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1.
Phosphorus-31 NMR studies of solutions of small L-alpha-dipalmitoyl phosphatidylcholine bilayer vesicles containing sodium dimethyl phosphate uniformly distributed between the continuous external and the intravesicular aqueous spaces, with the paramagnetic shift reagent Pr3+ present only in the external space, are reported. These studies give the distribution both of dipalmitoyl phosphatidylcholine in the vesicle inner and outer monolayers and of dimethyl phosphate in the aqueous spaces. With the third necessary parameter obtained from the vesicle sedimentation coefficient, the very different packing parameters of dipalmitoyl phosphatidylcholine in inner and outer monolayers can be determined. The vesicle outer radius is 109 A. Although the total bilayer thickness is virtually identical to that of planar bilayers, the outer monolayer is thicker (20 A) and the inner monolayer thinner (15 A). The area per head group at the inner surface, 68 A2, is like the planar value, but the tails are much more folded, so as to decrease the radial lengths and increase the tangential spreat (to 94A2). The reverse is true in the outer layer: the surface per head group is 76 A2, tapering to 51 A2 in the tail region, so that outer layer tails are relatively extended. The difference is equivalent to a shift of about two 2g1 kinks from outer to inner layers; the uneven packing certainly affects fluidity, and may have important biological consequences. 相似文献
2.
L I Barsukov Y E Shapiro A V Viktorov V I Volkova V F Bystrov L D Bergelson 《Chemistry and physics of lipids》1975,14(3):211-226
Phospholipid exchange between phosphatidylinositol and phosphatidylcholine vesicles has been studied by NMR spectroscopy with use of hydrophilic paramagnetic lanthanide probes (Pr3+ and Eu3+ ions). The dependence of the lanthanide induced shifts in the 1H and 31P NMR spectra on the phospholipid composition of the vesicles could be used for its quantitative evaluation. The method has been proved to be applicable for studying phospholipid exchange stimulated by soluble proteins (postmicrosomal supernatant fraction) from rat liver. Furthermore it has been shown that the phospholipid molecules newly introduced by protein-stimulated exchange are predominantly incorporated into the outer monolayer of the vesicular bilayer membrane. This makes it possible to produce liposomes with asymmetric distribution of the phospholipids across the bilayer. 相似文献
3.
The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg2+, Ca2+, Mn2+, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg2+, 7 mM for Sr2+, 6 mM for Ca2+, 3.5 mM for Ba2+ and 1.8 mM for Mn2+. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn2+ > Ca2+ > Mg2+ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn2+ and Ca2+, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The 相似文献
4.
P.J. Konigsberg 《生物化学与生物物理学报:生物膜》1982,685(3):355-366
Ca2+-ATPase and other membrane proteins of the sarcoplasmic reticulum membrane from rabbit skeletal muscle have been reconstituted into lipid vesicles with increasing amounts of phosphatidylcholine. The protein composition and phospholipid concentration of these vesicles were analyzed by determining the density of the reconstituted membrane vesicles on linear H2O-2H2O gradients, in a constant concentration of sucrose. In all combinations of the Ca2+-ATPase with a weight excess of phosphatidylcholine, the reconstituted vesicles had a phospholipid-to-protein ratio similar to that of the native sarcoplasmic reticulum membrane, even though both solubilization and mixing had occurred. These vesicles of low phospholipid and high protein content exhibited all the original Ca2+-ATPase activity and ATP-stimulated calcium transport. The Ca2+-ATPase, and the calcium-binding proteins to a lesser extent, may order the lipid in such a manner so as to maintain the initial stoichiometry of lipid to protein observed in the native sarcoplasmic reticulum membrane. 相似文献
5.
Use of an oriented transmembrane protein to probe the assembly of a supported phospholipid bilayer. 总被引:2,自引:1,他引:1
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Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer. 相似文献
6.
Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca2 + ATPase (SERCA) and helps to regulate Ca2 + flow. PLB inhibits SERCA impairing Ca2 + translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by 31P and 2H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The 31P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases 31P T1 values in the lipid headgroup region. 2H SCD order parameters of 2H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function. 相似文献
7.
