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1.
We report a single-vesicle approach to compare the all-or-none and graded mechanisms of lipid bilayer permeabilization by CpreTM and NpreTM, two peptides derived from the membrane-proximal external region of the HIV fusion glycoprotein gp41subunit. According to bulk requenching assays, these peptides permeabilize large unilamellar vesicles via all-or-none and graded mechanisms, respectively. Visualization of the process using giant unilamellar vesicles shows that the permeabilization of individual liposomes by these two peptides differs in kinetics, degree of dye filling, and stability of the permeabilized state. All-or-none permeabilization by CpreTM is characterized by fast and total filling of the individual vesicles. This process is usually accompanied by the formation of stably open pores, as judged from the capacity of the vesicles to incorporate a second dye added after several hours. In contrast, graded permeabilization by NpreTM is transient and exhibits slower kinetics, which leads to partial filling of the individual liposomes. Of importance, quantitative analysis of vesicle population distribution allowed the identification of mixed mechanisms of membrane permeabilization and the assessment of cholesterol effects. Specifically, the presence of this viral envelope lipid increased the stability of the permeating structures, which may have implications for the fusogenic activity of gp41. 相似文献
2.
Antimicrobial, cytolytic, and cell-penetrating peptides induce pores or perturbations in phospholipid membranes that result in fluxes of dyes into or out of lipid vesicles. Here we examine the fluxes induced by four of these membrane-active peptides in giant unilamellar vesicles. The type of flux is determined from the modality of the distributions of vesicles as a function of their dye content using the statistical Hartigan dip test. Graded and all-or-none fluxes correspond to unimodal and bimodal distributions, respectively. To understand how these distributions arise, we perform Monte Carlo simulations of peptide-induced dye flux into vesicles using a very simple model. The modality of the distributions depends on the rate constants of pore opening and closing, and dye flux. If the rate constants of pore opening and closing are both much smaller than that of dye flux through the pore, all-or-none influx occurs. However, if one of them, especially the rate constant for pore opening, increases significantly relative to the flux rate constant, the process becomes graded. In the experiments, we find that the flux type is the same in giant and large vesicles, for all peptides except one. But this one exception indicates that the flux type cannot be used to unambiguously predict the mechanism of membrane permeabilization by the peptides. 相似文献
3.
Leakage of membrane vesicle contents: determination of mechanism using fluorescence requenching. 总被引:2,自引:1,他引:1
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Agents such as antimicrobial peptides and toxins can permeabilize membrane vesicles to cause leakage of entrapped contents in either a graded or an all-or-none fashion. Determination of which mode of leakage is induced is an important step in understanding the molecular mechanism of membrane permeabilization. Wimley et al. (1994, Protein Sci. 3:1362-1378) have developed a fluorescence method for distinguishing the two modes that makes use of the dye/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) without the usual need for the physical separation of vesicles from released contents. Their "requenching" method establishes the mode of release through the fluorescence changes that occur when DPX is added externally to a solution of vesicles that have released some fraction of their contents. However, the requenching method as originally stated ignored the possibility of preferential release of dye or quencher. Here we extend the theory of the method to take into account preferential release and the effects of graded leakage. The ratio of the rates of release of the cationic quencher DPX and anionic dye 8-aminonapthalene-1,3,6 trisulfonic acid can be estimated by means of the theory. For graded leakage, we show that the release of the markers does not coincide with the fluorescence changes observed in the standard leakage assay. This is true for self-quenching dyes as well and means that 1) the amount of released material will be overestimated and 2) the kinetics will be nonexponential and have artificially high apparent rates.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Melittin-induced leakage from phosphatidylcholine vesicles is modulated by cholesterol: a property used for membrane targeting 总被引:4,自引:0,他引:4
Toni Benachir M. Monette Julie Grenier M. Lafleur 《European biophysics journal : EBJ》1997,25(3):201-210
Melittin, an amphiphathic peptide, affects the permeability of vesicles. This can be demonstrated using the dye release technique.
Calcein, a fluorescent marker, is trapped in large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) vesicles and
melittin-induced leakage of the dye can be monitored directly by increasing fluorescence intensity. First, we characterized
the effect of increasing cholesterol content in the membrane on melittin-induced leakage and our results reveal that cholesterol
inhibits the lytic activity of the peptide. Using intrinsic fluorescence of the single tryptophan of melittin and 2H-NMR of headgroup deuterated phosphatidylcholine, we demonstrated that the affinity of melittin for phosphatidylcholine vesicles
is reduced in the presence of cholesterol; this is associated with the tighter lipid packing of the cholesterol-containing
bilayer. This reduced binding is responsible for the reduced melittin-induced leakage from cholesterol-containing membranes.
