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1.
Amphotericin B (AmB) is the most widely used antibiotic to treat systemic fungal infections. However, the molecular mechanism of its activity is still not completely understood. In the present work we have used FTIR spectroscopy to investigate the conformational state of the aliphatic chains of DPPC liposomes using the 2850 cm(-1) band, associated with the methylene symmetric stretching mode. The liposomes were either binary mixtures of the lipid with AmB, cholesterol or ergosterol, or ternary systems of these constituents. The two sterols contribute to an ordering of the aliphatic chains of the lipid, this ordering being slightly more important with ergosterol. In the gel state, AmB does not change the conformational order of DPPC even at high concentration. In the fluid phase, however, the drug clearly structures its lipid environment. Our results show that AmB can initiate a redistribution of the ergosterol in the plane of the membrane, but not of the cholesterol molecules, which might constitute an additional mechanism to explain the activity of the antibiotic. 相似文献
2.
Amphotericin and model membranes. The effect of amphotericin B on cholesterol-containing systems as viewed by 2H-NMR 总被引:1,自引:0,他引:1
The interactions between amphotericin B and sterol-containing model membranes were monitored by 2H-NMR of deuterium-labelled dimyristoylphosphatidylcholine (DMPC), cholesterol and epicholesterol. The addition of amphotericin B to a cholesterol/DMPC (3:7) system was perceived differently by the lipid, depending upon the depth in the bilayer: no structural change was manifest in the acyl chain region associated with the plateau in molecular ordering (C4'), whereas the lipid clearly senses two environments near the center of the bilayer (C13', C14'). The amount as well as the ordering properties of the more ordered antibiotic-induced component, sensed at C14', increased with decreasing temperature. The structural parameters of deuterium-labelled cholesterol in cholesterol/DMPC mixtures were unchanged upon addition of amphotericin B, regardless of the bilayer depth. Upon addition of amphotericin B, the lipid T1 values are unchanged, whereas the T2 values are reduced by a factor of 2. The minimum in T1 observed for cholesterol in DMPC at 32-35 degrees C was shifted towards 38-40 degrees C in the presence of amphotericin B. Epicholesterol-containing dispersions of DMPC had properties similar to those of their cholesterol-containing analogs; a noticeable difference between the systems was an approx. 10% increase in the segmental order parameters on the addition of amphotericin B to the system containing the alpha-isomer of cholesterol. The concept of a dynamic complexation between amphotericin B and sterol is discussed. 相似文献
3.
The effects of pressure, up to 5 kbar, on multilamellar vesicles of 1,2-dipalmitoyl-sn-phosphatidylcholine perdeuterated in the acyl chains (DPPC-d62) were examined by using high-pressure NMR techniques. A deuterium probe was built, and the quadrupole splitting was measured against pressure at various temperatures. The experiments were performed on pure lipid bilayers in the liquid-crystalline state and on bilayers in the liquid-crystalline state containing the local anesthetic tetracaine. The results show that the order parameter of all segments of the acyl chains increases with pressure in the liquid-crystalline state. The more highly ordered regions of the chains are affected slightly more than the regions near the methyl ends. The addition of tetracaine increases the disorder of the chains, and pressure reverses the effect of anesthetic on the lipid as seen by the reversal of the changes in line shape and the measured order parameter. 相似文献
4.
The effects of amphotericin B upon the organization and dynamics of multibilayer membranes of dimyristoylphosphatidylcholine (DMPC) were investigated by means of 2H-NMR. At high amphotericin B concentrations (30 mol% with respect to the lipid) and at temperatures above 25 degrees C, DMPC experiences two different environments which are in slow exchange on the 2H-NMR time scale. In one of these, the lipid is immobilized by the antibiotic, in a molar ratio of approximately 1:1, whereas the lipid unsequestered by amphotericin B is more ordered than in its pure state. This ordering effect is perceived at relatively low antibiotic doses (4%). The local lipid order, and the relative percentage, of sequestered DMPC, are temperature-independent (up to 65 degrees C), whereas the ordering of the unsequestered lipid domain is not. The perturbation induced by amphotericin B is manifest similarly at the edges as well as in the center of the bilayer. Antibiotic addition leads to large decreases in the transverse relaxation time, T2, of the labelled lipid, but not in the spin-lattice relaxation time, T1. This indicates an increased density of slow motional modes and little change in rapid motions. 相似文献
5.
