1-2-oleoylphosphatidylcholine and 1-2-dipalmitoyl-3-phosphatidylethanolamine

Additive force fields are designed to account for induced electronic polarization in a mean-field average way, using effective empirical fixed charges. The limitation of this approximation is cause for serious concerns, particularly in the case of lipid membranes, where the molecular environment undergoes dramatic variations over microscopic length scales. A polarizable force field based on the classical Drude oscillator offers a practical and computationally efficient framework for an improved representation of electrostatic interactions in molecular simulations. Building on the first-generation Drude polarizable force field for the dipalmitoylphosphatidylcholine 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) molecule, the present effort was undertaken to improve this initial model and expand the force field to a wider range of phospholipid molecules. New lipids parametrized include dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The iterative optimization protocol employed in this effort led to lipid models that achieve a good balance between reproducing quantum mechanical data on model compound representative of phospholipids and reproducing a range of experimental condensed phase properties of bilayers. A parametrization strategy based on a restrained ensemble-maximum entropy methodology was used to help accurately match the experimental NMR order parameters in the polar headgroup region. All the parameters were developed to be compatible with the remainder of the Drude polarizable force field, which includes water, ions, proteins, DNA, and selected carbohydrates.

Topics: Diffusion; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Quantum Theory; Thermodynamics

Binding of amphiphilic molecules to supported lipid bilayers (SLBs) often results in lipid fibril extension from the SLBs. Previous studies proposed that amphiphiles with large and flexible hydrophilic regions trigger lipid fibril formation in SLBs by inducing membrane curvature via their hydrophilic regions. However, no experimental studies have verified this mechanism of fibril formation. In this work, we investigated the binding of lipopolysaccharide (LPS) and cholesterol-modified gelatin to SLBs using fluorescence microscopy. SLBs with restricted and unrestricted bilayer areas were employed to identify the mechanism of fibril generation. We show that the main cause of lipid fibril formation is an approximately 20% expansion in the bilayer area rather than increased membrane curvature. The data indicate that bilayer area confinement plays a critical role in morphological changes of SLBs even when bound amphiphilic molecules have a large hydrophilic domain. We also show that bilayer area change after LPS insertion is dependent on the patch shape of the SLB. When an SLB patch consists of a broad bilayer segment connected to a long thin streak, bilayer area expansion mainly occurs within the bilayer streak. The results indicate that LPS insertion causes net lipid flow from the broad bilayer region to the streak area. The differential increase in area is explained by the instability of planar bilayer streaks that originate from the large energetic contribution of line tension arising along the bilayer edge.

Topics: Biomimetic Materials; Cholesterol; Gelatin; Lipid Bilayers; Lipopolysaccharides; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Surface Tension; Unilamellar Liposomes; Xanthenes

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required to suppress phase separation decreases relative to longer polymers. Collectively, our results demonstrate that crowded, membrane-bound polymers are highly efficient suppressors of phase separation and suggest that the ability of lipid domains to resist steric pressure depends on both their lipid composition and the size and concentration of the membrane-bound polymers they incorporate.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Fluoresceins; Fluorescent Dyes; Membrane Microdomains; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Unilamellar Liposomes; Xanthenes

The fusion of lipid membranes is a key process in biology. It enables cells and organelles to exchange molecules with their surroundings, which otherwise could not cross the membrane barrier. To study such complex processes we use simplified artificial model systems, i.e., an optical fusion assay based on membrane-coated glass spheres. We present a technique to analyze membrane-membrane interactions in a large ensemble of particles. Detailed information on the geometry of the fusion stalk of fully fused membranes is obtained by studying the diffusional lipid dynamics with fluorescence recovery after photobleaching experiments. A small contact zone is a strong obstruction for the particle exchange across the fusion spot. With the aid of computer simulations, fluorescence-recovery-after-photobleaching recovery times of both fused and single-membrane-coated beads allow us to estimate the size of the contact zones between two membrane-coated beads. Minimizing delamination and bending energy leads to minimal angles close to those geometrically allowed.

