Interactions of phospholipid monolayers with carbohydrates

Surface pressure studies of phospholipid monomolecular films of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) formed at an air/water interface have been made and the effects on the films studied when various carbohydrates are present in the subphase. The results obtained show that at a given temperature, the area per molecule of DPPC increases with increasing concentration of the carbohydrate in the subphase. The carbohydrate which has the greatest expanding effect on the phospholipid monolayer is glycerol, followed in turn by trehalose, sucrose, glucose, raffinose, and inositol. The mechanism of monolayer expansion by glycerol is different from that observed in other carbohydrates, as the following experiments demonstrate. Below the phase transition temperature of DPPC, the area per molecule of DPPC at a pressure of 12.5 dyn/cm is the same with and without glycerol in the subphase. However, when the monolayer is heated to a temperature above the phase transition temperature for DPPC, the area/molecule on glycerol is considerably greater than the area/molecule on water at the same surface pressure. Cooling the monolayer back to the lower temperature produces an area/molecule of DPPC which is identical on both water and glycerol subphases. Glycerol therefore has no effect on the low-temperature (condensed) monolayers but causes expansion of the high-temperature (expanded) monolayers. By contrast with glycerol, both trehalose and sucrose interact with the DPPC monolayer producing an increased area/molecule over that observed on water, both with low-temperature (condensed) monolayers and with the high-temperature (expanded) monolayers. The efficiency of these carbohydrates at expanding the monolayer films (with the exception of glycerol) shows a strong correlation with their ability to stabilize membrane structure and function at low water contents.     

Cooperativity of phospholipid reorganization upon interaction of dipyridamole with surface monolayers on water

Results from various surface sensitive characterization techniques suggest a model for the interaction of the piperidinopyrimidine dipyridamole (DIP)--known as a vasodilator and inhibitor of P-glycoprotein associated multidrug resistance of tumor cells--with phospholipid monolayers in which the drug is peripherally associated with the membrane, binding (up to) five phospholipids at a time. These multiple interactions are responsible for a very strong association of the drug with the lipid monolayer even at exceedingly low concentrations (approximately 0.2 mol%). Electrostatic interactions and hydrogen bonding are likely involved in the binding of DIP to DPPC. Cooperative effects among the lipids are invoked to explain the macroscopically measurable changes of lipid monolayer properties even when only one out of 100 DPPC molecules is directly associated with a DIP molecule. A reversal of the observed changes upon drug association with the membrane as the DIP concentration surpasses a threshold concentration (c(crit)approximately 0.5 mol%) may be explained by cooperativity in a different context, the self-aggregation of drug molecules. With its implications for the interaction of DIP with phospholipid films, this work provides a first approach to the explanation of the high sensitivity of cell membranes to piperidinopyrimidine drugs on a molecular level.     

The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential.

The interaction of Pseudomonas aeruginosa exotoxin A (ETA) with lipid monolayers was studied by measuring the variation in surface pressure. ETA adsorbs to the monolayer, occupying an average area of approximately 4.6 nm2 per molecule, up to a maximum density of one molecule per 28 nm2 of lipid film, which corresponds roughly to the cross-sectional area of the toxin. This suggests that ETA molecules adsorb until they contact each other, but insert only a small portion into the lipid film. The kinetic process could be described by a Langmuir adsorption isotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membrane composition, pH, and ionic strength. Two parameters were found to be paramount for this interaction: pH and surface potential of the lipid. It appears that ETA binding occurs only in a conformational state induced by low pH and is promoted by an electrostatic interaction between a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient features of ETA involved in its adsorption were derived: 1) the existence of a conformational state induced by the protonation of a group with pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to interact with the surface potential of the membrane; 3) the fraction of potential experienced by the protein in the activated state that precedes binding, approximately 80%; 4) the intrinsic adsorption and desorption rate constants, k(a)0 = (4.8 +/- 0.3) x 10(3) M(-1) s(-1) and k(d)0 = (4.4 +/- 0.4) x 10(-4) s(-1). These rate constants are independent of pH and lipid and buffer composition, and provide a dissociation constant Kd approximately 90 nM.     