Amphotericin B (AmB) is a polyene antibiotic widely used in the treatment of deep-seated fungal infections. The mode of action of AmB is directly related to the effect of the drug on the lipid phase of biomembranes. In the present work the effect of AmB on the properties of lipid bilayers formed with dipalmitoylphosphatidylcholine (DPPC) and the effect of the lipid phase on the molecular organisation of AmB were studied with application of spectrophotometry in the UV-Vis region. The absorption spectra of AmB in lipid membranes display a complex structure with hypsochromically and bathochromically shifted bands indicative of formation of molecular aggregates of the drug. Formation of molecular aggregates was analysed at different concentrations of the drug in the lipid phase in the range 0.05--5 mol% and at different temperatures in the range 5--55 degrees C. The aggregation level of AmB in the ordered phase of DPPC displayed a minimum corresponding to a concentration of 1 mol% with respect to the lipid. An increase in the aggregation level was observed in the temperature region corresponding to the main phase transition. The structure of molecular aggregates of AmB is analysed on the basis of spectroscopic effects in terms of the exciton splitting model. Analysis of the position of the absorption maximum of AmB in the lipid phase of DPPC in terms of the theory of solvatochromc effects makes it possible to ascribe the refractive indices n=1.40 and n=1.49 to the hydrophobic core of the membrane in the L(alpha) and the P(beta)' phase respectively. Analysis of the aggregation of AmB in the lipid phase in relation to the physical state of the membrane reveals that the temperature range of the main phase transition of a lipid cluster in the immediate vicinity of AmB depends on its concentration. The termination of the phase transition temperature, as read from the AmB aggregation, varies between 42 degrees C at 1 mol% AmB in DPPC and 49 degrees C at 5 mol% AmB in DPPC. The exciton splitting theory applied to the analysis of the spectroscopic data makes it possible to calculate the diameter of the AmB pore as 2.8 A in the gel phase and 3.6 A in the fluid phase of the DPPC membrane, on the assumption that the pore is formed by nine AmB molecules. 相似文献
6.
The effects of amphotericin B upon the organization and dynamics of multibilayer membranes of dimyristoylphosphatidylcholine (DMPC) were investigated by means of 2H-NMR. At high amphotericin B concentrations (30 mol% with respect to the lipid) and at temperatures above 25°C, DMPC experiences two different environments which are in slow exchange on the 2H-NMR time scale. In one of these, the lipid is immobilized by the antibiotic, in a molar ratio of approximately 1:1, whereas the lipid unsequestered by amphotericin B is more ordered than in its pure state. This ordering effect is perceived at relatively low antibiotic doses (4%). The local lipid order, and the relative percentage, of sequestered DMPC, are temperature-independent (up to 65°C), whereas the ordering of the unsequestered lipid domain is not. The perturbation induced by amphotericin B is manifest similarly at the edges as well as in the center of the bilayer. Antibiotic addition leads to large decreases in the transverse relaxation time, T2, of the labelled lipid, but not in the spin-lattice relaxation time, T1. This indicates an increased density of slow motional modes and little change in rapid motions. 相似文献
7.
