanogens.

Unilamellar liposomes were formed by controlled detergent dialysis of mixed micelles consisting of acetone-insoluble total polar lipids extracted from various methanogens and the detergent n-octyl-beta-D-glucopyranoside. The final liposome populations were studied by dynamic light scattering and electron microscopy. Unilamellar liposomes with mean diameters smaller than 100 nm were obtained with lipid extracts of Methanococcus voltae, Methanosarcina mazei, Methanosaeta concilii, and Methanococcus jannaschii (grown at 50 degrees C), whereas larger (greater than 100-nm) unilamellar liposomes were obtained with lipid extracts of M. jannaschii grown at 65 degrees C. These liposomes were shown to be closed intact vesicles capable of retaining entrapped [14C]sucrose for extended periods of time. With the exception of Methanospirillum hungatei liposomes, all size distributions of the different liposome populations were fairly homogeneous.     

Solubilization and reconstitution of the sodium-and-chloride-coupled glycine transporter from rat spinal cord

Synaptic membranes from rat spinal cord were solubilized in the presence of 2% sodium cholate, phospholipids and 15% ammonium sulphate. The soluble extract was incorporated into liposomes consisting of asolectin and crude rat brain lipids. Reconstitution of the functional transporter protein was achieved by removal of detergent by gel filtration. Several parameters proved to be important for optimal reconstitution efficiency: (a) the lipid composition of the liposomes, (b) the type of detergent, and (c) the phospholipid/protein and detergent/protein ratio during reconstitution. In the reconstituted system, the transport of glycine showed a specific activity about twice that of native vesicles. The ionic dependence of the transport, the inhibitory effect of nigericin in the presence of external sodium and the stimulatory effect of valinomycin in the presence of internal potassium on glycine transport were preserved and more clearly observed in the reconstituted system. These results indicate that, in this preparation, the glycine transporter protein retains the same features displayed in the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity and inhibitor sensitivity.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s(o)) and liquid-disordered (l(d)) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l(o)) and l(d) phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s(o)-l(d) and l(o)-l(d) phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Solubilization and reconstitution of the renal phosphate transporter

Proteins from brush-border membrane vesicles of rabbit kidney cortex were solubilized with 1% octylglucoside (protein to detergent ratio, 1:4 (w/w). The solubilized proteins (80.2 +/- 2.3% of the original brush-border proteins, n = 10, mean +/- S.E.) were reconstituted into artificial lipid vesicles or liposomes prepared from purified egg yolk phosphatidylcholine (80%) and cholesterol (20%). Transport of Pi into the proteoliposomes was measured by rapid filtration in the presence of a Na+ or a K+ gradient (out greater than in). In the presence of a Na+ gradient, the uptake of Pi was significantly faster than in the presence of a K+ gradient. Na+ dependency of Pi uptake was not observed when the liposomes were reconstituted with proteins extracted from brush-border membrane vesicles which had been previously treated with papain, a procedure that destroys Pi transport activity. Measurement of Pi uptake in media containing increasing amounts of sucrose indicated that Pi was transported into an intravesicular (osmotically sensitive) space, although about 70% of the Pi uptake appeared to be the result of adsorption or binding of Pi. However, this binding of Pi was not dependent upon the presence of Na+. Both Na+-dependent transport and the Na+-independent binding of Pi were inhibited by arsenate. The initial Na+-dependent Pi transport rate in control liposomes of 0.354 nmol Pi/mg protein per min was reduced to 0.108 and 0 nmol Pi/mg protein per min in the presence of 1 and 10 mM arsenate, respectively. Future studies on reconstitution of Pi transport systems must analyze and correct for the binding of Pi by the lipids used in the formation of the proteoliposomes.     

The fusogenic effect of synthetic polycations on negatively charged lipid bilayers

The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.     

Ultrastructure of reconstituted membranes containing the muscarinic receptor

In this paper the demonstration is made that membrane vesicles (liposomes) containing the muscarinic receptor can be formed by polyethylene glycol (PEG) precipitation of detergent extracts of bovine atrial membranes. The incorporation of the muscarinic receptor in these vesicles may be related to the restoration of the heterogeneity and nucleotide modulation of muscarinic agonist binding by PEG precipitation of atrial detergent extracts, previously reported. Vesicles are also formed when detergent solubilized asolectin lipids, alone or in combination with membrane detergent extracts, are precipitated by PEG. The structure of the vesicles seems depend on their lipid and protein composition and the procedure employed for the removal of the dispersing medium. These results indicate that PEG precipitation could be used for the reconstitution of the muscarinic receptor into the liposomes of exogenous lipids.Special Issue dedicated to Prof. Eduardo De Robertis.     

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.     

