1-2-dilauroylphosphatidylcholine and 1-2-distearoyllecithin

The membrane properties of phospholipid mixtures supported on silica were studied by means of a quartz crystal microbalance with dissipation monitoring (QCM-D) technique, in situ soft-contact atomic force microscopy (AFM), and friction force microscopy (FFM). The phospholipids used in this study were di-stearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC). The phospholipid films were prepared by a vesicle-fusion method, in which DSPC/DLPC mixed liposomes dispersed in an aqueous medium are adsorbed on silica and their structure is transformed into a bilayer on the substrate. The changes in QCM-D (frequency and dissipation) and friction responses of DSPC single systems (gel state at 25°C) are relatively large compared with those of DLPC single systems (liquid-crystalline state at 25°C) and those of mixed DSPC/DLPC systems. This suggests that (i) the gel-state DSPC liposomes are somewhat flattened on the silica, by keeping their solid-like molecular rigidity, whereas (ii) both the liquid-crystalline DLPC and mixed liposomes experience instantaneous structural transformation at the silica/water interface and form a normally flattened bilayer on the substrate. The friction force response is dependent on the phase state of the phospholipids, and the liquid-crystalline DLPC has a more significant impact on the overall membrane properties (i.e., the degree of swelling and the friction response on the surface) than does the gel-state DSPC.

Topics: Friction; Lipid Bilayers; Liposomes; Microscopy, Atomic Force; Phosphatidylcholines; Quartz Crystal Microbalance Techniques; Silicon Dioxide

We have studied friction forces on binary mixtures of phospholipid monolayer films. The phospholipid monolayer films have been prepared via the Langmuir-Blodgett (LB) technique on mica. The two-component phospholipids are distearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC). At 25 degrees C the LB monolayer films give the gel-state DSPC domains surrounded by the liquid crystalline DLPC matrix. The friction forces measured on the DSPC domain region are significantly greater than those on the DLPC matrix region at this temperature. An increased temperature results in a decreased friction of the DSPC domain region, and above the gel-to-liquid crystalline phase transition temperature of DSPC, the difference in the friction forces measured on the two phospholipids becomes negligible. This means that the phase state is a key factor in determining friction forces on the phospholipid monolayer films.

Topics: Lipid Bilayers; Membrane Microdomains; Membranes, Artificial; Microscopy, Atomic Force; Phosphatidylcholines; Temperature; Thermodynamics

Lipid bilayers exhibit structural diversity that contributes to the complex properties of the cell membrane. We use interfacial force microscopy to correlate mechanical properties with the two-dimensional phase behavior of supported lipid bilayers (SLBs). Upon indentation by a 500 nm tungsten tip, a contrast in the mechanical response is observed for gel vs fluid phase SLBs. We measure the yield force and time scale for recovery for these films. Consistent with a gel phase, a DSPC SLB has a relatively high yield force and slow recovery. In the higher mobility fluid phase, a DLPC SLB has a lower yield force and completely recovers within the experimental time scale. Friction measurements offer further contrast between the two SLBs.

Topics: Elasticity; Friction; Gels; Lipid Bilayers; Phase Transition; Phosphatidylcholines; Silicon; Surface Properties

This work characterizes the impact of lipid symmetry/asymmetry on drying/rehydration reorganization in phase-separated dilauroylphosphatidylcholine (DLPC)/distearoylphosphatidylcholine (DSPC) supported lipid bilayers (SLBs) at the submicron and micron-scale. In addition the prevention of major drying/rehydration reorganization by the use of trehalose is demonstrated. Even though it was found using fluorescence microscopy that micrometer scale structure is preserved in the presence and absence of trehalose upon drying/rehydration, AFM and FRAP experiments successfully revealed major changes in the phase-separated structure such as defects, obstructions, lipid condensation, collapse structures, and complex incomplete DLPC-DSPC mixing/exchange in the absence of trehalose. In the presence of trehalose the membrane preserves its structure at the nanometer scale and mobility. We found that SLBs with asymmetric domain configurations underwent major rearrangements during drying and rehydration, whereas the symmetric domain configuration mainly rearranged during rehydration, that we hypothesize is related to lower transmembrane cohesiveness or lack of anchoring to the substrate in the case of the asymmetric domains.

