Interactions of hemin, antimalarial drugs and hemin-antimalarial complexes with phospholipid monolayers

Hemin, antimalarial drugs and complexes formed between them, have demonstrable effects on biological membranes. Using the phospholipid monolayer model, we show that hemin intercalates into the membrane and increases its surface pressure, depending on the lipid composition and the initial surface pressure: negative surface charges and particularly looser compaction of the phospholipids reduce the effect of hemin. With increasing surface pressure hemin tends to intercalate as a monomer, and the half-saturation concentration of its effect increases exponentially. The antimalarial monovalent drugs quinine and mefloquine, but not chloroquine, also penetrate into the membrane and expand it. All three drugs markedly increase the effect of hemin, but chloroquine reduces the effect in monolayers composed of unsaturated phospholipids. The drugs' effect is mostly due to an increase in the maximal surface pressure and suggests a complexation of hemin and drug within the membrane phase. Preformed hemin-drug complexes decrease the half-saturation concentration of the effect and suggest that the complexes adsorb to the membrane, releasing the hemin through an apolar continuum into the phospholipid phase. The implications of the results to the membrane toxicity mechanism proposed for the molecular mode of action of antimalarial drugs are discussed.     

Molecular packing of high-density and low-density lipoprotein surface lipids and apolipoprotein A-I binding

The surface pressure (pi)-molecular area (A) isotherms for monolayers of human high-density lipoprotein (HDL3) and low-density lipoprotein (LDL) phospholipids and of mixed monolayers of these phospholipids with cholesterol spread at the air-water interface were used to deduce the likely molecular packing at the surfaces of HDL3 and LDL particles. LDL phospholipids form more condensed monolayers than HDL3 phospholipids; for example, the molecular areas of LDL and HDL3 phospholipids at pi = 10 dyn/cm are 88 and 75 A2/molecule, respectively. The closer packing in the LDL phospholipids monolayer can be attributed to the higher contents of saturated phosphatidylcholines and sphingomyelin relative to HDL3. Cholesterol condenses both HDL3 and LDL phospholipid monolayers but has a greater condensing effect on the LDL phospholipid monolayer. The pi-A isotherms for mixed monolayer of HDL3 phospholipid/cholesterol and LDL phospholipid/cholesterol at stoichiometries similar to those at the surfaces of lipoprotein particles suggest that the monolayer at the surface of the LDL particle is significantly more condensed than that at the surface of the HDL3 particle. The closer lateral packing in LDL is due to at least three factors: (1) the difference in phospholipid composition; (2) the higher unesterified cholesterol content in LDL; and (3) a stronger interaction between cholesterol and LDL phospholipids relative to HDL3 phospholipids. The influence of lipid molecular packing on the affinity of human apolipoprotein A-I (apo A-I) for HDL3 and LDL surface lipids was evaluated by monitoring the adsorption of 14C-methylated apo A-I to monolayers of these lipids spread at various initial surface pressures (pi i).(ABSTRACT TRUNCATED AT 250 WORDS)     

IRIV-adjuvanted hepatitis A vaccine: in vivo absorption and biophysical characterization

Immunopotentiating reconstituted influenza virosomes (IRIV) are 150-nm proteoliposomes composed of influenza surface glycoproteins and a mixture of natural and synthetic phospholipids. Due to size, structure and composition of the IRIVs, they serve as an antigen carrier system for efficacious vaccination, as was demonstrated for hepatitis A and influenza. This paper reviews the unique properties of IRIVs and describes the in vivo biodistribution of model antigens using 14C-labeled IRIVs and 125I-labeled streptavidin. IRIV formulated streptavidin induced a strong depot effect after intra muscular (i.m.) vaccination of mice, whereas soluble streptavidin was soon eliminated via the kidney of the animals. A mixture of antigen and IRIVs yielded higher antibody titers after i.m. inoculation than streptavidin alone. The highest immunostimulation was achieved by the binding of the antigen to the investigated adjuvant. The potential penetration of inactivated hepatitis A virions into lipid membranes was assessed by measuring the area increase of a lipid monolayer kept at a constant surface pressure corresponding to that of lipid bilayer vesicles. The monolayers were composed of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) (75/25 mol/mol), thus resembling the lipid composition of the IRIV. The results suggested that the hepatitis A antigen may spontaneously bind to the reconstituted IRIV membranes.     

