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1.
In this study, we describe the effects of altered bilayer cholesterol content on reconstituted, protein-mediated sugar transport. The system used was the human erythrocyte sugar transporter (band 4.5) reconstituted into the bilayers of large unilamellar vesicles. Vesicle preparations were formed from synthetic lecithins whose bilayer cholesterol content ranged from 0 to 50 mol %. Transport was measured by microturbidimetric analysis over the temperature range of 0-65 degrees C while bilayer physical state was characterized by differential scanning calorimetry. Reconstituted transport activity was irreversibly lost between 62 and 65 degrees C. The Km for reconstituted transport was found to increase only slightly with increasing temperature and was not systematically affected by bilayer cholesterol content. The most striking observation of this study is that over certain critical cholesterol concentrations, as little as a 2.5% change in bilayer cholesterol can result in as much as a 100-fold change in Vmax per reconstituted protein. Our findings run counter to the view that increasing bilayer cholesterol content monotonically transforms a membrane into a state of "intermediate fluidity". Abrupt, cholesterol-induced bilayer reorganizations occurring at 15-20 and 30 mol % bilayer cholesterol are markedly reflected in altered sugar transport rates. Increasing the cholesterol content of crystalline distearoyllecithin bilayers inhibits the activity of the reconstituted transporter. It is apparent from these studies that bilayer "fluidity" is neither the sole nor a major determinant of the Indeed, we find the effect of cholesterol on transport activity is independent of its ability to fluidize membranes.  相似文献   

2.
The interaction of the galactocerebroside, N-palmitoylgalactosylsphingosine (NPGS), with cholesterol has been studied by differential scanning calorimetry (DSC) and x-ray diffraction. Thermal and structural studies demonstrate complex behavior characterized by two endothermic transitions: transition I (TI approximately equal to 50-60 degrees C) corresponding to an NPGS-cholesterol bilayer gel----bilayer liquid crystal transition II (TII where TI less than TII less than TNPGS) corresponding to an NPGS bilayer crystal (stable E form)----bilayer liquid crystal transition. For mixtures containing from 6 to 80 mol % cholesterol, x-ray diffraction studies at 22 degrees C (T less than TI) indicate two separate lamellar phases; an NPGS crystal bilayer phase and a cholesterol monohydrate phase. For cholesterol concentrations less than 50 mol % at TI less than T less than TII, NPGS-cholesterol liquid crystal bilayer and excess NPGS crystal bilayer phases are observed. For greater than 50 mol % cholesterol concentrations at these temperatures, an excess cholesterol monohydrate phase coexists with the NPGS-cholesterol liquid crystal bilayers. At T greater than TII, complete NPGS-cholesterol miscibility is only observed for less than 50 mol % cholesterol concentrations, whereas at greater than 50 mol % cholesterol an excess cholesterol phase is present. The solid phase immiscibility of cerebroside and cholesterol at low temperatures is suggested to result from preferential NPGS-NPGS associations via hydrogen bonding. The unique thermal and structural behavior of NPGS-cholesterol dispersions is contrasted with the behavior of cholesterol-phosphatidycholine and cholesterol-sphingomyelin bilayers. Thermal and structural studies of NPGS in dipalmitoylphosphatidylcholine (DPPC)/cholesterol (1:1, molar ratio) bilayers have been performed. For dispersions containing less than 20 mol % NPGS at 22 degrees C there are no observable calorimetric transitions and x-ray diffraction studies indicate complete lipid miscibility. At greater than 20 mol % NPGS, a high temperature transition is observed that is shown by x-ray diffraction studies to be due to an excess NPGS crystal bilayer----liquid crystal bilayer transition. Complete miscibility of NPGS in DPPC/cholesterol bilayers is observed at T greater than TNPGS. The properties of NPGS/DPPC/cholesterol bilayers are discussed in terms of the lipid composition of the myelin sheath.  相似文献   