Topics: Biophysics; Lipid Bilayers; Lipids; Membranes, Artificial; Microscopy, Atomic Force; Microscopy, Fluorescence; Models, Statistical; Nanoparticles; Nanotechnology; Phosphatidylcholines; Surface Properties; Trehalose

The miscibility of the solid-phase-forming distearoylphosphatidylcholine (DSPC) and the fluid-phase-forming dilauroylphosphatidylcholine (DLPC) at the air/water interface was investigated by the Langmuir film balance. Surface pressure-area isotherms suggest that mixtures containing 25.0-62.5-mol% DLPC (range of composition investigated) are phase-separated. The lateral structure of the DSPC/DLPC monolayers was imaged by Brewster angle microscopy (BAM) as a function of the surface pressure. Quasi-circular condensed domains appeared at pressures between 0 and 0.5mN m(-1), and these structures were already fully developed at approximately 1mN m(-1). Further compression of the monolayers above 1mN m(-1) merely brought the domains closer together. The mixed monolayers consisted of solid domains of DSPC, approximately 3-20 micro in size, in a fluid matrix of DLPC. BAM and the phase contrast mode of intermittent-contact atomic force microscopy (AFM) revealed that the quasi-circular DSPC domains are divided into segments of different reflectivities (BAM) or phase shift (AFM) that arise from abrupt changes in the long-range orientational order of the tilted hydrocarbon chains. The DSPC domains in DSPC/DLPC internally exhibited star and cardioid textures that were heretofore only reported for single-component lipid monolayers in the phase coexistence region.

Topics: Anisotropy; Membranes, Artificial; Microscopy, Atomic Force; Microscopy, Phase-Contrast; Molecular Conformation; Phosphatidylcholines

Giant vesicles generated from synthetic and natural lipids such as phosphatidylcholines are useful models for understanding mechanical properties of cell membranes. Line tension is the one-dimensional force enabling the closing of transient pores on cell membranes. Transient pores were repeatedly and reproducibly formed on the membrane edge of giant vesicles generated from synthetic and natural phosphatidylcholines employing a nitrogen-pumped coumarin dye laser (440 nm). Line tension was determined at room temperature from closing of these pores that occurred over several seconds when the radius of the vesicle could be considered to be constant. The value of line tension depends on the nature of the lipid for single lipid systems, which, at room temperature, yielded a vesicle bilayer region in the gel, fluid, or mixed gel and fluid phases. The line tension for vesicles generated from phosphatidylcholines with saturated acyl chains of lengths of 12-18 carbon atoms ranges from 1 to 12 pN, exhibiting an increase with chain length. Vesicles generated from the natural Egg-PC, which is a mixture of lipids, are devoid of phase transition and exhibited the largest value of line tension (32 pN). This value is much larger than that estimated from the line tensions of vesicles obtained from lipids with homologous acyl chains. This study, to our knowledge, is the first to employ laser ablation to generate transient pores and determine line tension from the rate of pore closure and demonstrate a relationship between line tension and acyl chain length.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Algorithms; Chemical Phenomena; Chemistry, Physical; Dimyristoylphosphatidylcholine; Elasticity; Kinetics; Lipid Bilayers; Phosphatidylcholines; Surface Tension; Unilamellar Liposomes; Viscosity

Phase and mixing behavior of dilauroylphosphatidylcholine (DLPC)/distearoylphosphatidylcholine (DSPC) lipid mixtures are studied by molecular dynamics simulations with use of a coarse-grained model over a wide range of concentrations. The results reveal that phase transformations from the fluid to the gel state can be followed over a microsecond time scale. The changes in structure suggest regions of phase coexistence allowing us to outline the entire phase diagram for this lipid mixture using a molecular based model. We show that simulations yield good agreement with the experimental phase diagram. We also address the effect of macroscopic phase separation on the determination of the transition temperature, different leaflet composition, and finite size effects. This study may have implications on lateral membrane organization and the associated processes dependent on these membrane regions on different time and length scales.

Topics: Computer Simulation; Lipid Bilayers; Models, Biological; Phase Transition; Phosphatidylcholines; Water

Variations in two-dimensional membrane structures on the molecular length scale are considered to have an effect on the mechanisms by which living cell membranes maintain their functionality. We created a molecular model of a patterned bilayer to asses the static and dynamic variations of membrane lateral and transbilayer distribution in two-component lipid bilayers on the molecular level. We study DSPC (distearoylphosphatidylcholine) nanometer domains in a fluid DLPC (dilauroylphosphatidylcholine) background. The system exhibits coexisting fluid and gel phases and is studied on a microsecond time scale. We characterize three different kinds of patterns: symmetric domains, asymmetric domains, and symmetric-asymmetric domains. Preferred bilayer configurations on the nanoscale are those that minimize the hydrophobic mismatch. We find nanoscale patterns to be dynamic structures with mainly lateral and rotational diffusion affecting their stability on the microsecond time scale.