The pathway of release was determined to be an all-or-none mechanism. Finally, we investigated the possibility of achieving
specific membrane targeting with melittin, when vesicles of different lipid composition are simultaneously present. Melittin
incubated together with vesicles made of pure POPC and POPC containing 30(mol)% cholesterol can empty nearly all the cholesterol-free
vesicles while the cholesterol-containing vesicles remain almost intact. Owing to the preferential interaction of melittin
with the pure POPC vesicles, we were able to achieve controlled release of encapsulated material from a specific vesicle population.
Received: 8 May 1996 / Accepted: 12 September 1996 相似文献
5.
Sonia M. Gregory 《Biophysical journal》2009,96(1):116-131
The all-or-none kinetic model that we recently proposed for the antimicrobial peptide cecropin A is tested here for magainin 2. In mixtures of phosphatidylcholine (PC)/phosphatidylglycerol (PG) 50:50 and 70:30, release of contents from lipid vesicles occurs in an all-or-none fashion and the differences between PC/PG 50:50 and 70:30 can be ascribed mainly to differences in binding, which was determined independently and is ∼20 times greater to PC/PG 50:50 than to 70:30. Only one variable parameter, β, corresponding to the ratio of the rates of pore opening to pore closing, is used to fit dye release kinetics from these two mixtures, for several peptide/lipid ratios ranging from 1:25 to 1:200. However, unlike for cecropin A where it stays almost constant, β increases five times as the PG content of the vesicles increases from 30 to 50%. Thus, magainin 2 is more sensitive to anionic lipid content than cecropin A. But overall, magainin follows the same all-or-none kinetic model as cecropin A in these lipid mixtures, with slightly different parameter values. When the PG content is reduced to 20 mol %, dye release becomes very low; the mechanism appears to change, and is consistent with a graded kinetic model. We suggest that the peptide may be inducing formation of PG domains. In either mechanism, no peptide oligomerization occurs and magainin catalyzes dye release in proportion to its concentration on the membrane in a peptide state that we call a pore. We envision this structure as a chaotic or stochastic type of pore, involving both lipids and peptides, not a well-defined, peptide-lined channel. 相似文献
6.
Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores. 总被引:19,自引:0,他引:19
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W. C. Wimley M. E. Selsted S. H. White 《Protein science : a publication of the Protein Society》1994,3(9):1362-1373
Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin. 相似文献
7.
B. Logan Spaller Julie M. Trieu Paulo F. Almeida 《The Journal of membrane biology》2013,246(3):257-262
Understanding the mechanisms of antimicrobial, cytolytic and cell-penetrating peptides is important for the design of new peptides to be used as cargo-delivery systems or antimicrobials. But these peptides should not be hemolytic. Recently, we designed a series of such membrane-active peptides and tested several hypotheses about their mechanisms on model membranes. To that end, the Gibbs free energy of binding to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was determined experimentally. Because the main lipid components of the outermost monolayer of erythrocyte membranes are zwitterionic, like POPC, we hypothesized that the Gibbs free energy of binding of these peptides to POPC would also be a good indicator of their hemolytic activity. Now, the hemolytic activity of those synthetic peptides was examined by measuring the lysis of sheep erythrocyte suspensions after peptide addition. Indeed, the Gibbs free energy of binding was in good correlation with the hemolytic activity, which was represented by the concentration of peptide in solution that produced 50 % hemolysis. Furthermore, with two exceptions, those peptides that caused graded dye release from POPC vesicles were also hemolytic, while most of those that caused all-or-none release were not. 相似文献
8.
The mechanism of leakage induced by surface active peptides is not yet fully understood. To gain insight into the molecular events underlying this process, the leakage induced by the peptide pardaxin from phosphatidylcholine/ phosphatidylserine/cholesterol large unilamellar vesicles was studied by monitoring the rate and extent of dye release and by theoretical modeling. The leakage occurred by an all-or-none mechanism: vesicles either leaked or retained all of their contents. We further developed a mathematical model that includes the assumption that certain peptides become incorporated into the vesicle bilayer and aggregate to form a pore. The current experimental results can be explained by the model only if the surface aggregation of the peptide is reversible. Considering this reversibility, the model can explain the final extents of calcein leakage for lipid/peptide ratios of > 2000:1 to 25:1 by assuming that only a fraction of the bound peptide forms pores consisting of M = 6 +/- 3 peptides. Interestingly, less leakage occurred at 43 degrees C, than at 30 degrees C, although peptide partitioning into the bilayer was enhanced upon elevation of the temperature. We deduced that the increased leakage at 30 degrees C was due to an increase in the extent of reversible surface aggregation at the lower temperature. Experiments employing fluorescein-labeled pardaxin demonstrated reversible aggregation of the peptide in suspension and within the membrane, and exchange of the peptide between liposomes. In summary, our experimental and theoretical results support reversible surface aggregation as the mechanism of pore formation by pardaxin. 相似文献
9.