Interaction of basic polypeptides with phospholipid monolayers

Adsorption of the polylysine and of the copolypeptides: L-lysine/L-serine and L-lysine/L-phenylalanine on phospholipid monolayers has been investigated. The charge density of the monolayers was varied by using the negatively charged phosphatidyl serine and the neutral phosphatidyl choline at different ratios. The surface concentrations of the adsorbed polypeptides was determined by measuring the surface radiation of their radioactive label.The adsorbing capacity of the monolayer surfaces increases with their negative charge, however with respect to polypeptides the surface activity sequence is pL < pLS < pLφ. From the dependence of adsorption on the ionic strength it was concluded that it is controlled by three types of interaction: (1) electrostatic attraction to the negatively charged surface; (2) electrostatic repulsion between adsorbed polybases; (3) hydrophobic interactions involving specific structural arrangements. This is true even of the apparently neutral PC monolayer where the fixed phosphate groups form an electrical double layer with the more mobile choline groups which can be interpenetrated by the charged groups of the basic polypeptides.     

NO2 interfacial transfer is reduced by phospholipid monolayers.

Nitrogen dioxide (NO2) is a ubiquitous, pollutant gas that produces a broad range of pathological and physiological effects on the lung. Absorption of inhaled NO2 is coupled to near-interfacial reactions between the solute gas and constituents of the airway and alveolar epithelial lining fluid. Although alveolar surfactant imparts limited resistance to respiratory gas exchange compared with that contributed by either the pulmonary membrane or uptake in red blood cells, resistance to NO2 flux could have a significant effect on NO2 absorption kinetics. To investigate the effect of interfacial surfactant on NO2 absorption, we designed an apparatus permitting exposure of variably compressed monolayers. Our results suggest that compressed monolayers enriched in 1,2-dipalmitoyl-sn-3-glycero-phosphocholine present significant resistance to NO2 absorption even at surface tensions greater than those achieved in vivo. However, monolayers composed of pure unsaturated phospholipids failed to alter NO2 absorption significantly when compressed, in spite of similar reductions in surface tension. The results demonstrate that phospholipid monolayers appreciably limit NO2 absorption and further that monolayer-induced resistance to NO2 flux is related to physicochemical properties of the film itself rather than alterations within the aqueous and gas phases. On the basis of these findings, we propose that pulmonary surfactant may influence the intrapulmonary gas phase distribution of inhaled NO2.     

The interaction of meso-tetraphenylporphyrin with phospholipid monolayers

In this article, we investigate the interaction of meso-tetraphenylporphyrin (TPP) with phospholipid monolayers. Pure TPP molecules form films at the air-water interface with large extension of aggregation, which is confirmed by UV-vis spectra of transferred monolayers. For mixed films of TPP with dipalmitoyl phosphatidyl choline (DPPC) or dipalmitoyl phosphatidyl glycerol (DPPG), on the other hand, aggregation is only significant at high surface pressures or high concentrations of TPP (above 0.1 molar ratio). This was observed via Brewster angle microscopy (BAM) for the Langmuir films and UV-vis spectroscopy for transferred layers onto solid substrates. TPP indeed causes the DPPC and DPPG monolayers to expand, especially at the liquid-expanded to liquid-condensed phase transition for DPPC. The effects from TPP cannot be explained using purely geometrical considerations, as the area per TPP molecule obtained from the isotherms is at least twice the expected value from the literature. Therefore, interaction between TPP and DPPC or DPPG should be cooperative, so that more phospholipid molecules are affected than just the first neighbors to a TPP molecule.     

