Receptor-mediated interaction of ricin with the lipid bilayer of ganglioside GM1-liposomes

The interaction of ricin with ganglioside GM1 or glycoprotein containing liposomes was investigated. At neutral pH, ricin bound to galactose moieties on the surface of the liposomes to form ricin-liposomes complexes, but did not associate with their lipid bilayers. When these ricin-liposomes complexes were exposed to a pH below 5, ricin bound to GM1-liposomes became associated with the lipid bilayer, whereas ricin bound to glycoprotein-liposomes (containing human erythrocyte Band 3) was only rarely associated. Association of ricin with the lipid bilayer of GM1-liposomes did not occur in the presence of lactose, which inhibits the binding of ricin to ganglioside GM1. Using a hydrophobic probe, 8-amino-1-naphthalene sulfonic acid (ANS), it was revealed that an acidity below pH 5 resulted in exposure of hydrophobic regions on the ricin molecule. These results strongly suggest that association of ricin with the lipid bilayer of GM1-liposomes at acidic pH is mediated by the binding of ricin to ganglioside GM1 at neutral pH and occurs through interaction between the exposed hydrophobic region on the ricin molecule and the lipid bilayer of GM1-liposomes at low pH.     

Mechanism of the interaction of hydrophobically-modified poly-(N-isopropylacrylamides) with liposomes

Interactions of hydrophobically-modified poly-(N-isopropylacrylamides) (HM PNIPAM) with phospholipid liposomes were studied as a function of the lipid type, the lipid bilayer fluidity, and the polymer conformation. Fluorescence experiments monitoring non-radiative energy transfer (NRET), between naphthalene attached to the HM PNIPAM and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the lipid bilayer, confirmed the direct penetration of hydrophobic anchor groups linked to the polymer into the liposome hydrophobic core. Contraction of the polymer backbone above the lower critical solution temperature (LCST) resulted in a partial withdrawal of the anchor groups from the lipid bilayer. Analysis of polymer/lipid mixtures by centrifugation and quasi-elastic light scattering (QELS) revealed the polymer-induced fission of liposomes in the liquid-crystalline state, resulting in the formation of vesicles 150–230 nm in diameter. The process is reversible and upon transition of the bilayer into the gel state these vesicles are converted into larger aggregates. According to the results of gel-filtration experiments the HM PNIPAM is in dynamic exchange between the liquid-crystalline lipid bilayer and the water solution, while the binding to the bilayer in the gel state is more static in nature. The binding constant for mixture of HM PNIPAM with DMPC liposomes, evaluated from the centrifugation experiments, was found to be 120 M⁻¹.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Effects of reconstitution of membrane-bound lipoprotein lipase and dispersity of substrate on its reaction characteristics

Reaction characteristics of a membrane-bound lipoprotein lipase acting on a hydrophobic substrate were investigated in aggregated structures—lipid bilayers of liposomes and mixed micelles of Triton X-100. The enzyme activity was enhanced with increases in Triton X-100 and phospholipid concentrations in micellar and liposomal structures. This higher activity was found to be due to both the solubilization state of the hydrophobic substrate and the hydrophobic interactions of the enzyme with either phospholipid or Triton X-100 molecules as a result of its incorporation into the aggregated systems. The enzyme reconstituted into lipid bilayers of liposomes prepared from 15 mM DMPC in the presence of 0.05% Triton X-100 showed a further 1.5-fold higher activity in comparison with the activity without reconstitution in micelles of 1.0% Triton X-100. These results indicate the necessity of the bilayer structure to retain the membrane-bound enzyme in an active conformation.     

