Translocation of phospholipids and dithionite permeability in liquid-ordered and liquid-disordered membranes

We present a detailed study of the translocation rate of two headgroup-labeled phospholipid derivatives, one with two acyl chains, NBD-DMPE, and the other with a single acyl chain, NBD-lysoMPE, in lipid bilayer membranes in the liquid-disordered state (POPC) and in the liquid-ordered states (POPC/cholesterol (Chol), molar ratio 1:1, and sphingomyelin (SpM)/Chol, molar ratio 6:4). The study was performed as a function of temperature and the thermodynamic parameters of the translocation process have been obtained. The most important findings are 1), the translocation of NBD-DMPE is significantly faster than the translocation of NBD-lysoMPE for all bilayer compositions and temperatures tested; and 2), for both phospholipid derivatives, the translocation in POPC bilayers is approximately 1 order of magnitude faster than in POPC/Chol (1:1) bilayers and approximately 2-3 orders of magnitude faster than in SpM/Chol (6:4) bilayers. The permeability of the lipid bilayers to dithionite has also been measured. In liquid disordered membranes, the permeability rate constant obtained is comparable to the translocation rate constant of NBD-DMPE. However, in liquid-ordered bilayers, the permeability of dithionite is significantly faster then the translocation of NBD-DMPE. The change in enthalpy and entropy associated with the formation of the activated state in the translocation and permeation processes has also been obtained.     

Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage

We have employed four lipids in the present study, of which two are cationic and two bear phosphatidylcholine (PC) headgroups. Unlike dipalmitoylphosphatidylcholine, the other lipids employed herein do not have any ester linkage between the hydrocarbon chains and the respective lipid backbones. Small unilamellar vesicles formed from each of the PC and cationic lipids with or without varying amounts of cholesterol have been examined using the steady-state fluorescence anisotropy method as a function of temperature. The anisotropy data clearly indicate that the order in the lipid bilayer packing is strongly affected upon inclusion of cholesterol. This effect is similar irrespective of the electrostatic character of the lipid employed. The influence of cholesterol inclusion on multi-lamellar lipid dispersions has also been examined by 1H-nuclear magnetic resonance spectroscopy above the phase transition temperatures. With all the lipids, the line widths of (CH2)n protons of hydrocarbon chains in the NMR spectra respond to the addition of cholesterol to membranes. The influence on the bilayer widths of various lipids upon inclusion of cholesterol was determined from X-ray diffraction studies of the cast films of the lipid-cholesterol coaggregates in water. The effect of cholesterol on the efflux rates of entrapped carboxyfluorescein (CF) from the phospholipid vesicles was determined. Upon incremental incorporation of cholesterol into the phospholipid vesicles, the CF leakage rates were progressively reduced. Independent experiments measuring transmembrane OH- ion permeation rates from cholesterol-doped cationic lipid vesicles using entrapped dye riboflavin also demonstrated that the addition of cholesterol into the cationic lipid vesicles reduced the leakage rates irrespective of lipid molecular structure. It was found that the cholesterol induced changes on the membrane properties such as lipid order, linewidth broadening, efflux rates, bilayer widths, etc., did not depend on the ability of the lipids to participate in the hydrogen bonding interactions with the 3beta-OH of cholesterol. These findings emphasize the importance of hydrophobic interaction between lipid and cholesterol and demonstrate that it is not necessary to explain the observed cholesterol induced effects on the basis of the presence of hydrogen bonding between the 3beta-OH of cholesterol and the lipid chain-backbone linkage region or headgroup region.     

Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.     

Effects of cholesterol and temperature on the permeability of dimyristoylphosphatidylcholine bilayers near the chain melting phase transition

The passive leakage of glucose across bilayers of dimyristoylphosphatidylcholine (DMPC), cholesterol (variable), and dicetyl phosphate (constant 5.9 mol%) has been measured as efflux over 30 min from multilamellar vesicles. Bilayer cholesterol was varied from 20 mol% to 40 mol%. Glucose permeation rates were measured from 10 degrees C to 36 degrees C, and showed a maximum in permeability at 24 degrees C, the DMPC phase transition temperature. Increasing the bilayer cholesterol content above 20 mol% reduced that permeability peak. These results are quite consistent with a large number of similar bilayer permeability studies over the past 25 years. However, they are not consistent with a previous study of these same systems, which reported increased glucose permeability with temperature, without any maximum at or near the lipid chain melting temperature (K. Inoue, Biochim. Biophys. Acta 339 (1974) 390-402).     

Physiochemical characterization of the nisin-membrane interaction with liposomes derived from Listeria monocytogenes.

