Effects of polypeptide-phospholipid interactions on bilayer reorganizations Raman spectroscopic study of the binding of polymyxin B to dimyristoylphosphatidic acid and dimyristoylphosphatidylcholine dispersions

The interactions of the antibiotic polymixin B, a polycationic cyclic polypeptide containing a branched acyl side chain, with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidic acid (DMPA) bilayers were investigated by Raman spectroscopy for a wide range of lipid/polypeptide mole fractions. Temperature profiles, constructed from peak height intensity ratios derived from the lipid methylene C-H stretching and acyl chain C-C stretching mode regions, reflected changes originating from lateral chain packing effects and intrachain trans / gauche rotamer formation, respectively. For DMPC/polymyxin B bilayers the temperature dependent curves indicate a broadening of the gel-liquid crystalline phase transition accompanied by an approx. 3 C deg. increase in the phase transition temperature from 22.8°C for the pure bilayer to 26°C for the polypeptide complex. For a 10:1 lipid/polypeptide mole ratio the temperature profile derived from the C-C mode spectral parameters displays a second order/disorder transition, at approx. 35.5°C, associated with the melting behavior of approximately three bilayer lipids immobilized by the antibiotic's charged cyclic headgroup and hydrophobic side chain. For the 10:1 mole ratio DMPA/polypeptide liposomes, the temperature profiles indicate three order/disorder transitions at 46, 36 and 24°C. Pure DMPA bilayers display a sharp lamellar-micellar phase transition at 51°C.     

Interaction of sporidesmin,a mycotoxin fromPithomyces chartarum,with lipid bilayers

Sporidesmin, a mycotoxin fromPithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A₂ (PLA₂). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations <10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA₂ kinetics are consistent with their ability to perturb bilayer organisation.     

Calcium-induced lateral phase separations in phosphatidylcholine-phosphatidic acid mixtures. A Raman spectroscopic study

The effects of calcium ions on mixed membranes of dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidylcholine (DMPC) with either the PA or the PC component deuterated have been studied by Raman spectroscopy. The spectra of the pure components show that the acyl chains of hydrated DMPA bilayers are less tightly packed and have more trans bonds than those of DMPC. This behavior appears to be due to the particular arrangement of the polar head groups of DMPA for which the glycerol chain is oriented parallel to the bilayer surface. In agreement with the calorimetrically determined phase diagram [Graham, I., Gagné, J., & Silvius, J. R. (1985) Biochemistry (preceding paper in this issue)], the Raman results show that, in the absence of calcium, DMPA and DMPC are completely miscible at an equimolar ratio but undergo extensive phase separation in the presence of excess calcium. DMPC in phase-separated DMPC-DMPA (Ca2+) mixtures has a conformation that is very similar to that of pure DMPC bilayers, but it is packed more tightly since, depending on the temperature, it is at least partly incorporated into either a solid solution in DMPA or a DMPA-Ca2+-rich "cochleate" phase. This latter shows the same characteristics as the cochleate phase of pure DMPA-Ca2+ which is highly ordered and does not give rise to a thermotropic transition between 5 and 100 degrees C. However, the cochleate phase in DMPA (Ca2+)-DMPC mixtures contains some 20 mol % of DMPC trapped in small domains. These clusters do not melt cooperatively but become as fluid as pure DMPC at 50 degrees C.     

Morphology of the intermediate stages in the lamellar to hexagonal lipid phase transition

Summary The addition of calcium to suspensions of egg phosphatidylcholine and cardiolipin converts multiwalled liposomes to the hexagonal (H_II) phase (Rand, R.P., Sengupta, S. (1972)Biochim. Biophys. Acta 255:484–492). We have studied this lamellar to hexagonal phase transition by freeze-fracture, thin-section electron microscopy, and X-ray diffraction and have morphologically characterized the intermediate stages. The first step in the transition involves the invagination and fusion of bilayers, marked by the appearance of lipidic intramembrane particles and crater-like indentations, as the large liposomes are converted to smaller flattened and elongated vesicles. The next step is the formation of tightly packed hexagonal arrays of tubules, each tubule being about 11 to 15 nm in diameter. These tubules are filled with fluid and a lipid bilayer forms the wall of each cylinder. Finally this tubular bilayer phase is converted to the hexagonal (H_II) phase, where the distance between tubes is 5.5 to 7.5 nm.     

