1-2-dielaidoylphosphatidylethanolamine and 1-2-oleoylphosphatidylcholine

It is clear that considerable conformational distortions may occur in peripheral proteins on interaction with anionic lipid bilayers. Specific lipid interactions do occur at least in the case of cytochrome c, and each perturbation and interaction may well take place before insertion into, or translocation across, a biomembrane.

Topics: Animals; Cytochrome c Group; Lipid Bilayers; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Phosphatidylethanolamines; Protein Conformation; Proteins

Desmin, an intermediate filament protein expressed in muscle cells, plays a key role in the integrity and regulation of the contractile system. Furthermore, the distribution of desmin in cells and its interplay with plasma and organelle membranes are crucial for cell functions; however, the fundamental properties of lipid-desmin interactions remain unknown. Using a water-in-oil method for a limited space system in vitro, we examined the distribution of desmin in three types of phospholipid droplets: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS). When fluorescent-labeled desmin was observed for 60 min after desmin assembly was initiated by adding 25 mM KCl, desmin accumulated on both the DOPE and DOPS layers; however, it did not accumulate on the DOPC layer of droplets. An increase in salt concentration did not moderate the accumulation. The initial form of either oligomer or mature filament affected the accumulation on each lipid layer. When liposomes were included in the droplets, desmin was associated with DOPE but not on DOPC liposomes. These results suggest that desmin has the potential for association with phospholipids concerning desmin form and lipid shape. The behavior and composition of living membranes may affect the distribution of desmin networks.

Topics: Animals; Chickens; Desmin; Intermediate Filaments; Lipid Droplets; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids

The interaction between the cinnamycin and the biomimetic membranes was studied using the atomic force microscope(AFM). The bilayer was composed of the monolayer tethered on the gold surface and the outer layer fused with the vesicles on the monolayer. The vesicles were prepared at the desired ratio of dioleoylphosphatidylethanolamine(DOPE) to dioleoylphosphatidylcholine(DOPC). On the bilayer, the surface force measurement was performed with the cinnamycin immobilized covalently on the tip surface. The immobilization led to the presence of the adhesion, which was found while the tip was retracted from the bilayer. In addition, the magnitude of the adhesive force was changed with respect to the composition of DOPE in the outer layer. The difference in the adhesion may be attributed to the mean-molecular-area of DOPE and the specific-binding density on the outer layer. Furthermore, the analysis of the rupture force with respect to the loading rate indicated that the rupture length was around 0.1∼0.13 nm, which was similar to that of a van der Waals bond.

Topics: Bacteriocins; Biomimetic Materials; Lipid Bilayers; Membranes, Artificial; Peptides, Cyclic; Phosphatidylcholines; Phosphatidylethanolamines

Parkinson's disease (PD), closely associated with the misfolding and aggregation of the neuronal protein α-synuclein (A-Syn), is a neurodegenerative disorder with no cure to date. Here, we show that the commercially available, inexpensive, aminoglycoside antibiotic kanamycin effectively inhibits both lipid-induced and solution-phase aggregation of A-Syn in vitro, pointing towards the prospective repurposing of kanamycin as a potential anti-PD drug.

Topics: alpha-Synuclein; Anti-Bacterial Agents; Cell Line, Tumor; Humans; Kanamycin; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Binding; Protein Conformation; Protein Multimerization; Unilamellar Liposomes

Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA-membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The K

Topics: Cholesterol; DNA, Single-Stranded; Lipid Bilayers; Molecular Dynamics Simulation; Nucleic Acid Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Static Electricity; Unilamellar Liposomes

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

This study describes the pore-forming properties of magainin 1 in planar lipid bilayers. These bilayers were prepared by the droplet contact method, which was executed on a microfabricated device for a high-throughput study. We arrayed four droplet chambers parallelly in the single device, and the current measurements were carried out simultaneously. Using this system, we measured the channel current conductance of magainin 1. We determined the pore size and the number of assembling monomers in magainin pores in mammalian and bacterial model membranes. This system is a powerful tool for analyzing transmembrane peptides and their antimicrobial activities.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Electric Conductivity; Lab-On-A-Chip Devices; Lipid Bilayers; Magainins; Membranes, Artificial; Microfluidic Analytical Techniques; Models, Theoretical; Molecular Sequence Data; Patch-Clamp Techniques; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Porosity; Xenopus laevis; Xenopus Proteins

The structural polymorphism and parameters of lyotropic phases formed in the mixed dioleoylphosphatidylcholine-dioleoylphosphatidylethanolamine (DOPC-DOPE) system upon heating and varying DOPC:DOPE composition were studied by means of small-angle X-ray diffraction (SAXD). In the temperature range 5-80°C a sequence of fluid lamellar Lα - inverse hexagonal HII - inverse cubic QII phases was detected at DOPE mole fractions XDOPE≥0.65. A superposition of two bicontinuous cubic QII phases of Pn3m and Ia3d space groups was identified. The Lα to HII phase transition temperature, the onset of the QII phase formation, as well as the lattice spacings of the Lα and HII phases were found to decrease with rising DOPE content. Moreover, evidence of structural rearrangement during the Lα to HII phase transition is given and change of transition mechanism with varying XDOPE is suggested.

Topics: Phase Transition; Phosphatidylcholines; Phosphatidylethanolamines; Scattering, Small Angle; Transition Temperature; X-Ray Diffraction

We report on the formation of planar lipid bilayer structures on mica where the bilayer contains the phosphocholine 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), cholesterol, sphingomyelin and sulforhodamine-tagged-1,2-dioleoyl-sn-phosphatidylethanolamine (SR-DOPE). Phase separation is seen for the cholesterol domains within the bilayer structure, and exposure of this supported bilayer to controlled concentrations of ethanol reveals organizational changes on both the micrometer- and molecular-length scales. We report steady state fluorescence imaging, fluorescence lifetime imaging, and fluorescence anisotropy decay imaging for these bilayers. These data are complementary to existing information on the interactions of lipid bilayers with ethanol and point to subtle but important changes in the molecular-scale organization of these structures.

Topics: Cholesterol; Ethanol; Fluorescence Polarization; Hydrogen Bonding; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins

Neutral lipids have received up to now a little attention as genetic material carriers, despite some valuable features, such as the absence of toxicity and the high stability in serum of their complexes with DNA. We have prepared two quaternary complexes of DNA and mixtures of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-hexanoylamine (6PE) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoylamine (12PE) with DOPC in aqueous dispersions of bivalent metal cations (PE/DOPC-DNA-M(2+)). The aim was to evaluate the effect of the amide moiety on the transfection efficiency. These complexes form in a self-assembled manner, the DNA condensation being promoted by the metal cations. Synchrotron X-ray diffraction analysis was used to determine the structure of the complexes, which exhibit the lamellar symmetry of the L(α)(c) phase. The size and surface charge of the complexes have also been measured, and promising results of DNA transfections in vitro have been reported.

Topics: Animals; Biophysical Phenomena; DNA; Drug Carriers; Metals; Mice; Models, Molecular; Molecular Conformation; NIH 3T3 Cells; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Transfection; X-Ray Diffraction

We report a fluorescence-based turn-on sensor for mapping the mechanical strain exerted by specific cell-surface proteins in living cells. The sensor generates force maps with high spatial and temporal resolution using conventional fluorescence microscopy. We demonstrate the approach by mapping mechanical forces during the early stages of regulatory endocytosis of the ligand-activated epidermal growth factor receptor (EGFR).

Topics: Biomechanical Phenomena; Biotin; Carbocyanines; Endocytosis; ErbB Receptors; Humans; Lipid Bilayers; Mechanoreceptors; Microscopy, Fluorescence; Nucleotides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylation; Polyethylene Glycols; Rhodamines

In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.

Topics: Chemistry Techniques, Analytical; Diffusion; Fluorescence; Fluorescent Dyes; Kinetics; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Time Factors

We have studied the interactions of the chromophore 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3-benzoxadiazol-4-yl (18:1 NBD-PE) imbedded in the headgroup region of bilayer lipid membranes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG). We have examined the molecular and mesoscale dynamics of the chromophore using time-correlated single photon counting (TCSPC) to measure rotational diffusion dynamics in lipid vesicles and fluorescence recovery after pattern photobleaching (FRAPP) to determine translational diffusion coefficients and mobile fractions in supported lipid bilayers. TCSPC data reveal that chromophore rotational diffusion rates in DOPG vesicles are statistically the same as in DOPC and mixed DOPC/DOPG vesicles, suggesting that the NBD-PE chromophore does not interact strongly with the headgroup region of these bilayers; however, FRAPP experiments show that lateral diffusion is statistically lower in mixed DOPC/DOPG-supported bilayers than in DOPC-supported bilayers. These results suggest that bilayers containing DOPG likely undergo interlipid headgroup hydrogen bonding interactions that suppress translational diffusion.

Topics: Anisotropy; Diffusion; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Hydrogen Bonding; Lipid Bilayers; Membrane Lipids; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Rotation

We show that cationic nanoparticles encapsulated within vesicles of phosphocholine lipid can induce pearling. The dynamic process occurs as two stages: formation of tubular protrusions followed by pearling instability. The breakup into individual vesicles can be controlled by nanoparticle concentration.

Topics: Liposomes; Nanoparticles; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylcholine; Rhodamines

Antifreeze proteins have been reported to be capable of maintaining the membrane integrity of cold sensitive mammalian cells when exposed to hypothermic temperatures. However the mechanism(s) whereby these proteins exert this protective effect is unknown. The present study used liposomes as a model system to examine the nature of the interactions between four antifreeze (glyco)protein types (AFP I, II, III and AFGP) and albumin, with lipid membranes. Fluorescein isothiocyanate labelling indicated that all of the proteins bound to the three liposome types (dielaidoylphosphatidylcholine (DEPC), dielaidoylphosphatidylethanolamine (DEPE) and dielaidoylphosphatidylglycerol (DEPG)). AFGP was found to be highly effective at preventing leakage from all three liposome compositions as they were cooled through their phase transition temperatures. This was not the case for the other proteins. All four antifreeze types prevented zwitterionic DEPC liposomes from leaking as they were cooled through their phase transition temperature. However, albumin was equally as effective, indicating that this capacity was not unique to antifreeze proteins. All of the proteins, except AFGP, induced the negatively charged DEPG liposomes to leak prior to cooling, and were less effective than AFGP in preventing phase transition leakage from DEPE liposomes. It is proposed that many proteins, including antifreeze proteins, can protect zwitterionic liposomes, such as DEPC, by binding to the lipid bilayer thereby maintaining the ordered structure of the membrane during phase transition. However, when the membrane contains a negatively charged polar group, such as with DEPE and DEPG, proteins, although bound to them, may not be able to maintain sufficient membrane organization to prevent leakage during phase transition or, they may gain entry into the lipid bilayer, disrupt the structure and induce leakage. These results imply that the efficacy of antifreeze proteins in the cold protection of mammalian cells will not only depend on protein structure, but also on the lipid composition of the cell membrane.

Topics: Antifreeze Proteins; Fluorescein-5-isothiocyanate; Lipid Bilayers; Liposomes; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Protein Binding; Protein Conformation

The effects of nonlamellar-prone lipids, diacylglycerol and phosphatidylethanolamine (PE), on the kinetic association of SecA with model membranes were examined by measuring changes in the intrinsic emission fluorescence with a stopped-flow apparatus. Upon interaction with standard liposomes composed of 50 mol% dioleolyphosphatidylcholine (DOPC) and 50 mol% of dioleoylphosphatidylglycerol (DOPG), the intrinsic fluorescence intensity of SecA was decreased after a lapse of time with a rate constant of 0.0049 s(-1). When the DOPC of the standard vesicles was gradually replaced with either dioeloyl PE (DOPE) or Escherichia coli (E. coli) PE, the rate constant increased appreciably as a function of PE concentration, in the order DOPE > E. coli PE. In addition, when the PE of E. coli PE/DOPG (50/50) vesicles was replaced with more than 5 mol% dioleoylglycerol (DOG), the rate constant further increased by 40%. The incorporation of nonlamellar-prone lipids in the vesicles also enhanced the binding of SecA to model membranes in the order DOPE > or = E. coli PE/DOG > E. coli PE > DOPC. These results provide the first kinetic evidence for the importance of nonlamellar-prone phospholipids for the association rate of SecA with membranes.