Topics: Algorithms; Cell Fusion; Computer Simulation; Diffusion; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Glass; Heterocyclic Compounds, 4 or More Rings; Lipopeptides; Membrane Fusion; Membranes, Artificial; Microscopy, Confocal; Models, Theoretical; Phosphatidylcholines; Phosphatidylethanolamines; Silicon Dioxide; Xanthenes

Colistin (Polymyxin E), an antimicrobial peptide, is increasingly put forward as salvage for severe multidrug-resistant infections. Unfortunately, colistin is potentially toxic to mammalian cells. A better understanding of the interaction with specific components of the cell membranes may be helpful in controlling the factors that may enhance toxicity. Here, we report a physico-chemical study of model phospholipid (PL) mono- and bilayers exposed to colistin at different concentrations by Langmuir technique, atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The effect of colistin on chosen PL monolayers was examined. Insights into the topographical and elastic changes in the PL bilayers within time after peptide injection are presented via AFM imaging and force spectra. Finally, changes in the PL bilayers' ATR-FTIR spectra as a function of time within three bilayer compositions, and the influence of colistin on their spectral fingerprint are examined together with the time-evolution of the Amide II and νCO band integrated intensity ratios. Our study reveals a great importance in the role of the PL composition as well as the peptide concentration on the action of colistin on PL model membranes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Anti-Bacterial Agents; Colistin; Elasticity; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylethanolamines; Spectroscopy, Fourier Transform Infrared; Unilamellar Liposomes

Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir-Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10(-8) mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Boron Compounds; Chickens; Cholesterol; Fluorescent Dyes; Microscopy, Fluorescence; Microscopy, Polarization; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins; Xanthenes

Hybrids composed of amphiphilic block copolymers and lipids constitute a new generation of biological membrane-inspired materials. Hybrid membranes resulting from self-assembly of lipids and polymers represent adjustable models for interactions between artificial and natural membranes, which are of key importance, e.g., when developing systems for drug delivery. By combining poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) amphiphilic copolymers (PDMS-b-PMOXA) with various phospholipids, we obtained hybrid films with modulated properties and topology, based on phase separation, and the formation of distinct domains. By understanding the factors driving the phase separation in these hybrid lipid-polymer films, we were able to use them as platforms for directed insertion of membrane proteins. Tuning the composition of the polymer-lipids mixtures favored successful insertion of membrane proteins with desired topological distributions (in polymer or/and lipid regions). Controlled insertion and location of membrane proteins in hybrid films make these hybrids ideal candidates for numerous applications where specific spatial functionality is required.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Dimethylpolysiloxanes; Membrane Proteins; Membranes, Artificial; Models, Biological; Phosphatidylcholines; Phosphatidylethanolamines; Polyamines; Polymerization; Thermodynamics

We report the experimental observation of osmotically induced transient pearling instabilities in vesicular membranes. Giant phospholipid vesicles subjected to negative osmotic gradient, which drives the influx of water in to the vesicular interior, produces transient cylindrical protrusions. These protrusions exhibit a remarkable pearling intermediate, which facilitates their subsequent retraction. The pearling front propagates from the distal free end of the protrusion toward the vesicular source and accompanies gradual shortening of the protrusion via pearl-pearl coalescence. Real-time introduction of a positive osmotic gradient, on the other hand, drives vigorous shape fluctuations, which in turn produce cylindrical, prolate- and pear-shaped intermediates presumably due to an increased vesicular area relative to the encapsulated volume. These intermediates transiently produce a pearled state prior to their fission. In both cases, the transient pearling state gives rise to an array of stable spherical daughter vesicles, which may be connected to one another by fine tethers not resolved in our experiments. These results may have implications for self-reproduction in primitive, protein-free, cells.