Mechanistic information about the bacteriocin nisin was obtained by examining the efflux of 5(6)-carboxy-fluorescein from Listeria monocytogenes-derived liposomes. The initial leakage rate (percentage of efflux per minute) of the entrapped dye was dependent on both nisin and lipid concentrations. At all nisin concentrations tested, 5(6)-carboxyfluorescein efflux plateaued before all of the 5(6)-carboxyfluorescein was released (suggesting that pore formation was transient), but efflux resumed when more nisin was added. Isotherms for the binding of nisin to liposomes constructed on the basis of the Langmuir isotherm gave an apparent binding constant of 6.2 x 10(5)M(-1) at pH 6.0. The critical number of nisin molecules required to induce efflux from liposomes at pH 6.0 was approximately 7,000 molecules per liposome. The pH affected the 5(6)-carboxyfluorescein leakage rates, with higher pH values resulting in higher leakage rates. The increased leakage rate observed at higher pH values was not due to an increase in the binding affinity of the nisin molecules towards the liposomal membrane. Rather, the critical number of nisin molecules required to induce activity was decreased (approximately 1,000 nisin molecules per liposome at pH 7.0). These data are consistent with a poration mechanism in which the ionization state of histidine residues in nisin plays an important role in membrane permeabilization.     

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9–10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA₂s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA₂s are key players.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state (31)P and (1)H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. (31)P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in (1)H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 degrees C and 24.0 degrees C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.     

Vertical fluctuations of phospholipid acyl chains in bilayers

The possibility of vertical displacement of acyl chains in lipid bilayers has been examined by quenching the fluorescence of 2-(9-anthroyloxy)palmitic acid with 5- and 16-doxylstearates in dipalmitoylphosphatidylcholine unilamellar vesicles. Measurement of lifetime and steady-state quenching showed that the dynamic component of quenching was independent of the transverse position of the quencher indicating that a quencher at the 16-position could interact with a fluorophore at the 2-position with high frequency. The differences in steady-state quenching could be accounted for by the differences in the static component of quenching. The results provide further evidence for rapid vertical displacements of acyl chains in phospholipid bilayers.     

Osmotic behavior of liposomes of phosphatidylcholine and phosphatidylsulfocholine as a function of lipid concentration

A relationship between the initial rate of liposome swelling, d(1/A)/dt and the reciprocal of the lipid concentration of the liposomes has been derived and then utilized to describe the osmotic swelling behavior of serially diluted liposomes and chloroplasts exposed to hypertonic urea solutions. The slopes of plots of d(1/A)/dt vs. the reciprocal of the lipid concentration of liposomes were not affected by differences in the initial absorbance of phosphatidylcholine-sterol bilayers, and were used to assess the ability of sterols to reduce the initial rates of urea permeation through dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline state. Multilamellar liposomes and sonicated vesicles were prepared from dimyristoylphosphatidylsulfocholine (DMPSC), in which the quaternary ammonium group of choline is replaced by -S⁺(CH₃)₂. Cholesterol reduced the initial rate of osmotic urea penetration into liposomes and the rate of 6-carboxyfluorescein efflux from vesicles at 35°C. The effect of cholesterol on bilayers of phosphatidylsulfocholine and phosphatidylcholine was very similar, suggesting that no strict structural requirements need be met in the choline moiety for lecithin-cholesterol interaction. The sulfonium analog could thus functionally replace phosphatidylcholine in natural membranes.     

The relationship between the antioxidant and the antibacterial properties of galloylated catechins and the structure of phospholipid model membranes

The effects of four catechins, (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), on the physical properties of phospholipid model membranes and the correlation to their antioxidant and antibacterial capacities have been studied by using differential scanning calorimetry (DSC), fluorescence spectroscopy, infrared spectroscopy (IR), AAPH-induced oxidation, and leakage experiments. DSC data revealed that galloylated catechins, especially ECG, affected the physical properties of both the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bilayers dramatically. Galloylated catechins showed higher phospholipid/water partition coefficients than their homologues and were immersed in the phospholipid palisade intercalating within the hydrocarbon chains, ECG being at the deepest position. In contrast, nongalloylated catechins presented a shallow location close to the phospholipid/water interface. ECG also exhibited the highest antioxidant capacity against lipid peroxidation, which correlated with its strong effect on DPH fluorescence anisotropy (as observed by the increase of the lipid order of fluid PC bilayers) and with the presence of highly cooperative transitions as seen by DSC. We propose that the high antioxidant capacity of some galloylated catechins such as ECG could be partially due to the formation of membrane structures showing resistance to detergent solubilization and in which the phospholipids have tightly packed acyl chains and highly hydrated phosphate groups. Significantly, PE was found to be essential to the promotion of carboxyfluorescein leakage from bacterial model membranes by galloylated catechins, indicating that their bactericidal activity, at least at the membrane level, could be due to the specific effect of these catechins on PE.     

Mechanism of magainin 2a induced permeabilization of phospholipid vesicles.

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)-induced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages--an initial rapid phase, with t1/2 approximately 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptide-lipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a.     

An infrared spectroscopic study of specifically deuterated fatty-acyl methyl groups in phosphatidylcholine liposomes.