Topics: Adenosine Triphosphatases; Bacterial Proteins; Diglycerides; Escherichia coli Proteins; Ethanolamines; Flow Injection Analysis; Glycerophospholipids; Kinetics; Lipid Bilayers; Lipids; Liposomes; Membrane Transport Proteins; Membranes, Artificial; Models, Biological; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; SEC Translocation Channels; SecA Proteins; Spectrometry, Fluorescence

Incorporation of the helical antimicrobial peptide alamethicin from aqueous phase into hydrated phases of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) was investigated within a range of peptide concentrations and temperatures by time-resolved synchrotron X-ray diffraction. It was found that alamethicin influences the organizations of the non-bilayer-forming (DOPE) and the bilayer-forming (DOPC) lipids in different ways. In DOPC, only the bilayer thickness was affected, while in DOPE new phases were induced. At low peptide concentrations (<1.10(-4) M), an inverted hexagonal (H(II)) phase was observed as with DOPE dispersions in pure buffer solution. A coexistence of two cubic structures was found at the critical peptide concentration for induction of new lipid/peptide phases. The first one Q224 (space group Pn3m) was identified within the entire temperature region studied (from 1 to 45 degrees C) and was found in coexistence with H(II)-phase domains. The second lipid/peptide cubic structure was present only at temperatures below 16 degrees C and its X-ray reflections were better fitted by a Q212 (P4(3)32) space group, rather than by the expected Q229 (Im3m) space group. At alamethicin concentrations of 1 mM and higher, a nonlamellar phase transition from a Q224 cubic phase into an H(II) phase was observed. Within the investigated range of peptide concentrations, lamellar structures of two different bilayer periods were established with the bilayer-forming lipid DOPC. They correspond to lipid domains of associated and nonassociated helical peptide. The obtained X-ray results suggest that the amphiphilic alamethicin molecules adsorb from the aqueous phase at the lipid head group/water interface of the DOPE and DOPC membranes. At sufficiently high (>1.10(-4) M) solution concentrations, the peptide is probably accommodated in the head group region of the lipids thus inducing structural features of mixed lipid/peptide phases.

Topics: Alamethicin; Amino Acid Sequence; Anti-Bacterial Agents; Lipid Bilayers; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylethanolamines; Protein Conformation; Thermodynamics; Water; X-Ray Diffraction

Phospholipids containing phosphoethanolamine (PE) headgroups within biological membranes have been suggested to be important with respect to the functional regulation of membrane proteins, including the Ca-ATPase in sarcoplasmic reticulum (SR). To investigate the role of PE headgroups in modulating the catalytic activity of the Ca-ATPase, we have reconstituted the Ca-ATPase into unilamellar liposomes containing defined amounts of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). The enzymatic activity of the Ca-ATPase progressively increases upon incorporation of increasing amounts of PE into reconstituted vesicles, and approaches that characteristic of native SR membranes. To identify structural changes that correlate with enzyme activation, we have used frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of erythrosin isothiocyanate covalently bound to Lys464 in the phosphorylation domain of the Ca-ATPase. Progressive increases in the rotational dynamics of the phosphorylation domain result from the incorporation of increasing amounts of DOPE, and correlate with enhanced enzymatic function. These results suggest that PE headgroups induce dynamic structural rearrangements involving the phosphorylation domain that modify the rates of nucleotide utilization. In contrast, no changes in the rotational dynamics of the lipid acyl chains are observed irrespective of the PE content. Therefore, the enhanced ATP hydrolytic activity associated with the incorporation of DOPE into these proteoliposomes is the result of specific noncovalent interactions involving PE phospholipid headgroups and the Ca-ATPase.

Topics: Calcium-Transporting ATPases; Calorimetry; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Enzyme Activation; Fluorescence Polarization; Kinetics; Liposomes; Luminescent Measurements; Lysine; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylation; Proteolipids; Recombinant Proteins; Sarcoplasmic Reticulum; Thermodynamics

In an effort to model the interaction of lipid-based DNA delivery systems with anionic surfaces, such as a cell membrane, we have utilized microelectrophoresis to characterize how electrokinetic measurements can provide information on surface charge and binding characteristics. We have established that cationic lipids, specifically N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), incorporated into liposomes prepared with 1, 2-dioleoyl-i-glycero-3-phosphoethanolamine (DOPE) or 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 50 mol%, change the inherent electrophoretic mobility of anionic latex polystyrene beads. Self-assembling lipid-DNA particles (LDPs), prepared at various cationic lipid to negative DNA phosphate charge ratios, effected no changes in bead mobility when the LDP charge ratio (+/-) was equal to or less than 1. Increasing the LDP concentration in a solution of 0.1% (w/v) anionic beads resulted in a charge reversal effect when a net charge of LDP to total bead charge ratio (+/-) of 1:1 was observed. LDP formulations, utilizing either DOPE or DOPC, showed similar titration profiles with a charge reversal observed at a 1:1 net LDP to bead charge ratio (+/-). It was confirmed through centrifugation studies that the DNA in the LDP was associated with the anionic latex beads through electrostatic interactions. LDP binding, rather than the binding of dissociated cationic lipids, resulted in the observed electrophoretic mobility changes of the anionic latex beads.

Topics: Anions; Cations; DNA; Drug Compounding; Drug Delivery Systems; Electricity; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Surface Properties; Surface-Active Agents

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.

Topics: Bacteriorhodopsins; Cholic Acids; Dimyristoylphosphatidylcholine; Halobacterium salinarum; Lipid Bilayers; Membrane Proteins; Micelles; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipid Ethers; Pressure; Protein Folding; Protein Structure, Secondary; Spectrophotometry, Ultraviolet

A peptide corresponding to the 23 N-terminal amino acid residues of the human immunodeficiency virus type-1 (HIV-1) gp41 has the capacity to induce intervesicular lipid mixing in large unilamellar liposomes composed of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol (CHOL) (molar ratio, 1:1:1). Cryo-transmission electron microscopy (cryo-TEM) of diluted vesicles to which peptides has been externally added reveals a morphology that is compatible with the formation of nonlamellar lipidic aggregates during the time-course of lipid mixing. 31P-nuclear magnetic resonance and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMADPH) steady-state anisotropy data at equilibrium indicate that the peptide is able to modulate the lipid polymorphism in pelletted membranes by: (i) promoting the thermotropic formation of inverted phases; and (ii) driving the lamellar-to-nonlamellar transition towards the formation of isotropic phases. Therefore, our combined morphological and spectroscopic data reveal the existence of a direct correlation between the ability of the externally added peptide to induce lipid-mixing in dilute liposome samples and its capacity to modulate lipid polymorphism in stacked bilayers.

Topics: Amino Acid Sequence; Cholesterol; Cryoelectron Microscopy; HIV Envelope Protein gp41; HIV-1; Humans; Kinetics; Lipid Bilayers; Molecular Sequence Data; Peptide Fragments; Phosphatidylcholines; Phosphatidylethanolamines

The effects of a series of normal alkanes (decane, dodecane, tetradecane, hexadecane, and octadecane) on the hexagonal H(II) structures containing dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) were studied using x-ray diffraction and osmotic stress. The alkanes affect structural dimensions and the monolayer intrinsic curvature and bending modulus. The alkane effects are chain-length dependent and are attributed to their different distribution within the H(II) structure. The data suggest that short-chain alkanes are more uniformly distributed within the H(II) hydrocarbon regions and change the curvature and bending modulus of the monolayer, whereas longer-chain alkanes appear confined more to the interstitial region and do not change the curvature and bending modulus.

Topics: Alkanes; Elasticity; Models, Chemical; Models, Molecular; Molecular Conformation; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines; Stress, Mechanical; Structure-Activity Relationship; Thermodynamics; X-Ray Diffraction

We have recently described a method for preparing lipid-based DNA particles (LDPs) that form spontaneously when detergent-solubilized cationic lipids are mixed with DNA. LDPs have the potential to be developed as carriers for use in gene therapy. More importantly, the lipid-DNA interactions that give rise to particle formation can be studied to gain a better understanding of factors that govern lipid binding and lipid dissociation. In this study the stability of lipid-DNA interactions was evaluated by measurement of DNA protection (binding of the DNA intercalating dye TO-PRO-1 and sensitivity to DNase I) and membrane destabilization (lipid mixing reactions measured by fluorescence resonance energy transfer techniques) after the addition of anionic liposomes. Lipid-based DNA transfer systems were prepared with pInexCAT v.2.0, a 4.49-kb plasmid expression vector that contains the marker gene for chloramphenicol acetyltransferase (CAT). LDPs were prepared using N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and either 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). For comparison, liposome/DNA aggregates (LDAs) were also prepared by using preformed DODAC/DOPE (1:1 mole ratio) and DODAC/DOPC (1:1 mole ratio) liposomes. The addition of anionic liposomes to the lipid-based DNA formulations initiated rapid membrane destabilization as measured by the resonance energy transfer lipid-mixing assay. It is suggested that lipid mixing is a reflection of processes (contact, dehydration, packing defects) that lead to formulation disassembly and DNA release. This destabilization reaction was associated with an increase in DNA sensitivity to DNase I, and anionic membrane-mediated destabilization was not dependent on the incorporation of DOPE. These results are interpreted in terms of factors that regulate the disassembly of lipid-based DNA formulations.

Topics: Animals; Chloramphenicol O-Acetyltransferase; Deoxyribonucleases; Detergents; DNA; Drug Carriers; Liposomes; Melanoma, Experimental; Mice; Models, Molecular; Molecular Conformation; Nucleic Acid Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Recombinant Proteins; Solubility; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured; Unithiol

The gradual hydration of phospholipid films can be effectively probed by Fourier transform infrared (FTIR) spectroscopy (cf. part I of this series). The hydration-induced changes observed for lipid IR-absorption bands are probably composed of contributions arising from the effects of both the direct binding of water molecules and the thereby caused conformational changes and phase transitions in the lipid molecules and assemblies, respectively. In this article, an attempt is made to attribute some of the more indicative spectroscopic results to these molecular and supermolecular processes with a view to separating their individual contributions to the relevant spectroscopic data. This is done by considering a series of suitable PLs consisting of the palmitoyl and oleoyl lecithins, DPPC, DOPC, POPC, and OPPC, and one cephalin, DOPE. This choice of PCs and DOPE means that at room temperature and different degrees of hydration, several phase states including lamellar gel and liquid crystalline as well as certain nonlamellar phases are covered. The separation of the water-binding and phase-transition contributions to the FTIR-spectroscopic data, we believe, is clearly demonstrated by interpreting the hydration-dependent wavenumber shifts of the nu C=O band of the PCs. Carbonyl groups are affected to a more significant degree for lipids arrayed in the L alpha phase than in the gel phase. A number of spectral features reveal the lyotropically triggered chain-melting transition as well as other structural rearrangements of PCs. This is discussed in detail and demonstrates the excellent sensitivity of the FTIR methodology for the study of such systems.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Kinetics; Phosphatidylcholines; Phosphatidylethanolamines; Spectroscopy, Fourier Transform Infrared; Structure-Activity Relationship; Thermodynamics; Water

Topics: Choline-Phosphate Cytidylyltransferase; Dimyristoylphosphatidylcholine; Lipid Bilayers; Membrane Lipids; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Stress, Mechanical

Cationic liposomes complexed with DNA (CL-DNA) are promising synthetically based nonviral carriers of DNA vectors for gene therapy. The solution structure of CL-DNA complexes was probed on length scales from subnanometer to micrometer by synchrotron x-ray diffraction and optical microscopy. The addition of either linear lambda-phage or plasmid DNA to CLs resulted in an unexpected topological transition from liposomes to optically birefringent liquid-crystalline condensed globules. X-ray diffraction of the globules revealed a novel multilamellar structure with alternating lipid bilayer and DNA monolayers. The lambda-DNA chains form a one-dimensional lattice with distinct interhelical packing regimes. Remarkably, in the isoelectric point regime, the lambda-DNA interaxial spacing expands between 24.5 and 57.1 angstroms upon lipid dilution and is indicative of a long-range electrostatic-induced repulsion that is possibly enhanced by chain undulations.

Topics: Bacteriophage lambda; Cations; Chemical Phenomena; Chemistry, Physical; DNA; DNA, Viral; Fatty Acids, Monounsaturated; Isoelectric Point; Light; Lipid Bilayers; Liposomes; Microscopy, Fluorescence; Microscopy, Interference; Nucleic Acid Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Scattering, Radiation; X-Ray Diffraction

Phospholipase A2 (PLA2)-mediated hydrolysis of membrane phospholipids was measured by ellipsometry, and the inhibition of this process by annexin V was studied. Planar membranes, consisting of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (PC/PE/PS; 54:33:13, on molar basis), were degraded by pancreatic PLA2, and the rate of hydrolysis was limited to about 0.7%/min. The influence of graded coverage of the membrane with annexin V was studied. The degree of PLA2 inhibition was nonlinearly related to the amount of membrane-bound annexin V, and binding of only 12% and 54% of full membrane coverage resulted in, respectively, 50% and 93% inhibition. These findings indicate that the inhibition of PLA2-mediated hydrolysis by annexin V cannot be simply explained by shielding of phospholipid substrates from the enzyme. Moreover, the present results leave room for a role of endogenous annexin V in regulating phospholipid turnover in the plasma membrane of parenchymal cells such as cardiomyocytes.

