Cholesterol Modulates the Interaction of β-Amyloid Peptide with Lipid Bilayers

The interaction of an amphiphilic, 40-amino acid β-amyloid (Aβ) peptide with liposomal membranes as a function of sterol mole fraction (X_sterol) was studied based on the fluorescence anisotropy of a site-specific membrane sterol probe, dehydroergosterol (DHE), and fluorescence resonance energy transfer (FRET) from the native Tyr-10 residue of Aβ to DHE. Without Aβ, peaks or kinks in the DHE anisotropy versus X_sterol plot were detected at X_sterol ≈ 0.25, 0.33, and 0.53. Monomeric Aβ preserved these peaks/kinks, but oligomeric Aβ suppressed them and created a new DHE anisotropy peak at X_sterol ≈ 0.38. The above critical X_sterol values coincide favorably with the superlattice compositions predicted by the cholesterol superlattice model, suggesting that membrane cholesterol tends to adopt a regular lateral arrangement, or domain formation, in the lipid bilayers. For FRET, a peak was also detected at X_sterol ≈ 0.38 for both monomeric and oligomeric Aβ, implying increased penetration of Aβ into the lipid bilayer at this sterol mole fraction. We conclude that the interaction of Aβ with membranes is affected by the lateral organization of cholesterol, and hypothesize that the formation of an oligomeric Aβ/cholesterol domain complex may be linked to the toxicity of Aβ in neuronal membranes.     

Effect of cholesterol on the lateral nanoscale dynamics of fluid membranes

Inelastic neutron scattering was used to study the effect of 5 and 40?mol% cholesterol on the lateral nanoscale dynamics of phospholipid membranes. By measuring the excitation spectrum at several lateral q _|| values (up to q _||?=?3 ?^?1), complete dispersion curves were determined of gel, fluid and liquid-ordered phase bilayers. The inclusion of cholesterol had a distinct effect on the collective dynamics of the bilayer’s hydrocarbon chains; specifically, we observed a pronounced stiffening of the membranes on the nanometer length scale in both gel and fluid bilayers, even though they were experiencing a higher degree of molecular disorder. Also, for the first time we determined the nanoscale dynamics in the high-cholesterol liquid-ordered phase of bilayers containing cholesterol. Namely, this phase appears to be “softer” than fluid bilayers, but better ordered than bilayers in the gel phase.     

Interaction of human serum albumin with membranes containing polymer-grafted lipids: spin-label ESR studies in the mushroom and brush regimes

The adsorption of human serum albumin (HSA) to dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was studied as a function of content and headgroup size of the polymer lipid. In the absence of protein, conversion from the low-density mushroom regime to the high-density brush regime of polymer-lipid content is detected by the change in ESR outer hyperfine splitting, 2A_max, of chain spin-labelled phosphatidylcholine in gel-phase membranes. The values of 2A_max remain constant in the mushroom regime, but decrease on entering the brush regime. Conversion between the two regimes occurs at mole fractions X_PEG(m→b)≈0.04, 0.01-0.02 and 0.005-0.01 for PEG-DPPE with mean PEG molecular masses of 350, 2000 and 5000 Da, respectively, as expected theoretically. Adsorption of HSA to DPPC membranes is detected as a decrease of the spin label 2A_max hyperfine splitting in the gel phase. Saturation is obtained at a protein/lipid ratio of ca. 1:1 w/w. In the presence of polymer-grafted lipids, HSA adsorbs to DPPC membranes only in the mushroom regime, irrespective of polymer length. In the brush regime, the spin-label values of 2A_max are unchanged in the presence of protein. Even in the mushroom regime, protein adsorption progressively becomes strongly attenuated as a result of the steric stabilization exerted by the polymer lipid. These results are in agreement with theoretical estimates of the lateral pressure exerted by the grafted polymer in the brush and mushroom regimes, respectively.     

N-palmitoyl-sulfatide participates in lateral domain formation in complex lipid bilayers

Sulfatides (galactosylceramidesulfates) are negatively charged glycosphingolipids that are important constituents of brain myelin membranes. These membranes are also highly enriched in galactosylceramide and cholesterol. It has been implicated that sulfatides, together with other sphingolipids, take part in lateral domain formation in biological membranes. This study was conducted to characterize the lateral phase behavior of N-palmitoyl-sulfatide in mixed bilayer membranes. Going from simple lipid mixtures with sulfatide as the only sphingolipid in a fluid matrix of POPC, to more complex membranes including other sphingolipids, we have examined 1) ordered domain formation with sulfatide, 2) sterol enrichment in such domains and 3) stabilization of the domains against temperature by the addition of calcium. Using two distinct phase selective fluorescent probes, trans-parinaric acid and cholestatrienol, together with a quencher in the fluid phase, we were able to distinguish between ordered domains in general and ordered domains enriched in sterol. We found that N-palmitoyl-sulfatide formed ordered domains when present as the only sphingolipid in a fluid phospholipid bilayer, but these domains did not contain sterol and their stability was unaffected by calcium. However, at low, physiologically relevant concentrations, sulfatide partitioned favorably into domains enriched in other sphingolipids and cholesterol. These domains were stabilized against temperature in the presence of divalent cations. We conclude that sulfatides are likely to affect the lateral organization of biomembranes.     