3.
We carried out comparative DSC and Fourier transform infrared spectroscopic studies of the effects of cholesterol and lanosterol on the thermotropic phase behavior and organization of DPPC bilayers. Lanosterol is the biosynthetic precursor of cholesterol and differs in having three rather than two axial methyl groups projecting from the β-face of the planar steroid ring system and one axial methyl group projecting from the α-face, whereas cholesterol has none. Our DSC studies indicate that the incorporation of lanosterol is more effective than cholesterol is in reducing the enthalpy of the pretransition. Lanosterol is also initially more effective than cholesterol in reducing the enthalpies of both the sharp and broad components of the main phase transition. However, at sterol concentrations of 50 mol %, lanosterol does not abolish the cooperative hydrocarbon chain-melting phase transition as does cholesterol. Moreover, at higher lanosterol concentrations (~30–50 mol %), both sharp and broad low-temperature endotherms appear in the DSC heating scans, suggestive of the formation of lanosterol crystallites, and of the lateral phase separation of lanosterol-enriched phospholipid domains, respectively, at low temperatures, whereas such behavior is not observed with cholesterol at comparable concentrations. Our Fourier transform infrared spectroscopic studies demonstrate that lanosterol incorporation produces a less tightly packed bilayer than does cholesterol, which is characterized by increased hydration in the glycerol backbone region of the DPPC bilayer. These and other results indicate that lanosterol is less miscible in DPPC bilayers than is cholesterol, but perturbs their organization to a greater extent, probably due primarily to the rougher faces and larger cross-sectional area of the lanosterol molecule and perhaps secondarily to its decreased ability to form hydrogen bonds with adjacent DPPC molecules. Nevertheless, lanosterol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers, although this phase is not as tightly packed as comparable cholesterol/DPPC mixtures.  相似文献   

4.
The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.  相似文献   

5.
Stearoylsphingomyelin (SSM) bilayers containing 0, 22, and 50 mol % cholesterol (Chol) and a pentadecanoyl-stearoylphosphatidylcholine (15SPC) bilayer containing 22 mol % Chol were molecular dynamics simulated at two temperatures (37 degrees C and 60 degrees C). 15SPC is the best PC equivalent of SSM. The Chol effect on the SSM bilayer differs significantly from that on the 15SPC bilayer. At the same temperature and Chol content, H-bonding of Chol with SSM is more extensive than with 15SPC. SSM-Chol H-bonding anchors the OH group of Chol in the lower regions of the SSM-Chol bilayer interface. Such a location strengthens the influence of Chol on the SSM chains. In effect, the phase of the SSM-Chol bilayer containing 22 mol % Chol at 37 degrees C is shifted from the gel to the liquid-ordered phase, and the bilayer displays similar properties below and above the main phase-transition temperature for a pure SSM bilayer of approximately 45 degrees C. In contrast, due to a higher location, Chol is not able to change the phase of the 15SPC-Chol bilayer, which at 37 degrees C remains in the gel phase. Chol affects both the core and interface of the SSM bilayer. With increasing Chol content, the order of SSM chains and hydration of SSM headgroups increase, whereas polar interactions between lipids decrease.  相似文献   

6.
J T Kim  J Mattai  G G Shipley 《Biochemistry》1987,26(21):6599-6603
Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melting) transition, TM = 44.2 degrees C; a pretransition, TP = 36.2 degrees C; and a subtransition, TS = 5.5 degrees C. DPPC shows two reversible transitions: TM = 41.3 degrees C and TP = 36.5 degrees C. TM decreases linearly from 44.2 to 41.3 degrees C as DPPC is incorporated into DHPC bilayers; TP exhibits eutectic behavior, decreasing sharply to reach 23.3 degrees C at 40.4 mol % DPPC and then increasing over the range 40-100 mol % DPPC; TS remains constant at 4-5 degrees C and is not observed at greater than 20 mol % DPPC. At 50 degrees C, X-ray diffraction shows a liquid-crystalline bilayer L alpha phase at all DHPC:DPPC mole ratios. At 22 degrees C, DHPC shows an interdigitated bilayer gel L beta phase (bilayer periodicity d = 47.0 A) into which approximately 30 mol % DPPC can be incorporated. Above 30 mol % DPPC, a noninterdigitated gel L beta' phase (d = 64-66 A) is observed. Thus, at T greater than TM, DHPC and DPPC are miscible in all proportions in an L alpha bilayer phase. In contrast, a composition-dependent gel----gel transition between interdigitated and noninterdigitated bilayers is observed at T less than TP, and this leads to eutectic behavior of the DHPC/DPPC system.  相似文献   