Topics: Animals; Annexin A5; Kinetics; Lipid Bilayers; Pancreas; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipases A; Phospholipases A2; Surface Properties; Swine

The behavior of dioleoylphosphatidylethanolamine (DOPE)/cholesterol/tetradecane and dioleoylphosphatidylcholine (DOPC)/cholesterol/tetradecane were examined using x-ray diffraction and the osmotic stress method. DOPE/tetradecane, with or without cholesterol, forms inverted hexagonal (HII) phases in excess water. DOPC/tetradecane forms lamellar phases without cholesterol at lower temperatures. With tetradecane, as little as 5 mol% cholesterol in DOPC induced the formation of HII phases of very large dimension. Increasing levels of cholesterol result in a systematic decrease in the HII lattice dimension for both DOPE and DOPC in excess water. Using osmotic pressure to control hydration, we applied a recent prescription to estimate the intrinsic curvature and bending modulus of the HII monolayers. The radii of the intrinsic curvature, RPO, at a pivotal plane of constant area within the monolayer were determined to be 29.4 A for DOPE/tetradecane at 22 degrees C, decreasing to 27 A at 30 mol% cholesterol. For DOPC/tetradecane at 32 degrees C, RPO decreased from 62.5 A to 40 A as its cholesterol content increased from 30 to 50 mol%. These data yielded an estimate of the intrinsic radius of curvature for pure DOPC of 87.3 A. The bending moduli kc of DOPE/tetradecane and DOPC/tetradecane, each with 30 mol% cholesterol, are 15 and 9 kT, respectively. Tetradecane itself was shown to have little effect on the bending modulus in the cases of DOPE and cholesterol/DOPE. Surprisingly, cholesterol effected only a modest increase in the kc of these monolayers, which is much smaller than estimated from its effect on the area compressibility modulus in bilayers. We discuss possible reasons for this difference.

Topics: Alkanes; Cholesterol; Elasticity; Liposomes; Models, Chemical; Models, Structural; Molecular Conformation; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines; Stress, Mechanical; X-Ray Diffraction

Freezing injury in rye and oat is a consequence of the formation of the inverted hexagonal (H(II)) phase in regions where the plasma membrane is brought into close proximity with cytoplasmic membranes during freeze-induced dehydration. Susceptibility to plasma membrane destabilization and H(II) phase formation during freezing is associated with alterations in plasma membrane lipid composition. This paper examines the influence of lipid composition and hydration on the propensity of lipid mixtures of DOPE:DOPC and DOPE:DOPC:sterols with added cerebrosides (CER) to form the H(II) phase during dehydration. The addition of CER to DOPE:DOPC:beta-sitosterol mixtures decreased the water content of the dispersions in a manner suggesting that most or all of the water in the dehydrated mixtures was associated with the phospholipids. The addition of CER significantly decreased the osmotic pressure at which the L(alpha) --> H(II) phase transition occurred from an osmotic pressure of 76.1 MPa for DOPE:DOPC (50:50) to 20 MPa in DOPE:DOPC:beta-sitosterol:CER (22.5:22.5:50:5) and 8 MPa in DOPE:DOPC:beta-sitosterol:CER (15:15:50:20). Experiments examining the effects of CER on the thermally-induced formation of the H(II) phase in fully hydrated mixtures and examining the influence of CER on the formation of the H(II) phase in DOPE:DOPC mixtures lacking beta-sitosterol suggested that CER facilitated the L(alpha) --> H(II) phase transition by effecting a decrease in bilayer hydration and by increased lateral packing pressures within the acyl domain of the bilayer. Taken in sum, these data indicate that the differential propensity of the rye and oat plasma membranes to undergo freeze-induced formation of the L(alpha) --> H(II) phase cannot be attributed to one lipid species. Rather, the propensity towards freeze-induced membrane destabilization is a consequence of the summation of physical characteristics of the membrane lipid components that included bilayer hydration, packing pressures within the hydrophobic domain of the membrane, the propensity of the lipid components to demix, and the relative proportions of the various lipid components.

Topics: Avena; Calorimetry, Differential Scanning; Cell Membrane; Cerebrosides; Freezing; Lipid Bilayers; Microscopy, Electron; Osmotic Pressure; Phosphatidylcholines; Phosphatidylethanolamines; Secale; Sitosterols; Sterols; Temperature; Water; X-Ray Diffraction

The structural and fusogenic properties of large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-phosphatidylethanotamine (DOPE) have been examined in the presence of pCMV5 plasmid and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between DOTMA/DOPE (1:1) LUVs and DOTMA/1,2-dioleoyl-3-phosphatidylcholine (DOTMA/DOPC) (1:1) LUVs such that at a cationic lipid-to-DNA charge ratio of 3.0, approximately 80% fusion is observed. The anions citrate and chloride can also trigger fusion, but at much higher concentrations. Freeze-fracture electron microscopy studies demonstrate the tendency of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels. 31P NMR studies of the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" 31P NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader "bilayer" 31P NMR spectra. In agreement with previous studies, the transfection potency of the DOPE-containing systems is dramatically higher than for the DOPC-containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. Interestingly, it was found that the complexes could be separated by centrifugation into a pellet fraction, which exhibits superior transfection potencies, and a supernatant fraction. Again, the pellet fraction in the DOPE-containing system exhibits a significantly narrower 31P NMR resonance than the corresponding DOPC-containing system. It is suggested that the 31P NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of nonbilayer lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection.

Topics: Animals; Anions; Cations; Cells, Cultured; Cricetinae; Fluorescent Dyes; Fluorometry; Freeze Fracturing; Kinetics; Liposomes; Magnetic Resonance Spectroscopy; Membrane Fusion; Microscopy, Electron; Phosphatidylcholines; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Structure-Activity Relationship; Transfection

Cationic liposomes are used to deliver genes into cells in vitro and in vivo. The present study is aimed to characterize the electrostatic parameters of cationic, large unilamellar vesicles, 110 +/- 20 nm in size, composed of DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, DMRIE/DOPE 1/1, or DC-CHOL/DOPE (mole ratio 1/1). {. DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; DC-CHOL, 3beta[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol}. The cationic liposomes had a large positive surface potential and a high pH at the liposomal surface in 20 mM Hepes buffer (pH 7.4) as monitored by the pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin. In contrast to DOTAP and DMRIE which were 100% charged, DC-CHOL in DC-CHOL/DOPE (1/1) liposomes was only about 50% charged in 20 mM Hepes buffer (pH 7.4). This might result in an easier dissociation of bilayers containing DC-CHOL from the plasmid DNA (which is necessary to enable transcription), in a decrease of the charge on the external surfaces of the liposomes or DNA-lipid complexes, and in an increase in release of the DNA-lipid complex into the cytosol from the endosomes. Other electrostatic characteristics found were that the primary amine group of DOPE in cationic liposomes dissociated at high (> 7.9) pHbulk and that a salt bridge was likely between the quaternary amine of DOTAP or DMRIE and the phosphate group of DOPE or DOPC, but not between the tertiary amine of DC-CHOL and the phosphate group of DOPE. The liposomes containing DOTAP were unstable upon dilution, probably due to the high critical aggregation concentration of DOTAP, 7 X 10(-5) M. This might also be a mechanism of the dissociation of bilayers containing DOTAP from the plasmid DNA.

Topics: Cholesterol; Chromatography, High Pressure Liquid; Fatty Acids, Monounsaturated; Fluorescent Dyes; Gene Transfer Techniques; Hydrogen-Ion Concentration; Kinetics; Lipids; Liposomes; Models, Structural; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Static Electricity; Structure-Activity Relationship; Surface Properties; Umbelliferones

The effect of a variety of gangliosides has been tested on the phospholipase C-induced fusion of large unilamellar vesicles. Bilayer composition was phosphatidylcholine:phosphatidylethanolamine: cholesterol (2:1:1 mole ratio) plus the appropriate amounts of glycosphingolipids. Enzyme phosphohydrolase activity, vesicle aggregation, mixing of bilayer lipids and mixing of liposomal aqueous contents were separately assayed. Small amounts ( < 1 mol %) of gangliosides in the lipid bilayer produce a significant inhibition of the above processes. The inhibitory effect of gangliosides increases with the size of the oligosaccharide chain in the polar head group. Inhibition depends in a nonlinear manner on the ganglioside proportion, and is complete at approximately 5 mol %. Inhibition is not due to ganglioside-dependent changes in vesicle curvature or size. Ganglioside inhibition of vesicle fusion is due to two different effects: inhibition of phospholipase C activity and stabilization of the lipid lamellar phase. Enzyme inhibition leads to a parallel decrease of vesicle aggregation and lipid mixing rates. Mixing of aqueous contents, though, is depressed beyond the enzyme inhibition levels. This is explained in terms of the fusion pore requiring a local destabilization of the lipid bilayer, the lamellar structure being stabilized by gangliosides. 31P-NMR and DSC experiments confirm the inhibitory effect of gangliosides in various lamellar-to-nonlamellar transitions.

Topics: Animals; Bacillus cereus; Brain; Calorimetry, Differential Scanning; Carbohydrate Sequence; Cattle; Ceramides; Gangliosides; Kinetics; Lipid Bilayers; Liposomes; Membrane Fusion; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylethanolamines; Structure-Activity Relationship; Thermodynamics; Type C Phospholipases

In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.

Topics: Animals; beta-Galactosidase; CHO Cells; Cricetinae; Drug Carriers; Elasticity; Escherichia coli; Fatty Acids, Monounsaturated; Freeze Fracturing; Light; Liposomes; Microscopy, Confocal; Microscopy, Electron; Phosphatidylcholines; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Recombinant Proteins; Scattering, Radiation; Transfection

The control of cytochrome c oxidase turnover in proteoliposomes by membrane potential (delta psi) and by pH gradient (delta pH) is probably kinetic in nature, and inhibition by valinomycin and stimulation by nigericin indicate that delta pH exerts a greater influence than does an equivalent delta psi. Oleic acid at 100 microM removes all delta psi and delta pH control, whereas a similar concentration of palmitic acid increases turnover but does not completely abolish control. Valinomycin acts synergistically with both fatty acids, indicating that the latter can act as H+/K+ exchangers, but neither fatty acid alone markedly affects delta pH, showing that they cannot fully mimic nigericin. Oleate, but not palmitate, diminishes delta psi, and can move electrophoretically as oleate anion. Submicromolar palmitic acid concentrations partly stimulate turnover in delta psi- and delta pH-controlled proteoliposomes, as reported by Labonia, Muller and Azzi [(1988) Biochem. J. 254, 130-145], which might represent a direct effect on cytochrome c oxidase. The ubiquity of fatty acids in biological membranes suggests that these substances might be responsible for limiting respiratory control and enzyme activity in vivo.

Topics: Animals; Cattle; Electron Transport Complex IV; Fatty Acids, Nonesterified; Hydrogen-Ion Concentration; Ionophores; Kinetics; Liposomes; Membrane Potentials; Mitochondria, Heart; Nigericin; Oleic Acid; Palmitic Acid; Phosphatidylcholines; Phosphatidylethanolamines; Proteolipids; Valinomycin

The intrinsic or spontaneous radius of curvature, R(o), of lipid monolayer assemblies is expressed in terms of a lipid molecular packing parameter, V/AI, for various geometries. It is shown that the equivalent lipid length, 1, in inverted hexagonal (HII) phases, defined by a cylindrical shell of equal total lipid volume, yields an expression for R o identical to that for inverted cylindrical micelles (or, equivalently, HII phases in the presence of excess hydrocarbon). This identity is used to obtain values of the effective packing parameter for various phosphatidylethanolamines. The temperature dependence of the intrinsic radius of curvature is predicted to be negative and to be considerably greater than that for the lipid length in nearly all cases. The thermal expansion coefficient is not constant but is found to vary, depending on the value of the lipid packing parameter. A possible addition rule is constructed for the intrinsic radius of curvature of lipid mixtures, based on the linear additivity of the effective molecular volumes, V, and molecular areas, A. This relation is found to hold for mixtures of dioleoyl phosphatidylcholine (DOPC) with dioleoyl phosphatidylethanolamine, and a value of R(o) of > or = 9 A (V/AI = 1.08) is obtained for DOPC. The energetics of the intrinsic curvature and lamellar-nonlamellar transitions are also discussed within the framework of the model.