R C Roozemond D C Urli J Jansen B Bonavida 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(4):1209-1216
NK cells exert their lytic action through the release of NK cytotoxic factors (NKCF) after stimulation by the bound target cell. NKCF may be related to granule-derived perforin/cytolysin on one hand and to the pleiotropic cytokine TNF on the other hand. In the present study, we show that NKCF can also lyse artificial lipid vesicles, as had been reported previously for cytotoxic granules and cytolysin. The lysis of large unilamellar vesicles was monitored by measuring the release of the encapsulated fluorescent dye carboxyfluorescein. NKCF-induced lysis was only observed with liposomes composed of a complex mixture of lipids including acidic phospholipids. No lysis could be demonstrated if the liposomes contained phosphatidylcholine as the only phospholipid, suggesting some kind of lipid specificity for the action of NKCF. A remarkable finding was that neither recombinant nor natural TNF were able to lyse large unilamellar vesicles, irrespective of their lipid composition, indicating different ways of interaction of NKCF and TNF with artificial (and presumably also biological) membranes. 相似文献
10.
Characterization of interaction between cationic lipid-oligonucleotide complexes and cellular membrane lipids using confocal imaging and fluorescence correlation spectroscopy 总被引:3,自引:0,他引:3
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Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers. 相似文献
11.
Fragment A of diphtheria toxin has been shown to insert into lipid bilayers at low pH (Montecucco, C., Schiavo, G., and Tomasi, M. (1985) Biochem. J. 231, 123-128; Zhao, J.-M., and London, E. (1988) J. Biol. Chem. 263, 15369-15377). In this report, evidence is provided which demonstrates that fragment A, like diphtheria toxin, can also cause the release of a fluorescent dye (calcein) from vesicles under acidic conditions and that this release parallels fragment A insertion into the membrane. Although the permeability changes are not as large as those obtained with whole toxin (Jiang, G.-S., Solow, R., and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429), molecular sieving experiments indicate that the lesion induced by fragment A increases in size with decreasing pH and reaches an upper limit of 30 A at pH 4.0. In addition to size differences, the lesion induced by fragment A releases calcein in a graded manner, whereas diphtheria toxin causes an all-or-none release. One possible interpretation of this result is that the fragment A lesion is transient in comparison to that induced by whole toxin. Although the molecular bases for the observed differences are not understood, these data suggest that fragment A interaction with the lipid bilayer may play a significant role in mediating its own translocation across membranes and that fragment B may aid this process by initiating, enlarging, and stabilizing the lesion formed. 相似文献
12.
Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes 总被引:1,自引:0,他引:1
Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that dissociation from the membrane is rate limiting, while other studies find that flip-flop is rate limiting and on the order of seconds. We have reinvestigated this problem and find that the methods used in studies reporting rapid flip-flop have not been interpreted correctly. We find that accurate information about transport of FFA across lipid vesicles requires that FFA be delivered to the vesicles as complexes with albumin (BSA). For example, we find that stopped-flow mixing of uncomplexed FFA with small unilamellar vesicles (SUV) containing pyranine yields the very fast influx rates reported previously (>100 s(-1)). However, these influx rates increase linearly with lipid vesicle concentration and can therefore not, as previously interpreted, represent flip-flop. In contrast, measurements of influx rates in SUV and giant unilamellar vesicles performed with oleate-BSA complexes reveal no dependence on vesicle concentration and yield influx rate constants of approximately 4 and approximately 0.5 s(-1), respectively. Rate constants for efflux and dissociation were determined from the transfer of oleate from vesicles to BSA and reveal similar influx and efflux but dissociation rate constants that are approximately 5-10-fold greater. We conclude that flip-flop is rate limiting for transport of FFA across lipid vesicles and slows with an increasing radius of curvature. These results, in contrast to those reporting that flip-flop is extremely fast, indicate that the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes. 相似文献
13.