The study on the interaction between phytosterols and phospholipids in model membranes

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.     

Direct observation of poloxamer 188 insertion into lipid monolayers

P188, a triblock copolymer of the form poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) helps seal electroporated cell membranes, arresting the leakage of intracellular materials from the damaged cells. To explore the nature of the interaction between P188 and cell membranes, we have constructed a model system that assesses the ability of P188 to insert into lipid monolayers. Using concurrent Langmuir isotherm and fluorescence microscopy measurements, we find that P188 changes the phase behavior and morphology of the monolayers. P188 inserts into both dipalmitoylphosphatidlycholine and dipalmitoylphosphatidylglycerol monolayers at surface pressures equal to and lower than approximately 22 mN/m at 30 degrees C; this pressure corresponds to the maximal surface pressure attained by P188 on a pure water subphase. Similar results for the two phospholipids indicate that P188 insertion is not influenced by headgroup electrostatics. Because the equivalent surface pressure of a normal bilayer is on the order of 30 mN/m, the lack of P188 insertion above 22 mN/m further suggests the poloxamer selectively adsorbs into damaged portions of electroporated membranes, thereby localizing its effect. P188 is also found to be "squeezed out" of the monolayers at high surface pressures, suggesting a mechanism for the cell to be rid of the poloxamer when the membrane is restored.     

Characterization of the binary mixed monolayers of α-tocopherol with phospholipids at the air-water interface

The mixed Langmuir monolayers composed of model constituents of biological membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were investigated to provide information on the intermolecular interactions between these membrane components and the physiologically active vitamin E–α-tocopherol (TF), as well as on the phase behavior of these mixed systems. Additionally, topography of these monolayers transferred onto the mica support was investigated by the inverted metallurgical microscope. Morphological characteristics were directly observed by Brewster angle microscopy (BAM). From the surface pressure–area isotherms and the analysis of physicochemical parameters (compressibility and mean molecular area at the maximum compressibility) it was found that depending on the acyl chains saturation degree, TF has different effect on the phospholipids. In the case of DPPC, the addition of TF to the phospholipid film causes destabilization of the ordered hydrocarbon chains, while in the POPC/DOPC–TF systems, the attractive interactions are responsible for the monolayer increased stability. Thus, the results of these studies confirm the hypothesis that α-tocopherol may play a role in the stabilization of biological membranes.     

Effect of bovine prothrombin fragment 1 on the translational diffusion of phospholipids in Langmuir-Blodgett monolayers.

Previous work has shown that bovine prothrombin fragment 1 binds to supported planar membranes composed of phosphatidylcholine and phosphatidylserine in a Ca(2+)-specific manner (Tendian et al. (1991) Biochemistry 30, 10991; Pearce et al. (1992) Biochemistry 31, 5983-5995). In the present work, fluorescence pattern photobleaching recovery has been used to examine the effect of membrane-bound fragment 1 on the translational diffusion coefficients of two fluorescent phospholipids in fluid-like phosphatidylserine/phosphatidylcholine Langmuir-Blodgett monolayers. The results show that saturating concentrations of fragment 1, in the presence of Ca2+, reduce the diffusion coefficient of nitrobenzoxadiazolyl-conjugated phosphatidylserine (NBD-PS) and nitrobenzoxadiazolyl-conjugated phosphatidylcholine (NBD-PC) by factors of approximately four and two, respectively. Ca2+ or fragment 1 alone do not have a statistically significant effect on NBD-PS or NBD-PC diffusion. In addition, a nonspecific protein (ovalbumin) does not change the diffusion coefficients of the fluorescent phospholipids either in the absence or presence of Ca2+. The fractions of the fluorescent phospholipids that are laterally mobile are approximately 0.9 for all samples. These results are interpreted with several models for possible mechanisms by which extrinsically bound proteins might retard phospholipid diffusion in membranes.     