The interaction of liposomes containing intrinsic erythrocyte membrane proteins with lipid monolayers at air/water and oil/water interfaces

The main intrinsic membrane proteins of the human erythrocyte membrane, glycophorin and the anion transporter, were isolated by extraction with Triton X-100 and ion-exchange chromatography. After removal of detergent the extract consisted of proteolipid vesicles with a lipid:protein molar ratio in the range 50-60 and a diameter of the order of 200 nm. The interaction between these vesicles and dipalmitoylphosphatidylcholine (DPPC), cholesterol and cholesterol:DPPC (2:1 molar ratio) monolayers at air/water and n-decane/water interfaces has been studied. The vesicles interact with the monolayers, rapidly causing large increases in surface pressure. Limiting values of surface pressure, 39.4-43 mN . m-1 at air/water and 31.5-33.4 mN . m-1 at the n-decane/water interface, were reached at protein levels above 1 microgram . ml-1. At the air/water interface, and probably at the n-decane/water, surface pressure increases were limited by monolayer collapse. Compression isotherms and surface potential measurements indicated that material from the proteolipid vesicles entered the monolayer phase. In contrast to proteolipid vesicles, injection of protein-free liposomes beneath the monolayer resulted in smaller, slower increases in surface pressure. Thus, the presence of intrinsic membrane proteins in vesicles greatly facilitated the transfer of material into the lipid monolayer.     

Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles.

1. Intact cells, cell fragments (membranes) and matrix vesicles were isolated from the proliferating and calcifying layers of epiphyseal cartilage by sequential hyaluronidase and collagenase digestion and differential centrifugation. Lipids were extracted and analyzed for various lipid classes and their fatty acid composition by column, thin-layer, paper and gas-liquid chromatography. 2. On a protein basis the isolated matrix vesicles had more total lipid than either the membrane or cell fractions, the vesicles and membranes being richer in non-polar lipids and containing smaller quantities of phospholipids than whole cells. Expressed as a percentage of the total lipid, the cells were richer in triacylglycerols and lower in free fatty acids than in the membrane or vesicle fractions. The proportion of free cholesterol and the cholesterol/phospholipid ratio were nearly twice as high in the matrix vesicles as in the other tissue fractions. Choline and ethanolamine phosphoglycerides progressively declined in the membrane and matrix vesicle fractions, whereas serine phosphoglycerides and sphinogomyelin increased. Non-phosphorus-containing polar lipids were present in all fractions, the vesicles being richer in polyhexosyl ceramides, cerebrosides, glycosyldiacylglycerols and certain uncharacterized acidic polar lipids. 3. Fatty acid patterns of the matrix vesicles were distinctive from those of isolated cells, being generally richer in 18 : 0 and 18 : 2, and lower in 16 : 1 and 18 : 1 fatty acids. Monoacyl forms were similarly increased in 16 : 0 and/or 18 : 0, and reduced in 16 : 1, 18 : 1 or 20 : 2 fatty acids, depending on the lipid class. The fatty acid composition of diphosphatidylglycerol from cells and matrix vesicles was markedly different, providing evidence that the cardiolipin in the vesicles was not from mitochondrial components. 4. Based on the fact that the matrix vesicles were significantly enriched in free cholesterol, sphingomyelin, glycolipids and serine-phosphoglycerides, it is concluded that they are derived from the plasma membrane of the cell, supporting earlier conclusions based upon morphological and enzymological evidence.     

Role of Cholesterol, DOTAP, and DPPC in Prostasome/Spermatozoa Interaction and Fusion

Prostasomes are membranous vesicles present in ejaculated human semen. They are very rich in cholesterol and can interact with spermatozoa. Their physiological roles are still under study. Prostasomes were mixed with liposomes prepared from various lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), DOTAP/1,2-dipalmytoyl-sn-glycero-3-phosphorylcholine (DPPC, 4:1 molar ratio) and DOTAP/cholesterol (4:1, molar ratio) at different pH values (5–8). The mixing of the lipid phases (fusion) was determined by the relief of octadecyl rhodamine B chloride (R₁₈) self-quenching and the radii of the vesicles, by light scattering measurements. The mixing of lipids and the radii of prostasomes were both influenced by the addition of liposome, although in a different manner. The ability of prostasomes (modified by previous treatment with liposomes) to transfer lipid to spermatozoa was also measured. Pretreatment with DOTAP decreased the phenomenon and addition of DPPC abolished it. On the other hand, pretreatment of prostasomes with DOTAP/cholesterol liposomes did not affect the transfer of lipid between prostasome and spermatozoa. Therefore, the ability of vesicles to fuse (or, at least, to exchange the lipid component) was affected by the enrichment in either natural or artificial lipid. This may open new possibilities for the modulation of spermatozoa capacitation and acrosome reaction.     

Preparation and characterization of F-protein vesicles isolated from Sendai virus by means of octyl glucoside

We have demonstrated that Triton X-100 is always present in F-protein vesicles at concentrations that can provoke cell lysis. In order to avoid any misinterpretation of the fusogenic capacity of this protein, we solubilized the Sendai virus using octyl glucoside, which can be totally removed from the F protein preparation in less than 16 h by dialysis in the presence of absorbent beads. F-glycoprotein preparations preserved their ability to lyse erythrocytes in the presence of lectins and to induce cell-vesicle fusion as demonstrated by ESR studies. These vesicles were characterized by electron microscopy and SDS-polyacrylamide gel electrophoresis. Lipid analysis of these preparations by thin-layer chromatography indicated that they had the same proportion of lipids as virus envelopes, with slight variations in the sphingomyelin content and the cholesterol/phospholipid molar ratio. F-protein vesicles of different sizes can be obtained by adding exogenous lipids before detergent removal. The hemolytic activity of the vesicles was retained over a large range of lipid concentrations. We conclude that F-protein vesicles prepared with octyl glucoside are convenient tools for studying the fusogenic mechanism of this protein and improving the fusion process between liposomes and cells.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.