Binding of a neuropeptide, substance P, to neutral and negatively charged lipids

The binding of substance P (SP), a positively charged neurotransmitter peptide, to neutral and to negatively charged phospholipids has been investigated by means of a monolayer technique. Monolayers formed at room temperature from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or mixtures of the two, were maintained throughout the course of a binding experiment at a constant surface pressure while the monolayer surface area was monitored. Injection of SP into the aqueous subphase (154 mM NaCl, 10 mM Tris adjusted to pH 7.4) led to an expansion of the monolayer surface area that was attributed to a spontaneous insertion of SP between the lipid molecules. A quantitative evaluation of the area increase at constant pressure yielded SP insertion isotherms that showed that levels of SP insertion increased directly with the monolayer POPG content and decreased to negligible levels at surface pressures above 35 +/- 1 mN/m. If electrostatic effects were ignored, these data showed biphasic behavior in Scatchard plots. The apparent binding constants ranged, at 20 mN/m, from (3.2 +/- 0.3) X 10(4) M-1 for 100% POPG monolayers to (2.0 +/- 0.05) X 10(3) M-1 for 25% POPG/75% POPC monolayers. At 32 mN/m, a monolayer surface pressure that mimics bilayer conditions, the apparent binding constant for a 100% POPG monolayer was measured to be (1.1 +/- 0.05) X 10(3) M-1. However, for a monolayer containing only 25% charged lipids, corresponding to a natural membrane composition, K app at 32 mN/m was estimated to be at most 41 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrin maintains the lateral order in phosphatidylserine monolayers

We investigate the effect of the skeletal protein spectrin on the lateral order in dipalmitoyl phosphatidylserine monolayers spread on aqueous surfaces using grazing incidence X-ray diffraction. Without spectrin, the condensed lipid monolayer exhibits two-dimensional hexagonal packing, characterized by monotonic decrease in the d-spacing and increase in the degree of order with increasing surface pressure between 17 and 36 mN/m. Addition of spectrin to the aqueous subphase at high pressures preserves the monolayers structural parameters unchanged from 36 to 25 mN/m. These results demonstrate for the first time that spectrin could participate in sustaining the two-dimensional order in lipid domains through a direct interaction with phosphatidylserine species.     

Interaction of plasma apolipoproteins with lipid monolayers.

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...     

Effect of calcium and temperature on mixed lipid-valinomycin monolayers. A comparison of glycosphingolipids (ganglioside GT1b, sulphatides) and phosphatidylcholine

The influence of calcium and temperature on pure lipid (bovine brain PC, sulphatides, ganglioside GT1b), valinomycin and mixed lipid-valinomycin monolayers at the air/water interface was studied. In mixed films, evidence was found that the two components were miscible. On the other hand, at higher surface pressures, phase separation occurs in the cases of PC and sulphatides. Measuring the area requirement and the collapse pressure the stability of both lipid and the peptide was increased in particular due to ganglioside-valinomycin interaction. The addition of 10(-5) M calcium into the subphase at 20 and 37 degrees C and surface pressures of 10 and 20 mN/m led to a condensing effect in ganglioside mixtures, with formation of aggregates as indicated also by the nearly ideal behaviour of two component monolayers.     

Differential effects of surfactant protein A on regional organization of phospholipid monolayers containing surfactant protein B or C

Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.     

Anaesthetic — phospholipid interaction

Binding of the positively charged drug chlorpromazine to phospholipid monolayers was investigated. A preferential uptake was observed near the phase transtion of the corresponding lipid. Cholesterol considerably diminishes the chlorpromazine uptake, again particularly near a lipid phase transition. The binding properties depend on the chlorpromazine concentration in the subphase. A critical concentration is 5·10^-5M, where higher uptake occurs in the liquid condensed than in the liquid expanded state of the monolayer at pressures of about 10 mN/m. Dipalmitoylphosphatidylcholine monolayers spread on a subphase containing chlorpromazine are comparable to monolayers at higher temperature but in the absence of chlorpromazine. These data are in agreement with previous fluorescence and electron paramagnetic resonance experiments on lipid bilayer membranes (Luxnat and Galla 1986).Abbreviations CPZ chlorpromazine - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - LE liquid expanded - LC liquid condensed     

Membrane binding of a lipidated N-Ras protein studied in lipid monolayers

The adsorption of doubly lipidated full-length N-Ras protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Adsorption of apolipoprotein A-IV to phospholipid monolayers spread at the air/water interface. A model for its labile binding to high density lipoproteins.