Topics: Gels; Lipid Bilayers; Membrane Fluidity; Membranes, Artificial; Models, Molecular; Phosphatidylcholines; Surface Properties; Time Factors

Anhydrobiotic preservation has the potential to allow the processing and storage of mammalian cells in a state of suspended animation at ambient conditions in trehalose glasses; however, stresses--particularly to the lipid bilayer--during desiccation and rehydration have thus far prevented the full realization of the promise of this technique. Giant gel-phase 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and liquid-crystalline-phase 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) vesicles provide a model cell system with which to elucidate the role of trehalose in surface-lipid bilayer interactions, as well as the part played by lipid phase. In the absence of trehalose, DSPC liposomes adsorbed to polystyrene, producing irreversible structural changes and apparent leakage of all intravesicular solute upon drying and rehydration. Addition of trehalose significantly reduced vesicle adsorption with only transient intravesicular solute leakage for the rehydrated vesicles; however, at very low moisture contents, the vesicles underwent permanent structural changes. In contrast to the results with DSPC vesicles, DLPC vesicles largely avoided adsorption and exhibited high intravesicular solute retention when dried and rehydrated even in the absence of trehalose, despite significant internal structural changes.

Topics: Adsorption; Phosphatidylcholines; Trehalose; Water

Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of approximately 100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.

Topics: Cell Membrane; Lipid Bilayers; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Nanotechnology; Phosphatidylcholines; Spectrometry, Mass, Secondary Ion

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Fluorescent Dyes; Lipid Bilayers; Phase Transition; Phosphatidylcholines; Phospholipids; Spectrometry, Fluorescence; Stress, Mechanical; Temperature

The effect of alpha-tocopherol on the structure and phase behavior of dilauroyl-, dimyristoyl-, dipalmitoyl-, and distearoyl-phosphatidylcholines was examined using X-ray diffraction and freeze-fracture electron microscopic methods. A ripple phase was observed in all of the mixtures at temperatures well below the pretransition temperature of the corresponding pure phospholipid. Freeze-fracture studies indicated that with proportion of alpha-tocopherol less than 5 mol% a ripple phase with large periodicity (50-150 nm) predominated and with about 10 mol% alpha-tocopherol a ripple phase of periodicity about 16 nm was formed. With more than 10 mol% alpha-tocopherol planar bilayers tended to be formed. Partial phase diagrams of mixed aqueous dispersions of saturated phosphatidylcholines and alpha-tocopherol over temperature ranges about the gel to liquid-crystal phase boundary have been constructed. Alpha-tocopherol-enriched domains form ripple phases that coexist with regions of lamellar gel phase of the pure phospholipid in mixtures containing less than 10 mol% alpha-tocopherol. The presence of increasing amounts of alpha-tocopherol in the phospholipid causes an increase in the proportion of ripple phase at the expense of pure phospholipid bilayer indicating that the alpha-tocopherol-enriched domains might possess a defined stoichiometry of the two constituents.

Topics: Dimyristoylphosphatidylcholine; Freeze Fracturing; Microscopy, Electron; Phosphatidylcholines; Synchrotrons; Temperature; Vitamin E; X-Ray Diffraction

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domai

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Dimyristoylphosphatidylcholine; Fluorescent Dyes; Laurates; Lipid Bilayers; Liposomes; Microscopy, Fluorescence; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Rhodamines; Temperature

A non-ideal lipid binary mixture (dilauroylphosphatidylcholine/distearoylphosphatidylcholine), which exhibits gel/fluid phase coexistence for wide temperature and composition ranges, was studied using photophysical techniques, namely fluorescence anisotropy, lifetime and resonance energy transfer (FRET) measurements. The FRET donor, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanol amine, and a short-tailed FRET acceptor, 1,1'-didodecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC12(3)), were shown to prefer the fluid phase by both intrinsic anisotropy, lifetime and FRET measurements, in agreement with published reports. The other studied FRET acceptor, long-tailed probe 1,1'-dioctadecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC18(3)), is usually reported in the literature as partitioning mainly to the gel. While intrinsic lifetime studies indeed indicated preferential partition of DiIC18(3) into a rigidified environment, FRET analysis pointed to an increased donor-acceptor proximity as a consequence of phase separation. These apparently conflicting results were rationalized on the basis of segregation of DiIC18(3) to the gel/fluid interphase. In order to fluid-located donors sense these interphase-located acceptors, fluid domains should be small (not exceed approximately 10-15 nm). It is concluded that membrane probes which apparently prefer the gel phase may indeed show a non-random distribution in this medium, and tend to locate in an environment which simultaneously leads to less strict packing constraints and to favorable hydrophobic matching interactions.