Cholesterol interactions with fluid-phase phospholipids: effect on the lateral organization of the bilayer

The lateral organization of lipids and proteins in cell membranes is recognized as an important factor in several cellular processes. Cholesterol is thought to function as a modulator of the lateral segregation of lipids into cholesterol-poor and cholesterol-rich domains. We investigated how the affinity of cholesterol for different phospholipids, as seen in cholesterol partitioning between methyl-β-cyclodextrin and large unilamellar vesicles, was reflected in the lateral organization of lipids in complex bilayers. We especially wanted to determine how the low-T_m lipid affected the lateral structure. Partition experiments showed that cholesterol had a higher affinity for N-oleoyl-sphingomyelin (OSM) than for palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers, but the highest preference was for N-palmitoyl-sphingomyelin (PSM)-containing bilayers. Partial phase diagrams of POPC/PSM/cholesterol and OSM/PSM/cholesterol bilayers at 23°C and 37°C were used to gain insight into the lateral organization of lipids in bilayers. Analysis of phase diagrams revealed that the phospholipid composition of cholesterol-poor and cholesterol-rich domains reflected the affinity that cholesterol exhibited toward bilayers composed of different lipids. Therefore, the determined affinity of cholesterol for different phospholipid bilayers was useful in predicting the cholesterol-induced lateral segregation of lipids in complex bilayers.     

Melatonin Alters Fluid Phase Coexistence in POPC/DPPC/Cholesterol Membranes

The structure and biophysical properties of lipid membranes are important for cellular functions in health and disease. In Alzheimer’s disease, the neuronal membrane is a target for toxic amyloid-β (Aβ). Melatonin is an important pineal gland hormone that has been shown to protect against Aβ toxicity in cellular and animal studies, but the molecular mechanism of this protection is not fully understood. Melatonin is a small membrane-active molecule that has been shown to interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components. To make it more relevant to neuronal membranes, we used a more complex ternary lipid mixture as our membrane model. In this study, we used ²H-NMR to investigate the effect of melatonin on the phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol lipid membranes. We used deuterium-labeled POPC-d₃₁ and DPPC-d₆₂,separately to probe the changes in hydrocarbon chain order as a function of temperature and melatonin concentration. We find that POPC/DPPC/cholesterol at molar proportions of 3:3:2 is close to liquid-disordered/liquid-ordered phase separation and that melatonin can induce phase separation in these ternary mixtures by preferentially incorporating into the disordered phase and increasing its level of disorder. At 5 mol% melatonin, we observed phase separation in samples with POPC-d₃₁, but not with DPPC-d₆₂, whereas at 10 mol% melatonin, phase separation was observed in both samples with either POPC-d₃₁ or DPPC-d₆₂. These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against Aβ, and that choice of chain perdeuteration is an important consideration from a technical point of view.     

La and Gd induce shape change of giant unilamellar vesicles of phosphatidylcholine

Lanthanides such as La³⁺ and Gd³⁺ are well known to have large effects on the function of membrane proteins such as mechanosensitive ionic channels and voltage-gated sodium channels, and also on the structure of phospholipid membranes. In this report, we have investigated effects of La³⁺ and Gd³⁺ on the shape of giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine (DOPC-GUV) and GUV of DOPC/cholesterol by the phase-contrast microscopy. The addition of 10-100 μM La³⁺ (or Gd³⁺) through a 10-μm diameter micropipette near the DOPC-GUV (or DOPC/cholesterol-GUV) triggered several kinds of shape changes. We have found that a very low concentration (10 μM) of La³⁺ (or Gd³⁺) induced a shape change of GUV such as the discocyte via stomatocyte to inside budded shape transformation, the two-spheres connected by a neck to prolate transformation, and the pearl on a string to cylinder (or tube) transformation. To understand the effect of these lanthanides on the shape of the GUV, we have also investigated phase transitions of 30 μM dipalmitoylphosphatidylcholine-multilamellar vesicle (DPPC-MLV) by the ultra-sensitive differential scanning calorimetry (DSC). The chain-melting phase transition temperature and the L_β′ to P_β′ phase transition temperature of DPPC-MLV increased with an increase in La³⁺ concentration. This result indicates that the lateral compression pressure of the membrane increases with an increase in La³⁺ concentration. Thereby, the interaction of La³⁺ (or Gd³⁺) on the external monolayer membrane of the GUV induces a decrease in its area (A^ex), whereas the area of the internal monolayer membrane (Aⁱⁿ) keeps constant. Therefore, the shape changes of the GUV induced by these lanthanides can be explained reasonably by the decrease in the area difference between two monolayers (ΔA=A^ex−Aⁱⁿ).     