7.
Aqueous dispersions of a porcine lung surfactant (PLS) extract with and without cholesterol supplementation were analyzed by X-ray scattering. Lamellar liquid-crystalline and gel-type bilayer phases are formed, as in pure phosphatidylcholine (PC)-cholesterol systems. This PLS extract, developed for clinical applications, has a cholesterol content of less than 1% (w/w). Above the limit of swelling, the bilayer structure shows a melting (main) transition during heating at about 34 degrees C. When 13 mol% cholesterol was added to PLS, so that the cholesterol content of natural lung surfactant was reached, the X-ray scattering pattern showed pronounced changes. The main transition temperature was reduced to the range 20-25 degrees C, whereas according to earlier studies of disaturated PC-cholesterol bilayers in water the main transition remains almost constant when the amount of solubilized cholesterol is increased. Furthermore, the changes in scattering pattern at passing this transition in PLS-cholesterol samples were much smaller than at the same transition in PLS samples. These effects of cholesterol solubilization can be related to phase segregation within the bilayers, known from pure PC-cholesterol systems. One phase, solubilizing about 8 mol% cholesterol, exhibits a melting transition, whereas the other bilayer phase, with a liquid-crystalline disordered conformation, has a cholesterol content in the range 20-30 mol% and this phase shows no thermal transition. The relative amount of bilayer lipids that is transformed at the main transition in the PLS-cholesterol sample is therefore only half compared to that in PLS samples. The reduction in transition temperature in the segregated bilayer of lung surfactant lipids is probably an effect of enrichment of disaturated PC species in the phase, which is poor in cholesterol. This work indicates that cholesterol in lung surfactant regulates the crystallization behavior.  相似文献   

8.
We have examined the effects of cholesterol on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylserines by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. We find that the incorporation of increasing quantities of cholesterol progressively reduces the temperature, enthalpy, and cooperativity of the gel-to-liquid-crystalline phase transition of the host phosphatidylserine bilayer, such that a cooperative chain-melting phase transition is completely or almost completely abolished at 50 mol % cholesterol, in contrast to the results of previous studies. We are also unable to detect the presence of a separate anhydrous cholesterol or cholesterol monohydrate phase in our binary mixtures, again in contrast to previous reports. We further show that the magnitude of the reduction in the phase transition temperature induced by cholesterol addition is independent of the hydrocarbon chain length of the phosphatidylserine studied. This result contrasts with our previous results with phosphatidylcholine bilayers, where we found that cholesterol increases or decreases the phase transition temperature in a chain length-dependent manner (1993. Biochemistry, 32:516-522), but is in agreement with our previous results for phosphatidylethanolamine bilayers, where no hydrocarbon chain length-dependent effects were observed (1999. Biochim. Biophys. Acta, 1416:119-234). However, the reduction in the phase transition temperature by cholesterol is of greater magnitude in phosphatidylethanolamine as compared to phosphatidylserine bilayers. We also show that the addition of cholesterol facilitates the formation of the lamellar crystalline phase in phosphatidylserine bilayers, as it does in phosphatidylethanolamine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of cholesterol. We ascribe the limited miscibility of cholesterol in phosphatidylserine bilayers reported previously to a fractional crystallization of the cholesterol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. In general, the results of our studies to date indicate that the magnitude of the effect of cholesterol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipid dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.  相似文献   

9.
The association of ethanol with unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes of varying cholesterol content has been investigated by isothermal titration calorimetry over a wide temperature range (8-45 degrees C). The calorimetric data show that the interaction of ethanol with the lipid membranes is endothermic and strongly dependent on the phase behavior of the mixed lipid bilayer, specifically whether the lipid bilayer is in the solid ordered (so), liquid disordered (ld), or liquid ordered (lo) phase. In the low concentration regime (<10 mol%), cholesterol enhances the affinity of ethanol for the lipid bilayer compared to pure DMPC bilayers, whereas higher levels of cholesterol (>10 mol%) reduce affinity of ethanol for the lipid bilayer. Moreover, the experimental data reveal that the affinity of ethanol for the DMPC bilayers containing small amounts of cholesterol is enhanced in the region around the main phase transition. The results suggest the existence of a close relationship between the physical structure of the lipid bilayer and the association of ethanol with the bilayer. In particular, the existence of dynamically coexisting domains of gel and fluid lipids in the transition temperature region may play an important role for association of ethanol with the lipid bilayers. Finally, the relation between cholesterol content and the affinity of ethanol for the lipid bilayer provides some support for the in vivo observation that cholesterol acts as a natural antagonist against alcohol intoxication.  相似文献   