The mechanisms that mediate the labile binding of apolipoprotein A-IV (apoA-IV) to high density lipoproteins (HDL) are not known. We therefore used a surface balance and surface radioactivity detector to investigate the adsorption of apoA-IV to egg phosphatidylcholine monolayers spread at the air/water interface. ApoA-IV bound rapidly and reversibly to phospholipid monolayers and generated a maximum increase in surface pressure of 19 millinewtons (mN)/m at a subphase concentration of 2 x 10(-5) g/dl. Binding decreased linearly with increasing initial surface pressure; at pressures greater than 28-29 mN/m, apoA-IV could no longer penetrate the lipid monolayer. The area occupied by the amino acid residues in apoA-IV reached an unusually low limiting molecular area of 10-12 A2/residue at surface saturation. The surface pressure of native HDL3 was calculated to be 33 mN/m, and it rapidly decreased with the action of lecithin:cholesterol acyltransferase on the particle surface. We conclude that the surface activity of apoA-IV is lower than that of any other human apolipoprotein; its binding and surface conformation are particularly sensitive to pressure; and at saturation, a significant portion of the molecule is excluded from the interface. The exclusion pressure of apoA-IV may be only slightly lower than the surface pressure of HDL; in vivo, the action of lecithin:cholesterol acyltransferase and lipid transfer proteins may cause the HDL3 surface pressure to oscillate about a narrow range that spans the exclusion pressure of apoA-IV. The resultant labile association of apoA-IV and HDL may be of central importance to its role in lipoprotein metabolism.     

Interaction of plasma apolipoproteins with lipid monolayers

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidyl[¹⁴C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipid by apolipoprotein C-III.The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface.     

Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air-fluid interface of the lung. The Wilhelmy plate method was used to study the involvement of SP-B and SP-C in the formation of phospholipid monolayers. The proteins were either present in the phospholipid vesicles which were injected into the subphase or included in a preformed phospholipid monolayer. In agreement with earlier investigators, we found that SP-B and SP-C, present in phospholipid vesicles, were able to induce the formation of a monolayer, as became apparent by an increase in surface pressure. However, when the proteins were present in a preformed phospholipid monolayer (20 mN/m) at similar lipid to protein ratios, the rate of surface pressure increase after injection of pure phospholipid vesicles into the subphase at similar vesicle concentrations was 10 times higher. The process of phospholipid insertion from phospholipid vesicles into the protein-containing monolayers was dependent on (1) the presence of (divalent) cations, (2) the phospholipid concentration in the subphase, (3) the size of the phospholipid vesicles, (4) the protein concentration in the preformed monolayer, and (5) the initial surface pressure at which the monolayers were formed. Both in vesicles and in preformed monolayers, SP-C was less active than SP-B in promoting the formation of a phospholipid monolayer. The use of preformed monolayers containing controlled protein concentrations may allow more detailed studies on the mechanism by which the proteins enhance phospholipid monolayer formation from vesicles.     

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.     