Membrane fusion induced by Ca2+ and Mg2+ in large unilamellar vesicles composed of mixtures of phosphatidylethanolamine with phosphatidate and phosphatidylinositol was studied by means of a fluorescence assay for the intermixing of internal aqueous contents of the vesicles. The threshold concentrations of Ca2+ or Mg2+ required for fusion increased only moderately when up to 80 mol% phosphatidylethanolamine was included with phosphatidate at pH 7.4, but no fusion could be detected in vesicles containing 70 mol% phosphatidylcholine even at high concentrations of Ca2+ or Mg2+. Phosphatidate-phosphatidylethanolamine (1 : 4) vesicles could be induced to fuse by 0.1 mM Ca2+ in the presence of a Mg2+ concentration which alone was insufficient for fusion. When equimolar amounts of phosphatidylethanolamine was included with phosphatidylinositol, the vesicles were susceptible to fusion by Ca2+, although pure phosphatidylinositol vesicles themselves merely aggregate and do not fuse (Sundler, R. and Papahadjopoulos, D. (1981) Biochim. Biophys. Acta 649, 743–750, accompanying paper). The role of phosphatidylethanolamine acyl chains, and hence the possible involvement of the bilayer-hexagonal (HII) transition in membrane fusion, was examined by the temperature dependence of Ca2+-induced fusion in phosphatidylinositol-dimyristoylphosphatidylethanolamine (1 : 1) vesicles. Fusion was strictly dependent on the gel-liquid crystalline transition of the mixture and not on the phase behavior of the phosphatidylethanolamines. Comparable fusion rates were obtained for both egg yolk phosphatidylethanolamine and dimyristoylphosphatidylethanolamine at 50°C. As the dimyristoylphosphatidylethanolamine does not convert to a non-bilayer phase in this temperature range, we conclude that the bilayer-hexagonal transition is not necessary for membrane fusion. We propose that the dehydration characteristics of the phospholipids and their metal ion complexes are the critical factors determining fusion suceptibility of phospholipid membranes. 相似文献
8.
《Chemistry and physics of lipids》1986,39(3):193-202
19F-labeled phospholipids, L-α-bis-(ω-fluoro palmitoyl)phosphatidylcholine, L-α-bis-(12,12-difluoro stearoyl)phosphatidylcholine and L-α-bis-(6,6-difluoro palmitoyl)phosphatidylcholine, were incorporated in phospholipid vesicles by sonication of aqueous lipid emulsions. Vesicles were prepared both from the pure fluorine substituted phospholipids as well as from lipid mixtures obtained by combining the fluorine substituted lipids with the synthetic phospholipids, L-α-dilauroyl-, L-α-dimyristoyl-, L-α-dipalmitoyl- and L-α-distearoyl-phosphatidylcholine. Characterization by gel permeation chromatography showed that stable unilamellar vesicles with diameters of ≌200 Å could be obtained with a minimum or absence of multilamellar material. The vesicles give rise to two 19F resonances in most cases as observed previously [K.J. Longmuir and F.W. Dahlquist, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2716]. The chemical shift differences undergo systematic changes that confirm the interpretations that the dual 19F resonances arise from the inner and outer halves of the vesicle bilayer. The shift separation increases systematically as the fluorine label is positioned closer to the phospholipid headgroup and decreases systematically with increasing temperature. Both observations agree with what is currently known about phospholipid vesicle structure. Anomalous results are obtained with DSPC as host vesicle since only a single resonance of inbedded fluorinated phospholipids is found. 相似文献
9.
Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration. 相似文献
10.