10.
Properties of hydrated unsaturated phosphatidylcholine (PC) lipid bilayers containing 40 mol % cholesterol and of pure PC bilayers have been studied. Various methods were applied, including molecular dynamics simulations, self-consistent field calculations, and the pulsed field gradient nuclear magnetic resonance technique. Lipid bilayers were composed of 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules. Lateral self-diffusion coefficients of the lipids in all these bilayers, mass density distributions of atoms and atom groups with respect to the bilayer normal, the C-H and C-C bond order parameter profiles of each phospholipid hydrocarbon chain with respect to the bilayer normal were calculated. It was shown that the lateral self-diffusion coefficient of PC molecules of the lipid bilayer containing 40 mol % cholesterol is smaller than that for a corresponding pure PC bilayer; the diffusion coefficients increase with increasing the degree of unsaturation of one of the PC chains in bilayers of both types (i.e., in pure bilayers or in bilayers with cholesterol). The presence of cholesterol in a bilayer promoted the extension of saturated and polyunsaturated lipid chains. The condensing effect of cholesterol on the order parameters was more pronounced for the double C=C bonds of polyunsaturated chains than for single C-C bonds of saturated chains.  相似文献   

11.
In this study, we have examined how the headgroup size and properties affect the membrane properties of sphingomyelin and interactions with cholesterol. We prepared N-palmitoyl ceramide phosphoethanolamine (PCPE) and compared its membrane behavior with D-erythro-N-palmitoyl-sphingomyelin (PSM), both in monolayers and bilayers. The pure PCPE monolayer did not show a phase transition at 22 degrees C (in contrast to PSM), but displayed a much higher inverse isothermal compressibility as compared to the PSM monolayer, indicating stronger intermolecular interactions between PCPEs than between PSMs. At 37 degrees C the PCPE monolayer was more expanded (than at 22 degrees C) and displayed a rather poorly defined phase transition. When cholesterol was comixed into the monolayer, a condensing effect of cholesterol on the lateral packing of the lipids in the monolayer could be observed. The phase transition from an ordered to a disordered state in bilayer membranes was determined by diphenylhexatriene steady-state anisotropy. Whereas the PSM bilayer became disordered at 41 degrees C, the PCPE bilayer main transition occurred around 64 degrees C. The diphenylhexatriene steady-state anisotropy values were similar in both PCPE and PSM bilayers before and after the phase transition, suggesting that the order in the hydrophobic core in both bilayer types was rather similar. The emission from Laurdan was blue shifted in PCPE bilayers in the gel phase when compared to the emission spectra from PSM bilayers, and the blue-shifted component in PCPE bilayers was retained also after the phase transition, suggesting that Laurdan molecules sensed a more hydrophobic environment at the PCPE interface compared to the PSM interface both below and above the bilayer melting temperature. Whereas PSM was able to form sterol-enriched domains in dominantly fluid bilayers (as determined from cholestatrienol dequenching experiments), PCPE failed to form such domains, suggesting that the size and/or properties of the headgroup was important for stabilizing sphingolipid/sterol interaction. In conclusion, our study has highlighted how the headgroup in sphingomyelin affect its membrane properties and interactions with cholesterol.  相似文献   

12.
Tran R  Ho S  Dea P 《Biophysical chemistry》2004,110(1-2):39-47
Differential scanning calorimetry (DSC) and fluorescence spectroscopy are useful techniques for investigating the phase transitions of phospholipid bilayers. In this study, these methods have been extended to determine the effects of ethanol on DSPC and DSPC/2 mol.% cholesterol bilayers. The biphasic effect of the main transition was observed on the DSC heating scans above 0.60 M ethanol. In addition, the concentration at which the biphasic effect occurs is not significantly changed in the presence of 2 mol.% cholesterol. For the fluorescence studies, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into the bilayer to monitor the phase transitions through the displacement of DPH. This fluorescent probe is used to directly determine the onset of interdigitation in the bilayer systems as indicated by a large decrease in the DPH fluorescence intensity. The addition of cholesterol lowered and broadened the transition temperatures of the phosphatidylcholine (PC) system. However, 2 mol.% cholesterol did not have a significant effect on the induction of the interdigitated phase in DSPC as observed from the small difference in ethanol threshold concentration for the two systems. This suggests that DSPC forms a more stable interdigitated gel phase than other PCs with shorter acyl chains.  相似文献   

13.
Ali MR  Cheng KH  Huang J 《Biochemistry》2006,45(41):12629-12638
The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.  相似文献   