Morphological behavior of acidic and neutral liposomes induced by basic amphiphilic alpha-helical peptides with systematically varied hydrophobic-hydrophilic balance

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

Lipogels: single-lipid-bilayer-enclosed hydrogel spheres

We have fabricated Lipogels consisting of a single POPC lipid bilayer supported by a micrometer-sized, thermoresponsive, hydrophobically modified (HM), hydrogel sphere. The hydrogel consists of a lightly cross-linked poly(N-isopropylacrylamide) (pNIPAM) core surrounded by a highly cross-linked acrylic acid (AA)-rich p(NIPAM-co-AA) shell. The lipid bilayer was assembled by binding liposomes to HM microgels, followed by several cycles of freeze-thaw. The pNIPAM volume phase transition (VPT) at ～32 °C was present both before and after hydrophobic modification and after lipid bilayer coating. Fluorescence studies confirmed the fusion of liposomes into a continuous single bilayer. At a temperature above the VPT, it was found that the volume decrease in the hydrogel was coupled to the appearance of highly curved obtrusions of the uncompromised lipid bilayer into the surroundings. It is anticipated that these properties of Lipogels will prove to be useful in drug delivery applications and in fundamental biophysical studies of membranes.     

From liposomes to supported, planar bilayer structures on hydrophilic and hydrophobic surfaces: an atomic force microscopy study

The sequence of events involved in the transition from attached liposomes to bilayer patches on hydrophilic and hydrophobic solid supports were visualized in situ by Tapping Mode atomic force microscopy in liquid. In a smooth manner, the attached liposomes spread and flattened from the outer edges toward the center until the two membrane bilayers were stacked on top of each other. The top bilayer then either rolls or slides over the bottom bilayer, and the adjacent edges join to form a larger membrane patch. This is clearly visible from the apparent height of 6.0-7.5 nm of the single bilayer, measured in situ. The addition of calcium appeared to increase the rate of the processes preventing the visualization of the intermediate stages. The same intermediate steps appeared to be present on hydrophobic surfaces, although the attached liposomes seemed to be distorted and the resultant membrane edges were uneven. This work has provided visual and detailed information on liposome coalescence (fusion) onto solid supports and demonstrated how the atomic force microscope can be used to study the process.     

The structure and function of gramicidin A embedded in interdigitated bilayer

The effects of phase transition from normal to interdigitated lipid bilayer on the function and structure of membrane proteins were studied using linear gramicidin (gramicidin A) as a model. Interdigitated bilayer structure of dipalmitoylphosphatidylglycerol (DPPG) liposomes that was induced by atropine could not be changed notably by intercalating of gramicidin. The K+ transportation of gramicidin in both normal and interdigitated bilayer was assayed by measuring the membrane potential. Results showed that gramicidin in interdigitated bilayer exhibited lower transport capability. Intrinsic fluorescence spectrum of gramicidin in interdigitated bilayer blue-shifted 2.8 nm from the spectrum in normal bilayer, which means that interdigitation provides a more hydrophobic environment for gramicidin. Circular dichroism measurement results indicated that the conformation of gramicidin in interdigitated bilayer is not the typical beta6.3 helix as in the normal bilayer. The results suggested that the interdigitated lipid bilayer might largely affect the structure and function of membrane proteins.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

膜融合剂对脂质体及血影膜膜流动性的影响

本文用荧光探针ANS,DPH与A研究了几种膜融合剂对脂质体与血影膜流动性的影响.蔗糖使PS脂质体的脂双层流动性降低,探针越是在极性区流动性越小,说明蔗糖主要作用于脂双层的极性区;蔗糖也使血影膜流动性降低,此作用是可逆的.油酸甘油脂(GMO)使PS脂质体的流动性增加,且越是在疏水区内部,流动性增加得越大,说明GMO主要是作用于脂双层的非极性区:GMO也使血影膜流动性增加,此作用是不可逆的.二甲亚砜(DMSO)对血影膜的作用,两种不同荧光探针不一样,对DPH的作用出现双相让,低浓度与高浓度的作用结果分别与蔗糖和GMO的作用一致.     

Structurally related glucosylated liposomes: Correlation of physicochemical and biological features

Liposomes functionalized on their surface with carbohydrates (glycoliposomes) represent an optimal approach for targeting of drugs to diseased tissues in vivo, thanks to biocompatibility, low toxicity and easy manufacturing of these lipid nanoparticles. Here we report on the study of liposomes including a novel glycosylated amphiphile and on the comparison of their features with those of glycosylated analogues described previously. Further, the capability of the different glucosylated formulations to interact with three breast cancer cell lines was investigated. Our results show that the hydrophobic portion of the lipid bilayer strongly influences both the properties and the internalization of glycosylated liposomes.     