Palmitoylation of a pulmonary surfactant protein C analogue affects the surface associated lipid reservoir and film stability

Surfactant protein C (SP-C) is a lipopeptide that contains two thioester-linked palmitoyl groups and is considered to be important for formation of the alveolar surface active lipid film. Here, a non- or dipalmitoylated SP-C analogue (SP-C(Leu)), in which all helical Val residues were replaced with Leu and Cys-5 and Cys-6 were replaced with Ser, was tested for surface activity in a captive bubble system (CBS). SP-C(Leu), either palmitoylated at Ser-5 and Ser-6 or non-palmitoylated, was added to mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidyl glycerol (PG)/palmitic acid (PA), 68:22:9, (by mass) at a concentration of 2 and 5%. With 2% peptide, surface film formation was rapid, reaching a surface tension below 25 mN/m within 5 s, but the samples with 5% SP-C(Leu) required more than 20 s to reach values below 25 mN/m. Minimum surface tension for the samples with dipalmitoylated SP-C(Leu) was below 1.5 mN/m and very stable, as the surface tension increased by less than 0.5 mN/m within 10 min at constant bubble volume. Minimum surface tension for the non-palmitoylated SP-C(Leu) was approximately 2 and 5 mN/m for 2 and 5% peptide, respectively, but the films were less stable as seen by frequent bubble clicking at low surface tensions. Films with dipalmitoylated SP-C(Leu) that were dynamically cycled at 20-30 cycles/min were substantially less compressible at a surface tension of 20 mN/m (0.007 m/mN) than those that contained the non-palmitoylated peptide (0.02 m/mN). After subphase depletion, the incorporation of lipids into the surface active film during initial bubble expansion occurred at a relatively low surface tension (about 35 mN/m) for the samples with dipalmitoylated SP-C(Leu) compared to approximately 45 mN/m for those containing the non-palmitoylated peptide. Furthermore, for samples that contained non-palmitoylated SP-C(Leu), the ability to reach near zero stable surface tension was lost after a few adsorption steps, whereas with the dipalmitoylated peptide the film quality did not deteriorate even after more than 10 expansion steps and the incorporation of reservoir material equivalent to more than two monolayers. It appears that the covalently linked palmitoyl groups of the SP-C analogue studied are important for the mechanical stability of the lipid film, for the capacity to incorporate material from the reservoir into the surface active film upon area expansion, and for the low film compressibility of dynamically cycled films.     

Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.     

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.     

Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films

Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 wt%, and 10 wt% of both) under conditions of cyclic compression and expansion. Matrix-assisted laser desorption/ionization (MALDI) spectroscopy of R-SP-B and F-SP-C indicated that the proteins were intact and labeled with the appropriate fluorescent probe. The monolayers were compressed and expanded for four cycles at an initial rate of 0.64 A2 x mol(-1) x s(-1) (333 mm2 x s x [-1]) up to a surface pressure pi approximately 65 mN/m, and pi-area per residue (pi-A) isotherms at 22 +/- 1 degrees C were obtained. The monolayers were microscopically observed for the fluorescence emission of the individual proteins present in the film lipid matrix, and their visual features were video recorded for image analysis. The pi-A isotherms of the DPPC/protein monolayers showed characteristic "squeeze out" effects at pi approximately 43 mN/m for R-SP-B and 55 mN/m for F-SP-C, as had previously been observed for monolayers of the native proteins in DPPC. Both proteins associated with the expanded (fluid) phase of DPPC monolayers remained in or associated with the monolayers at high pi (approximately 65 mN/m) and redispersed in the monolayer upon its reexpansion. At comparable pi and area/molecule of the lipid, the proteins reduced the amounts of condensed (gel-like) phase of DPPC monolayers, with F-SP-C having a greater effect on a weight basis than did R-SP-B. In any one of the lipid/protein monolayers the amounts of the DPPC in condensed phase were the same at equivalent pi during compression and expansion and from cycle to cycle. This indicated that only minor loss of components from these systems occurred between compression-expansion cycles. This study indicates that hydrophobic PS proteins associate with the fluid phase of DPPC in films, some proteins remain at high surface pressures in the films, and such lipid-protein films can still attain high pi during compression.     

Preparation and characterization of monolayer and multilayer Langmuir-Blodgett films of a series of 6-O-alkylcelluloses

Monolayer and multilayer Langmuir-Blodgett (LB) films of 6-O-alkylcelluloses with various chain lengths were prepared and studied. The surface pressure (pi)-area (A) isotherms of 6-O-alkylcelluloses exhibited characteristic behaviors depending on the length of the alkyl chain and temperature. 6-O-Stearylcellulose on the subphase formed a homogeneous monolayer at 10 mN m(-1). By transfer ratio, FT-IR, and contact angle measurements, it was proved that the monolayer of 6-O-stearylcellulose on the water surface was transferred successfully onto a substrate by a vertical dipping method to form a Z-type LB film. The transmission and reflection absorption IR spectrum indicated that the hydrocarbon chains had all-trans rotamers and were oriented nearly perpendicular to the surface in the film. AFM section analysis revealed that the thickness per layer was calculated to be 2.35 nm. These results suggested that the hydrocarbon chains were inclined at an angle of about 25.3 degrees to have high packing density in the alkyl region.     

Hisactophilin-mediated binding of actin to lipid lamellae: a neutron reflectivity study of protein membrane coupling.

The neutron reflectivity technique is applied to determine the adsorptive interaction of the 13.5-kDa actin-binding protein hisactophilin from Dictyostelium discoideum with lipid monolayers at a lateral pressure of 21 mN/m < or = pi < or = 25 mN/m at the air-water interface. We compare binding of natural hisactophilin exhibiting a myristic acid chain membrane anchor at the N-terminus (DIC-HIS) and a fatty acid-deficient genetic product expressed in Escherichia coli (EC-HIS). It is demonstrated that only the natural hisactophilin DIC-HIS is capable of mediating the strong binding of monomeric actin to the monolayer, where it forms a layer of about 40 A thickness corresponding to the average diameter of actin monomers. Monolayers composed of pure dimyristoyl phosphatidylcholine with fully deuterated hydrocarbon tails and headgroup (DMPC-d67) and 1:1 mixtures of this lipid with chain deuterated dimyristoyl phosphatidylglycerol (DMPG-d54) are studied on subphases consisting either of fully deuterated buffer (D2O) or of a 9:1 H2O/D2O buffer that matches the scattering length density of air (CMA buffer). The reflectivity data are analyzed in terms of layer models, consisting of one to three layers, depending on the contrast of the buffer and the system. We show that both protein species bind tightly to negatively charged 1:1 DMPC-d67/DMPG-d54 monolayers, thereby forming a thin and most probably monomolecular protein layer of 12-15 A thickness. We find that the natural protein (DIC-HIS) partially penetrates into the lipid monolayer, in contrast to chain-deficient species (EC-HIS), which forms only an adsorbed layer. The coverage of the monolayer with DIC-HIS strongly depends on the presence of anionic DMPG in the monolayer. At a bulk protein concentration of 1.5 micrograms/ml, the molar ratio of bound protein to lipid is about 1:45 for the 1:1 lipid mixture but only 1:420 for the pure DMPC.     

Characterization of mixed monolayers of phosphatidylcholine and a dicationic gemini surfactant SS-1 with a langmuir balance: effects of DNA

Monolayers of a cationic gemini surfactant, 2,3-dimethoxy-1,4-bis(N-hexadecyl-N;N-dimethyl-ammonium)butane dibromide (abbreviated as SS-1) and its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (pi-A) curves and determined the elastic area compressibility modulus (C) as a function of lateral packing pressure and the mole fraction of the cationic lipid (X(SS-1)), with and without DNA in the subphase. Both SS-1 and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (X(SS-1) < 0.05) of SS-1 in a POPC monolayer condensed the film dramatically, up to 20% at 30 mN/m. This effect is suggested to reflect reorientation of the P(-)-N(+) dipole of the POPC headgroup. Accordingly, the magnitude of the condensing effect diminishes with X(SS-1) and is not observed for mixed films of dioleoylglycerol and SS-1. Reorientation of the P(-)-N(+) dipole is further supported by the pronounced increase in monolayer dipole potential psi due to SS-1. The presence of DNA in the subphase affected the mixed POPC/SS-1 monolayers differently depending on the constituent lipid stoichiometry as well as on the DNA/SS-1 charge ratio. At a DNA concentration of 0.63 microM (in base pairs) condensation of neat POPC monolayers was evident, and this effect remained up to X(SS-1) < 0.5, corresponding to DNA/SS-1 charge ratio of 1.25. An expansion due to DNA, evident as an increase in DeltaA/molecule, was observed at X(SS-1) > 0.5. At a higher concentration of DNA (1.88 microM base pairs) in the subphase corresponding to DNA/SS-1 charge ratio of 3.75 at X(SS-1) = 0.5, condensation was observed at all values of X(SS-1).     