Topics: Cell Membrane; Microscopy, Fluorescence; Osmosis; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Sucrose; X-Ray Diffraction

The fusion between two lipid bilayers involves crossing a complicated energy landscape. The limiting barrier in the process appears to be between two closely opposed bilayers and the intermediate state where the outer leaflets are fused. We have performed molecular dynamics simulations to characterize the free energy barrier for the fusion of two liposomes and to examine the molecular details of barrier crossing. To capture the slow dynamics of fusion, a model using coarse-grained representations of lipids was used. The fusion between pairs of liposomes was simulated for four systems: DPPC, DOPC, a 3:1 mixture of DPPC/DPPE, and an asymmetric lipid tail system in which one tail of DPPC was reduced to half the length (ASTail). The weighted histogram method was used to compute the free energy as a function of separation distance. The relative barrier heights for these systems was found to be ASTail >> DPPC > DPPC/DPPE > DOPC, in agreement with experimental observations. Further, the free energy curves for all four can be overlaid on a single curve by plotting the free energy versus the surface separation (differing only in the point of fusion). These simulations also confirm that the two main contributions to the free energy barrier are the removal of water between the vesicles and the deformation of the vesicle. The most prominent molecular detail of barrier crossing in all cases examined was the splaying of lipid tails, where initially a single splayed lipid formed a bridge between the two outer leaflets that promotes additional lipid mixing between the vesicles and eventually leads to fusion. The tail splay appears to be closely connected to the energetics of the process. For example, the high barrier for the ASTail is the result of a smaller distance between terminal methyl groups in the splayed molecule. The shortening of this distance requires the liposomes to be closer together, which significantly increases the cost of water removal and bilayer deformation. Before tail splay can initiate fusion, contact must occur between a tail end and the external water. In isolated vesicles, the contact fraction is correlated to the fusogenicity difference between DPPC and DOPC. Moreover, for planar bilayers, the contact fraction is much lower for DPPC, which is consistent with its lack of fusion in giant vesicles. The simulation results show the key roles of lipid tail dynamics in governing the fusion energy landscape.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Liposomes; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics

This work presents the investigations of the interactions between nystatin, a polyene antibiotic, and phospholipids with various head groups (phosphatidylcholine and phosphatidylethanolamine) and acyl chains of different length and saturation degree. The experiments were performed with the Langmuir monolayer technique. Among phosphatidylethanolamines, DMPE, DPPE and DSPE were studied, while phosphatidylcholines were represented by DSPC and DOPC. The influence of the antibiotic on the molecular organization of the phospholipid monolayer was analysed with the compression modulus values, while the strength of nystatin/phospholipid interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The results obtained proved a high affinity of nystatin towards phospholipids. Nystatin was found to interact more strongly with phosphatidylcholines than with phosphatidylethanolamines. The most negative values of the excess free energy of mixing observed for the antibiotic and DOPC mixtures prove that nystatin favors the phospholipid with two unsaturated acyl chains. The results imply that nystatin/phospholipid interactions compete in the natural membrane with nystatin/sterol interactions, thereby affecting the antifungal activity of nystatin and its toxicity towards mammalian cells.

Topics: Antifungal Agents; Membranes; Models, Chemical; Models, Statistical; Nystatin; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Polyenes; Pressure; Surface Properties; Thermodynamics

This paper compares six phospholipidic monolayers at the water/chloroform interface by performing dilational rheological measurements with a drop tensiometer apparatus. The chosen lipids differ both in their headgroup structure and fatty acyl chain saturation or symmetry. The study concentrated on monolayers formed with DPPC, DPPE, DOPC, DOPE, POPC and POPE. Using a generalized Maxwell rheological model, transposed at the interface, the intimate intermolecular interactions between amphiphilic molecules are studied on and off the monolayer plane. The equilibrium and nonequilibrium phenomena are analyzed and, respectively, correlated with monolayer cohesion and with monolayer/sub-surface interactions. The purpose of this work is to gain further insights into the influences (as slight as they are) of the weak changes in phospholipid structure and on the behavior of the monolayers. The results, widely described, provide further details on nuances existing between very similar molecules, and likewise, on the synergies created between the different effects.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biochemistry; Chloroform; Fatty Acids; Membrane Lipids; Models, Chemical; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Rheology; Surface Properties

Lipid bilayer membranes--ubiquitous in biological systems and closely associated with cell function--exhibit rich shape-transition behaviour, including bud formation and vesicle fission. Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles. Detailed theoretical frameworks link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains.