An internal NMR monitor for the study of lanthanide ion (Ln3+) binding to phospholipid bilayer membranes has been developed. The dimethylphosphate anion, DMP?, forms labile complexes with Ln3+ in aqueous solution and in solutions also containing bilayer dispersions. The hyperfine shift in the DMP? resonance induced by Pr3+ ions has been used to determine the overall thermodynamic formation constants for the Pr(DMP)2+ and Pr(DMP)2+ complexes: 81 (M?1) and 349 (M?2) at 52°C; the limiting hyperfine shift (31P) at 52°C is 91.5 ppm downfield. These parameters, applied to the observed DMP? hyperfine shift in the presence of the membrane, establish both the free Pr3+ concentration and the amount of Pr3+ bound to the phospholipid surface. Extensive data for the binding of Pr3+ to the outer surfaces of sonicated vesicles yield a limiting hyperfine shift per Pr3+ of 181.6 ppm downfield for the dipalmitoylphosphatidylcholine 31P resonance at 52°C, clearly demonstrating that the binding stoichiometry is two DPPCs per Pr3+. A Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherm incorporating electrostatic considerations at the Debye-Hückel level. Fittings to specific models lead to a cooperative model in which tense (T) sites, with low affinity for Pr3+, present in the absence of metal ions, quickly give way to relaxed (R) sites (two DPPCs per site), with much higher affinity for Pr3+, as the amount of Pr3+ bound to the surface increases. The intrinsic equilibrium constants for the binding of Pr3+ to DPPC vesicles are 2 M?1 and 3 000 M?1 for the T and R sites, respectively, at 52°C. The distribution coefficient between these sites ([R]/[T]) in the absence of Ln3+ is 0.14 at 52°C. We picture the binding site conversion as a head-group conformational change involving mostly the choline moiety. Sketchy results for binding on the inside vesicle surface indicate that the overall affinity for Pr3+ is significantly greater and suggest that the site stoichiometry may be different. 相似文献
11.
M Ueno 《Biochimica et biophysica acta》1987,904(1):140-144
Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside. 相似文献
12.
R J Kurland M Hammoudah S Nir D Papahadjopoulos 《Biochemical and biophysical research communications》1979,88(3):927-932
Phosphorus-31 NMR lines corresponding to inner and outer surfaces of sonicated phosphatidylserine vesicles can be distinguished by the effects of added Ca2+ or Mg2+ at low bulk concentrations (millimolar or less). The changes in chemical shift and relaxation times indicate that Ca2+ binds directly to the PS phosphate, neutralizing at least a portion of the negative charge and restricting the motion of this group. Mg2+ ion also binds to the head group, but apparently not as strongly as Ca2+, nor is the mobility of the headgroup affected as much. 相似文献
13.
Sarcoplasmic reticulum membranes from skeletal muscle contain a proteolipid () which reduces both the nonspecific ion and water permeabilities of artificial planar phospholipid bilayers. The proteolipid does not show any ionophoric effect or specific pore formation for Ca2+. The a.c. capacitance of the bilayers is unaffected whereas the refractive index is increased by the presence of proteolipid. The results support the view that the proteolipid interacts with the phospholipids in the bilayer interior and causes a condensation in the packing of the alkyl chains. 相似文献
14.
Dan A. Klaerke Steven J. D. Karlish Peter L. Jørgensen 《The Journal of membrane biology》1987,95(2):105-112
Summary A barium-sensitive Ca-activated K+ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K+ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K+ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K+-channel activity is assayed by measurement of86Rb+ uptake against a large opposing K+ gradient. The reconstituted K+ channel is activated by Ca2+ in the physiological range of concentration (K1/22×10–7
m at pH 7.2) as found for the K+ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba2+, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K+ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K+-channel protein.Titration with Ca2+ shows that most of the active K+ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca2+ ionophore in order to observe Ca2+ stimulation. The reconstituted K+ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K+ channel in absence of Ca2+, to the level of activity seen with saturating concentrations of Ca2+. This tryptic split is located in a cytoplasmic aspect of the K+ channel that appears to be involved in opening and closing the K+ channel in response to Ca2+ binding. 相似文献
15.
The translocation of Ca2+ across phospholipid bilayers induced by a synthetic neutral Ca2+-ionophore
The effect of a neutral synthetic Ca2+-ligand, which induces selective Ca2+-transport in electrodialysis experiments in bulk membranes, on the Ca2+ permeability of phospholipid bilayers has been investigated. The ligand is able to promote the transport of Ca2+ across synthetic phospholipid bilayers and can therefore be classified as a Ca2+-ionophore. Its activity is enhancedby the uncoupler carbonyl cyanide (FCCP). The efficiency of the neutral carrier-mediated Ca2+ transport is rather low as compared with that of the charged Ca2+-ionophore X537A.The Ca2+ selectivity of the neutral ionophore is decreased by its incorporation in the low dielectric ambient of the phospholipid bilayer. 相似文献
16.