The effect of ergosterol on dipalmitoylphosphatidylcholine bilayers: a deuterium NMR and calorimetric study
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We have studied the effect of ergosterol, an important component of fungal plasma membranes, on the physical properties of dipalmitoylphosphatidylcholine (DPPC) multibilayers using deuterium nuclear magnetic resonance ((2)H NMR) and differential scanning calorimetry (DSC). For the (2)H NMR experiments the sn-1 chain of DPPC was perdeuterated and NMR spectra were taken as a function of temperature and ergosterol concentration. The phase diagram, constructed from the NMR spectra and the DSC thermograms, exhibits both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase coexistence regions with a clear three-phase line. This is the first demonstration that lo domains exist in liquid crystalline membranes containing ergosterol. The domain sizes in the ld+lo phase coexistence region were estimated by analyzing the exchange of labeled DPPC between the two regions, and depend on ergosterol concentration. The DPPC-ergosterol phase diagram is similar to that of the DPPC-cholesterol multibilayer system except that the so+lo and ld+lo phase coexistence regions are considerably broader. 相似文献
8.
The methyl-d(3) amide derivative of the polyene antibiotic amphotericin B was synthesized, assayed for biological activity, incorporated into mechanically aligned bilayers of dipalmitoylphosphatidylcholine (DPPC), and examined by deuterium and phosphorus NMR. The amide derivative has a lesser, but qualitatively similar, biological activity relative to amphotericin B. Incorporation of the amide derivative and ergosterol into aligned DPPC bilayers resulted in a single, stable bilayer phase, as shown by phosphorus NMR of the DPPC headgroups. Deuterium NMR spectra revealed one major (2)H quadrupolar splitting and one major (2)H-(1)H dipolar splitting in the liquid-crystalline phase, consistent with a high degree of alignment and a single, averaged physical state for amphotericin B methyl-d(3) amide in the bilayer. Variations of the quadrupolar and dipolar splittings as a function of macroscopic sample orientation and temperature indicated that the amide derivative undergoes fast rotation about a motional axis that is parallel to the bilayer normal. 相似文献
9.
One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium 总被引:16,自引:0,他引:16
The inducement of K+ permeability through membranes by the polyene antibiotic amphotericin B (AmB) has been analyzed as a measure of the antibiotic activity. Dose-response curves have been obtained with cholesterol- and ergosterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUVs), human erythrocytes, and Saccharomyces cerevisiae cells. Conductance changes induced by AmB in sterol-containing planar bilayer membranes have also been studied. AmB self-association in aqueous buffer was determined by circular dichroism (CD) as a function of the antibiotic concentration. Electronic absorption and CD spectra of AmB were recorded in the presence of LUVs. For given AmB concentrations, the extent of permeability inducement is dependent on the lipid concentration. On the other hand, for cholesterol-containing LUVs or erythrocytes, a critical AmB concentration had to be reached before any permeability is observed. Independent of lipid concentration, this concentration was directly related to antibiotic self-association in the aqueous buffer. The same observation was made for erythrocytes and nystatin. The AmB absorption and CD spectra were totally different for ergosterol- and cholesterol-containing LUVs. Formation of single channels by one-sided addition of AmB could be observed only in ergosterol-containing membranes. These data lead us to propose that the permeability pathways induced by amphotericin B or nystatin, in ergosterol- and in cholesterol-containing membranes, are of different natures. In the latter case the antibiotics are only active, by single-sided addition, in the self-associated form. These findings offer important clues for the design of less toxic derivatives of AmB: they should have a low degree of self-association in water. 相似文献
10.
The effects of ethylene oxide containing lipopolymers and tri-block copolymers on lipid bilayers of dipalmitoylphosphatidylcholine.
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A comparative study is conducted on the influence of two types of polymeric compounds on the phase behavior of 1,2-dihexadecanoyl-s,n-glycero-3-phosphotidylcholine (DC16PC) lipid bilayers. The first polymeric compound is a lipopolymer, with two different lengths of a hydrophilic polyethylene oxide moity, anchored to the bilayer by a 1,2-dioctadecanoyl-s,n-glycero-3-phosphoethanolamine (DC18PE) lipid. The second type, which is a novel type of membrane-spanning object, is an amphiphilic tri-block copolymer composed of two hydrophilic stretches of polyethylene oxide separated by a hydrophobic stretch of polystyrene. Hence the tri-block copolymer may act as a membrane-spanning macromolecule mimicking an amphiphilic protein or polypeptide. Differential scanning calorimetry is used to determine a partial phase diagram for the lipopolymer systems and to assess the amount of lipopolymer that can be loaded into DC16PC lipid bilayers before micellization takes place. Unilamellar and micellar phase structures are investigated by fluorescence quenching using bilayer permeating dithionite. The chain length-dependent critical lipopolymer concentration, denoting the lamellar-to-micellar phase transition, compares favorably with a theoretical prediction based on free-energy considerations involving bilayer cohesion and lateral pressure exerted by the polymer chains. 相似文献
11.