Topics: 4-Chloro-7-nitrobenzofurazan; Carbocyanines; Fluorescent Dyes; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Temperature

We have investigated the effect of a series of hydrophobic polypeptides (WALP peptides) on the mean hydrophobic thickness of (chain-perdeuterated) phosphatidylcholines (PCs) with different acyl chain length, using 2H NMR and ESR techniques. The WALP peptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked, e.g., FmAW2(LA)nW2AEtn. 2H NMR measurements showed that the shortest peptide with a total length of 16 amino acids (WALP16) causes an increase of 0.6 A in bilayer thickness in di-C12-PC, a smaller increase in di-C14-PC, no effect in di-C16-PC, and a decrease of 0.4 A in di-C18-PC, which was the largest decrease observed in any of the peptide/lipid systems. The longest peptide, WALP19, in di-C12-PC caused the largest increase in thickness of the series (+1.4 A), which decreased again for longer lipids toward di-C18-PC, in which no effect was noticed. WALP17 displayed an influence intermediate between that of WALP16 and WALP19. Altogether, incorporation of the WALP peptides was found to result in small but very systematic changes in bilayer thickness and area per lipid molecule, depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. ESR measurements with spin-labeled lipid probes confirmed this result. Because thickness is expected to be influenced most at the lipids directly adjacent to the peptides, also the maximal adaptation of these first-shell lipids was estimated. The calculation was based on the assumption that there is little or no aggregation of the WALP peptides, as was supported by ESR, and that lipid exchange is rapid on the 2H NMR time scale. It was found that even the maximal possible changes in first-shell lipid length were relatively small and represented only a partial response to mismatch. The synthetic WALP peptides are structurally related to the gramicidin channel, which was therefore used for comparison. In most lipid systems, gramicidin proved to be a stronger perturber of bilayer thickness than WALP19, although its length should approximate that of the shorter WALP16. The effects of gramicidin and WALP peptides on bilayer thickness were evaluated with respect to previous 31P NMR studies on the effects of these peptides on macroscopic lipid phase behavior. Both approaches indicate that, in addition to the effective hydrophobic length, a

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amino Acid Sequence; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Gramicidin; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Peptides; Phosphatidylcholines; Protein Structure, Secondary

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Crystallization; Diffusion; Dimyristoylphosphatidylcholine; Lipid Bilayers; Models, Theoretical; Molecular Conformation; Permeability; Phosphatidylcholines; Solubility; Surface Properties; Thermodynamics

A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.

Topics: Bacteriorhodopsins; Biophysical Phenomena; Biophysics; Fluorescence Polarization; In Vitro Techniques; Lipid Bilayers; Models, Chemical; Models, Molecular; Phosphatidylcholines; Spectrometry, Fluorescence

Fourier-transform infrared-spectroscopic and fluorescence measurements have been combined to examine the effect of cholesterol on the intermixing of short-chain dilauroyl phosphatidylcholine (DLPC) and its bromo-substituted derivative (12BrPC) with longer-chain (C16- or C18-) phosphatidylcholines (PCs) in hydrated lipid bilayers. Infrared spectroscopy of mixtures combining protonated DLPC or 12BrPC with chain-perdeuterated dipalmitoyl PC reveals that cholesterol at lower concentrations in the bilayer modifies the resolved thermal melting profiles for both phospholipid components and, at high bilayer concentrations, produces a convergence of the thermal transitions for the two PC species. Fluorescence-quenching measurements using a short-chain fluorescent PC (1-dodecanoyl-2-[8-[N-indolyl]octanoyl] PC) in ternary mixtures combining 12BrPC, dipalmitoyl or distearoyl PC, and cholesterol confirm that very high cholesterol levels (50 mol %) abolish the lateral segregation of the PC components at 25 degrees C, a temperature where the phospholipids extensively phase-separate in the absence of sterol. By contrast, under these same conditions cholesterol at lower concentrations in the bilayer is found to enhance the tendency of the PC components to exhibit lateral segregation. We show that these seemingly contradictory effects of cholesterol can be readily explained in the light of a ternary phase diagram that is fully consistent with out current understanding of the nature of cholesterol-phospholipid interactions in binary mixtures.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Lipid Bilayers; Phosphatidylcholines; Phospholipids; Spectroscopy, Fourier Transform Infrared; Temperature

