Phospholipid vesicles promote human hemoglobin oxidation.

We have studied the heme oxidation kinetics of purified human hemoglobin (Hb) in the presence of lipid vesicles of dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine that exhibited minimal lipid peroxidation. We showed that the lipid vesicles enhanced Hb oxidation and that small unilamellar vesicles (SUVs) exerted a larger effect than large unilamellar vesicles (LUVs). We have determined pseudo first-order rate constants for the initial disappearance of oxygenated ferrous Hb (k0) and for the initial formation of several ferric Hb species (methemoglobin, hemichrome, and choleglobin) in the presence of SUVs and LUVs. k0 and other rate constants depended linearly on lipid-to-hemoglobin molar ratio (lipid/Hb), with k0SUV (h-1) = k0auto (h-1) + 3.7 x 10(-3) x lipid/Hb, and k0LUV (h-1) = k0auto (h-1) + 0.2 x 10(-3) x lipid/hb, where k0auto is the rate constant for Hb autoxidation in the absence of vesicles. Thus, in the absence of lipid peroxidation products, lipid vesicles themselves promote Hb oxidation by enhancing the rate of Hb oxidation. The enhanced oxidation was inhibited by catalase, but not by butylated hydroxytoluene. The rate constants were independent of Hb concentration, in the range of about 3.1 to 100 microM. We suggest that the lipid surface properties, including surface curvature, surface energy, and hydrophobicity, promote hemoglobin oxidation.     

Free energy potential for aggregation of erythrocytes and phosphatidylcholine/phosphatidylserine vesicles in Dextran (36,500 MW) solutions and in plasma.

The free energy potential (affinity) for aggregation of human red blood cells and lipid vesicles in Dextran solutions and blood plasma has been quantitated by measuring to what extent a vesicle is encapsulated by the red cell surface. The free energy reduction per unit area of contact formation (affinity) was computed from the observation of the fractional extent of encapsulation at equilibrium with the use of a relation based on the elastic compliance of the red cell membrane as it is deformed to adhere to the vesicle surface. Micromanipulation methods were used to select and transfer single lipid vesicles (2-3 X 10(-4) cm diameter) from a chamber that contained the vesicle suspension to a separate chamber on the microscope stage that contained red cells in an EDTA buffer with Dextran or whole plasma. The vesicle and a red cell were maneuvered into close proximity and contact allowed to take place without forcing the cells together. To evaluate the effects of surface charge density and steric interactions on aggregation, vesicles were made from mixtures of egg phosphatidylcholine (PC) and bovine phosphatidylserine (PS) over a range of mole ratios (PC/PS)from (1:0) to (1:1); the vesicles were formed by rehydration in buffer. The Dextran solutions were made with a sharp-cut fraction of 36,500 MW in a concentration range of 0-10% by weight in grams (wt/wt).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids.

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions.     

Macrophage catabolism of lipid A is regulated by endotoxin stimulation

Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.     

Membrane solubilization by a hydrophobic polyelectrolyte: surface activity and membrane binding.

We have previously observed that the hydrophobic polyelectrolyte poly(2-ethylacrylic acid) solubilizes lipid membranes in a pH-dependent manner, and we have exploited this phenomenon to prepare lipid vesicles that release their contents in response to pH, light, or glucose (Thomas, J. L., and D. A. Tirrell. Acc. Chem. Res. 25:336-342, 1992). The physical basis for the interaction between poly(2-ethylacrylic acid) and lipid membranes has been explored using surface tensiometry and fluorimetry. Varying the polymer concentration results in changes in surface activity and membrane binding that correlate with shifts in the critical pH for membrane solubilization. Furthermore, the binding affinity is reduced as the amount of bound polymer increases. These results are consistent with a hydrophobically driven micellization process, similar to those observed with apolipoproteins, melittin, and other amphiphilic alpha-helix-based polypeptides. The absence of specific secondary structure in the synthetic polymer suggests that amphiphilicity, rather than structure, is the most important factor in membrane micellization by macromolecules.     

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.     

Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes

Lipopolysaccharide (LPS) endotoxin is implicated as the bacterial product responsible for the clinical syndrome of Gram-negative septicemia. Although the lipid A domain of LPS appears to be responsible for the toxicity of endotoxin, lipid A from the photosynthetic bacterium Rhodobacter sphaeroides (RSLA) and a disaccharide precursor of lipid A from enteric bacteria, termed lipid IVA, have little activity on human cells. Using the human promonomyelocytic cell line THP-1 and human monocytic cells, we now show that both lipid IVA and RSLA are antagonists of LPS. Complete, apparently competitive, inhibition of LPS activity is possible at a 10-100-fold excess of antagonist, as judged by measuring the release of cytokines and prostaglandin E2. Both antagonists prevent monocyte stimulation by endotoxin extracted from a variety of Gram-negative bacteria. Cells pretreated with either inhibitor and subsequently washed still show attenuated responses to LPS. Stimulation of monocytes by whole Gram-negative bacteria is also antagonized in a dose-dependent manner. Lipid X has no inhibitory effect in the same dose range as lipid IVA and RSLA. These findings rule out LPS sequestration as the explanation for the observed antagonism. Neither inhibitor alters monocyte stimulation by phorbol 12-myristate 13-acetate, Staphylococcus aureus, or purified protein derivative, demonstrating specificity for LPS. Although RSLA appears to inhibit LPS when tested with macrophages from both humans and mice, lipid IVA had the unique ability to act as an LPS antagonist with human-derived cells but to exhibit LPS-like effects with murine-derived cells. Like LPS, lipid IVA stimulated the release of both tumor necrosis factor alpha and arachidonic acid from murine-derived RAW 264.7 macrophage tumor cells. The range of concentrations necessary for lipid IVA to induce LPS-like effects in murine cells was similar to that necessary to antagonize the actions of LPS in human monocytes. The agonist activities of lipid IVA were completely inhibitable by RSLA. This unique species-dependent pharmacology observed with lipid IVA may reflect differences between human and murine LPS receptors. RSLA and lipid IVA may be useful in defining the role of LPS in Gram-negative bacterial infections and may prove to be prototypical therapeutic agents for the treatment of Gram-negative septicemia.     

Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3.

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain. This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.     

Diffusion behavior of lipid vesicles in entangled polymer solutions.

Dynamic light scattering was used to follow the tracer diffusion of phospholipid/cholesterol vesicles in aqueous polyacrylamide solutions and compared with the diffusive behavior of polystyrene (PS) latex spheres of comparable diameters. Over the range of the matrix concentration examined (Cp = 0.1-10 mg/ml), the diffusivities of the PS spheres and the large multilamellar vesicles exhibited the Stokes-Einstein (SE) relation, while the diffusivity of the unilamellar vesicles did not follow the increase of the solution's viscosity caused by the presence of the matrix molecules. The difference between the diffusion behaviors of unilamellar vesicles and hard PS spheres of similar size is possibly due to the flexibility of the lipid bilayer of the vesicles. The unilamellar vesicles are capable of changing their shape to move through the entangled polymer solution so that the hindrance to their diffusion due to the presence of the polymer chains is reduced, while the rigid PS spheres have little flexibility and they encounter greater resistance. The multilamellar vesicles are less flexible, thus their diffusion is similar to the hard PS spheres of similar diameter.     

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.     

Interaction of poly(ethylene-glycols) with air-water interfaces and lipid monolayers: investigations on surface pressure and surface potential.

We have characterized the surface activity of different-sized poly(ethylene-glycols) (PEG; M(r) 200-100,000 Da) in the presence or absence of lipid monolayers and over a wide range of bulk PEG concentrations (10(-8)-10% w/v). Measurements of the surface potential and surface pressure demonstrate that PEGs interact with the air-water and lipid-water interfaces. Without lipid, PEG added either to the subphase or to the air-water interface forms relatively stable monolayers. Except for very low molecular weight polymers (PEGs < 1000 Da), low concentrations of PEG in the subphase (between 10(-5) and 10(-4)% w/v) increase the surface potential from zero (with respect to the potential of a pure air-water interface) to a plateau value of approximately 440 mV. At much higher polymer concentrations, > 10(-1)% (w/v), depending on the molecular weight of the PEG and corresponding to the concentration at which the polymers in solution are likely to overlap, the surface potential decreases. High concentrations of PEG in the subphase cause a similar decrease in the surface potential of densely packed lipid monolayers spread from either diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine (DPPC), or dioleoyl phosphatidylserine (DOPS). Adding PEG as a monolayer at the air-water interface also affects the surface activity of DPhPC or DPPC monolayers. At low lipid concentration, the surface pressure and potential are determined by the polymer. For intermediate lipid concentrations, the surface pressure-area and surface potential-area isotherms show that the effects due to lipid and PEG are not always additive and that the polymer's effect is distinct for the two lipids. When PEG-lipid-mixed monolayers are compressed to surface pressures greater than the collapse pressure for a PEG monolayer, the surface pressure-area and surface potential-area isotherms approach that of the lipid alone, suggesting that for this experimental condition PEG is expelled from the interface.     