14.
A dynamic light scattering setup was used to study the undulations of freely suspended planar lipid bilayers, the so-called black lipid membranes, over a previously inaccessible range of frequency and wave number. A pure synthetic lecithin bilayer, 1,2-dielaidoyl-sn-3-glycero-phoshatidylcholine (DEPC), and binary mixtures of DEPC with 40 mol % of cholesterol, ergosterol, or lanosterol were studied. By analyzing the dynamic light scattering data (oscillation and damping curves) in terms of transverse shear motion, we extracted the lateral tension and surface viscosity of the composite bilayers for each sterol. Cholesterol gave the strongest increase in lateral tension (approximately sixfold) with respect to the DEPC control, followed by lanosterol (approximately twofold), and ergosterol (1.7-fold). Most interestingly, only cholesterol simultaneously altered the surface viscosity of the bilayer by almost two orders of magnitude, whereas the other two sterols did not affect this parameter. We interpret this unique behavior of cholesterol as a result of its previously established out-of-plane motion which allows the molecule to cross the bilayer midplane, thereby effectively coupling the bilayer leaflets to form a highly flexible but more stable composite membrane.  相似文献   

15.
Membrane interactions with β-amyloid peptides are implicated in the pathology of Alzheimer's disease and cholesterol has been shown to be key modulator of this interaction, yet little is known about the mechanism of this interaction. Using atomic force microscopy, we investigated the interaction of monomeric Aβ(1-40) peptides with planar mica-supported bilayers composed of DOPC and DPPC containing varying concentrations of cholesterol. We show that below the bilayer melting temperature, Aβ monomers adsorb to, and assemble on, the surface of DPPC bilayers to form layers that grow laterally and normal to the bilayer plane. Above the bilayer melting temperature, we observe protofibril formation. In contrast, in DOPC bilayers, Aβ monomers exhibit a detergent-like action, forming defects in the bilayer structure. The kinetics of both modes of interaction significantly increases with increasing membrane cholesterol content. We conclude that the mode and rate of the interaction of Aβ monomers with lipid bilayers are strongly dependent on lipid composition, phase state and cholesterol content.  相似文献   

16.
T Y Tson- 《Biochemistry》1975,14(25):5415-5417
The transport of 8-anilino-1-naphthalenesulfonate in dimyristoyl-L-alpha-lecithin bilayers has been found to be extremely sensitive to the crystalline state of the phospholipid dispersions. Thus this reaction may be used for probing the membrane structures. In binary mixtures of cholesterol and phospholipid the fluorescence enhancement of the dye completely disappears when the mole fraction of cholesterol reaches 33%. At temperatures below and above the phase transition of the lipid bilayers, the rate of the probe transport increases significantly in the binary mixtures. It reaches a maximum at 17 mol % of cholestero. The rate at this cholesterol content approaches the maximum value obtained for the probe transport in pure phospholipis, e.i., the rate at the midpoint of the phase transition. These observations indicate that the effect of cholesterol in the phospholipid dispersion is to maintain the bilayer structure close to the melting temperature of the lipid phase transition. In other words, cholesterol may be an effective buffer for membrane crystalline state when its concentration is near 17 mol %.  相似文献   

17.
Villar AV  Alonso A  Goñi FM 《Biochemistry》2000,39(46):14012-14018
Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.  相似文献   

18.
We performed differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopic measurements to study the effects of lathosterol (Lath) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes and compared our results with those previously reported for cholesterol (Chol)/DPPC binary mixtures. Lath is the penultimate intermediate in the biosynthesis of Chol in the Kandutsch-Russell pathway and differs from Chol only in the double bond position in ring B, which is between C7 and C8 in Lath and between C5 and C6 in Chol. Our DSC studies indicate that the incorporation of Lath is more effective than Chol in reducing the temperature and enthalpy of the DPPC pretransition. At lower sterol concentrations (≤10 mol %), incorporation of both Lath and Chol decreases the temperature, enthalpy, and cooperativity of the sharp component of the main phase transition of DPPC to a similar extent, but at higher sterol concentrations, Lath is more effective at decreasing the phase transition temperature, enthalpy, and cooperativity than Chol. These results indicate that at higher concentrations, Lath is more disruptive of DPPC gel-state bilayer packing than Chol is. Moreover, incorporation of Lath decreases the temperature of the broad component of the main phase transition of DPPC, whereas Chol increases it; this difference in the direction and magnitude of the temperature shift is accentuated at higher sterol concentrations. Although at sterol concentrations of ≤20 mol % Lath and Chol are almost equally effective at reducing the enthalpy and cooperativity of the broad component of the main phase transition, at higher sterol levels Lath is less effective than Chol in these regards and does not completely abolish the cooperative hydrocarbon chain melting phase transition at 50 mol %, as does Chol. These latter results indicate that Lath both is more disruptive with respect to the low-temperature state of the sterol-enriched domains of DPPC bilayers and has a lower lateral miscibility in DPPC bilayers than Chol. Our FTIR spectroscopic studies suggest that Lath incorporation produces a less tightly packed bilayer than does Chol at both low (gel state) and high (liquid-crystalline state) temperatures, which is characterized by increased H-bonding between water and the carbonyl groups of the fatty acyl chains in the DPPC bilayer. Overall, our studies indicate that Lath and Chol incorporation can have rather different effects on the thermotropic phase behavior and organization of DPPC bilayers and thus that the position of the double bond in ring B of a sterol molecule can have an appreciable effect on the physical properties of sterol molecules.  相似文献   