Phospholipid Monolayers Probed by Vibrational Sum Frequency Spectroscopy: Instability of Unsaturated Phospholipids

The surface specific technique vibrational sum frequency spectroscopy has been applied to in situ studies of the degradation of Langmuir monolayers of 1,2-diacyl-phosphocholines with various degrees of unsaturation in the aliphatic chains. To monitor the degradation of the phospholipids, the time-dependent change of the monolayer area at constant surface pressure and the sum frequency intensity of the vinyl CH stretch at the carbon-carbon double bonds were measured. The data show a rapid degradation of monolayers of phospholipids carrying unsaturated aliphatic chains compared to the stable lipids carrying fully saturated chains when exposed to the ambient laboratory air. In addition, the degradation of the phospholipids can be inhibited by purging the ambient air with nitrogen. This instability may be attributed to spontaneous degradation by oxidation mediated by various reactive species in the air. To further elucidate the process of lipid oxidation in biological membranes artificial Langmuir monolayers probed by a surface specific spectroscopic technique as in this study can serve as a model system for studying the degradation/oxidation of cell membrane constituents.     

Surface activity of plasma membranes

Plasma membranes of rabbit intestinal smooth muscle cells manifest low surface activity on the boundary of the electrolyte-air phases. This activity undergoes essential changes if the electrolyte surface is covered with the lecithin monolayer. According to the experimental data, the interaction of plasma membranes with the lecithin monolayers is hydrophobic in nature and essentially depends on the density of molecular packing of phospholipids in the monolayer and on the status of the membrane preparation. A possible mechanism of the formation of the monolayer patterns from cell membranes is discussed.     

Poliovirus 2b insertion into lipid monolayers and pore formation in vesicles modulated by anionic phospholipids

Enterovirus 2B viroporin has been involved in membrane permeabilization processes occurring late during cell infection. Even though 2B lacks an obvious signal sequence for translocation, the presence of a Lys-based amphipathic domain suggests that this product bears the intrinsic capacity for partitioning into negatively charged cytofacial membrane surfaces. Pore formation by poliovirus 2B attached to a maltose-binding protein (MBP) has been indeed demonstrated in pure lipid vesicles, a fact supporting spontaneous insertion into and direct permeabilization of membranes. Here, biochemical evidence is presented indicating that both processes are modulated by phosphatidylinositol and phosphatidylserine, the main anionic phospholipids existing in membranes of target organelles. Insertion into lipid monolayers and partitioning into phospholipid bilayers were sustained by both phospholipids. However, MBP-2B inserted into phosphatidylserine bilayers did not promote membrane permeabilization and addition of this lipid inhibited the leakage observed in phosphatidylinositol vesicles. Mathematical modelling of pore formation in membranes containing increasing phosphatidylserine percentages was consistent with its inhibitory effect arising from a higher reversibility of MBP-2B surface aggregation. These results support that 2B insertion and pore-opening are mechanistically distinguishable events modulated by the target membrane anionic phospholipids.     

Protein insertion and patterning of PEG bearing langmuir monolayers

Protein adsorption to multicomponent lipid monolayers is presented as a means of inducing protein-specific binding pockets or imprints in membranes. Adsorption of the acidic protein ferritin to Langmuir monolayers of cationic dioctadecyldimethylammonium bromide (DOMA), nonionic methyl stearate (SME), and poly(ethylene glycol) (PEG) bearing phospholipids is investigated as a model system. The number, size, and distribution of protein binding pockets (domains of SME and DOMA in a PEG matrix) are defined by controlling the molar ratios, miscibility, and lateral mobility of the lipids. Protein patterning of binary SME:DOMA monolayers is limited by protein-protein interactions that hinder desorption to regenerate the imprint site. The incorporation of PEG bearing phospholipids as a third lipid component provides a successful approach to prevent protein surface aggregation during imprinting. Atomic force microscopy reveals a user-defined distribution of protein molecules where protein-protein interactions on the monolayer are eliminated, thus facilitating protein desorption and regeneration of the protein binding pockets.     

The interaction of bacterial lipopolysaccharide with phospholipid bilayers and monolayers

The association of bacterial lipopolysaccharide with artificial membranes was studied in an attempt to understand the mechanism of binding of lipopolysaccharide to cell surfaces and to look for an effect on membrane stability. The membrane models used were phospholipid bilayers and monolayers. As measured by survival time, lipopolysaccharide was found to decrease the stability of bilayers at a concentration of 300 μg/ml. When assayed by dielectric breakdown, an effect of lipopolysaccharide was noticeable at concentrations of 50 μg/ml. In studies involving the penetration of monomolecular films of various phospholipids, native and alkali-treated lipopolysaccharide both caused increases in surface pressure, and therefore penetrated the films. However, alkali-treated lipopolysaccharide was at least ten times more efficient than the native product in penetration. Alkali-treated lipopolysaccharide had a greater degree of surface activity than native lipopolysaccharide, since alkali-treated lipopolysaccharide formed monomolecular films by itself, whereas native lipopolysaccharide did not. The changes in the surface pressure and surface potential of phospholipid films produced by lipopolysaccharide in the subsolution suggested that the interaction of lipopolysaccharide with phospholipid monolayers was by a combination of penetration and adsorption to the undersurface.     