Topics: Cell Membrane; Fluorescent Dyes; Lipid Bilayers; Liposomes; Microscopy, Fluorescence; Models, Biological; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Photons; Temperature

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Animals; Cholesterol; Fluorescent Dyes; G(M1) Ganglioside; Kidney Cortex; Laurates; Lipid Bilayers; Membrane Lipids; Microscopy, Fluorescence; Microvilli; Models, Biological; Models, Molecular; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Rats; Rats, Sprague-Dawley; Sphingomyelins

In this study, the mechanism by which an amphipathic negatively charged peptide consisting of 11 amino acids (WAE) induces fusion of liposomal phosphatidylcholine membranes is investigated. WAE-induced fusion, which only occurs when the peptide is covalently attached to the bilayer, shows a highly remarkable dependence on naturally occurring phosphatidylcholine species. The initial rate of fusion increased in the order 1-palmitoyl 2-arachidonoyl PC (PAPC) > 1-palmitoyl 2-oleoyl PC (POPC) > 1-stearoyl 2-oleoyl PC (SOPC) > dioleoyl PC (DOPC) > egg yolk PC. Interestingly, the susceptibility of the various PC species toward WAE-induced fusion matched a similar order of increase in intrinsic lipid headgroup spacing of the target membrane. The degree of spacing, in turn, was found to be related to the extent by which the fluorescence quantum yield of the Trp residue increased, which occurred upon the interaction of WAE with target membranes. Therefore, these results demonstrate an enhanced ability for WAE to engage in hydrophobic interactions when headgroup spacing increases. Thus, this latter parameter most likely regulates the degree of penetration of WAE into the target membrane. Apart from penetrating, WAE oligomerizes at the site of fusion as revealed by monitoring the self-quenching of the fluorescently derivatized lipid anchor to which WAE is attached. Clustering appears specifically related to the process of membrane fusion and not membrane aggregation. This is indicated by the fact that fusion and clustering, but not aggregation, display the same strict temperature dependence. However, evidence is presented indicating that clustering is an accompanying event rather than a prerequisite for fusion. The notion that various biologically relevant fusion phenomena are accompanied by protein clustering and the specific PC-species-dependent regulation of membrane fusion emphasize the biological significance of the peptide in serving as a model for investigating mechanisms of protein-induced fusion.

Topics: Egg Yolk; Glycerides; Lipid Bilayers; Lysophosphatidylcholines; Membrane Fusion; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Temperature

We have studied in detail the effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on the thermotropic properties of model membranes using differential scanning calorimetry (DSC). The results obtained showed that, for model membranes from hydrated dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and egg sphingomyelin, the biologically active analog significantly broadened the phase transition, in contrast to the inactive one which produced only marginal effects. Also, alphaxalone abolished the pretransition in these preparations whereas its delta 16-analog only broadened it. However, in DPPE bilayers almost no differences were observed in the effects produced by the two analogs. These results suggest that the ability of the two steroids to perturb membranes is lipid dependent. Comparisons between the effects of the two steroids on lipid/cholesterol model membranes revealed that delta 16-alphaxalone excluded cholesterol from lipid/cholesterol/delta 16-alphaxalone ternary systems whereas alphaxalone enhanced the effects of cholesterol and reduced the cooperativity in the binary phospholipid/cholesterol system. In an attempt to determine whether the different thermotropic effects of the two steroids on model membranes were due to (a) differences in their ability to perturb the bilayers; (b) different extents of incorporation into the bilayer, solid state 2H-NMR was applied using specifically deuterated steroids. The 2H-NMR data showed that alphaxalone incorporated fully into the membrane bilayer up to a molar concentration of 20%, while its inactive analog did only up to a concentration of 1%. To compare the abilities of the two steroids to perturb membrane preparations when both analogs were present in equal amounts in the membrane, the effects of very low steroid concentrations on DPPC bilayers were studied using DSC. The experiment showed that alphaxalone perturbed the membrane bilayers more effectively than its inactive analog. These results strongly suggest that the small structural differences between the two steroids are responsible for the observed differences in their abilities to perturb membranes, possibly because of differences in the packing of these two molecules within the bilayers.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Anesthetics; Calorimetry, Differential Scanning; Cholesterol; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylethanolamines; Pregnanediones; Sphingomyelins