Interaction of cytochromec with phospholipid monolayers and bilayer membranes

Summary For the study of the interaction between oxidized cytochromec and phosphatidylinositide, two different model systems were used: (1) monolayers which were deposited after the method of Langmuir and Blodgett onto glass plates, and (2) bimolecular (“black”) membranes in aqueous phase. The amount of bound protein was determined with a sensitive spectrophotometer. It was found that at low ionic strength about 10¹³ cytochromec molecules per cm² are bound to the lipid surface, which nearly corresponds to a densely packed monolayer. At high ionic strength (∼ 0.1m) or low pH (pH<3), the adsorbed protein layer becomes unstable. This result indicates that the interaction is mainly electrostatic. In accordance with this conclusion is the observation that the rate of adsorption is diffusion controlled; i.e., almost every protein molecule hitting the surface is bound. The cytochromec monolayer can be reduced by ascorbate. In contrast to ferrocytochromec in solution, the bound ferrocytochrome was found to be autoxidable.     

Interaction of two phenothiazine derivatives with phospholipid monolayers

This paper addresses the cooperative interaction of two phenothiazine drugs, viz. trifluoperazine (TFP) and chlorpromazine (CPZ), with phospholipid monolayers as the model membrane system. Surface pressure and surface potential isotherms were obtained for mixed Langmuir monolayers of either dipalmitoyl-phosphatidyl-choline (DPPC) or dipalmitoyl-phosphatidyl-glycerol (DPPG) co-spread with TFP or CPZ. The changes in monolayer behavior caused by incorporation of a few molar ratio of drug molecules were practically within the experimental dispersion for the zwitterionic DPPC, and therefore a more refined analysis will be required to probe the interactions in an unequivocal way. For the charged DPPG, on the other hand, the surface pressure and the dipole moment were significantly affected even for TFP or CPZ concentrations as low as 0.002 molar ratio. Overall, the effects from CPZ and TFP are similar, but small differences exist which are probably due to the different protonation properties of the two drugs. For both drugs, changes are more prominent at the liftoff of the surface pressure, i.e. at the gas-condensed phase transition, with the surface pressure and surface potential isotherms becoming more expanded with the drug incorporation. With DPPG/CPZ monolayers, in particular, an additional phase transition appears at higher CPZ concentrations, which resembles the effects from increasing the subphase temperature for a pure DPPG monolayer. The dipole moment for DPPG/CPZ and DPPG/TFP monolayers decreases with the drug concentration, which means that the effects from the charged drugs are not associated with changes in the double-layer potential. Otherwise, the effective dipole moment should increase with the drug concentration. The changes caused in surface pressure and dipole moment by small concentrations of TFP or CPZ can only be explained by some cooperative effect through which the contribution from DPPG molecules changes considerably, i.e. even DPPG molecules that are not neighbor to a CPZ or TFP molecule are also affected. Such changes may occur either through a significant reorientation of the DPPG molecules or to a change in their hydration state. We discuss the cooperativity semi-quantitatively by estimating the number of lipid molecules affected by the drug interaction. CPZ and TFP also affect the morphology of DPPG monolayers, which was confirmed with Brewster angle microscopy. The biological implications from the cooperative, non-specific interaction of CPZ and TFP with membranes are also commented upon.     

The displacement of calcium ions from phospholipid monolayers by pharmacologically active and other organic bases

1. The binding of (45)Ca(2+) to a monolayer of phosphatidylinositol at the air-water interface was maximal when the separation of the phospholipid head groups approximated to the diameter of a hydrated Ca(2+) ion. 2. The displacement of Ca(2+) adsorbed on monomolecular films of phosphatidylinositol by a series of drugs (both narcotic and excitatory) and other organic bases was related to the ability of the bases to penetrate into the film. 3. With films of phosphatidylinositol at constant area, and at an initial surface pressure of 10dynes/cm., the displacement of Ca(2+) by increasing concentrations of the local anaesthetic, tetracaine, was linearly related to the change in surface pressure (Deltapi) caused by the penetration of the drug. 4. Deltapi and the displacement of Ca(2+) showed a related fall when the initial surface pressure of the phosphatidylinositol film was increased from 4 to 40dynes/cm. both at a constant bulk tetracaine concentration and when this latter concentration was adjusted to keep it at a constant ratio to the surface density of phosphatidylinositol molecules. 5. The displacement of Ca(2+) from phosphatidylinositol films by cetyltri-methylammonium ions was directly compared with the surface concentration of the base in the film, measured by using labelled base and a surface-radioactivity technique. 6. The ability of a series of straight-chain aliphatic amines to displace Ca(2+) from phosphatidylinositol films increased with the number of carbon atoms up to C(12). However, there was a marked jump in the displacing activity after hexylamine, and this could probably be correlated with the carbon chain's being of sufficient length to just reach the hydrophobic fatty acid chains of the orientated phospholipid molecules with the charges on both substances in juxtaposition.     