Topics: Kinetics; Lipid Bilayers; Membrane Lipids; Micelles; Models, Theoretical; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics

The roles of acyl chain unsaturation and curvature in the excimer formation efficiency (EFE) of site-specific conjugated pyrene molecules in lipid membranes have been investigated by steady-state and time-resolved fluorescence spectroscopy. Six 1-2-(pyrenyl-n-acyl)-phosphatidylcholine (dipy(n)PC) probes, with pyrenyl chains of varying methylene units n from 4 to 14 carbons, were incorporated separately into dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE) lipid membranes at 0.1 mol%. Both the excimer-to-monomer fluorescence intensity ratio and association-to-dissociation rate constant ratio of conjugated pyrenes were used to quantify EFE. At all temperatures (T = 0-30 degrees C) and for n = 4 and 6, the EFE for DOPE was always smaller than EFE for DOPC. At T < 10 degrees C (where DOPE and DOPC are in the liquid crystalline L alpha phase) and for n > 8, the EFE for curvature frustrated DOPE was significantly greater than EFE for nonfrustrated DOPC (control), and the difference increased gradually with n. At T> 18 degrees C (where DOPE is in the inverted hexagonal H(II) phase and DOPC is in the L alpha phase) and for n > 8, EFE for the curvature-relaxed DOPE was again smaller than the EFE for DOPC control. The contributions of splay conformation and internal dynamics of pyrenyl chains to EFE were examined separately using a lattice model. Our results suggest that i) the cis double bonds of the host lipid matrix strongly perturb both the conformation and dynamics of conjugated pyrenes at the specific location around n = 8, and ii) the lateral stress at the upper part (n < 8) of the curvature frustrated bilayer membranes (DOPE) may be significantly relaxed once the membrane surface adopts a favorable negative interfacial curvature.

Topics: Kinetics; Lipid Bilayers; Models, Structural; Models, Theoretical; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Pyrenes; Spectrometry, Fluorescence; Structure-Activity Relationship; Thermodynamics

Freeze-induced injury of protoplasts of non-acclimated rye and oat is associated with the formation of the inverted hexagonal (HII) phase in regions where the plasma membrane and various endomembranes are brought into close apposition as a result of freeze-induced dehydration. The influence of lipid composition and hydration on the propensity of mixtures of DOPE:DOPC containing either sterols or acylated steryl glucosides to form the HII phase was determined by DSC, freeze-fracture electron microscopy and X-ray diffraction. The addition of plant sterols to a mixture of DOPE/DOPC (either 1:1:1 or 1:1:2 mole ratio of DOPE/DOPC/sterols) reduced the total hydration of the mixture (expressed as wt% water) after desorption over a range of osmotic pressures of 2.8 to 286 MPa. However, most or all of the water remaining in the dehydrated lipid mixtures was associated predominantly with the phospholipids. Both sterols and acylated steryl glucosides significantly promoted both the dehydration-induced and thermally induced L alpha-->HII phase transitions in DOPE/DOPC mixtures however, acylated steryl glucosides were much more effective. In mixtures containing plant sterols, the HII phase occurred after dehydration at 20 MPa (20 degrees C), which resulted in a water content of 11.7 wt%. In contrast, mixtures containing acylated steryl glucosides were in the HII phase in excess water, i.e., they did not require dehydration to effect the L alpha-->HII phase transition. The results indicate that genotypic differences in the lipid composition of the plasma membrane of rye and oat leaves have a significant influence on the propensity for formation of the HII phase during freeze-induced dehydration.

Topics: Acylation; Calorimetry, Differential Scanning; Cell Membrane; Freeze Fracturing; Glucosides; Phosphatidylcholines; Phosphatidylethanolamines; Phytosterols; Water; X-Ray Diffraction

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Topics: Animals; Endocytosis; Lipid Bilayers; Liposomes; Membrane Fusion; Phosphatidylcholines; Phosphatidylethanolamines; Rats; Rats, Sprague-Dawley; Reticulocytes; Transferrin

The response of coagulation tests to heparin can be expressed as the coagulation time of plasma containing heparin divided by the coagulation time of the same plasma without heparin (CT ratio). The purpose of the present study was to assess the influence of liposomes on these response to heparin of four coagulation tests: the kaolin-induced coagulation time, the tissue factor-induced coagulation time, the factor Xa-induced coagulation time, and the thrombin-induced coagulation time. Liposomes were prepared from dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and dioleoylphosphatidylserine (DOPS). High concentrations of DOPS/DOPE/DOPC (20:40:40) liposomes enhanced the CT ratio of the four coagulation tests, more than DOPS/DOPC (20:80) or DOPE/DOPC (40:60) or a mixture of DOPS/DOPC and DOPE/DOPC liposomes. These experiments demonstrate that there is synergism between DOPS and DOPE in promoting heparin's anticoagulant effect if both phospholipids are incorporated into the same liposome surface.

Topics: Blood Coagulation; Blood Coagulation Tests; Drug Synergism; Heparin; Humans; Kaolin; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Thromboplastin

Capsaicin is a natural compound with pharmacological and toxicological effects, which given its hydrophobicity, can influence the structure of membranes. The interaction of capsaicin with model membranes of dipalmitoylphosphatidylcholine and dielaidoylphosphatidylethanolamine has been studied by using differential scanning calorimetry, fluorescent probe spectroscopy and 31P-nuclear magnetic resonance. Capsaicin remarkably affects the phase transition of dipalmitoylphosphatidylcholine, shifting the transition temperature to lower values, and giving rise, at relatively high capsaicin concentrations, to the appearance of two peaks in the thermogram. These peaks may correspond to separated phases as indicated by the partial phase diagram. Whereas capsaicin did not affect the fluorescence polarization of the probes diphenylhexatriene and trimethylammonium-diphenylhexatriene, it clearly affected that of the probe 2-anthroyloxystearic acid, indicating that the perturbation produced by capsaicin on the membrane would be mainly at the position where this fluorophore is located. On the other hand, capsaicin, at relatively low concentrations, gives rise to immiscible phases in the presence of dielaidoylphosphatidylethanolamine and decrease the temperature of the lamellar to hexagonal HII phase transition. At concentrations of capsaicin higher than 0.3 mol fraction, isotropic phases were detected. The possible implications of the effects of capsaicin on biological membranes are discussed.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Calorimetry, Differential Scanning; Capsaicin; Indicators and Reagents; Lysophosphatidylcholines; Magnetic Resonance Spectroscopy; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Structure-Activity Relationship

The interaction of cationic liposomes prepared using either dioleoyltrimethylammonium propane (DOTAP) or 3 beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-CHOL) with model membranes and with cultured mammalian cells was examined using an assay developed for monitoring virus-cell fusion (Stegmann et al. (1993) Biochemistry 32, 11330-11337). Lipid mixing between cationic liposomes and liposomes composed of DOPE/dioleoylphosphatidylglycerol (DOPG) or dioleoylphosphatidylcholine (DOPC)/DOPG was insensitive to pH in the range of pH 4.5-7.0 and was not affected by sodium chloride concentration in the range of 0-150 mM. Lipid mixing was dependent on dioleoylphosphatidylethanolamine (DOPE), since cationic liposomes prepared using dioleoylphosphatidylcholine (DOPC) were incapable of lipid mixing with DOPC/DOPG liposomes. The interaction of cationic liposomes with Hep G-2 and CHO D- cells was also studied. For both cell types, liposome-cell lipid mixing was rapid at 37 degrees C, beginning within minutes and continuing for up to 1 hour after uptake. The extent of lipid mixing was decreased at 15 degrees C, especially at later (> or = 20 min) time points. This suggests that at least part of the observed lipid mixing occurred after reaching cellular lysosomes. No lipid mixing was seen at 4 degrees C. Monensin inhibited lipid mixing between cationic liposomes and the cells, despite having no effect on liposome uptake. Inhibition of endocytic uptake of liposomes, either by incubation in hypertonic media or by depletion of cellular ATP with sodium azide and 2-deoxyglucose abolished liposome-cell fusion in both cell types. These data demonstrate that binding to the cell surface is insufficient for cationic liposome-cell fusion and that uptake into the endocytic pathway is required for fusion to occur.

Topics: Animals; Azides; Cations; CHO Cells; Cholesterol; Cricetinae; Deoxyglucose; Endocytosis; Fatty Acids, Monounsaturated; Hydrogen-Ion Concentration; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Monensin; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Saline Solution, Hypertonic; Sodium Azide

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.

Topics: Biological Transport; Calcium; Cations, Divalent; Cell Membrane; Escherichia coli; Escherichia coli Proteins; Magnesium; Membrane Lipids; Membrane Potentials; Mutation; Phosphatidylcholines; Phosphatidylethanolamines; Porins; Protein Precursors

Assays on vesicle aqueous content leakage are widely used in the study of peptide-lipid interactions. We found this assay to be affected by the mode of mixing vesicle and peptide solutions. This effect can lead to artifactual conclusions regarding the lytic activity of peptides. We demonstrate that the source of this artifact is that fast (millisecond range) peptide-membrane association creates a nonhomogeneous distribution which exists sufficiently long after peptide addition to markedly alter the course of leakage. Mixing problems can be overcome by using a stopped flow apparatus. It can also be diminished by switching to injecting a small volume of vesicle suspension into a large volume of peptide solution. Mixing rates are rarely considered in literature reports of peptide effects on liposomes. The same artifacts can also take place in a number of other assays of the activity of membrane active peptides on liposomes and thus their consideration is of general importance.

Topics: Artifacts; Hydrolysis; Kinetics; Liposomes; Naphthalenes; Peptides; Phosphatidylcholines; Phosphatidylethanolamines

Topics: Animals; Fluorescence; Humans; Hydrolysis; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipases A; Phospholipases A2; Rats; Recombinant Proteins; Substrate Specificity

Topics: Cryoprotective Agents; Dimethyl Sulfoxide; Freezing; Ice; In Vitro Techniques; Membrane Lipids; Membranes, Artificial; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics; Water; X-Ray Diffraction

We studied the effect of gangliosides GD1a and GM1 on the lamellar-to-hexagonal II phase transition of mixtures of dioleoylphosphatidylethanolamine/dioleoylphosphatidyl choline, 3:1, and of transphosphatidylated phosphatidylethanolamine with dioleoylglycerol by high-sensitivity differential scanning calorimetry, 31P-NMR, and pyrene fluorescence of a phosphatidylcholine probe. Gangliosides had a dual effect. Below 1 mol % ganglioside the hexagonal II phase transition was affected but still occurred at lower temperature than in the absence of gangliosides. The presence of between 1 and 2 mol % gangliosides increased the temperature for formation of the hexagonal II phase and progressively decreased its cooperativity. Above 3 mol % gangliosides totally inhibited the formation of both the temperature-induced and composition-induced hexagonal phase, probably by opposing the geometric distortions necessary for the inverted micellar structures.

Topics: Diglycerides; Gangliosides; Lipid Bilayers; Magnetic Resonance Spectroscopy; Micelles; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorus; Thermodynamics

Molecular dynamics (MD) simulations at 37 degrees C have been performed on three phospholipid bilayer systems composed of the lipids DLPE, DOPE, and DOPC. The model used included 24 explicit lipid molecules and explicit waters of solvation in the polar head group regions, together with constant-pressure periodic boundary conditions in three dimensions. Using this model, a MD simulation samples part of an infinite planar lipid bilayer. The lipid dynamics and packing behavior were characterized. Furthermore, using the results of the simulations, a number of diverse properties including bilayer structural parameters, hydrocarbon chain order parameters, dihedral conformations, electron density profile, hydration per lipid, and water distribution along the bilayer normal were calculated. Many of these properties are available for the three lipid systems chosen, making them well suited for evaluating the model and protocols used in these simulations by direct comparisons with experimental data. The calculated MD behavior, chain disorder, and lipid packing parameter, i.e. the ratio of the effective areas of hydrocarbon tails and head group per lipid (a(t)/ah), correctly predict the aggregation preferences of the three lipids observed experimentally at 37 degrees C, namely: a gel bilayer for DLPE, a hexagonal tube for DOPE, and a liquid crystalline bilayer for DOPC. In addition, the model and conditions used in the MD simulations led to good agreement of the calculated properties of the bilayers with available experimental results, demonstrating the reliability of the simulations. The effects of the cis unsaturation in the hydrocarbon chains of DOPE and DOPC, compared to the fully saturated one in DLPE, as well as the effects of the different polar head groups of PC and PE with the same unsaturated chains on the lipid packing and bilayer structure have been investigated. The results of these studies indicate the ability of MD methods to provide molecular-level insights into the structure and dynamics of lipid assemblies.