Evidence for superlattice arrangements in fluid phosphatidylcholine/phosphatidylethanolamine bilayers.

Recently, evidence for cholesterol and phosphatidylcholine (PC) molecules to adapt superlattice arrangements in fluid lipid bilayers has been presented. Whether superlattice arrangements exist in other biologically relevant lipid membranes, such as phosphatidylethanolamine (PE)/PC, is still speculative. In this study, we have examined the physical properties of fluid 1-palmitoyl-2-oleoyl-PC (POPC) and 1-palmitoyl-2-oleoyl-PE (POPE) binary mixtures as a function of the POPE mole fraction (X(PE)) using fluorescence and Fourier transform infrared spectroscopy. At 30 degrees C, i.e., above the Tm of POPE and POPC, deviations, or dips, as well as local data scattering in the excimer-to-monomer fluorescence intensity ratio of intramolecular excimer forming dipyrenylphosphatidylcholine probe in POPE/POPC mixtures were detected at X(PE) approximately 0.04, 0.11, 0.16, 0.26, 0.33, 0.51, 0.66, 0.75, 0.82, 0.91, and 0.94. The above critical values of X(PE) coincide (within +/-0.03) with the critical mole fractions X(HX,PE) or X(R,PE) predicted by a headgroup superlattice model, which assumes that the lipid headgroups form hexagonal or rectangular superlattice, respectively, in the bilayer. Other spectroscopic data, generalized polarization of Laurdan and infrared carbonyl and phosphate stretching frequency, were also collected. Similar agreements between some of the observed critical values of X(PE) from these data and the X(HX,PE) or X(R,PE) values were also found. However, all techniques yielded critical values of X(PE) (e.g., 0.42 and 0.58) that cannot be explained by the present headgroup superlattice model. The effective cross-sectional area of the PE headgroup is smaller than that of the acyl chains. Hence, the relief of "packing frustration" of PE in the presence of PC (larger headgroup than PE) may be one of the major mechanisms in driving the PE and PC components to superlattice-like lateral distributions in the bilayer. We propose that headgroup superlattices may play a significant role in the regulation of membrane lipid compositions in cells.     

Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase

Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S_A, vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S_A, was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.     

Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2

The ability of secretory phospholipase A₂ (sPLA₂) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA₂ activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA₂. To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl-β-cyclodextrin. Treatment of cells with methyl-β-cyclodextrin increased the hydrolysis rate and total substrate hydrolyzed by sPLA₂. In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA₂ activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the hydrolysis rate and altered the relationship with membrane order. These data demonstrated that interactions with sPLA₂ observed in artificial bilayers apply to biological membranes. It is also proposed that the high level of cholesterol in erythrocyte membranes is a protective mechanism to guard against hydrolytic enzymes.     

Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.     

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C₁₂-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L_o domains but induces the formation of a gel-like phase. The activation of phospholipase A₂ by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.     

Differential dynamics of membrane proteins in yeast

Lateral diffusion of lipids and proteins in yeast plasma membranes has been reported to be anomalously slow, and implicated as a possible reason for polarization in yeast. In order to gain insight into the observed slow diffusion in yeast membranes, we explored lateral diffusion of two proteins of different origin. We compared lateral dynamics of the Candida drug resistance protein-1 (Cdr1p), and the human serotonin_1A receptor (5-HT_1AR) by fluorescence recovery after photobleaching (FRAP). Our results show that while Cdr1p-GFP displays slow diffusion, the diffusion of 5-HT_1AR-EYFP is significantly faster. Interestingly, upon ergosterol depletion, the mobility of Cdr1p-GFP did not exhibit appreciable change, while 5-HT_1AR-EYFP mobility showed an increase. On the other hand, upon actin cytoskeleton destabilization, the mobile fraction of 5-HT_1AR-EYFP showed considerable increase, while the mobility of Cdr1p-GFP was not altered. Our results represent the first report on the dynamics of the important drug resistance protein Cdr1p and provide novel insight on diffusion of membrane proteins in yeast membranes.     