Interaction of cholesterol with sphingomyelins and acyl-chain-matched phosphatidylcholines: a comparative study of the effect of the chain length.

In this study we have synthesized sphingomyelins (SM) and phosphatidylcholines (PC) with amide-linked or sn-2 linked acyl chains with lengths from 14 to 24 carbons. The purpose was to examine how the chain length and degree of unsaturation affected the interaction of cholesterol with these phospholipids in model membrane systems. Monolayers of saturated SMs and PCs with acyl chain lengths above 14 carbons were condensed and displayed a high collapse pressure ( approximately 70 mN/m). Monolayers of N-14:0-SM and 1(16:0)-2(14:0)-PC had a much lower collapse pressure (58-60 mN/m) and monounsaturated SMs collapsed at approximately 50 mN/m. The relative interaction of cholesterol with these phospholipids was determined at 22 degreesC by measuring the rate of cholesterol desorption from mixed monolayers (50 mol % cholesterol; 20 mN/m) to beta-cyclodextrin in the subphase (1.7 mM). The rate of cholesterol desorption was lower from saturated SM monolayers than from chain-matched PC monolayers. In SM monolayers, the rate of cholesterol desorption was very slow for all N-linked chains, whereas for PC monolayers we could observe higher desorption rates from monolayers of longer PCs. These results show that cholesterol interacts favorably with SMs (low rate of desorption), whereas its interaction (or miscibility) with long chain PCs is weaker. Introduction of a single cis-unsaturation in the N-linked acyl chain of SMs led to faster rates of cholesterol desorption as compared with saturated SMs. The exception was monolayers of N-22:1-SM and N-24:1-SM from which cholesterol desorbed almost as slowly as from the corresponding saturated SM monolayers. The results of this study suggest that cholesterol is most likely capable of interacting with all physiologically relevant (including long-chain) SMs present in the plasma membrane of cells.     

Polymorphism and interactions of a viral fusion peptide in a compressed lipid monolayer.

With a view toward possible new insights into viral fusion mechanisms, we have investigated the HIV-1 gp41 fusion peptide in a monomolecular film of the biomembrane lipid palmitoyloleoylphosphatidylcholine. Its surface activity at an air/water interface was measured under equilibrium conditions, using the conventional Langmuir trough technique. Through a novel thermodynamic analysis, the partial molecular area of the peptide in the lipid moiety could be determined as a function of the lateral pressure and the interfacial peptide/lipid ratio. This indicates an orientation of the peptide backbone parallel to the lipid hydrocarbon tails. The molecular area decreases significantly upon monolayer compression, suggesting a conformational transition from a somewhat compact configuration to a more extended, presumably beta-strand structure when a lipid packing density is approached that is generally believed to mimic the physical state of a biological membrane. Up to a lateral pressure of approximately 15 mN/m, practically all peptide inserts into the lipid monolayer. At higher compression a distinct partitioning into the aqueous subphase is observed. Under these conditions the data also reflect a strong aggregation of the lipid-associated peptide. Beyond a critical peptide/lipid ratio, the peptide's area requirement was found to become substantially enhanced, possibly because of the formation of water-filled pores.