Topics: Algorithms; Computer Simulation; Lipid Bilayers; Models, Molecular; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Stereoisomerism; Temperature

The manner in which ice forms in lamellar suspensions of dielaidoylphosphatidylethanolamine, dielaidoylphosphatidylcholine, and dioleoylphosphatidylcholine in water depends strongly on the water fraction. For weight fractions between 15 and 9%, the freezing and melting temperatures are significantly depressed below 0 degree C. The ice exhibits a continuous melting transition spanning as much as 20 degrees C. When the water weight fraction is below 9%, ice never forms at temperatures as low as -40 degrees C. We show that when water contained in a lamellar lipid suspension freezes, the ice is not found between the bilayers; it exists as pools of crystalline ice in equilibrium with the bound water associated with the polar lipid headgroups. We have used this effect, together with the known chemical potential of ice, to measure hydration forces between lipid bilayers. We find exponentially decaying hydration repulsion when the bilayers are less than about 7 A apart. For larger separations, we find significant deviations from single exponential decay.

Topics: Freezing; Lipid Bilayers; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics; X-Ray Diffraction

The intramolecular dynamics of the excimer-forming dipyrenyl lipids (DipynPE) of different chain lengths (n) in fully hydrated dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) binary mixtures was investigated by the use of frequency-domain fluorescence intensity dcay technique. Using a 3-state model (see companion paper), the extent of aggregation and rotational rate of the two covalently attached pyrene moieties in DipynPE were estimated from the frequency-domain data. At 1 degrees C, the rotational rate and aggregation for Dipy4PE and Dipy10PE were insensitive to DOPE% of the lipid bilayer. At 27 degrees C, the rotational rate decreased, whereas the aggregation increased steadily for Dipy10PE as the DOPE% of the bilayer increased from 0 to 80. However, an abrupt increase in the rotational rate and a decrease in the aggregation for Dipy10PE were detected as the DOPE% reached 100, at which point the membranes are in the inverted hexagonal (HII) phase. No similar changes were found for Dipy4PE. These results indicate that the presence of PE with large intrinsic-curvature increases the lateral stress at the region near the center of the bilayer, and that this stress can be relieved as the membranes enter the highly curved HII phase.

Topics: Kinetics; Lipid Bilayers; Models, Theoretical; Phosphatidylcholines; Phosphatidylethanolamines; Pyrenes; Spectrometry, Fluorescence

Antioxidant reactions of mixtures of vitamin E, vitamin C and phospholipids in autoxidizing lipids at 90 degrees C have been studied by ESR spectroscopy. When the phospholipid contained a tertiary amine (e.g. phosphatidylcholine), the vitamin C and the vitamin E radicals were successively observed as these two vitamins were sequentially oxidised during lipid oxidation. In the presence of the primary amine contained in phosphatidylserine, the vitamin E oxidation was delayed for a few hours. In this case neither the vitamin C, nor the vitamin E radicals but a nitroxide radical derived from the phospholipid was observed. Similar results to those obtained with PS were obtained in the presence of either phosphatidylethanolamine or soybean lecithin. The participation in the radical reactions of phospholipids possessing a primary amine can therefore explain the synergistic effect of these phospholipids in a mixture of vitamins E and C.

Topics: Ascorbic Acid; Electron Spin Resonance Spectroscopy; Fatty Acids, Unsaturated; Free Radicals; Hot Temperature; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Vitamin E

We have monitored the fusion of intact A/PR/8/34 influenza virus with glycophorin-bearing liposomes and with ganglioside- (GD1a-) containing liposomes. The lipid bilayers of the glycophorin-bearing liposomes had several compositions, including pure dioleoylphosphatidylethanolamine (DOPE), pure egg phosphatidylethanolamine (EPE), and pure dioleoylphosphatidylcholine (DOPC). Examination of the temperature dependence of fusion for these and other compositions showed that even if the lipids are competent to form inverted hexagonal phases (HII), there is no enhancement of the fusion rate constant at the L alpha-HII phase transition temperature of the lipids, TH. Thus, the HII phase transition is not involved in the HA-mediated fusion mechanism. However, this mechanism is sensitive to lipid composition, in that PC bilayers fused more slowly than PE-containing bilayers above 20 degrees C. These results show that the HA-mediated fusion mechanism depends primarily upon specific lipid-protein interactions, although the fundamental parameters of lipid phase stability (interstice stabilization and monolayer spontaneous radius of curvature) may also be important. The fact that HII phase-component lipid bilayers in the glycophorin liposomes do not enhance the HA-mediated fusion rate strongly suggests that substantial bilayer-bilayer contact is not involved in HA-mediated fusion. Previously, we have shown that glycoprotein-bearing liposomes bind to HA-expressing cells specifically through HA-glycophorin interactions and that fusion is mediated by HAs not bound to glycophorin. Thus, with respect to the target membrane, the fusion site involves just the lipid bilayer. Our results with GD1a-containing liposomes strongly suggest that HAs bound to this sialic acid-bearing molecule are likewise incapable of participating in the fusion site. This could be due to a diminished lateral mobility of the HAs simultaneously bound to both closely apposed membranes. Finally, we find that the low-pH-induced viral inactivation is inhibited by binding to either glycophorin- or GD1a-containing target membranes.

Topics: Animals; Chick Embryo; Influenza A virus; Kinetics; Lipid Bilayers; Liposomes; Membrane Fusion; Microscopy, Electron; N-Acetylneuraminic Acid; Phosphatidylcholines; Phosphatidylethanolamines; Sialic Acids; Virion

The lamellar/inverted hexagonal (L alpha/HII) phase transition can be very fast, despite the drastic change in the topology of the lipid/water interfaces. The first structures to form in this transition may be similar to those that mediate membrane fusion in many lipid systems. To study the transition mechanism and other dynamic phenomena in membrane dispersions, we constructed an apparatus to rapidly trigger the transition and then vitrify the specimens to preserve the structure of transient intermediates. The apparatus applies millisecond-long temperature jumps of variable size to aqueous dispersions of lipids on electron microscope grids at times 9-16 ms before specimen vitrification. The vitrified specimens are then examined by cryo-transmission electron microscopy. Dispersions of egg phosphatidylethanolamine completed the transition within 9 ms when superheated by 20 K. Similar transition times have been observed in dioleoylphosphatidylethanolamine via time-resolved x-ray diffraction. N-monomethylated dioleoylphosphatidylethanolamine dispersions superheated to lesser extent exhibited slower transitions and more complex morphology. The structure of the first intermediates to form in the transition process could not be determined, probably because the intermediates are labile on the time scale of sample cooling and vitrification (< 1 ms) and because of the poor contrast developed by some of these small structures. However, the results are more compatible with a transition mechanism based on "stalk" intermediates than a mechanism involving inverted micellar intermediates. Temperature-jump cryo-transmission electron microscopy should be useful in studying dynamic phenomena in biomembranes, large protein complexes, and other colloidal dispersions. It should be especially helpful in studying the mechanism of protein-induced membrane fusion.

Topics: Biophysical Phenomena; Biophysics; Freezing; Image Processing, Computer-Assisted; Kinetics; Liposomes; Membrane Lipids; Microscopy, Electron; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines

These experiments were designed to observe specific binding of fluorescein-conjugated FAB'2 secondary antibodies to epitopes on the surface of isolated hepatocytes. The hepatocytes were attached as monolayers on microscope cover slips and an antigenic adduct known to be formed during metabolism of halothane, -trifluoroacetyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, was exchanged into their surface. Then the monolayers of hepatocytes were incubated with primary rabbit antibodies specific for the trifluoroacetyl group. Each coverslip was mounted in a perfusion chamber on a fluorescence microscope and a set of digital fluorescence images was made. Then fluorescein-conjugated goat-anti-rabbit FAB'2 secondary antibodies were flowed over the monolayer, the perfusion chamber was washed with buffer, and a second set of digital fluorescence images was made. The difference of these two sets of images demonstrated intense fluorescence superimposed on the outline of the cells. This intense fluorescence was not observed in control experiments in which the primary antibodies were omitted.

Topics: Animals; Antibodies; Cell Membrane; Fluoresceins; Goats; Halothane; Immunoglobulin Fab Fragments; Liver; Male; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Rabbits; Rats; Rats, Sprague-Dawley; Trifluoroacetic Acid

To understand the mechanism of membrane fusion, we have to infer the sequence of structural transformations that occurs during the process. Here, it is shown how one can estimate the lipid composition-dependent free energies of intermediate structures of different geometries. One can then infer which fusion mechanism is the best explanation of observed behavior in different systems by selecting the mechanism that requires the least energy. The treatment involves no adjustable parameters. It includes contributions to the intermediate energy resulting from the presence of hydrophobic interstices within structures formed between apposed bilayers. Results of these calculations show that a modified form of the stalk mechanism proposed by others is a likely fusion mechanism in a wide range of lipid compositions, but a mechanism based on inverted micellar intermediates (IMIs) is not. This should be true even in the vicinity of the lamellar/inverted hexagonal phase transition, where IMI formation would be most facile. Another prediction of the calculations is that traces of apolar lipids (e.g., long-chain alkanes) in membranes should have a substantial influence on fusion rates in general. The same theoretical methods can be used to generate and refine mechanisms for protein-mediated fusion.

Topics: Biophysical Phenomena; Biophysics; Elasticity; Energy Metabolism; In Vitro Techniques; Lipid Bilayers; Membrane Fusion; Micelles; Models, Biological; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics

Topics: Diglycerides; Energy Metabolism; Lipid Bilayers; Membrane Fusion; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids

With few exceptions, membrane lipids are usually regarded as a kind of filler or passive solvent for membrane proteins. Yet, cells exquisitely control membrane composition. Many phospholipids found in plasma membrane bilayers favor packing into inverted hexagonal bulk phases. It was suggested that the strain of forcing such lipids into a bilayer may affect membrane protein function, such as the operation of transmembrane channels. To investigate this, we have inserted the peptide alamethicin into bilayer membranes composed of lipids of empirically determined inverted hexagonal phase "spontaneous radii" Ro, which will have expectably different degrees of strain when forced into bilayer form. We observe a correlation between measured Ro and the relative probabilities of different conductance states. States of higher conductance are more probable in dioleoylphosphatidylethanolamine, the lipid of highest curvature, 1/Ro, than in dioleoylphosphatidylcholine, the lipid of lowest curvature.

Topics: Alamethicin; Biophysical Phenomena; Biophysics; Electric Conductivity; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Phosphatidylcholines; Phosphatidylethanolamines

Plasma membranes of protoplasts isolated from non-acclimated rye plants undergo a transition from the bilayer to the inverted hexagonal (HII) phase during freeze-induced dehydration at -10 degrees C. It has been suggested (Bryant, G. and Wolfe, J. (1989) Eur. Biophys. J. 16, 369-372) that the differential hydration of various membrane components may induce fluid-fluid demixing of highly hydrated (e.g., PC) from poorly hydrated (PE) components during dehydration. This could yield a PE-enriched domain more prone to form the HII phase. We have examined the lyotropic phase behavior of mixtures of DOPE and DOPC at 20 degrees C by freeze-fracture electron microscopy, differential scanning calorimetry, and X-ray diffraction. HII phase formation was favored by higher proportions of DOPE and lower water contents. Mixtures of 1:1 and 1:3 DOPE/DOPC had a hydration-dependent appearance of two L alpha phases at water contents just above those at which the HII phase occurred. The hydration-dependence of the lamellar repeat spacings suggested that the DOPE-enriched domains preferentially underwent the L alpha-to-HII phase transition. Mixtures of 3:1 DOPE/DOPC did not separate into two L alpha phases during dehydration. These data suggest that the differential hydration characteristics of various membrane components may induce their lateral fluid-fluid demixing during dehydration.

Topics: Chemical Phenomena; Chemistry, Physical; Freeze Fracturing; Phosphatidylcholines; Phosphatidylethanolamines; Protoplasts; Secale; Water

Topics: Animals; Cattle; Electron Transport Complex IV; Kinetics; Liposomes; Microscopy, Electron; Myocardium; Phosphatidylcholines; Phosphatidylethanolamines; Proteolipids

We investigated the effect of the antineoplastic drug doxorubicin on the order of the acyl chains in liquid-crystalline mixed bilayers consisting of dioleoylphosphatidylserine (DOPS) or -phosphatidic acid (DOPA), and dioleoylphosphatidylcholine (DOPC) or -phosphatidylethanolamine (DOPE). Previous 2H-NMR studies on bilayers consisting of a single species of di[11,11-2H2]oleoyl-labeled phospholipid showed that doxorubicin does not affect the acyl chain order of pure zwitterionic phospholipid but dramatically decreases the order of anionic phospholipid [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 1096, 67-80]. In the present work, we studied mixed bilayers in which alternatively the anionic or the zwitterionic phospholipid component was 2H-labeled so as to monitor its individual acyl chain order. Doxorubicin decreased the order parameter of the mixed anionic and zwitterionic lipids by approximately the same amount and did not induce a clear segregation of the lipid components into extended, separate domains. The drug had a comparable disordering effect on mixed bilayers of unlabeled cardiolipin and 2H-labeled zwitterionic phospholipid, indicating the absence of extensive segregation also in that case. Upon addition of doxorubicin to bilayers consisting of 67 mol% DOPE and 33 mol% anionic phospholipid, a significant part of the lipid adopted the inverted hexagonal (HII) phase at 25 degrees C. This bilayer destabilization, which occurred only in mixtures of anionic phospholipid and sufficient amounts of DOPE, might be of physiological importance. Even upon formation of extended HII-phase domains, lipid segregation was not clearly detectable, since the relative distribution of 2H-labeled anionic phospholipid and [2H]DOPE between the bilayer phase and HII phase was very similar. Our findings argue against a role of extensive anionic/zwitterionic lipid segregation in the mechanism of action and toxicity of doxorubicin.