Interfacial binding sites for cholesterol on GABAA receptors and competition with neurosteroids

γ-Aminobutyric acid type A (GABA_A) receptors in the brain are located in the outer membranes of brain cells where the concentration of cholesterol is high. Of the 25 available high-resolution structures available for GABA_A receptors, none were determined in the presence of cholesterol, but four include resolved molecules of cholesterol hemisuccinate (CHS). Here, a molecular docking procedure is used to sweep the transmembrane (TM) surfaces of the receptors for cholesterol binding sites. Cholesterol docking poses determined in this way match 89% of the resolved CHS when CHS molecules deemed unlikely to represent typical bound cholesterols are excluded. The receptors are pentameric, and their TM surfaces consist of a set of five facets, each including pairs of TM helices from two adjacent subunits. Each facet contains hydrophobic hollows running from one side of the membrane to the other, within which are six potential binding sites for cholesterol, three on each side of the membrane. High-resolution structures of GABA_A receptors with bound neurosteroids show that neurosteroids bind in these cholesterol binding sites, so the binding of neurosteroids and cholesterol will be competitive.     

Chemically induced lipid phase separation in model membranes containing charged lipids: A spin label study

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca²⁺ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, W_ex. By measuring W_ex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithinphosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers.In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8·10^?8 cm²/s at 59°C.Addition of Ca²⁺ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca²⁺-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of W_ex on the Ca²⁺ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca²⁺ concentration clusters containing about 30 mol% lecithin are formed. At high lecithin or Ca²⁺ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn²⁺ using ESR spectroscopy.Polylysine leads to the same strong increase in the lecithin segregation as Ca²⁺. The transition of the phosphatidic acid bound by the polypeptide is shifted from T_t = 47.5° to T_t = 62°C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Interaction of cholesterol with photoactivable phospholipids in sonicated vesicles

Photoactivable phospholipids containing either α-diazo-β-trifluoropropionyloxy or m-diazirinophenoxyl groups in the ω-positions of sn-2 fatty acyl chains were synthesized and incorporated into sonicated vesicles containing 33 mol% of cholesterol. Photolysis of the vesicles at 350 nm produced covalent cross-links between the synthetic phospholipids and cholesterol. The cross-linked products obtained using [¹⁴C]cholesterol were characterized by their chromatographic behavior, cleavage on phospholipase A₂ treatment, base-catalyzed transesterification and mass spectral measurements. The cross-linking was shown not to involve the 3-β-hydroxyl group of cholesterol, and it was concluded that the reactive carbene intermediates formed from the photolabels inserted into the hydrocarbon skeleton of cholesterol in the bilayer. The extent of cross-linking obtained was comparable to that observed previously using phospholipids alone, indicating that no lateral phase separation occurred. The present approach is promising for further precise studies of the molecular interactions between cholesterol and phospholipids in biological membranes.     

Distribution of cholesterol between the outer and inner halves of the lipid bilayer of mycoplasma cell membranes

Rapid kinetic studies of filipin binding to intact cells and isolated membranes were performed with a stopped-flow apparatus to determine the distribution of cholesterol in the outer and inner surfaces of mycoplasma membranes. The initial rates of association of filipin with cholesterol in Mycoplasma gallisepticum and Mycoplasma capricolum intact cells were slower than those obtained with isolated membrane preparations. Ratios of the second-order rate constants for filipin binding to cells relative to membranes indicate that cholesterol is distributed symmetrically in membranes of M. gallisepticum cells whereas in M. capricolum ～66% of the free cholesterol is localized in the outer half of the lipid bilayer.     

Phosphatidylcholine-cholesterol interactions: bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentration

Model membranes composed of cholesterol plus one of two phosphatidylcholines (PC), each containing a saturated and a dienoic acyl chain, have been studied by differential scanning calorimetry. The gel to liquid-crystalline phase transition temperature of 1-palmitoyl-2-linoleoyl PC was -19.5 degrees C and that of 1-stearoyl-2-linoleoyl PC was -13.7 degrees C. The addition of cholesterol to the phosphatidylcholines in aqueous dispersion resulted in the progressive removal of the phase transition as observed by differential scanning calorimetry. Per mole of sterol in the membrane, cholesterol was more effective at reducing the enthalpy change of the phase transitions of these bilayers containing dienoic phosphatidylcholines than it is in eliminating the transition of membranes made with other phospholipids that contain more saturated chains. No transitions in membranes made with palmitoyl-linoleoyl PC or stearoyl-linoleoyl PC could be detected calorimetrically when 17 mol% cholesterol was present.