Incorporation of the antimicrobial protein seminalplasmin into lipid bilayer membranes

The interaction between seminalplasmin, an antimicrobial protein from bull semen, and lipid bilayers has been investigated. The fluorescence of the single tryptophan residue of the protein was measured. In the presence of phosphatidylcholine or phosphatidic acid bilayer vesicles the fluoresence maximum was shifted to shorter wavelengths, indicating transfer of the tryptophan to a more apolar environment. Circular dichroism spectra show an increased -helical content for the protein in the presence of lipid. Quenching experiments clearly show the incorporation of the protein with the tryptophan localized near the bilayer surface. The shift of the tryptophan fluorescence emission was used to monitor the lipid phase transition in phosphatidylcholine membranes.Abbreviations TEMPOL 2,2,6,6-Tetramethyl-4-hydroxy-piperidine-1-oxyl - DMPC 1,2-Dimyristoylphosphatidylcholine - DMPA 1,2-Dimyristoylphosphatidic acid - SL 5 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl - SL 12 2-(10-Carboxydecyl)-4,4-dimethyl-2-hexyl-3-oxazolinoxyl     

Sterol chemical configuration influences the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayers containing 5α-cholestan-3β- and 3α-ol

It is commonly believed that all membrane sterols are rigid all-trans ring systems with a fully extended alkyl side-chain and that they similarly influence phospholipid bilayer physical properties. Here, we report the sterol concentration-dependent, thermotropic phase behaviour of binary dipalmitoylphosphatidylcholine (DPPC)/sterol mixtures containing two similar 5α-H sterols with different functional group orientations (3α-OH or 3β-OH), which adopt an ideal all-trans planar ring conformation but lack the deformed ring B conformation of cholesterol (Chol) and epicholesterol (Echol), using differential scanning calorimetry (DSC). Our deconvolution of the DSC main phase transition endotherms show differences in the proportions of sterol-poor (sharp) and sterol-rich (broad) domains in the DPPC bilayer with increasing sterol concentration, which delineate gel/liquid-crystalline (P_β′/L_α) and disordered gel (L_β)/liquid-ordered (l_o) phase regions. There are similarities in the DPPC main phase transition temperature, cooperativity and enthalpy for each 3β-ol and 3α-ol pair with increasing sterol concentration and differences in the parameters obtained for both the sterol-poor and sterol-rich regions. The sterol-poor domain persists over a greater concentration range in both 3α-ol/DPPC mixtures, suggesting that either those domains are more stable in the 3α-ols or that those sterols are less miscible in the sterol-rich domain. Corresponding parameters for the sterol-rich domain show that at sterol concentrations up to 20 mol%, the 5α-H,3β-ol is more effective at reducing the phase transition enthalpy of the broad component () than Chol, but is less effective at higher concentrations. Although mixtures containing Echol and 5α-cholestan-3α-ol have similar positive slopes below 7 mol% sterol, suggesting that they abolish the L_β/l_o phase transition equally effectively at low concentrations, Echol is more effective than the saturated 3α-ol at higher sterol concentrations. A comparison of obtained for the saturated and unsaturated pairs suggests that the latter sterols stabilize the l_o phase and broaden and abolish the DPPC main phase transition more effectively than the saturated sterols at physiologically relevant concentrations, supporting the idea that the double bond of Chol and Echol promotes greater sterol miscibility and the formation of l_o phase lipid bilayers relative to corresponding saturated sterols in biological membranes.     