Topics: Doxorubicin; Membranes, Artificial; Models, Biological; Molecular Structure; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Structure-Activity Relationship

Covalent attachment of methoxypoly(ethylene glycol) (MPEG) 5000 to the surface of unilamellar liposomes composed of egg phosphatidylcholine and dioleoylphosphatidylethanolamine (DOPE) (8:2) containing paramagnetic chelates, either entrapped within the interior volume of the liposomes, or associated with the membrane surface, had no effect upon the measured spin-lattice relaxation rates (1/T1) for water in these systems. 31P-NMR studies indicate no destabilization of dioleoylphosphatidylcholine (DOPC)/(DOPE) (1:1) vesicles following attachment of MPEG. However, in DOPC/DOPE (1:3) mixtures, covalent modification with MPEG results in a destabilization of multilamellar vesicles into smaller vesicular structures. These results indicate that covalent attachment of poly(ethylene glycol) to liposomal magnetic resonance agents may prove a useful method for increasing their utility as vascular MR agents by extending their lifetime in the circulation, without decreasing the relaxivity of paramagnetic species associated with the liposome, but that the presence of PEG covalently attached to the membrane surface may modify the polymorphic phase behavior of the lipid system to which it is covalently linked.

Topics: Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorus Isotopes; Polyethylene Glycols

Incubation of small unilamellar vesicles consisting of dioleoyl phosphatidylcholine-dioleoyl phosphatidylethanolamine (3:1) and 2 mol% [3H]dolichol-19 with postheparin plasma from rat resulted in the formation of dolichyl oleate. Normal plasma or heat-treated postheparin plasma contained no activity and, hence, the results indicate the presence of a cell surface associated dolichol acyltransferase that can be released into the blood by heparin. The reaction is strongly stimulated by phosphatidylethanolamine and Ca2+, whereas no stimulation with triglycerides or acyl-CoA was observed. Together with the fact that the only product formed was dolichyl oleate, these results strongly suggest that a transacylation mechanism from the phospholipids to dolichol is operative in the liposomes. Gel chromatography of postheparin plasma yielded a molecular mass of about 350 kilodaltons for the active enzyme and density gradient centrifugation indicated that this high molecular mass complex consists mainly of proteins. Finally, we conclude that this enzyme is not unique to the rat, but is also present in human postheparin plasma.

Topics: Acyltransferases; Animals; Chromatography, Gel; Dolichols; Fatty Acids; Heparin; Humans; Lipoproteins; Liposomes; Liver; Membrane Proteins; Molecular Weight; Phosphatidylcholine-Sterol O-Acyltransferase; Phosphatidylcholines; Phosphatidylethanolamines; Rats

The biodistribution and immunotargetability of liposomes composed primarily of dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC) in mice injected via the tail vein were examined and compared. The ganglioside GM1 (7 mol%) prolonged the circulation of DOPC but not DOPE liposomes. Gangliosides GD1a and GT1b (7 mol%) also increased the amount of DOPC liposomes remaining in circulation, and to a similar extent as GM1, at 15 min post injection. However, these liposomes were cleared from the circulation by 2.5 h. Monoclonal antibody 34A, which specifically binds to a surface glycoprotein (gp 112) of the pulmonary endothelial cell surface, was coupled with N-glutarylphosphatidylethanolamine and incorporated into liposomes by a dialysis procedure. These 34A-immunoliposomes, composed of DOPE and GM1 (7 mol%), but not the antibody-free liposomes, accumulated efficiently (approximately 24% of the injected dose) in the lungs. Inclusion of cholesterol (31 mol%) enhanced the lung accumulation of both DOPE/GM1 immunoliposomes and DOPC/GM1 immunoliposomes to 33% and 51% of the injected dose, respectively. The transient increase in DOPC liposome circulation provided by GD1a and GT1b was sufficient to enhance DOPC immunoliposome binding, where 44% and 43% of the injected dose of DOPC/Chol/GD1a and DOPC/Chol/GT1b immunoliposomes accumulated in lung at 15 min after injection, respectively. In general, cholesterol-containing DOPC liposomes were more targetable than DOPE liposomes, and the degree to which these liposomes avoid RES uptake influences their targetability. The results presented here are relevant to the design of targetable drug delivery vehicles.

Topics: Animals; Cholesterol; Gangliosides; Liposomes; Liver; Lung; Mice; Phosphatidylcholines; Phosphatidylethanolamines; Spleen

Frequency-resolved fluorescence measurements have been performed to quantitate the lateral stress of the lipid layer containing nonbilayer phase preferring dioleoylphosphatidylethanolamine (DOPE). On the basis of a new rotational diffusion model, the wobbling diffusion constant (Dw), the curvature-related hopping diffusion constant (DH), and the two local orientational order parameters ([P2] and [P4]) of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC) in fully hydrated DOPE and DOPE/dioleoylphosphatidylcholine (DOPC) mixtures were calculated from the frequency-domain anisotropy data. The values of [P2], [P4], and DH for DOPE were found to increase significantly at approximately 12 degrees C, the known lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition temperature of DOPE. Similar features as well as a decline of Dw were detected in the DOPE/DOPC mixtures as the DOPE content was increased from 85% to 90% at 23 degrees C, corresponding to the known lyotropic phase transition of the DOPE/DOPC. In contrast, for DOPC (0-40 degrees C) and DOPE/DOPC (0-100% DOPE at 3 degrees C), which remained in the L alpha phase, these changes were not detected. The most probable local orientation of DPH-PC in the DOPE/DOPC mixtures shifted progressively toward the normal of the lipid/water interface as the content of DOPE increased. We concluded that the curvature-related lateral stress in the lipid layer increases with the content of the nonbilayer phase preferring lipids.

Topics: Diffusion; Fluorescence Polarization; Lipid Bilayers; Mathematics; Phosphatidylcholines; Phosphatidylethanolamines; Temperature

One- and two-dimensional 31P-exchange NMR has been used to investigate chemical exchange between coexisting lamellar (L alpha) and non-lamellar (hexagonal HII and cubic I2) lipid phases. Samples of DOPE, DOPE/DOPC (9:1 and 7:3), DOPE/cholesterol sulfate (9:1), DOPC/monoolein (MO) (3:7 and 1:1), and DOPC/DOPE/cholesterol (1:1:2) were macroscopically oriented on glass plates and studied at the 0 degree orientation (angle between the bilayer normal and the external magnetic field), where the L alpha, HII, and I2 resonances are resolved. A reversible L alpha to HII transition was observed for all of the samples except for the DOPC/MO mixtures, which displayed a reversible L alpha to I2 transition. Near-equilibrium mixtures of L alpha and either HII or I2 were obtained after prolonged incubation at a given temperature. Two-dimensional exchange experiments were performed on DOPE at 9-14 degrees C for mixing times ranging from 500 ms to 2 s. For all samples, one-dimensional exchange experiments were performed for mixing times ranging from 100 ms to 4 s, at temperatures ranging from 3 degrees C to 73 degrees C. No evidence of lipid exchange between lamellar and non-lamellar phases was observed, indicating that if such a process occurs it is either very slow on the seconds' timescale, or involves an undetectable quantity of lipid. The results place constraints on the stability or kinetic behaviour of proposed transition intermediates (Siegel, D.P. (1986) Biophys. J. 49, 1155-1170).

Topics: Chemical Phenomena; Chemistry, Physical; Cholesterol Esters; Glycerides; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylethanolamines; Temperature

The stability of two-component liposomes composed of the polymerizable 1,2-bis-[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphati dylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC-containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC; photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents.

Topics: Drug Stability; Fluoresceins; Liposomes; Magnetic Resonance Spectroscopy; Models, Structural; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Photolysis; Structure-Activity Relationship; Ultraviolet Rays

Unilamellar dioleoylphosphatidylcholine (DOPC) liposomes (250 microM) incorporated 2 mol% of [3H]pristane at 37 degrees C after addition of 50 microM pristane solubilized with beta-cyclodextrin. Conventional solubilization in dimethyl sulphoxide resulted in much lower uptake. Premixing of perdeuterated pristane with DOPC and dipalmitoylphosphatidylcholine (DPPC) prior to the formation of multilamellar liposomes resulted in homogeneous incorporation of up to 5 mol% pristane at 22 degrees C and 50 degrees C, respectively, as observed by 2H-NMR. Lipid order parameters measured by 31P and 2H-NMR remained unchanged after pristane uptake. Pristane induced the transformation of part of the dioleoylphosphatidylethanolamine (DOPE)/DOPC (3:1, mol/mol) liquid crystalline lamellar phase into an inverse hexagonal phase. 5 mol% pristane in DPPC bilayers decreased the midpoint of the main phase transition temperature of DPPC from 41.5 degrees C to 40.9 degrees C. Upon cooling in the temperature range from 41 degrees C to 36 degrees C, pristane was either displaced from the DPPC bilayer or the mode of incorporation changed. These results may aid in defining the mechanisms whereby pristane, an isoprenoid C19-isoalkane, induces plasmacytomagenesis in mice.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Calorimetry, Differential Scanning; Carcinogens; Lipid Bilayers; Magnetic Resonance Spectroscopy; Models, Biological; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Radioisotope Dilution Technique; Terpenes; Tritium

Topics: Animals; Fourier Analysis; Kidney; Lipid Peroxidation; Magnetic Resonance Spectroscopy; Mercuric Chloride; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Rats

Lipid polymorphism was studied with the aim to gain more insight in bilayer to non-bilayer phase transitions, with particular emphasis on the development of cubic structures on one hand and inverted hexagonal structures on the other hand. Thin vitrified films prepared from aqueous lipid suspensions were used in this study. The entire hydrated contents of these films can be visualized in their two-dimensional projection by cryo-electron microscopy. As the starting material, unilamellar vesicles were prepared from mixtures of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol. By heating of the suspension, vesicle fusion (Frederik et al. (1989) Biochim. Biophys. Acta 979, 275-278) and lipid polymorphism was induced. From these suspensions thin films were prepared at various temperatures, and vitrified for low temperature observation. In a parallel series of experiments samples were fast-frozen for freeze-fracture analysis. In vitrified thin films bilayer structures were often observed in coexistence with an inverted hexagonal structure. The bilayer areas were frequently of a complex structure because multiple contacts between stacked membranes were found. These contact points were variable in size and shape and usually had the form of a diabolo (when viewed side-on) giving the impression of a bilayer contact with an aqueous channel. This structure is compatible with the interlamellar attachment site (ILA) proposed by Siegel ((1986) Biophys. J. 49, 1155-1170). In some specimens ILA's seemed to merge into arrays. After thermal cycling of the suspension, arrays of packed globules were observed, which are likely the result of close packing of ILA's. The arrays probably represent a cubic structure. A comparison of freeze-fracture replicas and vitrified thin films indicated that both techniques may provide valuable structural information on lipid polymorphism. Most of the lipidic particles observed by freeze-fracturing probably correspond to the ILA's (fractured around their waist region) as observed in vitrified thin films. The results obtained with vitrified thin films were interpreted in relation to the principles of thin-film formation. Finally, we speculate that lipid structures occurring close to each other in space may represent a developmental series of structures occurring successively in time.