J Milhaud 《Biochimica et biophysica acta》1992,1105(2):307-318
The binding of the pentaene antibiotic filipin to egg-yolk phosphatidylcholine (EPC) and dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles, has been studied by ultraviolet (UV) absorption and circular dichroism (CD). A stoichiometry of one molecule of filipin for five molecules of phospholipid was demonstrated by CD when phospholipids were in fluid phase. The similarity of the CD spectra with EPC and DMPC established a similar filipin-phospholipid assemblage in both membranes. We therefore postulated that filipin incorporation leads to the formation of gel-like domains in fluid EPC membranes as previously demonstrated for fluid DMPC membranes (Milhaud, J., Mazerski J., Bolard, J. and Dufoure, E.J. (1989) Eur. Biophys. J. 17, 151-158). The release of fluorescent probes (carboxyfluorescein (CF) or calcein (CC)), entrapped in EPC small unilamellar vesicles (SUV), due to the action of filipin, was followed by fluorescence and CD measurements concomitantly. The following observations were made. (1) The percentage of released probe, as a function of the filipin/phospholipid molar ratios, was the same whether or not membranes contained cholesterol. (2) The permeabilization of vesicles proceeded concomitantly with filipin-phospholipid binding while filipin-cholesterol binding leveled off. (3) The release of the content of vesicles occurred by an all-or-none mechanism leaving the depleted vesicles intact. From these observations and from the previous structural findings, a new interpretation of the action of filipin is proposed. Precluding any disruptive effect, inducement of permeability would result from the high intrinsic permeability of the interfacial region at the boundaries of the gel-like domains corresponding to the filipin-phospholipid aggregates. Additionally, we obtained the permeability coefficients for the anionic forms of CC and CF across EPC SUV, 0.6.10(-10) cm s-1 and 2.10(-10) cm s-1, respectively, as compared to 2.5.10(-14) cm s-1 for the counterion Na+ (Hauser, H, Oldani, D. and Phillips, M.C. (1973) Biochemistry 12, 4507-4517). 相似文献
14.
Sandoval CM Salzameda B Reyes K Williams T Hohman VS Plesniak LA 《FEBS letters》2007,581(28):5464-5468
Peptides that target tissue for an apoptotic death have potential as therapeutics in a variety of disease conditions. The class of peptides described herein enters the cell through a specific receptor-mediated interaction. Once inside the cell, the peptide migrates toward the mitochondria, where the membrane barrier is disrupted. These experiments demonstrate that upon treatment with these short peptides large unilamellar vesicles are not lysed, a graded mode of leakage is observed and the transient pores formed by these peptides are large enough to release entrapped cytochrome c from the vesicles. 相似文献
15.
R L Richards R C Habbersett I Scher A S Janoff H P Schieren L D Mayer P R Cullis C R Alving 《Biochimica et biophysica acta》1986,855(2):223-230
Complement-dependent antibody-mediated damage to multilamellar lipid vesicles (MLVs) normally results in a maximum release of 50-60% of trapped aqueous marker. The most widely accepted explanation for this is that only the outermost lamellae of MLVs are attacked by complement. To test this hypothesis, complement damage to two different types of large unilamellar vesicles (LUVs), large unilamellar vesicles prepared by the reverse-phase evaporation procedure (REVs) and large unilamellar vesicles prepared by extrusion techniques (LUVETs), were determined. In the presence of excess antibody and complement the LUVs released a maximum of only approx. 25 to 40% of trapped aqueous marker, instead of close to 100% that would be expected. Since small unilamellar vesicles apparently differ from LUVs in that they can release 100% of trapped aqueous marker it appeared that the size of the vesicles was an important factor. Because of these observations the influence of MLV size on marker release was examined. Three populations of MLVs of different sizes were separated by a fluorescence activated cell sorter. Assays of the separated MLV populations showed that the degree of complement-dependent marker release was inversely related to MLV size. No detectable glucose was taken up by MLVs when glucose was present only outside the liposomes during complement lysis. Our results can all be explained by the closing, or loss, of complement channels. We conclude that complement channels are only transiently open in liposomes, and that loss of channel patency may be due to either channel closing or to loss of channels. 相似文献
16.
We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites. 相似文献
17.