Ca2+-induced fusion of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles.

The fusogenic properties of sulfatide-containing 1,2-dioleoyl-3-sn -phosphatidylethanolamine (DOPE) small unilamellar vesicles (SUVs) in the presence of CaCl2 were studied by mixing membrane lipids based on an assay of fluorescence resonance energy transfer (FRET). Fusion of the vesicles was also confirmed by mixing aqueous contents with the Tb/dipicolinate (DPA) assay. The half-times of lipid mixing revealed that the fusion rate decreased with increasing molar concentration of sulfatide. This inhibitory effect was more obvious at sulfatide concentrations higher than 30 mol%, where hydration at the membrane surface reached its maximum and the fusion was no longer pH-sensitive in the range of pH 6.0 - 9.0. Similar inhibitory effect was also observed in Ca2+-induced fusion of DOPE/ganglioside GM1 vesicles but at a lower concentration of the glycosphingolipid (20 mol%). In contrast, increasing the concentration of phosphatidylserine (PS) in DOPE/PS SUVs resulted in an increase in the rate of Ca2+-induced lipid mixing and the pH sensitivity of this system was not affected.These results are consistent with an increasing steric hindrance to membrane fusion at higher molar concentration and larger headgroup size of the glycosphingolipids. Interestingly, the pH sensitivity of the sulfatide-containing liposomes was retained when they were allowed to fuse with synaptosomes in the absence of Ca2+ by a mechanism involving protein mediation.     

Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli.

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that overproduces the transferase approximately 500-fold, we have purified the enzyme to near homogeneity. The subunit molecular mass is approximately 43 kDa. Activity is stimulated by Triton X-100, is maximal at pH 7, but does not require Mg2+. The apparent Km values for lipid IVA and CMP-Kdo are 52 and 88 microM, respectively. Vmax is 15-18 mumol/min/mg when both substrates are added near saturation at pH 8. The purified enzyme transfers 2 Kdo residues to lipid A precursors or analogs bearing four to six fatty acyl chains and a 4'-monosphosphate moiety. Activity is inhibited by polymixin B and Re endotoxin. At low Kdo concentrations small amounts of the intermediate, (Kdo)1-IVA, accumulate. When this substance is isolated and incubated with purified enzyme in the presence of CMP-Kdo, it is converted to (Kdo)2-IVA. Formation of (Kdo)1-IVA is also observed when purified enzyme is incubated with (Kdo)2-IVA and 5 mM CMP, demonstrating that Kdo transfer is reversible. In summary, Kdo transferase consists of a single bifunctional polypeptide that incorporates the 2 innermost Kdo residues common to all lipopolysaccharide molecules in E. coli.     

A deacylase in Rhizobium leguminosarum membranes that cleaves the 3-O-linked beta-hydroxymyristoyl moiety of lipid A precursors

Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4'-phosphate groups but is derivatized with a galacturonic acid substituent at position 4'. R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked beta-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleave E. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVA and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked beta-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.     

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.     

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.     

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.     

Acid-base equilibria at interface separating electrolyte solution and lipid bilayer formed from phosphatidylcholine

The dependence of the interfacial tension of a lipid membrane formed from phosphatidylcholine on the pH of the aqueous solution has been studied. The model describing the H(+) and OH(-) ions adsorption in the bilayer lipid surface has been presented in this work. We take suitable equations to describe the dependence of interfacial tension of a lipid bilayer membrane on H(+) and OH(-) ion concentrations. A theoretical equation is derived to describe this dependence in the whole pH range.     

Fragmentation of dimyristoylphosphatidylcholine vesicles by apomyoglobin.

Previously we have reported results of a preliminary study on the micellization of phosphatidylcholine vesicles by apomyoglobin at pH 4 (J. W. Lee and H. Kim, 1988, FEBS Lett. 241, 181-184). The micellization study has been extended here to investigate the effect of the lipid to protein ratio, temperature, size of vesicles, and pH. The pH-dependent study indicated that micellization occurs when the protein assumes either a molten globular or random coil structure. Time-dependent hydrophobic labeling by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine showed that there is an initial increase in contact between the protein and hydrophobic acyl chain of lipid followed by a decrease in the interaction. This may be explained as the initial stage of vesicle aggregation which is subsequently superseded by the fragmentation. These reactions are discussed in term of protein unfolding at low pH.