Polar-group behaviour in mixed monolayers of phospholipids and fusogenic lipids.

1. The surface potentials of mixed monolayers of synthetic phospholipids with lipids that are fusogenic for hen erythrocytes were investigated. 2. At pH 5.6 and 10, but not at pH2, mixed monolayers of the fusogenic lipid, glycerol mono-oleate, with phosphatidylcholine exhibited negative deviations from the ideality rule in surface potential per molecule which were accompanied by negative deviations in mean molecular area. 3. Interactions of this type were not seen with chemically related but non-fusogenic lipids, nor were they found in mixed monolayers of any of the lipids with phosphatidylethanolamine. 4. Experiments with dihexadecyl phosphate and hexadecyltrimethyl-ammonium indicated that the complete head group of phosphatidylcholine is required for its observed behaviour with fusogenic lipids. 5. Bivalent cations (Ca2+, UO2(2+) or Zn2+) in the subphase at pH 5.6 significantly modified the behaviour of mixed monolayers of fusogenic lipids with phospholipids; there was a parallel perturbing effect of fusogenic lipids on interactions between monolayers of phospholipids and bivalent cations. 6. Possible molecular interactions of fusogenic lipids with membrane phospholipids, and the role of Ca2+, are discussed which may be relevant to cell fusion in erythrocytes induced by low-melting lipids in the presence of Ca2+.     

Study of hydrophobic interactions between acylated proteins and phospholipid bilayers using BIACORE

Intracellular proteins of eukaryotic cells are frequently covalently modified by the addition of long chain fatty acids. These modifications are thought to allow otherwise soluble proteins to associate with membranes by lipid-lipid based hydrophobic interactions. The purpose of this work was to quantify the effect of acyl chain length on hydrophobic interactions between acylated proteins and phospholipid monolayers. The binding of an artificially acylated model protein to electrically neutral phospholipids was studied by surface plasmon resonance, using BIACORE. Kinetic rates for the binding of bovine pancreatic ribonuclease A (RNase A), monoacylated on its N-terminal lysine with fatty acids of 10, 12, 14, 16 or 18 carbon atoms, to phospholipids on hydrophobic sensor chips, were measured. Unlike unmodified ribonuclease, acylated RNase A bound to the phospholipids, and the association level increased with the acyl chain length to reach a maximum for C16. Reproducible kinetics were obtained which did not fit a 1:1 Langmuir model but rather a two-step binding profile.     

Studies on mixed monolayers of phospholipids and fusogenic lipids.

1. The behaviour of mixed monolayers of 14 different lipids with preparations of erythrocyte lipids, purified natural and synthetic phospholipids, cholesterol and galactosylceramide was investigated. 2. The mean areas occupied per molecule in mixed films containing lipids that are fusogenic for hen erythrocytes were compared with those for corresponding films containing lipids that are inactive as fusogens. 3. Fusogenic lipids were found to exhibit interactions, which were not shown by non-fusogenic lipids, in mixed monolayers with several species of phospholipid, particularly those containing a choline head group. 4. Heterogeneity in the hydrophobic chains of phosphatidylcholine, their degree of unsaturation and the presence of cholesterol had little effect on the interaction of phosphatidylcholine with fusogenic lipids. 5. Fusogenic lipids showed little specific interaction with natural or synthetic preparations of phosphatidylethanolamine. 6. The possible significance of these observations in relation to the action of fusogenic lipids on biological membranes is discussed in the light of the asymmetrical distribution of phospholipids in erythrocyte membranes.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

A model for the packing of lipids in bilayer membranes

A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle.Phosphotidylcholine and phosphotidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphotidylethanolamine is almost cylindrical. The “condensing” effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accomodation. The lipid distribution in erythrocyte membranes is shown to be conductive to a stable strain-free membrane.