Boll A Jatho A Czudnochowski N Geyer M Steinem C 《Biochimica et biophysica acta》2011,1808(11):2685-2693
The mechanism of how full length Tat (aa 1-86) crosses artificial lipid membranes was elucidated by means of fluorescence spectroscopy and fluorescence microscopy. It was shown that full length Tat (aa 1-86) neither forms pores in large unilamellar vesicles (LUVs) nor in giant unilamellar vesicles (GUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In contrast, an N-terminally truncated Tat protein (aa 35-86) that lacks the structurally defined proline- and cysteine-rich region as well as the highly conserved tryptophan residue at position 11 generates pores in artificial POPC-membranes, through which a water-soluble dye up to a size of 10kDa can pass. By means of fluorescence microscopy, the transfer of fluorescently labeled full length Tat across POPC-bilayers was unambiguously visualized with a concomitant accumulation of the protein in the membrane interface. However, if the dye was attached to the protein, also pore formation was induced. The size of the pores was, however smaller than the protein size, i.e. the labeled protein with a mass of 11.6kDa passed the membrane, while a fluorescent dye with a mass of 10kDa was excluded from the vesicles' interior. The results demonstrate that pore formation is not the prime mechanism by which full length Tat crosses a membrane. 相似文献
18.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2022,1866(10):130199
BackgroundThe permeability of a lipid bilayer is a function of its phase state and depends non-linearly on thermodynamic variables such as temperature, pressure or pH. We investigated how shear forces influence the phase state of giant unilamellar vesicles and their membrane permeability.MethodsWe determined the permeability of giant unilamellar vesicles composed of different phospholipid species under shear flow in a tube at various temperatures around and far off the melting point by analyzing the release of fluorescently labelled dextran. Furthermore, we quantified phase state changes of these vesicles under shear forces using spectral decomposition of the membrane embedded fluorescent dye Laurdan.ResultsWe observed that the membrane permeability follows a step function with increasing permeability at the transition from the gel to the fluid phase and vice versa. Second, there was an all-or-nothing permeabilization near the main phase transition temperature and a gradual dye release far off the melting transition. Third, the Laurdan phase state analysis suggests that shear forces induce a reversible melting temperature shift in giant unilamellar vesicle membranes.Major conclusionsThe observed effects can be explained best in a scenario in which shear forces directly induce membrane pores that possess relatively long pore lifetimes in proximity to the phase transition.General significanceOur study elucidates the release mechanism of thermo-responsive drug carriers as we found that liposome permeabilization is not continuous but quantized. Furthermore, the shear force induced melting temperature shift must be taken into consideration when thermo-responsive liposomes are designed. 相似文献
19.
《生物化学与生物物理学报:生物膜》1986,859(2):165-170
Carboxyfluorescein is the most commonly used probe to measure the rate of release of vesicle contents. The validity of the data obtained by this method depends on obtaining an end point based on the complete release of the dye on treatment of the liposomes with a detergent, usually Triton X-100. However, Triton does not completely release entrapped carboxyfluorescein from multilamellar liposomes and the amount and rate of release of marker upon detergent treatment is a function of lipid composition of the liposome, Triton concentration and temperature and duration of detergent incubation. The fluorescence ‘end point’ for distearoyl-l-α-phosphatidylcholine/cholesterol (2:1, mol%) multilamellar liposomes treated with 0.5% Triton at 22°C (a condition often used) is only about one-fifth the value for liposomes treated with 5% Triton at 72°C. The conditions of treatment appear to affect the release of carboxyfluorescein from the lipid of the partially or completely disrupted liposome and the subsequent partitioning of the free dye into the aqueous phase. This effect can lead to serious errors in the interpretation of multilamellar liposome stability data. However, Triton allows complete release of entrapped dye from small unilamellar vesicles under all conditions tested. 相似文献
20.
Diphtheria toxin interaction with membranes has been studied by following the release of a fluorescent dye (calcein) encapsulated within large unilamellar vesicles. Results showed that diphtheria toxin induced temperature- as well as pH-dependent permeability changes in these model membranes. Interestingly, insertion of the "channel-forming" B domain was not sufficient for calcein release, since dye release from vesicles composed of dimyristoyllecithin:cholesterol:dicetylphosphate 4:3:0.8) was completely inhibited at low temperatures which permitted B insertion. Rather, the temperature dependence of calcein release from and A domain insertion into dimyristoyllecithin:cholesterol:dicetyl phosphate vesicles suggest some relationship between "channel formation" and fragment A translocation across membranes. However, the nature of the toxin channel is called into question by our observations that channel size, in addition to activity, was pH-dependent. On the basis of these experiments, it is proposed that the toxin "channel" is the result of localized perturbations in the lipid bilayer at the interface between lipids and inserted toxin molecules that are sufficiently large in fluid membranes at low pH to allow the translocation of fragment A across the bilayer. 相似文献