The region of the infrared spectrum corresponding to C-2H stretching vibrations (2050-2250 cm-1) has been examined for liposomes composed of dimyristoylphosphatidylcholine deuterated specifically at the methyl ends of either one (sn-2) or both the fatty acyl chains. This label is intended to provide information on lipid dynamics in the contact region between monolayers. The two most prominent bands observed correspond, respectively, to antisymmetric (2212 cm-1) and symmetric (2075 cm-1) C-2H stretching vibration. The antisymmetric band consists of two overlapping peaks, whose positions vary with the gel or liquid-crystalline state of the lipid. The separation between the peaks making up the antisymmetric band increases with temperature, and is maximum above the Tc transition temperature; this rules out the previously proposed assignment of these two peaks to different rotational modes of the methyl group relative to the adjacent methylene. The position and width of the symmetric band at 2075 cm-1 are also sensitive to the physical state of the lipid. The presence of cholesterol at an equimolar ratio with the phospholipid abolishes all the phase-dependent changes observed. The intrinsic polypeptide gramicidin A, at a 5:1 lipid/peptide mol ratio, is seen to enlarge the lipid thermotropic transition, with small effects above Tc. Cytochrome c, an extrinsic protein, at a 10:1 mole ratio, does not modify the phase-dependent behaviour of the terminal methyl groups, but consistently shifts all the observed bands to lower-frequency positions, which suggests a long-range effect of the protein along the phospholipid fatty acyl chains.     

The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex.

1. The role of length and unsaturation of phospholipid acyl chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467--2474). 2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low. 3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chain-length was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length. 4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers. 5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidylglycerol was independent on acyl-chain composition and occurred at very low amounts of phospholipid. 6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gel-to-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids. 7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interfacial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.     

Stabilization of large multilamellar liposomes by human serum in vitro

The leakage of 5,6-carboxyfluorescein from large multilamellar liposomes prepared from dipalmitoylphosphatidylcholine (without or with cholesterol) was investigated in vitro in the presence of human serum. Below the phospholipid phase transition temperature, the rate of dye release is retarted 3–8-fold in the presence of up to 25% human serum in the incubation medium, as compared to the release in isotonic phosphate-buffered saline. This effect is significantly augmented by incorporation of 50 mol% cholesterol into the lipid bilayer. At and above the phase transition temperature, the initial rapid dye leakage in the presence of serum is followed by a slow long-term release. Incubation of the liposomes with serum is assumed to result in the association of serum proteins with the outermost lipid bilayer which in turn will lead to their stabilization, while the inner lamellae are not immediately accessible to the serum proteins. The permeability of the outer protein-rich lipid bilayer appears to be restricted, as concluded from the decreased dye release in the presence of serum. Massive leakage from multilamellar liposomes appears to be primarily due to bilayer defects occurring in the lipid transition region rather than being caused by protein-lipid interactions. The results of our in vitro experiments are discussed in terms of the potential usefulness of multilamellar liposomes as drug carriers in vivo for local and topical applications.     

Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin

Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and possesses several other interesting biological activities. This work deals with the molecular mechanism of membrane permeabilization by incorporation of surfactin. The surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC). The effect of the addition of cholesterol, dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylethanolamine (POPE) was also checked. It was observed that surfactin was able to induce content leakage at concentrations far below the onset surfactin/lipid ratio for membrane solubilization to occur, which in our system was around 0.92. Electron microscopy showed that vesicles were present after addition of surfactin at a ratio below this value, whereas no vesicles could be observed at ratios above it. Cholesterol and POPE attenuated the membrane-perturbing effect of surfactin, whereas the effect of DPPC was to promote surfactin-induced leakage, indicating that bilayer sensitivity to surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that surfactin destabilizes the inverted-hexagonal structure. Fourier-transform infrared spectroscopy (FTIR) was used to specifically follow the effect of surfactin on different parts of the phospholipid bilayer. The effect on the C=O stretching mode of vibration of POPC indicated a strong dehydration induced by surfactin. On the other hand, the C-H stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization. The reported effects could be useful to explain surfactin-induced 'pore' formation underlying the antibiotic and other important biological actions of this bacterial lipopeptide.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state ³¹P and ¹H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. ³¹P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in ¹H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 °C and 24.0 °C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.     

Incorporation of spin-labeled fatty acids into bovine brain clathrin coated vesicles

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. An abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.     

Phospholipid interactions of synthetic peptides representing the N-terminus of HIV gp41

Peptides representing the N-terminal 23 residues of the surface protein gp41 of LAV1a and LAVmal strains of the human immunodeficiency virus were synthesized and their interactions with phospholipid vesicles studied. The peptides are surface-active and penetrate lipid monolayers composed of negatively charged but not neutral lipids. Similarly, the peptides induce lipid mixing and solute (6-carboxyfluorescein) leakage of negatively charged, but not neutral, vesicles. Circular dichroism and infrared spectroscopy show that at low peptide:lipid ratios (approximately 1:200), the peptides bind to negatively charged vesicles as alpha-helices. At higher peptide:lipid ratios (1:30), a beta conformation is observed for the LAV1a peptide, accompanied by a large increase in light scattering. The LAVmal peptide showed less beta-structure and induced less light scattering. With neutral vesicles, only the beta conformation and a peptide:lipid ratio-dependent increase in vesicle suspension light scattering were observed for both peptides. We hypothesize that the inserted alpha-helical form causes vesicle membrane disruption whereas the surface-bound beta form induces aggregation.