The phase behavior of bovine rod outer segment disk lipids has been investigated using freeze-fracture and 31P nuclear magnetic resonance (NMR) techniques. 31P-NMR spectra of isolated disk membranes were taken as a function of temperature between 25°C and 45°C. The 31P-NMR spectrum characteristic of phospholipid bilayers was observed at all temperatures both in the absence of Ca2+ and in the presence of 10 mM and 50 mM Ca2+. A similar study was performed on lipids isolated from the disk membranes. In the absence of Ca2+ only lamellar phase behavior was observed. In the presence of less than 10 mM Ca2+, however, there was a change in morphology to non-lamellar structures. Removal of the Ca2+ caused the system to reassume the lamellar form. 相似文献
17.
The exchange of phosphatidylcholine between [32P]phosphatidylcholine liposomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of [32P]phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homogeneous pool of small single walled vesicles. Treatment of phosphatidyl [14C]choline liposomes with phospholipase D (phosphatidylcholine phosphatidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed could not be hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of Phosphatidylcholine from the inner to the outer layer of the liposome bilayer. 相似文献
18.
L I Barsukov Y E Shapiro A V Viktorov V I Volkova V F Bystrov L D Bergelson 《Biochemical and biophysical research communications》1974,60(1):196-203
NMR spectroscopy with the use of non-penetrating paramagnetic probes permits in situ determination of the composition of the outer surface of phospholipid vesicles. The method was employed to follow the phospholipid exchange between phosphatidylinositol and phosphatidylcholine vesicles induced by a postmicrosomal protein fraction from rat liver. The effects of these proteins on the lipid bilayer and the structure of the vesicles produced by exchange were studied. 相似文献
19.
Resealing of erythrocyte ghosts in the presence of 4.5 mm Ca2+ induces the segregation of small membrane vesicles with a very high phospholipid:protein ratio and a high lysolecithin content. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the vesicles consist mainly of the high molecular spectrin peptides, the Ca2+-induced increase of band IIa (Mr 198,000) which is not extractable at low ionic strength, and a weak peptide band in the 72,000 Mr region. Ca2+ ghosts and vesicles show significant differences with regard to the specific activities of several membrane-associated enzymes. The segregated vesicles dispose of an efficient outwarddirected Ca2+-transport system. 相似文献
20.
The interaction of the 5-14, 1-14, and 1-24 fragments of ACTH with sonicated phospholipid bilayers containing egg yolk phosphatidylcholine (EPC) either pure or mixed with 10 mole % phosphatidic acid (EPA), was investigated by proton nuclear magnetic resonance (1H-nmr). The effects observed with zwitterionic EPC vesicles were small, indicating a low binding of the ACTH derivatives. The N-terminal aromatic resonances of the ACTH peptides were markedly broadened in the presence of negatively charged vesicles (EPC/EPA 9:1 M/M), while those of the C-terminal end were barely affected, showing that ACTH interacts with its N-terminal fragment. The choline resonance of the EPC molecules of the outer monolayer was shifted and broadened upon ACTH binding to the lipid vesicles, while that of the inner layer was not affected, suggesting that the peptide molecules interact only with the external leaflet of the lipid bilayer. The C2H and C4H resonances of the histidine-6 side chain were both shifted downfield upon peptide binding to the negatively charged lipid interface. In the case of the 1–24 derivative, these resonances were also split into two signals reflecting two different species of membrane-bound ACTH 1–24. Analysis of the line width and chemical shift variations of the ACTH and lipid resonances observed upon peptide binding shows that the membrane-binding potency of the shorter 5–14+1 fragment, which presents a +1 net charge, is roughly similar to that of the highly cationic 1–24+6 (net charge +6) derivative, implying that the 15–24+5 segment is not essential for membrane binding. The nmr measurements at a fixed lipid-to-peptide ratio in the presence of increasing amounts of spin-labeled lipids demonstrate that the N-terminal fragment of ACTH does not penetrate the hydrophobic core of the bilayer, and should lie parallel to the membrane surface. © 1997 John Wiley & Sons, Inc. Biopoly 42: 731–744, 1997 相似文献