Transferrin was incorporated into the external surface of liposomes that were previously derivatized with ceramides. This new hydrophobic derivative of transferrin was incubated with liposomes at different protein-to-lipid ratios. The surface activity of both native and derivatized transferrin was determined with monomolecular layers as a membrane model. The maximal interaction was found with dipalmitoylphosphatidylcholine in all the experiences.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Ceramides; Chemical Phenomena; Chemistry, Physical; Kinetics; Liposomes; Membranes, Artificial; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylethanolamines; Schiff Bases; Spectrophotometry, Ultraviolet; Surface Properties; Thermodynamics; Transferrin

CTP:phosphocholine cytidylyltransferase present in rat liver cytosol was activated almost 30-fold when assayed in the presence of liposomes containing 60 mole % dioleoyl phosphatidylethanolamine (DOPE). During the assay, some of the DOPE was degraded to lysoPE and oleic acid. Whereas cytidylyltransferase activity was not affected when assayed in the presence of liposomes containing lysoPE, liposomes containing oleic acid activated the enzyme. Activation by oleic acid could be eliminated by the addition of fatty acid-free bovine serum albumin (BSA) to the assay. When cytidylyltransferase activity was measured in the presence of both BSA and liposomes containing DOPE, enzyme activity was increased almost 20-fold, as compared with assays performed in the absence of added lipid. The 1.5-fold difference in cytidylyltransferase activity observed when cytosol was assayed with DOPE containing liposomes in the absence or presence of BSA (30-fold stimulation vs 20-fold stimulation) cannot be explained by the loss of activation attributable to oleic acid alone. Activation of the enzyme in the presence of liposomes containing DOPE and oleic acid is several-fold greater than the sum of the activations caused by the individual compounds. These data suggest that PE and oleic acid act synergistically in activating the cytidylyltransferase.

Topics: Animals; Choline-Phosphate Cytidylyltransferase; Cytosol; Drug Synergism; Enzyme Activation; Lipids; Liver; Nucleotidyltransferases; Oleic Acid; Oleic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Rats; Tissue Preservation

CaATPase from rabbit sarcoplasmic reticulum has been isolated, purified, stripped of its native lipids, and reconstituted into binary lipid mixtures of dielaidoylphosphatidylcholine (DEPC) and dipalmitoylphosphatidylethanolamine (DPPE) or acyl-chain perdeuterated DPPE (DPPE-d62). The partitioning properties of the protein were determined from differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. Acyl-chain perdeuteration allows the separate determination of the order and melting characteristics of each lipid species with FT-IR. The binary lipid mixture has been shown to be phase separated in the gel state (Brauner, J. W., and R. Mendelsohn, 1986, Biochim. Biophys. Acta, 861:16-24). The solid phases present at low temperatures correspond to a pure DEPC phase and a mixed phase of DEPC/DPPE-d62. Insertion of protein at 37 degrees C leads to a domain of relatively protein-free DPPE-d62 and a phase containing both lipids plus CaATPase. We suggest that CaATPase selects a fixed composition (60% DEPC, 40% DPPE-d62) for its immediate environment. The composition of the lipid in the immediate vicinity of protein is largely independent of the initial DEPC/DPPE-d62 ratios in the reconstitution protocol. The relevance of these results to observations of discrete domains in native membranes is discussed.

Topics: Animals; Calcium-Transporting ATPases; Lipid Bilayers; Muscles; Phosphatidylcholines; Phosphatidylethanolamines; Protein Conformation; Rabbits; Sarcoplasmic Reticulum; Spectrophotometry, Infrared; Thermodynamics