We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, (2)H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S(mol)) and dynamics (T(1)) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a "transient site", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel. 相似文献
12.
Tilt angles of transmembrane model peptides in oriented and non-oriented lipid bilayers as determined by 2H solid-state NMR
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Strandberg E Ozdirekcan S Rijkers DT van der Wel PC Koeppe RE Liskamp RM Killian JA 《Biophysical journal》2004,86(6):3709-3721
Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method. 相似文献
13.
Interaction of the neuropeptide met-enkephalin with zwitterionic and negatively charged bicelles as viewed by 31P and 2H solid-state NMR
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The interaction of the neuropeptide methionine-enkephalin (Menk) with bicelles was investigated by solid-state NMR. Bicelles composed of dimyristoylphosphatidylcholine (DMPC) and dicaproylphosphatidylcholine (DCPC) were modified to investigate the effect of the lipid headgroup and electrostatic charges on the association with Menk. A total of 10 mol % of DMPC was replaced by zwitterionic phosphatidylethanolamine (DMPE), anionic phosphatidylglycerol (DMPG), or phosphatidylserine (DMPS). The preparation of DMPE-doped bicelles (Bic/PE) is reported for the first time. The (31)P and (2)H NMR results revealed changes in the lipid dynamics when Menk interacts with the bicellar systems. (2)H NMR experiments showed a disordering effect of Menk on the lipid chains in all the bicelles except Bic/PG, whereas the study of the choline headgroups indicated a decreased order of the lipids only in Bic/PE and Bic/PG. Our results suggest that the insertion depth of Menk into bicelles is modulated by their composition, more specifically by the balance between hydrophobic and electrostatic interactions. Menk would be buried at the lipid polar/apolar interface, the depth of penetration into the hydrophobic membrane core following the scaling Bic > Bic/PE > Bic/PS at the slightly acidic pH used in this study. The peptide would not insert into the bilayer core of Bic/PG and would rather remain at the surface. 相似文献
14.
(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes. 相似文献
15.
The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers 总被引:2,自引:0,他引:2
R P Rand V A Parsegian J A Henry L J Lis M McAlister 《Canadian journal of biochemistry》1980,58(10):959-968
We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC)--cholesterol-water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ?ngstr?ms (1 A = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart of 35-A separation from the 19-A characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability. 相似文献
16.
The interfacial region of dipalmitoylphosphatidylcholine bilayers is perturbed by fusogenic amphipaths.
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Several structural methods were used to probe the influence of three fusogenic and four nonfusogenic amphipaths on large, unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles. For four of these structural measurements there was a correlation observed between the ability of an amphipath to favor poly(ethylene glycol) (PEG)-induced fusion and the structural perturbation reported by each method. First, the fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenyhexa-1,3,5-triene (TMA-DPH), which probes the upper region of the bilayer, decreased in the range of PEG concentrations previously found to cause fusion of membranes containing fusogenic amphipaths. For nonfusogenic amphipaths, the anisotropy increased monotonically with PEG concentration. The properties of similar probes that locate in the hydrophobic core of the bilayer showed no correlation with fusogenicity, nor did the properties of probes purported to sense the aqueous surface of the membrane. Second, the frequency of the C=O stretch increased and then decreased dramatically as fusogenic but not nonfusogenic membranes were heated through their phase transition. Third, there was a dramatic increase in the frequency of the C-O-C ester stretch at the membrane order/disorder phase transition for membranes containing fusogenic amphipaths, twice the increase observed for nonfusogenic amphipaths. The spectral characteristics of phosphate, choline, and acyl chain motions showed no such correlation with fusogenicity. Finally, calorimetric measurements showed that low levels of fusogenic amphipaths eliminated the "pretransition" (L beta-->P beta) in DPPC membranes, whereas other amphipaths shifted but did not eliminate this transition. Taken together, these results indicate that fusogenic amphipaths perturb the interface or "backbone" region of the bilayer rather than the hydrophobic core, the headgroup, or the water interface regions of DPPC bilayers. 相似文献
17.