On the permeability of water molecules across vesicular lipid bilayers

Summary The permeation of water molccules across single-component lecithin or lecithin-cholesterol bilayers is studied by a new technique. The new technique makes use of the different fluorescence quantum yields of appropriate molecules in D₂O and H₂O. Water-soluble indole derivatives which by experimental manipulation reside almost entirely within the aqueous (H₂O) intravesicular compartment thus can monitor D₂O molecules permeating the bilayer by virtue of an increased quantum yield of the fluorescence. In a stopped-flow instrument, a vesicle solution containing the fluorescent chromophore in the intravesicular space is rapidly mixed with the deuterated solvent. The approach to the steady state, where the intra- and extravesicular D₂O and H₂O concentrations are equal, proceeds in a single-exponential manner. Consequently, the exchange relaxation time for the D₂O molecules passing the bilayer can be deduced from the time-dependent increase of the fluorescence intensity. The method and results on lecithin and lecithin-cholesterol bilayer vesicles are discussed. The exchange relaxation times of temperature-dependent studies are interpreted within the framework of the solubility-diffusion theory. Below the crystalline to liquid-crystalline phase transition temperature and for cholesterol-free vesicles, the rate-limiting step for the D₂O permeation is attributed to the intracore diffusion. Above the phase transition and for cholesterol-containing vesicles, the intracore diffusion seems not to be rate-limiting. Deviations from the linearity below the phase transition in the Arrhenius-type presentation of the data are related to changes of the partition coefficient of water between the solvent and the lipid phase at the premelting temperature.     

Softening of lipid bilayers

The softening of wet lipid bilayer membranes during their gel-to-fluid first-order phase transition is studied by computer simulation of a family of two-dimensional microscopic interaction models. The models include a variable number, q, of lipid chain conformational states, where 2q10. Results are presented as functions of q and temperature for a number of bulk properties, such as internal energy, specific heat, and lateral compressibility. A quantitative account is given of the statistics of the lipid clusters which are found to form in the neighborhood of the transition. The occurrence of these clusters is related to the softening and the strong thermal density fluctuations which dominate the specific heat and the lateral compressibility for the high-q models. The cluster distributions and the fluctuations behave in a manner reminiscent of critical phenomena and percolation. The findings of long-lived metastable states and extremely slow relaxational behavior in the transition region are shown to be caused by the presence of intermediate lipid chain conformational states which kinetically stabilize the cluster distribution and the effective phase coexistence. This has as its macroscopic consequence that the first-order transition apperas as a continuous transition, as invariably observed in all experiments on uncharged lecithin bilayer membranes. The results also suggest an explanation of the non-horizontal isotherms of lipid monolayers. Possible implications of lipid bilayer softening and enhanced passive permeability for the functioning of biological membranes are discussed.Abbreviations PC phosphatidvlcholine - DMPC dimyristoyl PC - DPPC dipalmitoyl PC - ac alternating current - DSC differential scanning calorimetry - T _m lipid gel-to-fluid phase transition temperature - TEMPO 2,2,6,6-tetramethylpiperidine-N-oxyl Supported by the Danish Natural Science Research Council and A/S De Danske Spritfabrikkers JubilæumslegatSupported in part by the NSERC of Canada and Le FCAC du Quebec     

Temperature-dependent osmotic permeability in glycoproteion containing liposomes

The osmotic water outflow of large multilamellar liposomes containing ₁-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.     

Thermal transitions in dimyristoylphosphatidyleholine foam bilayers

Thermal transitions in the system dimyristoylphosphatidylcholine/water/ethanol/sodium chloride were studied in the temperature range 10–31 °C. The water-ethanol dispersions were investigated by differential scanning calorimetry and the foam bilayers by the microinterferometric method for investigation of thin liquid films. Calorimetry showed that an increase in ethanol content (up to 47.5 vol.% — the concentration used in the experiments with foam bilayers) did not significantly influence the temperature of the main phase transition and led to the disappearance of the pretransition. The microinterferometric study of the foam bilayer thickness showed that there were two thermal transitions — at 13 and 23 °C. An Arrhenius type dependence was obtained for the critical concentration of dimyristoylphosphatidylcholine (DMPC) in the solution, which was necessary for the formation of the foam bilayer. A steep change in the slope of the linearized Arrhenius dependence was found at 23 °C. Values of the binding energy of a DMPC molecule in the foam bilayers were calculated using the hole-nucleation theory of stability and permeability of bilayers. It was proved that the phase transition at 23 °C was due to melting of the hydrocarbon tails of phospholipid molecules. The low-temperature phase transition was assumed to be due to a change in the tilt of the hydrocarbon tails. These experiments demonstrate for the first time the occurrence of phase transitions in foam bilayers. Correspondence to: D. Exerowa     