Incorporation of P0 Protein into Liposomes: Demonstration of a Two-Domain Structure by Immunochemical and PAGE Analysis

The amphiphilic nature of P0, the major glycoprotein of peripheral nerve myelin, has been suggested previously. In the present study, purified P0 from human peripheral nerve myelin was incorporated into an artificial lipid bilayer consisting of dimyristoyl lecithin and cholesterol. The liposomes were fractionated on a sucrose gradient. The continued expression of P0 antigenicity by the liposomes was shown by specific complement consumption with a multivalent antiserum against P0 or with an IgM monoclonal antibody. Both antibodies recognized P0 expressed on the surface of peripheral nerve myelin and the P0 liposomes. P0 liposomes and peripheral nerve myelin treated with trypsin lost the surface determinant that reacted with the monoclonal antibody. Analysis of the trypsin-treated liposomes and peripheral nerve myelin by polyacrylamide gel electrophoresis revealed molecular weights for this protein of 19,500 and 20,500, respectively. Similar treatment of the P0 in the fluid phase resulted in many smaller fragments. These results indicate that P0 consists of two domains, a hydrophilic domain accessible to trypsin digestion and a hydrophobic domain, which is potentially trypsin-sensitive, but shielded by the lipid bilayer. Binding studies with an anti-P0 monoclonal antibody and polyacrylamide gel analysis of the lipid-shielded P0 fragment in liposomes and peripheral nerve myelin suggest that the orientation of the protein in the liposome is similar to that in peripheral nerve myelin.     

S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Glycoprotein-protein interaction examined by kinetic studies of pyrene transfer

The transfer of pyrene between ₁-acid glycoprotein, acethylcholinesterase and sonicated liposomes was used to monitor glycoprotein-protein interaction on the lipid bilayer. When a density solution of glycoprotein or protein labeled with pyrene was mixed with unlabeled suspension of free-phospholipid liposomes, or suspensions containing the complexes of glycoprotein-lipid, protein-lipid, or glycoprotein-protein-lipid, pyrene excimer fluorescence increased with a half-time of approximately 30–50 msec. Since the increase in excimer fluorescence indicates an increase in the microscope concentrations of pyrene, the observed fluorescence change reflects pyrene transfer. The half-times for the increase in excimer fluorescence were determined in the presence of glycoprotein and protein in the liposomes. On the basis of the determined half-times it was concluded that both, glycoprotein and protein are bound on the lipid bilayer. Our data also suggest that the thickness of the lipid bilayer is significantly changed in this case. The observation suggests strongly that the limiting step in the transfer of pyrene is not the dissociation of pyrene, but the uptake of the pyrene monomers by the lipid phase.     

Changes Caused by Fruit Extracts in the Lipid Phase of Biological and Model Membranes

The aim of the study was to determine changes incurred by polyphenolic compounds from selected fruits in the lipid phase of the erythrocyte membrane, in liposomes formed of erythrocyte lipids and phosphatidylcholine liposomes. In particular, the effect of extracts from apple, chokeberry, and strawberry on the red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC liposomes was studied. In the erythrocyte population, the proportions of echinocytes increased due to incorporation of polyphenolic compounds. Fluorimetry with a laurdan probe indicated increased packing density in the hydrophilic phase of the membrane in presence of polyphenolic extracts, the highest effect being observed for the apple extract. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The polyphenolic extracts slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The studies have shown that the phenolic compounds contained in the extracts incorporate into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The compounds also penetrate the outer part of the external lipid layer of liposomes formed of natural and DPPC lipids, changing its packing order.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Interaction of the herbicide atrazine with model membranes. I: Physico-chemical studies on dipalmitoyl phosphatidylcholine liposomes

Atrazine (2-chloro-4 ethylamino-6-(isopropylamino)-s-triazine) is one of the most widely used herbicides. Fourier transform infrared spectroscopy, differential scanning calorimetry and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its derivative 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were used to study the interaction of atrazine with dipalmitoyl phosphatidylcholine liposomes used as a model for biological membranes. The results show that atrazine does not perturb the hydrophobic core of the lipid bilayer and suggest that the herbicide localizes near the glycerol backbone of the lipid.