A fluorescent assay based on concentration-dependent self-quenching of the fluorescent phospholipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine was used to measure the rate of phospholipid exchange between taurocholate/phosphatidylcholine mixed micelles. Two NBD-labeled phosphatidylethanolamine probes (dilauryl and dimyristoyl) were tested in taurocholate/phosphatidylcholine mixed micelles prepared from phosphatidylcholine molecules varying in saturated chain length from 12 to 18. All combinations of probes and micellar phosphatidylcholines gave kinetic results that were best described by a transfer model in which phospholipids exchange predominantly through the water phase at low micellar concentrations and through transient micelle fusions at higher concentrations. Increasing the chain length of the micellar-saturated diacylphosphatidylcholine from 12 to 18 carbons resulted in a decrease in the overall rate of exchange by a factor of 127 for NBD-labeled dilaurylphosphatidylethanolamine and a factor of 2490 for NBD-labeled dipalmitoylphosphatidylethanolamine. The reduction in the overall rate resulted from decreases in both mechanisms of transfer. These results argue that the hydrophobicity of the lipophilic core of bile salt/phospholipid mixed micelles is the predominant determinant of the rate of formation of transfer-competent, transient micelle fusions and a major determinant of the rate of micelle to water phospholipid dissociation.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 4-Chloro-7-nitrobenzofurazan; Dimyristoylphosphatidylcholine; Kinetics; Micelles; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Taurocholic Acid

In the present study the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1 has been investigated. The results demonstrate that (a) the maximum rate of O-dealkylation (V) for both P-450 enzymes was about two times higher in the L-alpha-dilauroyl-sn-glycero-3-phosphocholine (Lau2GroPCho) system and (b) changes in the fatty acyl moiety of phosphatidylcholine (acyl2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Kms) of P-450 IA1 and IIB1 in a different way. In addition, (c) the kinetic parameters appeared to be dependent on the acyl2GroPCho/P-450 ratio and a change in this ratio affected the kinetic parameters of P-450 IA1 and IIB1 in a different manner. From these last two observations it was concluded that the mechanism by which phospholipids influence P-450-IIB1-dependent O-dealkylation of ethoxycoumarin is different from that by which they influence P-450-IIB1-dependent O-dealkylation of this substrate. Furthermore, the results of the present study demonstrate that the increase in the rate of O-dealkylation of ethoxycoumarin, reported in the literature for reconstituted systems in the presence of Lau2GroPCho, results from an effect of Lau2GroPCho on both the Kms and the V. In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Kms and V of P-450 IA1 and IIB1 were investigated. This was done by studying the effect of the two acyl2GroPCho species on the kinetic parameters of some of the different steps of the P-450 cycle, namely substrate binding, oxygen binding and the rate of electron transfer. The results demonstrate an influence of Lau2GroPCho and Ste2GroPCho on (a) substrate binding to cytochrome P-450, (b) the affinity of cytochromes P-450 for NADPH-cytochrome reductase and thus on (c) the electron flow through the reconstituted system. Based on the results from these experiments it was concluded that the increased V of P-450 IA1 and IIB1 in the presence of Lau2GroPCho compared to the systems with Ste2GroPCho was at least in part due to an increased affinity of both P-450 enzymes for NADPH-cytochrome reductase in the presence of Lau2GroPCho compared to Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Coumarins; Cytochrome P-450 CYP2B1; Cytochrome P-450 Enzyme System; Dealkylation; Kinetics; Microsomes, Liver; Oxazines; Oxidoreductases; Phosphatidylcholines; Rats; Rats, Wistar

The difference in pentoxyresorufin O-dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau2GroPCho) or distearoylglycerophosphocholine (Ste2GroPCho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P-450b (IIB1) system. The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P-450 system, mainly based on the comparison of systems without phospholipid and with Lau2GroPCho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau2GroPCho system as compared to the Ste2GroPCho system cannot be ascribed to (a) an increased affinity of cytochrome P-450 for the NADPH-cytochrome reductase in the Lau2GroPCho system, also not to (b) a Lau2GroPCho-dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme. We found a different apparent Km for pentoxyresorufin in the Lau2GroPCho system compared with the Ste2GroPCho system. Furthermore, we found a difference between the cytochrome P-450b tryptophan fluorescence polarization of the Lau2GroPCho system and the Ste2GroPCho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau2GroPCho system compared with the Ste2GroPCho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P-450 molecules in these systems. Furthermore, the different effects of both phosphatidylcholines on the Km and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P-450 molecule, but also on the efficiency of electron transfer from NADPH-cytochrome reductase to cytochrome P-450.

Topics: Animals; Chromatography, Affinity; Cytochrome P-450 CYP2B1; Cytochrome P-450 Enzyme System; Kinetics; Macromolecular Substances; Male; Microsomes, Liver; Molecular Weight; NADPH-Ferrihemoprotein Reductase; Oxidoreductases; Phenobarbital; Phosphatidylcholines; Rats; Rats, Inbred Strains