Molecular motion of small nonelectrolyte molecules in lecithin bilayers

Summary We have used magnetic resonance spectroscopy, both ESR and¹³C spin relaxation, to measure translational and rotational mobilities and partition coefficients of small nitroxide solutes in dipalmitoyl lecithin liposomes. Above the bilayer transition temperature,T _c, the bilayer interior is quite fluid, as determined from the solutes' rapid rotational and moderately rapid translational motion; the rotational and translational viscosities within the bilayer are _R <1cP and =6–10cP, respectively. and _R are independent of molecular size for all solutes studied, but all were small compared to the size of the phospholipids. , and probably _R , are relatively independent of temperature aboveT _c, but both increase very sharply as temperature is lowered belowT _c; at 32°C, _R increases to 6cP and is greater than 1000 cP. Anisotropy of rotational motion increases gradually as temperature is lowered toT _c, and changes little belowT _c; anisotropy of translational motion was not investigated.¹³C nuclear spin relaxation measurements indicate that translational motion of nitroxide solutes is more rapid in the center of the bilayer than near the polar interface. It takes at least 100 nsec for a solute molecule to cross the bilayer/water interface. We estimate a lower limit of 2 sec/cm for the interfacial resistance to solute diffusion; this result indicates that interfacial resistance dominates permeation across the membrane. The relative solubility, or partition coefficient, is a strong function of solute structure, and decreases abruptly on cooling through the transition temperature. From the partition coefficient and its temperature dependence we calculate the free energy, enthalpy, and entropy of partition. Effects of cholesterol on partition and diffusion coefficients are compatible with the interpretation that bilayers containing cholesterol consist of two phases.     

Comparative study on the properties of saturated phosphatidylethanolamine and phosphatidylcholine bilayers: Barrier characteristics and susceptibility to phospholipase A2 degradation

Comparative studies on bilayer systems of saturated phosphatidylcholines and phosphatidylethanolamines revealed a maximum in ionic permeability in phosphatidylcholine bilayers at the temperature of the gel to liquid-crystalline phase transition but such an increase in permeability was not detectable in bilayers of phosphatidylethanolamine. Furthermore, it was found that at the phase transition temperature the phosphatidylcholine bilayers are subject to rapid hydrolysis by pancreatic phospholipase A₂ whereas phosphatidylethanolamine bilayers are not. These differences are discussed in view of detailed information on the molecular organization in the gel and liquid crystalline phases of the two phospholipid classes.     

Model of interaction between a cardiotoxin and dimyristoylphosphatidic acid bilayers determined by solid-state 31P NMR spectroscopy.

The interaction of cardiotoxin IIa, a small basic protein extracted from Naja mossambica mossambica venom, with dimyristoylphosphatidic acid (DMPA) membranes has been investigated by solid-state 31P nuclear magnetic resonance spectroscopy. Both the spectral lineshapes and transverse relaxation time values have been measured as a function of temperature for different lipid-to-protein molar ratios. The results indicate that the interaction of cardiotoxin with DMPA gives rise to the complete disappearance of the bilayer structure at a lipid-to-protein molar ratio of 5:1. However, a coexistence of the lamellar and isotropic phases is observed at higher lipid contents. In addition, the number of phospholipids interacting with cardiotoxin increases from about 5 at room temperature to approximately 15 at temperatures above the phase transition of the pure lipid. The isotropic structure appears to be a hydrophobic complex similar to an inverted micellar phase that can be extracted by a hydrophobic solvent. At a lipid-to-protein molar ratio of 40:1, the isotropic structure disappears at high temperature to give rise to a second anisotropic phase, which is most likely associated with the incorporation of the hydrophobic complex inside the bilayer.     