Topics: Cholesterol; Freeze Fracturing; Membrane Fusion; Membrane Lipids; Microscopy, Electron; Phosphatidylcholines; Phosphatidylethanolamines; Suspensions; Thermodynamics

The interactions between unsaturated phospholipid bilayers deposited on mica were measured in aqueous solution using a surface forces apparatus. The bilayers were made of L-alpha-dioleoylphosphatidyl-choline (DOPC), L-alpha-dioleoylphosphatidyl ethanolamine (DOPE), and mixtures of the two, and were formed on mica by Langmuir-Blodgett deposition after the lipids were spread on an aqueous substrate from a chloroform solution. The forces are interpreted as electrostatic double-layer and van der Waals forces with long range, and a strong repulsion (hydration or steric force) at distances of several nm. Together they produce a region of weak attraction (a secondary minimum) at 5 nm (DOPE) and 6 nm (DOPE). Fusion of two bilayers into one was observed when the local force per unit area was 2-3 MPa. Other researchers report that phosphatidylethanolamine in vesicles enhances fusion. In this study using deposited bilayers, the presence of DOPE in a DOPC bilayer did not promote fusion, nor did DOPE bilayers fuse more easily than DOPC. The value of the force per unit area at which the two bilayers fuse into one was however decreased by several orders of magnitude when the bilayers were formed from lipids kept in chloroform solution for several days or more. Chromatography showed traces of lipid degradation products in such chloroform solutions.

Topics: Chemical Phenomena; Chemistry, Physical; In Vitro Techniques; Lipid Bilayers; Membrane Fusion; Phosphatidylcholines; Phosphatidylethanolamines

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.

Topics: Apoproteins; Biological Transport; Cytochrome c Group; Cytochromes c; Ethanol; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Mitochondria; Phenylethyl Alcohol; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Protein Precursors; Trypsin

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

Amphiphiles respond both to polar and to nonpolar solvents. In this paper X-ray diffraction and osmotic stress have been used to examine the phase behavior, the structural dimensions, and the work of deforming the monolayer-lined aqueous cavities formed by mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) as a function of the concentration of two solvents, water and tetradecane (td). In the absence of td, most PE/PC mixtures show only lamellar phases in excess water; all of these become single reverse hexagonal (HII) phases with addition of excess td. The spontaneous radius of curvature R0 of lipid monolayers, as expressed in these HII phases, is allowed by the relief of hydrocarbon chain stress by td; R0 increases with the ratio DOPC/DOPE. Mixtures with very large R0's can have water contents higher than the L alpha phases that form in the absence of td. The drive for hydration is understood in terms of the curvature energy to create large water cavities in addition to direct hydration of the polar groups. Much of the work of removing water to create hexagonal phases of radius R less than R0 goes into changing monolayer curvature rather than dehydrating polar groups. Single HII phases stressed by limited water or td show several responses. (a) The molecular area is compressed at the polar end of the molecule and expanded at the hydrocarbon ends. (b) For circularly symmetrical water cylinders, the degrees of hydrocarbon chain splaying and polar group compression are different for molecules aligned in different directions around the water cylinder. (c) A pivotal position exists along the length of the phospholipid molecule where little area change occurs as the monolayer is bent to increasing curvatures. (d) By defining R0 at the pivotal position, we find that measured energies are well fit by a quadratic bending energy, K0/2 (1/R-1/R0); the fit yields bilayer bending moduli of Kc = (1.2-1.7) X 10(-12) ergs, in good agreement with measurements from bilayer mechanics. (e) For lipid mixtures, enforced deviation of the HII monolayer from R0 is sufficiently powerful to cause demixing of the phospholipids in a way suggesting that the DOPE/DOPC ratio self-adjusts so that its R0 matches the amount of td or water available, i.e., that curvature energy is minimized.

Topics: Chemical Phenomena; Chemistry, Physical; Lipids; Membranes, Artificial; Molecular Conformation; Osmotic Pressure; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Solubility; Thermodynamics; Water; X-Ray Diffraction

The characteristic temperature dependence of the lattice basis vector length d of phospholipid-water systems in the inverted hexagonal (HII) phase has been investigated with X-ray diffraction. For 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), d falls sharply from 78.1 A at 10 degrees C to 62.5 A at 90 degrees C. When used in conjunction with the volume fractions of the constituents, d can be used to determine the dimensions within the lipid and water regions. These data showed that a reduction in the radius of the HII-phase water cylinders Rw accounted for most of the reduction in d. From geometrical relationships between the dimensions in the HII phase, it was shown that both d and Rw are sensitive functions of the thickness of the lipid monolayer dHII. The characteristic shape of d(T) could be parameterized with the small temperature dependence of dHII along with the ratio v/a, which is the ratio of the specific volume to the area per lipid molecule at the polar interface. The ratio v/a was found to be independent of temperature for the fully hydrated HII system. Additional measurements made with a mixture of DOPE and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), mole ratio 5.07:1, produced a similar parameterization of d(T). The larger basis vector lengths for this mixture compared to those for DOPE can be attributed to a smaller ratio of v/a, which was also found to be temperature independent for this mixture. The smaller value of v/a is due to the larger effective headgroup area of DOPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Membranes, Artificial; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Temperature; Thermodynamics; Water; X-Ray Diffraction

The importance of the protein-linked carbohydrates for the stabilization of dioleoylphosphatidylethanolamine (DOPE) bilayers has been investigated using glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, as a stabilizer. Two major types of glycophorin, differing in the sialic acid content, were used in the study. Type MM contains 19.2 +/- 2.5 sialic residues per molecule of glycophorin, and type NN contains 10.8 +/- 1.2. Type MM could stabilize DOPE bilayers at 0.5 mol%, whereas type NN was unable to do so even at 1 mol%. The importance of the sialic acid content to the stabilization activity of glycophorin was further confirmed by the observation that the neuraminidase-treated type MM showed a lower stabilization activity than the untreated type. Since type NN had no stabilizing activity, we attempted to couple a trisaccharide, NeuNAc----Gal----Glc, to type NN by reductive amination. 2.5 +/- 0.8 saccharide chains were added per molecule of type NN. The trisaccharide-attached type NN showed a greater stabilization activity than the parent type NN molecule, indicating again that the sialic acid content of the stabilizer molecule determines the stabilization activity. Addition of wheat-germ agglutinin (WGA), which binds to the sialic acid residues of a glycoprotein, to type MM-stabilized liposomes caused rapid aggregation and destabilization of liposomes, resulting in leakage of an entrapped marker, calcein. The aggregation increased with increasing amount of the lectin; however, the leakage rate was maximum at an optimum concentration of WGA. These results are discussed in terms of the role of sialic acid in the interfacial hydration and charge repulsion which determines the DOPE bilayer stability.

Topics: Carbohydrate Sequence; Chromatography, Affinity; Fluoresceins; Glycophorins; Humans; Indicators and Reagents; Lipid Bilayers; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Sialoglycoproteins

Lipidic phases, containing 'lipidic particles' (dioleoylphosphatidylethanolamine/cholesterol/dioleoylphosphatidylcho lin e and cardiolipin/dimyristoylphosphatidylcholine in the presence of Ca2+) have been investigated by preparing thin films from a suspension of sonicated vesicles. These thin films were vitrified and observed 'directly' by cryo-electron microscopy in their hydrated form. The thin films show various fusion products and fusion intermediates such as lipidic particles.

Topics: Calcium; Cardiolipins; Cholesterol; Dimyristoylphosphatidylcholine; Freezing; Hot Temperature; Liposomes; Membrane Fusion; Microscopy, Electron; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Sonication

Several derivatives of cholesterol containing oxyethylene headgroups with and without a terminal galactose have been synthesized in order to examine the effects of immobilizing a cryoprotectant at a membrane surface. In this work, we have studied the ability of the triethoxycholesterol (TEC) and triethoxycholesterol galactose (TEC-Gal) derivatives to modulate the phase behavior of phosphatidylcholine and phosphatidylethanolamine membranes. Methods of fluorescence polarization, 31P-NMR and freeze-fracture electron microscopy were employed to monitor these changes in lipid phase behavior. Fluorescence polarization data demonstrated the ability of the derivatives to fluidize gel state and rigidify liquid-crystalline state phosphatidylcholines in a manner similar to that observed for cholesterol. Unlike cholesterol, however, the Tm of dipalmitoylphosphatidylcholine (DPPC) was reduced in a concentration-dependent manner with each of the derivatives. Freeze-fracture electron microscopy and 31P-NMR of DOPE dispersions indicate an increase in the lamellar to hexagonal phase-transition temperature on the order of 10-20 C degrees above room temperature for mixtures with 20 mol% of the derivatives. These results are discussed in terms of the properties exhibited by compounds such as carbohydrates, which are known to serve as cryoprotectants for synthetic and biological membranes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Cryoprotective Agents; Freeze Fracturing; Galactosides; Liposomes; Magnetic Resonance Spectroscopy; Microscopy, Electron; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Thermodynamics

Factors affecting the hydrolytic activity of purified rat hepatic lipase have been examined in mixed-monolayer systems. When nonsubstrate lipids [either egg sphingomyelin or beta-O-hexadecyl-gamma-O-(1-ocadec-9-enyl)-DL-phosphatidylcholine (OPPC-ether)] were used as inert matrices, hydrolytic activity for both triolein and dioleoylphosphatidylethanolamine was shown to decrease with increasing surface pressure (pi); negligible activity occurred at pi greater than or equal to 30 mN/m. Examination of the effect of introduction of cholesterol into either matrix containing 2 mol % triolein indicated that the mean molecular area decreased with increasing cholesterol and that, at pi = 24 mN/m, triolein was fully miscible in the sphingomyelin matrix at cholesterol concentrations less than or equal to 32.5 mol % and in the OPPC-ether matrix at cholesterol concentrations less than or equal to 49 mol %. Above these critical concentrations of cholesterol, the phase diagrams indicate transitions that suggest that triolein is forced out of the monolayer. Introduction of increasing amounts of cholesterol into either inert matrix increased the rate of hydrolysis of triolein by hepatic lipase, although by different degrees. There are at least two factors contributing to these effects: (1) condensation of the monolayer by cholesterol, thus increasing the total surface concentration of triolein at pi = 24 mN/m in the constant area surface balance, and (2) some change in triolein conformation and/or accessibility since at identical surface concentrations of triolein (8.7 +/- 0.1 pmol/cm2) and pi (24 mN/m) the rate of hydrolysis of triolein by hepatic lipase is 1.5-fold higher in the OPPC-ether matrix than in the egg sphingomyelin matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cholesterol; Kinetics; Lipase; Liposomes; Liver; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipid Ethers; Pressure; Rats; Sphingomyelins; Surface Properties

The polymorphic phase behavior of aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and its N-methylated analogues, DOPE-Me, DOPE-Me2, and DOPC, has been investigated by X-ray diffraction. In the fully hydrated lamellar (L alpha) phase at 2 degrees C, the major structural difference is a large increase in the interlamellar water width from DOPE to DOPE-Me, with minor increases with successive methylation. Consistent with earlier reports, inverted hexagonal (HII) phases are observed upon heating at 5-10 degrees C in DOPE and at 65-75 degrees C in DOPE-Me and are not observed to at least 85 degrees C in DOPE-Me2 or DOPC. In DOPE, the L alpha-HII transition is facile and is characterized by a relatively narrow temperature range of coexistence of L alpha and HII domains, each with long-range order. DOPE-Me exhibits complex nonequilibrium behavior below the occurrence of the HII phase: Upon heating, the L alpha lattice spontaneously disorders on a time scale of days; on cooling from the HII phase, the disorder rises on a time scale of minutes. It is shown that, in copious water, the disordered state transforms very slowly into phases with cubic symmetry. This process is assisted by the generation of small amounts of lipid degradation products. The relative magnitudes of the monolayer spontaneous radius of curvature, R0 [Kirk, G. L., Gruner, S. M., & Stein, D. L. (1984) Biochemistry 23, 1093; Gruner, S. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3665], are inferred from the HII lattice spacings vs temperature and are shown to increase with increasing methylation. The relative magnitudes of R0 are categorized as small for DOPE, intermediate for DOPE-Me, and large for DOPC. It is suggested, and examples are used to illustrate, that small R0 lipid systems exhibit facile, low-temperature L alpha-HII transitions, intermediate R0 systems exhibit complex nonequilibrium transition behavior and are likely to form cubic phases, and large R0 systems are stable as L alpha phases. The relationship between the cubic phases and minimal periodic surfaces is discussed. It is suggested that minimal periodic surfaces represent geometries in which near constant, intermediate R0 values can be obtained concomitantly with monolayers of near constant thickness, thereby leading to equilibrium cubic phases. Thus, the relative magnitude of the spontaneous radius of curvature may be used to predict mesomorphic behavior.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Calorimetry, Differential Scanning; Lipid Bilayers; Methylation; Models, Biological; Models, Molecular; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics; X-Ray Diffraction

Charge-pulse experiments were performed in the presence of structural analogues of tetraphenylborate (TPB) on membranes made of dioleoyl phosphatidylethanolamine and dioleoyl phosphatidylcholine. The analysis of the experimental results using a previously proposed model allowed the calculation of the partition coefficient, beta, and of the translocation rate constant, kappa i. The temperature dependence of the partition coefficients was used to calculate the thermodynamics of the adsorption of the lipophilic ions to the membranes. The analysis of the translocation rate constants obtained at different temperatures yielded detailed information on the free energy of the TPB-analogues within artificial lipid bilayer membranes, and on the activation energy of the translocation rate constants. The adsorption of the different TPB-analogues to the membranes was only slightly affected by their structure, whereas a dramatic influence of the structure on the free energy of the lipophilic ions within the membranes was observed. The free energy of the ions in the membranes decreased from triphenylcyanoborate (TPCB) to tetrakis(3-trifluoromethylphenyl)borate (TTFPB) by more than 31 kJ/mol (7.4 kcal/mol). This could be concluded from the observed increase in the translocation rate constant by almost six orders of magnitude. The change of the free energy in the membrane was used for the estimation of an effective radius of the TPB-analogues with respect to TPB.