19.
Sphingomyelins (SMs) are order-imposing phospholipids in cell membranes which interact favorably with cholesterol. The hydrophobic part of SM constitutes a long-chain base with an amide-linked acyl chain, whereas the polar head group is phosphocholine. The long-chain base has a free hydroxyl group in position 3, which is an important donor/acceptor in hydrogen bonding. In newborn mammals, a SM in which a palmitic acid is esterified to the 3-OH has been reported. We have synthesized this SM analog (3O-P-PSM) and studied its properties in bilayer membranes, and also determined its interactions with cholesterol. Fully hydrated 3O-P-PSM bilayers underwent a gel-to-liquid crystalline phase transition at 55.5 °C (ΔH 8 kcal/mol), which is about 15 °C higher than the phase transition temperature of PSM. The 3O-P-PSM displayed rather poor miscibility with PSM in mixed bilayers, suggesting that the third acyl chain interfered significantly with lateral interactions. Bilayers made from 3O-P-PSM were much more resistant to detergent-induced solubilization than bilayers made from PSM. In binary bilayers, cholesterol was able to destabilize the gel phase, and order the fluid phase of 3O-P-PSM, in a concentration-dependent manner. Cholesterol was also able to form sterol-enriched ordered domains with 3O-P-PSM in fluid POPC bilayers. The interaction between cholesterol and 3O-P-PSM was not, however, as favorable as the interaction between cholesterol and PSM. It is unclear what physiological role 3O-P-PSM could play in newborn mammalian membranes. However, it is clear that 3O-P-PSM will form more highly ordered domains than PSM while still having a limited ability to interact with cholesterol.  相似文献   

20.
M Straume  B J Litman 《Biochemistry》1987,26(16):5121-5126
The influence of cholesterol on equilibrium and dynamic bilayer structure in minimally to highly unsaturated phosphatidylcholine (PC) vesicles has been examined by characterization of the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Large, unilamellar egg PC, palmitoyloleoyl-PC (POPC), dioleoyl-PC (DOPC), palmitoylarachidonoyl-PC (PAPC), and palmitoyldocosahexaenoyl-PC (P-22:6-PC) vesicles containing no cholesterol or approximately 15 or 30 mol % cholesterol have been examined. Equilibrium and dynamic DPH orientational properties were analyzed according to an orthogonal, bimodal orientational distribution function [Straume, M., & Litman, B.J. (1987) Biochemistry (preceding paper in this issue)]. The same mathematical formalism was applied to TMA-DPH except that probe orientational probability was permitted only in the distribution peak aligned parallel to the bilayer normal. TMA-DPH fluorescence lifetimes were consistently increased by incorporation of cholesterol into these vesicles. Greater acyl chain unsaturation and increasing temperature each promoted reduction of lifetimes in the presence or absence of cholesterol. DPH lifetimes were much less sensitive than those of TMA-DPH to changes in composition or temperature. This behavior is consistent with reduced water penetrability into liquid-crystalline bilayers as cholesterol content is increased and as acyl chain unsaturation and temperature are reduced. Cholesterol also induces substantial equilibrium ordering of the bilayer both at the hydrophobic core and at the bilayer-water interface. DPH orientational distributions were shifted in favor of alignment parallel to the acyl side chains. The distributions of both probes were narrowed in response to incorporation of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

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