Interactions of cytochromes b5 and c with phospholipid monolayers

Monolayers of charged and neutral phospholipids at the air/water interface containing the cytochromes b5 and c are studied by film balance techniques and by fluorescence microscopy. A new technique is introduced to obtain a defined and homogeneous protein distribution within the membrane. It is shown that both proteins preferentially partition into the fluid membrane phases coexisting with solid lipid domains, thus allowing formation of periodic protein distributions. Protein reconstitution in protein/lipid ratios up to 1:50 does not change the pressure, pi c, corresponding to the main lipid transition but changes the slope in the pressure/area isotherms. It also affects the pressure-induced lipid crystallization, in that the monolayer can be viewed as segregated into a protein-free and a protein-enriched phase. Whereas penetration of cytochrome c into the monolayer is highly dependent on lipid head group charge, this does not hold for cytochrome b. In both cases, monolayer penetration is monotonously reduced with increasing surface pressure, pointing to the dependence of hydrophobic protein-lipid interactions on hydrocarbon chain density.     

Surface activity and interaction of StarD7 with phospholipid monolayers

StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface showing marked surface activity. The latter is enhanced by penetration into phospholipid films at an initial surface pressure above the protein’s own equilibrium adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid, with preference for phosphatidylserine, cholesterol, and phosphatidylglycerol, and the increases of lateral surface pressure generated are comparable to those of other membrane-active proteins. The surface activity of StarD7 is strong enough to thermodynamically drive and retain StarD7 at the lipid membrane interface where it may undergo lipid-dependent reorganization as indicated by changes of surface pressure and electrostatics.     

c-Fos activated phospholipid synthesis is required for neurite elongation in differentiating PC12 cells

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.     

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.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Simulations of zwitterionic and anionic phospholipid monolayers

Results of atomistic molecular dynamics simulations of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol monolayers at the air/water interface are presented. Dipalmitoylphosphatidylcholine is zwitterionic and dipalmitoylphosphatidylglycerol is anionic at physiological pH. NaCl and CaCl2 water subphases are simulated. The simulations are carried out at different surface densities, and a simulation cell geometry is chosen that greatly facilitates the investigation of phospholipid monolayer properties. Ensemble average monolayer properties calculated from simulation are in agreement with experimental measurements. The dependence of the properties of the monolayers on the surface density, the type of the headgroup, and the ionic environment are explained in terms of atomistically detailed pair distribution functions and electron density profiles, demonstrating the strength of simulations in investigating complex, multicomponent systems of biological importance.     

Mixed monolayers of linear gramicidins and phospholipid. Surface pressure and surface potential studies.

The behavior of two gramicidins incorporated into lipid monolayers is analyzed on the basis of the force and surface potential area curves. It is shown that the position of the gramicidins (helical axis parallel or perpendicular to the interface) depends on the monolayer pressure and that these molecules are not miscible with dioleoylphosphatidylcholine. Surface potential measurements suggest the existence of a relationship between the single channel characteristics and the surface potential and indicate that the tryptophans are essential for lowering the lipid surface potential in agreement with the single channel behaviour of both gramicidin A and gramicidin M.     

HAMLET forms annular oligomers when deposited with phospholipid monolayers

Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.