Structure and phase behaviour of dimyristoylphosphatidic acid/poly(L-lysine) systems

Summary

Differential scanning calorimetry (DSC) and X-ray diffraction studies on (DMPA)/poly(L-lysine) systems are reported. DSC studies revealed that addition of poly(L-lysine) to DMPA bilayers raises the gel to liquid-crystalline phase transition of the systems, and that this effect depends on the molecular weight of the poly(L-lysine). Small-angle X-ray diffraction measurements showed that, in the liquid-crystalline phase, the lamellar spacing of a DMPA/short-poly(L-lysine) (～4000 mol. wt.) system is shorter than that of a DMPA/long-poly(L-lysine) (～22 000 mol. wt.). In this connection wide-angle X-ray diffraction measurements indicate that the long-poly(L-lysine) adopts a β-sheet conformation on the DMPA bilayers in both the gel and the liquid-crystalline phases, but the short-poly(L-lysine) adopts this conformation only on gel phase DMPA bilayers. We found that the spacings of the hydrocarbon chain packing in a DMPA bilayer in the gel phase increases with temperature, while the spacing between neighbouring polypeptide chains in long-poly(L-lysine) in the β-sheet conformation remains almost constant. These observations indicate that the positively charged lysine residues are structurally independent of the negatively charged head groups of the phospholipid. On the basis of the present results we propose a model to explain the elementary behaviour of extrinsic membrane proteins in biomembranes.     

The effects of acyl chain ordering and crystallization on the main phase transition of wet lipid bilayers

The main gel-fluid phase transition of wet lipid bilayers is examined in terms of a microscopic interaction model which incorporates both trans-gauche isomerism of the lipid acyl chains and crystal orientation variables for the lipid molecules. The model gives two scenarios for the phase behavior of wet lipid bilayers in terms of temperature: (i) chain melting occurs at a higher temperature than crystallization, or (ii) chain melting and crystallization occur at the same temperature. Experimental data for lipid bilayers is consistent with the second scenario. In this case, computer simulation is used to investigate the non-equilibrium behaviour of the model. The numerical data is intepreted in terms of interfacial melting on heating and grain formation on cooling through the main phase transition. Interfacial melting is a non-equilibrium process in which the grains of a polycrystalline bilayer melt inwards from the boundaries. The prediction of interfacial melting in wet lipid bilayers is examined in relation to data from both equilibrium and nonequilibrium measurements, to corresponding phase behavior in monolayers, and to previous theoretical work.Abbreviations DHPE dihexadecyl phosphatidylethanolamine - DMPA dimyristoyl phosphatidic acid - DMPC dimyristoyl phosphatidylcholine - DPPC dipalmitoyl phosphatidylcholine - DSC differential scanning calorimetry - MCS/S Monte Carlo steps per site Supported in part by the NSERC of Canada and FCAC du QuébecSupported by the Danish Natural Science Research Council under grant J.nr. 5.21.99.72     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids Permeability and 13C-nuclear magnetic resonance measurements

¹³C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of ¹³C-NMR relaxation enhancement measurements using cholesterol enriched with ¹³C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-¹³C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether-phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated ⁴⁵Ca²⁺ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether-phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for ⁴⁵Ca²⁺ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether-, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. These results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether-phosphatidylcholine bilayers, the 4-¹³C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.     

The effect of the polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation

Summary Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2-chloro,4-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as carriers for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases.     

Effect of bilayer cholesterol content on reconstituted human erythrocyte sugar transporter activity

The influence of altered bilayer cholesterol content on the catalytic activity of the human red cell hexose transporter was examined by reconstitution of the transport protein (band 4.5) into bilayers of large unilamellar vesicles formed from dipalmitoyl lecithin and varying amounts of cholesterol. The physical state of the bilayers was characterized by differential scanning calorimetry. The major findings are as follows: changes in bilayer phase behavior occur at membrane cholesterol levels of 15 to 20 mol % and 30 to 40 mol %; and the catalytic activity of the reconstituted transporter (Vmax/transporter) correlates with bilayer phase behavior. In crystalline bilayers, this is seen as an abrupt, stimulation of activity at 15 mol % cholesterol (which is reversed at 17.5 mol %) and a gradual acceleration of activity between 30 to 40 mol % cholesterol. In fluid bilayers (where activity is high), activity is unaffected by 10, 20, and 30 mol % cholesterol. However, 12.5 and 17.5 mol % cholesterol reduce activity by 100-fold. These studies demonstrate that small changes in bilayer cholesterol content result in drastic alterations in transporter activity. Transporter sensitivity to cholesterol is a complex rather than monotonic function of bilayer cholesterol content and appears to be primarily determined by bilayer composition rather than by bilayer "fluidity."     

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study.

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.