Topics: Boron Compounds; Kinetics; Lipid Bilayers; Models, Biological; Phosphatidylcholines; Phosphatidylethanolamines; Tetraphenylborate; Thermodynamics

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.

Topics: Deuterium; Gramicidin; Magnetic Resonance Spectroscopy; Models, Biological; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics

Interactions of antibody stabilized phosphatidylethanolamine (PE) immunoliposomes with Herpes Simplex virus (HSV) and virus infected cells were studied by detecting the immune-dependent lysis of liposomes. Employing PE immunoliposomes bearing anti-HSV glycoprotein D (gD) IgG, immune-specificity of these liposomes were documented by the sole ability of HSV and the HSV-infected L cells to induce immunoliposome lysis. In addition, inhibition of PE immunoliposome lysis by free anti-gD IgG, but not anti-HSV glycoprotein B IgG, indicated the target antigen specificity of these immunoliposomes. Based on these observations, alkaline phosphate encapsulated PE liposomes were used to directly detect HSV in fluid phase. This immunoliposome assay which does not require washing was shown to be very rapid and sensitive: 35pfu of HSV-1 in 5ul could be detected within 1.5hr.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens, Viral; Drug Stability; Immunoglobulin G; Liposomes; Mice; Phosphatidylcholines; Phosphatidylethanolamines; Simplexvirus

Measurements of membrane capacitance, Cm, were performed on lipid bilayers of different lipidic composition (diphytanoyl phosphatidylcholine PPhPC, dioleoyl phosphatidylcholine DOPE, glycerylmonooleate GMO) and containing n-decane as solvent. In the same membranes, the absorption of the lipophilic ions dipicrylamine (DPA-) and tetraphenylborate (TPhB-), and the kinetics of their translocation between the two membrane faces have been studied. The data were obtained from charge pulse relaxation measurements. Upon increasing pressure the specific capacity Cm increased in a fully reversible and reproducible way reflecting a thinning of the membrane that is attributed to extrusion of n-decane from the black membrane area. High pressure decreased the rate constant, ki, for lipophilic ion translocation. After correcting for changes in the height of the energy barrier for translocation due to membrane thinning the pressure dependence of ki yields an apparent activation volume for translocation of approximately 14 cm3/mol both for DPA- and TPhB-. Changes in lipophilic ion absorption following a step of pressure developed with a rather slow time course due to diffusion limitations in solution. The stationary concentration of membrane absorbed lipophilic ions increased with pressure according to an apparent volume of absorption of about -10 cm3/mol. The relevance of the results for the interpretation of the effects of pressure on nerve membrane physiology is discussed.

Topics: Hydrostatic Pressure; Lipid Bilayers; Models, Biological; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines

The initial kinetics of fusion and leakage of liposomes composed of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) have been correlated with the phase behavior of this lipid. Gagné et al. [Gagné, J., Stamatatos, L., Diacovo, T., Hui, S. W., Yeagle, P., & Silvius, J. (1985) Biochemistry 24, 4400-4408] have shown that this lipid is lamellar (L alpha) below 20 degrees C, is hexagonal (HII) above 70 degrees C, and shows isotropic 31P NMR resonances at intermediate temperatures. This isotropic state is also characterized by complex morphological structures. We have prepared DOPE-Me liposomes at pH 9.5 and monitored the temperature dependence of the mixing of aqueous contents, leakage, and changes in light scattering upon reduction of the pH to 4.5. At and below 20 degrees C, where the lipid is in the L alpha phase, there is very little aggregation or destabilization of the liposomes. Between 30 and 60 degrees C, i.e., where the lipid is in the isotropic state, the initial rates of liposome fusion (mixing of aqueous contents) and leakage increase. At temperatures approaching that where the hexagonal HII phase transition occurs, the initial rates and extents of fusion decrease, whereas leakage is enhanced. Similar results were found for dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine (2:1) liposomes. These results clearly establish a common mechanism between the appearance of the isotropic state (between the L alpha and HII phases) and the promotion of liposome fusion. We propose a simple model to explain both the observed behavior of phosphatidylethanolamine-containing membranes with respect to liposome fusion and/or lysis and the beginning of the L alpha-HII phase transition.

Topics: Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Liposomes; Magnetic Resonance Spectroscopy; Models, Biological; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics

The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles comprised of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was studied using a fluorescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilayer movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the phosphatidylcholine-specific transfer protein. 31P-NMR and freeze-fracture electron microscopy were employed to study the macroscopic organization of DOPC and DOPE containing model membranes in the absence or presence of dolichyl phosphate. The results indicate that both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; the amount of exchangeable PC from MLVs is increased by dolichyl phosphate, probably as a result of fusion processes; dolichyl phosphate destabilizes the bilayer organization in MLVs comprised of DOPE and DOPC, resulting in the formation of hexagonal (HII) phase and 'lipidic' particles.

Topics: Diterpenes; Dolichol Phosphates; Dolichols; Freeze Fracturing; Magnetic Resonance Spectroscopy; Membrane Fluidity; Membrane Fusion; Membrane Lipids; Membranes, Artificial; Phosphatidylcholines; Phosphatidylethanolamines; Polyisoprenyl Phosphates

Liposomes composed of either dioleoylphosphatidylethanolamine and oleic acid (pH-sensitive) or dioleoylphosphatidylcholine and oleic acid (pH-insensitive) were injected into C3H and Balb/c mice in order to determine the tissue distribution of both the lipid and the aqueous content. The lipid component was monitored by use of [3H]cholestanyl ether and the aqueous content was monitored by use of encapsulated 125I-tyraminyl-inulin. The pH-insensitive liposomes injected into both types of mice were rapidly cleared from the blood stream followed by accumulation primarily in the liver, followed by the spleen. The presence of a monoclonal antibody on the liposome surface caused a slight acceleration in liver accumulation, though generally gave the same profile as the antibody-free liposomes. pH-sensitive liposomes were leaky upon exposure to the mouse plasma following injection. The lipid component, though, displayed a large amount (e.g., 50-70% in C3H mice) of accumulation in the lung for up to 6 h, followed by a subsequent appearance in the liver and spleen. The presence of monoclonal antibody had no effect on the tissue distribution profile. These results indicate that the pH-sensitive liposomes, although ineffective as an aqueous drug delivery agent, may be effective as a means of delivering lipophilic drugs to the lung.

Topics: Animals; Antibodies, Monoclonal; Hydrogen-Ion Concentration; Inulin; Iodine Radioisotopes; Liposomes; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Oleic Acid; Oleic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Tissue Distribution

Cytochrome c oxidase has been reconstituted with two synthetic phospholipids, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine. Vesicles prepared from either of these two lipids alone showed no stimulation of enzyme activity upon addition of carbonyl cyanide (trifluoromethoxy)phenylhydrazone and valinomycin, indicating that they were leaky to small ions. However, when mixtures of the two lipids were used for the reconstitution, tightly coupled vesicles could be obtained. The coupling ratio was dependent upon the ratio of dioleoylphosphatidylcholine to dioleoylphosphatidylethanolamine and also on the lipid-to-protein ratio. Maximal rates of enzyme activity were not significantly different with different lipid mixtures. The results are discussed in terms of both the size distribution of the reconstituted vesicles and the possible requirement for a variety of lipid species to ensure tight sealing at the lipid-protein interface.

Topics: Animals; Electron Transport Complex IV; Freeze Fracturing; Horses; Kinetics; Liposomes; Microscopy, Electron; Mitochondria; Phosphatidylcholines; Phosphatidylethanolamines; Structure-Activity Relationship

Lipid activation data for (Na+ + K+)-ATPase (Ottolenghi, P. (1979) Eur. J. Biochem. 99, 113-131) have been subjected to a regression and fitting analysis based on a recent kinetic model (Sandermann, H. (1982) Eur. J. Biochem, 127, 123-128). The observed kinetic cooperativity could be generated from strictly non-cooperative binding events involving the known number of 30 boundary lipid-binding sites per ATPase monomer. Apparent lipid dissociation equilibrium constants of between 0.3 and 5 microM were obtained, enzyme activity being associated only with the fully lipid-substituted enzyme and enzyme-lipid complexes with less than six unoccupied lipid-binding sites. The enzyme appeared to operate close to a maximum of cooperativity.

Topics: Animals; Binding Sites; Dogfish; Enzyme Activation; Kinetics; Membrane Lipids; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Rectum; Sebaceous Glands; Sodium-Potassium-Exchanging ATPase

Glycophorin was incorporated into large unilamellar dioleoylphosphatidylcholine vesicles by either a detergent dialysis method using octylglucoside or a method avoiding the use of detergents. The vesicles were characterized and the permeability properties and transbilayer movement of lipids in both vesicles were investigated as a function of the protein concentration and were compared to protein-free vesicles. An insight in the permeability properties of the vesicles was obtained by monitoring the ratio potassium (permeant): dextran (impermeant) trap immediately after separation of the vesicles from the external medium. Glycophorin incorporated without the use of detergents in 1:300 protein:lipid molar ratio induces a high potassium permeability for the majority of the vesicles as judged from the low potassium trap (K+:dextran trap = 0.21). In contrast, the vesicles in which glycophorin is incorporated via the octylglucoside method (1:500 protein:lipid molar ratio) are much less permeable to potassium (K+:dextran trap = 0.67 and t12 of potassium efflux at 22 degrees C is 7.5 h.). The relationship between protein-induced bilayer permeability and lipid transbilayer movement in both vesicle preparations is discussed. Addition of wheat-germ agglutinin to glycophorin-containing vesicles comprised of dioleoylphosphatidylcholine and total erythrocyte lipids caused no or just a small effect (less than 20% release of potassium) on the potassium permeability of these vesicles. Also, addition of lectin to dioleoylphosphatidylethanolamine-glycophorin bilayer vesicles in a 25:1 lipid:glycophorin molar ratio had no effect on the permeability characteristics of the vesicles. In contrast, addition of wheat-germ agglutinin to bilayer vesicles made of dioleoylphosphatidylethanolamine and glycophorin in a 200:1 molar ratio resulted in a release of 74% of the enclosed potassium by triggering a bilayer to hexagonal (HII) phase transition. The role of protein aggregation and the formation of defects in the lipid bilayer on membrane permeability and lipid transbilayer movement is discussed.

Topics: Erythrocyte Membrane; Glycophorins; Humans; Kinetics; Lectins; Lipid Bilayers; Lysophosphatidylcholines; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylethanolamines; Potassium; Pulmonary Surfactants; Sialoglycoproteins; Wheat Germ Agglutinins

The polymorphic phase behavior of mixtures of synthetic dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) and the influence of cholesterol on these phase preferences have been investigated by employing nuclear magnetic resonance (NMR) techniques. In particular, 31P NMR procedures are utilized to study the overall phase preferences of these mixed systems, whereas 2H NMR is employed to monitor the structural preferences of individual components of these systems by using versions of DOPE and DOPC which are deuterium (2H) labeled at the C11 position of the acyl chains. The results obtained show that DOPE-DOPC systems containing as little as 20 mol % DOPC initially assume lamellar structure at 40 degrees C, even though DOPE in isolation prefers the hexagonal (HII) organization at this temperature. However, this lamellar organization appears to represent a metastable state, as incubation for extended periods at 40 degrees C results in formation of a structure, possibly the cubic phase, in which the phospholipids experience isotropic motional averaging. The addition of cholesterol induces hexagonal (HII) phase organization. 2H NMR studies of appropriately labeled versions of these systems indicate that cholesterol does not produce such effects by associating preferentially with either DOPE or DOPC. Further, in situations where bilayer, hexagonal, or "isotropic" phases coexist in the same sample, the phospholipids exhibit apparently ideal mixing behavior.

Topics: Cholesterol; Magnetic Resonance Spectroscopy; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines