Unilamellar Phosphatidylcholine Vesicles Research Articles

Absorption of Monoacylglycerols by Small Intestinal Brush Border Membrane

The absorption of monoacylglycerol by small intestinal brush border membrane is a passive process, i.e., the movement of monoacylglycerol from small unilamellar phospholipid vesicles as donor particles through the aqueous medium and the incorporation into the outer monolayer of the lipid bilayer of the brush border membrane are passive processes involving diffusion of the lipid along a concentration gradient. Small unilamellar vesicles of egg phosphatidylcholine containing 1 mol% of radiolabeled hexadecylglycerol were used as donor, and rabbit small intestinal brush border membrane vesicles or intact enterocytes isolated from pig jejunum, as acceptor. Hexadecylglycerol was employed as a lipase-resistant model compound for monoacylglycerols. Both acceptor membranes behave similarly in terms of hexadecylglycerol absorption: the kinetics of hexadecylglycerol absorption are biphasic. The initial fast phase is due to the movement of hexadecylglycerol from the donor particle through the aqueous medium to the outer lipid monolayer of the acceptor membrane, and the second slow phase probably involves the flip-flop motion of hexadecylglycerol from the outer to the inner monolayer of the acceptor membrane. The values for the pseudo-first-order rate constants of the initial fast phase for hexadecylglycerol absorption are relatively large and primarily determined by the high solubility (cmc) of hexadecylglycerol in aqueous media. The pseudo-first-order rate constants depend linearly on the protein (lipid) concentration of the acceptor membrane, indicating that the on rate of the hexadecylglycerol into the brush border membrane is rate limiting. The mechanism of the hexadecylglycerol absorption involves mainly monomer diffusion and probably collision-induced transfer.

Incorporation of D-glucosamine alkyi glucopyranosides into the bilayer of small unilamellar vesicles of egg phosphatidyl choline

Alkyl 2-acetamido-2-deoxy-β- d-glucopyranosides were incorporated into the phospholipid bilayer of small unilamellar vesicles. Depending on the lengths of the alkyl aglycone group (C 8–C 14), the carbohydrate was either found in the form of micelles (C 8) or was effectively incorporated in the bilayer (C 14). The use of radiolabelled lipids and/or carbohydrates enabled the quantitative determination of the amounts of lipid and carbohydrate recovered into the vesicles. An enzymatic assay has been carried out to evaluate the outer and inner distribution of the carbohydrate moieties on the liposomes.

The membrane potential has no detectable effect on the phosphocholine headgroup conformation in large unilamellar phosphatidylcholine vesicles as determined by 2H-NMR

In this study the effect of a transmembrane electrical potential on the phospholipid headgroup conformation was investigated using the 2H-NMR technique. Large unilamellar vesicles were prepared of dioleoylphosphatidylcholine, specifically 2H-labeled at the α- or β-position of the choline group. No conformational change of the phosphocholine headgroup could be detected after induction of a valinomycin-induced K +-diffusion potential across the bilayer. However, this method could be used to measure the redistribution of tetraphenylphosphonium across the bilayer in response to Δψ, which reorients the phosphocholine headgroups in the opposite bilayer—water interfaces.

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.

Thermodynamic stability and osmotic sensitivity of small unilamellar phosphatidylcholine vesicles

Evidence is presented to show that small unilamellar phosphatidylcholine vesicles with a diameter of approx. 20 nm are osmotically sensitive. Such vesicles respond to osmotic pressure by swelling or shrinking depending on the direction of the applied salt gradient. This is true for small unilamellar vesicles of egg phosphatidylcholine and dimyristoylphosphatidylcholine below and above their crystal-to-liquid crystal transition temperature. At the transition temperature the vesicles are osmotically insensitive due to the increased bilayer permeability resulting in rapid dissipation of salt gradients. Positive salt gradients produce shrinking and collapse of spherical phospholipid vesicles to disks. Shrinking of vesicles is associated with H 2O and solute efflux, but only limited solute influx. Clustering of lipid molecules in the bilayers of the resulting disks can be detected by EPR spin labeling. Negative salt gradients produce swelling of vesicles which is associated with H 2O and solute influx. Our experiments are consistent with an osmotically perturbed bilayer. In the presence of osmotic gradients the influx and efflux of H 2O is coupled with the movement of ions and small molecules which in the absence of salt gradients or osmotic stress cannot pass the phospholipid bilayer. However, during osmotically induced shrinking and swelling of SUV the integrity of the phospholipid bilayer is maintained to the extent that vesicles do not break, and therefore equilibration between external medium and vesicle cavity does not take place.

Tight insertion of cytochrome [formula omitted] into large unilamellar vesicles

Cytochrome b 5 spontaneously binds to liposomes in a ‘loose’, or transferable form, whereas in vivo b 5 binds post-translationally to the ER in the ‘tight’ or nontransferable form. The mechanism of tight insertion is unknown, except that it does not require SRP or energy input. The present study shows that prolonged incubation of b 5 with large unilamellar vesicles (LUVs) of phosphatidylcholine results in slow conversion of the loose to the tight form, with a halftime of days. However, the process is complex. When the b 5- LUVs are depleted of loose b 5 , by transfer of b 5 to sonicated vesicles, the tight b 5 is found to be concentrated to near saturating levels in a small fraction of the LUVs. If the LUVs devoid of tight b 5 are recovered and then reincubated with fresh b 5 , the same slow transformation recurs. Apparently, a new population of vesicles, containing tight b 5 , is generated during the prolonged incubation with the protein. The b 5- enriched LUVs contain about the same level of trapped sucrose as does the original vesicle preparation, indicating that vesicle integrity is maintained throughout the process. When fresh b 5 is added to these tight b 5- containing LUVs, all the freshly bound protein rapidly inserts ( < 2 h ) into the tight configuration. Apparently, the newly formed tight-b 5/ LUV vesicle population is ‘insertion-active’. A model for these complex transformations is proposed.

Interbilayer transfer of phospholipid-anchored macromolecules via monomer diffusion.

A series of conjugates has been prepared by linking various hydrophilic macromolecules [poly-(ethylene glycols), polylysine, aminodextrans, or apotransferrin] to synthetic phosphatidylethanolamines via linker moieties incorporating a fluorescent bimane group. Using a fluorescence energy transfer-based assay, the rate of transfer of these species between phospholipid vesicles has been monitored as a function of the nature and size of the coupled macromolecule and of the acyl chain composition of the lipid anchor. Conjugates in which the phospholipid anchor is linked to a small hydrophilic terminal residue (e.g., ethanolamine or ethylenediamine) transfer between large unilamellar vesicles of egg phosphatidylcholine with half-times ranging from tens of minutes (for dimyristoyl lipid conjugates) to a few tens of hours (for dipalmitoyl and 1-palmitoyl-2-oleoyl lipid conjugates), in agreement with previous results for unlabeled phospholipids. Conjugation of these same lipid anchors to larger hydrophilic molecules markedly accelerates their rates of intermembrane transfer, by factors ranging from 5-7-fold (for conjugates with apotransferrin and aminodextrans of molecular weight 10,000-70,000) to over 25-fold [for conjugates with poly(ethylene glycol)-5000]. In all cases the observed transfer appears to reflect the diffusion of lipid monomers through the aqueous phase. Our results suggest that substantial intermembrane transfer can occur, on a time scale of several hours or less, for hydrophilic macromolecules conjugated to diacyl(/alkyl) lipids with 14- to 18-carbon chains unless portions of the conjugate other than the lipid anchor also interact strongly with the membrane.

Structure and orientation of a bilayer-bound model tripeptide: a proton NMR study

The conformation and orientation of a model peptide, A-F-A-O-tert-butyl, was studied in sonicated unilamellar phosphatidylcholine (PC) vesicles by proton NMR. Intramolecular nuclear Overhauser enhancements (NOEs) were measured for the tripeptide in free solution and with PC vesicles. The peptide has no stable tertiary structure in free solution but adopts a structure in the presence of PC vesicles. Restrained molecular dynamics using NOE constraints were used to deduce a preferred structure for the bound tripeptide

Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles

Cholesterol is transported both in unilamellar phosphatidylcholine vesicles and in bile salts-mixed micelles in native bile. The vesicular carrier of biliary lipids apparently has a well defined protein profile with a potent cholesterol crystallization-promoting activity. This study was conducted to identify and further characterize these vesicular proteins and to test the effect of isolated vesicular proteins on the cholesterol crystal formation in supersaturated model bile. The results confirmed that proteins are a constant component of highly purified biliary vesicles both in hepatic and gallbladder bile. Immunoglobulins (IgA, IgG and IgM) and albumin are associated to the purified hepatic biliary vesicles. Furthermore, four different hydrophobic glycoproteins with a molecular mass of 130, 114, 86, and 62–67 kDa were isolated. These glycoproteins showed no reactivity with anti-human whole serum or anti-immunoglobulin antibodies, suggesting that these proteins are biliary-specific. Isolated 130, 114 and 62–67 kDa vesicular glycoproteins significantly decreased the cholesterol nucleation time in artificial model bile. We concluded that some, but not all, vesicular-bound hydrophobic glycoproteins have cholesterol pronucleating activity and they may be involved in the pathogenesis of cholesterol gallstone disease.

Effects of 1,2-diacylglycerol and cholesterol on the hydrolysis activity of phospholipase D in egg-yolk phosphatidylcholine bilayers

Effects of cholesterol (Chol) and 1,2-diacylglycerol (DAG) on the hydrolysis activity of phospholipase D (from Streptomyces chromofuscus) were studied in small unilamellar vesicles (SUV) of egg-yolk phosphatidylcholine (PC). 1,2-Diacylglycerol used here is derived from PC. Choline produced in the reaction was monitored by using a choline oxidase-oxygen electrode. Addition of 18.3 mol% Chol into SUV (2 mM PC) led to a small increase in the reaction rate. On the other hand, 18.3 mol% DAG in SUV brought about at 5–6-fold rate of choline production. The apparent maximum velocity, V max(app), increased by addition of DAG and Chol in SUV. In PC/Chol-SUV, the effect of increase in V max(app) was largely compensated by the increase in the apparent Michaelis constant, K m(app). The Chol and DAG molecules did not have significant effects on the kinetic parameters, when PC was solubilized in the micelles of heptaethylene glycol dodecyl ether. The effects of Chol and DAG are, therefore, not due to specific ones on the enzyme itself, but rather upon the bilayer-organization of the substrate. We discuss the activation of phospholipase D in terms of the influences of DAG and Chol on the structure of hydrophilic region and fluidity of the bilayers.

Biomimetic metal-sorbing vesicles: Cd2+ uptake by phosphatidylcholine vesicles doped with ionophore A23187.

Unilamellar phosphatidylcholine vesicles, harboring the ionophore, A23187, in the bilayer and the water-soluble chelating agent, nitrilotriacetate, in the vesicle interior, rapidly sequester and concentrate Cd2+ from dilute aqueous solution. Metal-sorbing vesicle permeabilities for cadmium ion at 5 ppm (42.8 microM) ranged from 8.09 x 10(-7) to 1.27 x 10(-4) cm/s for surface A23187 concentrations of 0.22-2.27 pmol/cm2 (which correspond to lipid:carrier molar ratios of 2000:1 to 200:1) and pH's from 5.5 to 8.5. The Cd2+ permeability shows linear variation with carrier concentration under the conditions studied. As pH is decreased, an increasing fraction of the A23187 becomes protonated, and the permeability exhibits a positive linear relationship with a function related to that for the fraction of unprotonated carrier. These noncovalently assembled, metal-sorbing vesicles exhibit shelf lives of several months and remain stable throughout typical metal sorption studies.

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles have been investigated by nuclear Overhauser effects (NOEs) in 1H nuclear magnetic resonance spectroscopy. Two-dimensional NOE and chemical exchange correlated spectroscopy (NOESY) and rotating frame NOE spectroscopy (ROESY) methods have been applied for obtaining the NOEs. In the NOESY spectra, NOEs between protons within the drug were overwhelmed by spin diffusion even at a short mixing time. This observation reduced the usefulness of the NOESY method on the one hand, however, on the other hand it facilitated remarkably in revealing signals due to the drug, hidden in the broad resonances of the membranes. In the ROESY spectra, the spin diffusion phenomena were less effective; accordingly the conformations of the drugs interacting with membranes were determined by the ROESY method. The observed NOE data showed that dibucaine takes more than two conformations and that both dibucaine and tetracaine are present as a dimer in the membranes. Molecular dynamics calculations supported these findings.

Microbial conversions in a liposomal medium. Part 2: Cholesterol oxidation by Rhodococcus erythropolis

The oxidation of cholesterol (5 g l −1) to cholestenone by Rhodococcus erythropolis ATCC 25544 was investigated in liposomal media made up of multilamellar vesicles (MLVs) and small unilamellar vesicles (SUVs) of phosphatidylcholine. While MLVs practically inhibited the biotransformation, SUVs promoted it at a faster rate and to a higher conversion than those obtained in an aqueous sterol dispersion. The reverse, however, was observed when the same conversion in SUVs was catalysed by free soluble cholesterol oxidase. A lower phospholipid:cholesterol ratio in SUVs enhanced the rate of the microbial conversion, but a negative charge slowed it down. The mechanism of the bioconversion in the liposomal medium was interpreted as a direct interaction between the microbial cells and SUVs. During the first 24 h, the bioconversion proceeded to completion in intact liposomes, and by then Rhodococcus cells had adapted to the phospholipids and could partly metabolize them with extended incubation.

Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles.

Unconjugated bilirubin (bilirubin-IX alpha), the hydrophobic end product of heme degradation, is esterified in the hepatocyte endoplasmic reticulum to water-soluble conjugates prior to excretion in bile. To characterize the process of intracellular bilirubin transport, the kinetic and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were determined. Bilirubin-IX alpha was added to donor vesicles labeled with the fluorescent phospholipid probe, (5-(dimethylamino)naphthalene-1-sulfonyl) dipalmitoyl-L-alpha-phosphatidylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirubin quenches the dansyl probe fluorescence through resonance energy transfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle transfer of the dansyl-PE probe were excluded by quasielastic light scattering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37 degrees C. The rate of bilirubin transfer was independent of acceptor vesicle concentration and decreased with increasing buffer ionic strength, indicating that intermembrane transfer occurred via aqueous diffusion, rather than vesicle collisions. The free energy of activation (delta G++) for the dissociation of bilirubin from donor vesicles was 14.2 kcal.mol-1. These studies suggest that bilirubin is associated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intracellular membranes occurs via spontaneous transfer through the aqueous phase.

A 31P-NMR spin-lattice relaxation and 31P{1H} nuclear Overhauser effect study of sonicated small unilamellar phosphatidylcholine vesicles

The motional properties of the inner and outer monolayer headgroups of egg phosphatidylcholine (PC) small unilamellar vesicles (SUV) were investigated by 31P-NMR temperature-dependent spin-lattice relaxation time constant (T1) and 31P{1H} nuclear Overhauser effect (NOE) analyses. Three different aspects of the dynamics of PC headgroups were investigated using the T1 analysis. First, differences in the dynamics of the headgroup region of both surfaces of the SUV were measured after application of a chemical shift reagent, PrCl3, to either the extra- or intravesicular volumes. Second, the ability of the T1 experiment to resolve the different motional states was evaluated in the absence of shift reagent. Third, comparison between correlation times obtained from a resonance frequency dependent 31P{1H} NOE analysis allowed a determination of the applicability of a simplified motional model to describe phosphorus dipolar relaxation. Temperature-dependent 31P-NMR T1 values obtained for the individual monolayers at 81.0 and 162.0 MHz were modelled assuming that phosphorus undergoes both a dipolar and an anisotropic chemical shielding relaxation mechanism, each being described by the same correlation time, τ. At 162.0 MHz, the position of the T1 minimum for the inner monolayer was 9° higher than that of the outer region, indicating a higher level of motional restriction for the inner leaflet, in agreement with 31P{1H} NOE measurements. The 162.0 MHz T1 profile of the combined SUV monolayers exhibited a smooth minimum located at the midpoint of the monolayer minima positions, effectively masking the presence of the individual surfaces. 31P{1H} NOE results obtained at 32.3, 81.0 and 162.0 MHz did not agree with those predicted from a simple dipolar relaxation model. These results suggest a T1-temperature method can neither discriminate two or more closely related motional time scales in a heterogeneous environment (such as incorporation of protein into lipid bilayers) nor allow accurate determination of the correlation time at the position of the minimum when the dipolar relaxation rate makes a significant contribution to the overall rate.

Lipid asymmetry in rabbit small intestinal brush border membrane as probed by an intrinsic phospholipid exchange protein

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.

Formation and structure of stably dispersed small particles composed of phosphatidylcholine and ubiquinone-10: Coexistence of emulsion particles with bilayer vesicles

Stable aqueous dispersions of ubiquinone-10 (Q 10 ) were produced by cosonication with egg yolk phosphatidylcholine (PC) in a Q 10 mole fraction range of lipid mixture of 0.1–0.7. Electron microscopic and dynamic light scattering measurements showed the diameter of the dispersed particles to be 65–75 nm. The trapped aqueous volume inside the particles was determined fluorometrically with an aqueous space marker, calcein. Trapped volume decreased remarkably with the addition of Q 10 into small unilamellar vesicles of PC. The decline in the fraction of vesicular particles was also confirmed by 1 H-NMR measurements of the choline methyl of PC in the presence of a paramagnetic cation, Pr 31 . These results indicate that the excessive amount of Q 10 separated from PC bilayers is stabilized as emulsion particles by the PC monolayer on the surface. Monolayer-bilayer equilibrium of the PC-Q 10 mixture was estimated by measurements of spreading and collapse pressures. Results show that the coexistence of the emulsion particles (surface monolayer of PC + core of Q 10 ) with the vesicular particles (PC bilayers) is responsible for the maximum surface pressure of the monolayer. The coexistence is probably critically important to the production of stably dispersed particles of the lipid mixture.

Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures.

We have examined the mechanism of the inhibition of cholesterol synthesis in cells treated with exogenous sphingomyelinase. Treatment of rat intestinal epithelial cells (IEC-6), human skin fibroblasts (GM-43), and human hepatoma (HepG2) cells in culture with sphingomyelinase resulted in a concentration- and time-dependent inhibition of the activity of HMG-CoA reductase, a key regulatory enzyme in cholesterol biosynthesis. The following observations were obtained with IEC-6 cells. Free fatty acid synthesis or general cellular protein synthesis was unaffected by the addition of sphingomyelinase. Addition of sphingomyelinase to the in vitro reductase assay had no effect on activity, suggesting that an intact cell system is required for the action of sphingomyelinase. The products of sphingomyelin hydrolysis, e.g., ceramide and phosphocholine, had no effect on reductase activity. Sphingosine, a further product of ceramide metabolism, caused a stimulation of reductase activity. Examination of the incorporation of [3H]acetate into the nonsaponifiable lipid fractions in the presence of sphingomyelinase showed no changes in the percent distribution of radioactivity in the post-mevalonate intermediates of the cholesterol biosynthetic pathway, but there was increased radioactivity associated with the polar sterol fraction. Pretreatment of cells with ketoconazole, a known inhibitor of oxysterol formation, prevented the inhibition of reductase activity by sphingomyelinase and decreased the incorporation of [3H]acetate in the polar sterol fraction. Ketoconazole had no effect on exogenous sphingomyelinase activity in vitro in the presence or absence of cells. Endogenous sphingomyelinase activity was also unaffected by ketoconazole. Addition of inhibitors of endogenous sphingomyelinase activity, e.g., chlorpromazine, desipramine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), to the culture medium caused a dose-dependent stimulation of reductase activity. However, these agents had no effect on the inhibition of reductase activity by exogenous sphingomyelinase. Treatment of cells with small unilamellar vesicles of dioleyl phosphatidylcholine or high density lipoprotein3 resulted in increased efflux of cholesterol and stimulation of reductase activity. Under similar conditions, the inhibitory effect of exogenous sphingomyelinase on reductase activity was prevented by incubation with small unilamellar vesicles of phosphatidylcholine or high density lipoprotein. These results support the hypothesis that alteration of the ratio of sphingomyelin:cholesterol in the plasma membrane plays a modulatory role on the flow of membrane cholesterol to a site where it may be converted to a putative regulatory molecule, possibly an oxysterol.

Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers

The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter wavelengths. Binding of the peptide to the vesicles was measured by the increase in the fluorescence emission yield. A surface partition constant of (3.9 +/- 0.5) x 10(3) M-1 was derived from these titrations. When the membrane contained, in addition to POPC, negatively charged 1-palmitoyl 2-oleoyl phosphatidylglycerol (POPG), the NBD fluorescence spectra were further shifted to shorter wavelengths and exhibited increased quantum yields. The apparent partition constants were increased to 10(4)-10(5) M-1 for vesicles with 20 or 100 mol% POPG. Lateral diffusion of the peptide was measured by fluorescence recovery after photobleaching in multibilayers of POPC, POPG, POPC/POPG (4:1) and 1,2-dimyristoyl phosphatidylcholine. The lateral diffusion coefficients of the peptide in bilayers of POPC (8 x 10(-8) cm2/s at 21 degrees C) were 1.5-1.6-fold greater than those of NBD-labelled phospholipids (5 x 10(-8) cm2/s at 21 degrees C), but 1.5-1.8-fold smaller (3 x 10(-8) cm2/s in 20% POPG and at 21 degrees C) than the lipid diffusion coefficients in the negatively charged bilayers. It is concluded that the signal peptide associates with phospholipid bilayers in two different forms, which depend on the lipid charge. The experiments with POPC bilayers are well explained by a model in which the peptide partitions into the region of the phospholipid head-groups and diffuses along the membrane/water interface. If POPG is present in the membrane, electrostatic attractions between the basic residues of the peptide and the acidic lipid head-groups result in a deeper penetration of the bilayer. For this case, two models that are both consistent with the experimental data are discussed, in which the peptide either forms an oligomer of three to six partially helical membrane-spanning monomers, or inserts into the bilayer with its amphiphilic helical segment aligned parallel to the plane of the membrane and located near the head-group and outer hydrocarbon region of the bilayer.

Changes in lipid ordering of model phospholipid membranes treated with chrysotile and crocidolite asbestos

A variety of inorganic particles bind to cell membranes and cause alterations in phagocytosis, migration, and metabolism leading to eventual cell death. The mechanism which mediate these events at the complex membrane level are not known. In attempting to define the mechanisms of asbestos-induced membrane changes, we have prepared small unilamellar vesicles (SUV) of dipalmitoyl phosphatidylcholine as a simplified model of the cell membrane. Negatively stained preparations for electron microscopy demonstrated that the SUVs bind to both chrysotile and crocidolite asbestos fibers. Electron spin resonance showed that both asbestos types caused increased membrane rigidity at the level of the 12th carbon, whereas chrysotile induced increased rigidity at the 5th carbon level as well. Studies using DPPC membranes compared to SUVs made of phosphatidylcholine from egg yolk support the view that lipid peroxidation may play no significant role in alterations of membrane rigidity induced by the asbestos particle binding. Similar alterations of membrane rigidity and lipid peroxidation at the depth of the 12th carbon have been reported in complex naturally occurring erythrocyte membranes. This suggests that our observations may be relevant to biological membranes and that the model system of SUVs is appropriate for additional studies on the mechanisms of particle-induced membrane injury.