The effects of the polyene antibiotic filipin on the conductance and permeability of planar lipid bilayers were investigated under voltage-clamp conditions. The membrane conductance of lipid bilayers containing no cholesterol was not affected by filipin. In the presence of cholesterol containing lipid bilayers, filipin induced a 10(4)-10(5)-fold increase in transmembrane conductance. This conductance increase was dependent on the ionic species present in solution, decreasing in the following order: GCsCl greater than GNaAc greater than GKCl greater than GNaCl greater than CaCl2 greater than GNa2SO4 greater than GBaCl2 greater than GMgCl2. Reversal potential measurements in simple biionic conditions revealed the following relative permeability sequence: PK greater than PCl greater than PNa approximately Pac approximately PBa greater than PCs greater than PMg approximately PCa greater than Psulphate. The filipin-sterol mediated increase in membrane conductance was independent of the membrane potential. The increase in membrane current following a step alteration in membrane potential occurred instantaneously and had no dependence on the previous value of the holding membrane potential. We propose that the filipin-sterol complex forms ion channels in lipid membranes. These channels are found in a single configuration (open state) and select preferentially monovalent cations or anions over divalent ions. Our experimental results are discussed in relation to the effects of other polyene antibiotics on the membrane permeability, and also in relation to experimental problems previously reported with the use of filipin in planar lipid bilayers. 相似文献
18.
In this study amphotericin B released the divalent trace metals Zn2+, Co2+, Cu2+, Ni2+, Mn2+, Fe2+, Cd2+ and Pb2+ from multilamellar liposomes containing cholesterol. This observation is consistent with amphotericin B channels being permeable to these metals, and it is proposed, therefore, that the antibiotic may be useful in investigating the metabolism of these elements. 相似文献
19.
The (1)H NMR technique was applied to study binding of AmB, an antifungal drug, to lipid membranes formed with egg yolk phosphatidylcholine. The analysis of (1)H NMR spectra of liposomes, containing also cholesterol and ergosterol (at 40 mol%), shows that AmB binds preferentially to the polar headgroups. Such a binding restricts molecular motion of the choline fragment in the hydrophilic region at the surface of liposomes but increases the segmental motional freedom in the hydrophobic core. The same effects are also observed in the sterol-containing membranes, except that the effect on the hydrophobic core was exclusively observed in the membranes containing ergosterol. 相似文献
20.
Jacques Bolard Aline Vertut-Croquin Barbara E. Cybulska Claude M. Gary-Bobo 《生物化学与生物物理学报:生物膜》1981,647(2):241-248
Amphotericin B transfer between single-walled vesicles of dipalmitoylphosphatidylcholine (DPPC) and of egg phosphatidylcholine, both containing 10 mol% cholesterol, has been studied concurrently by circular dichroism spectroscopy and permeability measurements. At 22°C amphotericin B is rapidly transferred from DPPC to DPPC vesicles as well as from egg phosphatidylcholine to egg phosphatidylcholine vesicles. On the other hand, although amphotericin B is rapidly transferred from egg phosphatidylcholine to DPPC vesicles, it is not transferred from DPPC to egg phosphatidylcholine vesicles. At 48°C, above the transition temperature of DPPC, transfer occurs rapidly both ways. These results are interpreted in terms of difference of association constant of amphotericin B with vesicle membranes in the gel and liquid-crystalline state. 相似文献