Influence of stearic acid monolayers upon the procaine adsorption from underlying alkaline aqueous solutions

Adsorption of procaine at the air/water interface and its penetration into stearic acid monolayers from aqueous subphase of pH 8 are studied by measuring surface tension of aqueous procaine solutions and by recording surface pressure vs. mean molecular area curves for stearic acid monolayers spread onto procaine solutions of different concentrations. The amount of procaine in the interface is derived by means of Gibbs' equation. Results are compared to those obtained earlier at pH 2 and on unbuffered subphases. With increasing pH an increasing procaine adsorption and procaine penetration is observed. This phenomenon is interpreted in terms of protolytic equilibria in which participate both surfactants procaine and stearic acid.     

Lung surfactant protein SP-B promotes formation of bilayer reservoirs from monolayer and lipid transfer between the interface and subphase

We investigated the possible role of SP-B proteins in the function of lung surfactant. To this end, lipid monolayers at the air/water interface, bilayers in water, and transformations between them in the presence of SP-B were simulated. The proteins attached bilayers to monolayers, providing close proximity of the reservoirs with the interface. In the attached aggregates, SP-B mediated establishment of the lipid-lined connection similar to the hemifusion stalk. Via this connection, a lipid flow was initiated between the monolayer at the interface and the bilayer in water in a surface-tension-dependent manner. On interface expansion, the flow of lipids to the monolayer restored the surface tension to the equilibrium spreading value. SP-B induced formation of bilayer folds from the monolayer at positive surface tensions below the equilibrium. In the absence of proteins, lipid monolayers were stable at these conditions. Fold nucleation was initiated by SP-B from the liquid-expanded monolayer phase by local bending, and the proteins lined the curved perimeter of the growing fold. No effect on the liquid-condensed phase was observed. Covalently linked dimers resulted in faster kinetics for monolayer folding. The simulation results are in line with existing hypotheses on SP-B activity in lung surfactant and explain its molecular mechanism.     

Enzyme-catalyzed oxidation of cholesterol in pure monolayers at the air/water interface.

Mean molecular area vs. lateral surface pressure isotherms were determined for monolayers containing cholesterol, 4-cholesten-3-one (cholestenone), or binary mixtures of the two. At all lateral surface pressures examined, cholestenone had a larger mean molecular area requirement than cholesterol. Results with the binary mixtures of cholesterol and cholestenone suggested that the sterols did not mix ideally (non additive mean molecular area) with each other in the monolayer; the observed mean molecular area for mixtures was less than would be expected based on ideal mixing. The mixed sterol monolayers also displayed a reduction in the lateral collapse pressure which appeared to be a linear function of the mole fraction of cholestenone in the monolayer, suggesting that cholesterol and cholestenone were completely miscible in the mixed monolayer. The pure cholesterol monolayer was next used to examine the cholesterol oxidase-catalyzed (Brevibacterium sp.) oxidation of cholesterol to cholestenone at different lateral surface pressures at 22 degrees C. The difference in mean molecular area requirements of cholesterol and cholestenone was directly used to convert monolayer area changes (at constant lateral surface pressure) into average reaction rates. It was observed that the average catalytic activity of cholesterol oxidase increased linearly with increased lateral surface pressure in the range of 1 to 20 mN/m. In addition, the enzyme was capable to oxidize cholesterol in monolayers with a lateral surface pressure close to the collapse pressure of cholesterol monolayers (collapse pressure 45 mN/m; oxidation was observed at 40 mN/m). The adsorption of cholesterol oxidase to an inert sterol monolayer film at low surface pressures (around 9 mN/m) was marginal, although clearly detectable at very low (0.5-4 mN/m) lateral surface pressures, suggesting that the enzyme did not penetrate deeply into the monolayer in order to reach the 3 beta-hydroxy group of cholesterol. This interpretation is further supported by the finding that a maximally compressed cholesterol monolayer (40 mN/m) was readily susceptible to enzyme-catalyzed oxidation. It is concluded that cholesterol oxidase is capable of oxidizing cholesterol in laterally expanded monolayers as well as in tightly packed monolayers, where the lateral surface pressure is close to the collapse pressure. The kinetic results suggested that the rate-limiting step in the overall process was the substrate availability per surface area (or surface concentration) at the water/lipid interface.     

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions

Owing to its distinct chemico-biological properties, chitosan, a cationic biopolymer, offers a great potential in multifarious bioapplications. One such application is as a dietary antilipidemic supplement to be used to reduce obesity/overweight and to lower cholesterol. The lipid-binding efficiency of chitosan, however, remains debatable. Accordingly, in this study we investigated the interactions of chitosan with selected lipids, cholesterol and fatty acids, the latter including saturated (stearic acid) and unsaturated (oleic, linoleic, alpha-linolenic) acids. The experiments were performed with the Langmuir monolayer technique, in which surface pressure-area isotherms were recorded for the lipid monolayers spread on the acetate buffer pH 4.0 subphase in the absence and presence of chitosan. We found that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and alpha-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results we tentatively propose a mechanism of the chitosan action that includes both electrostatic and hydrophobic lipid-chitosan interactions as well as hydrogen bonding between them.     

Hydrophobic membrane protein from chromatophores of Rhodospirillum rubrum. Structural and spectroscopic studies of monolayers and multilayers.

A hydrophobic, lipid- and pigment-free polypeptide from the chromatophore membrane of Rhodospirillum rubrum was spread from chloroform/methanol, pyridine and formic acid solutions at an air-water interface. Surface pressure versus area isotherms of the monolayers formed at the interface were partially dependent upon the spreading solvent used. From the surface area at 20 dynes/cm compression, an average molecular area of 12.9 nm2/molecule was calculated for a polypeptide monolayer spread from chloroform/methanol. Multilayers built up on germanium plates at different surface pressures were subjected to attenuated total reflection infrared spectroscopy. In all cases the amide I and II absorption bands were typical of alpha-helical and random conformations. Electron microscopy of transferred monolayers replicated by rotary platinum shadowing revealed domains of regular texture in specimens prepared at 20 dynes/cm. Such domains were virtually absent in specimens prepared at 10 and 30 dynes/cm. Light optical diffractometry of the ordered arrays yielded a smallest repetitive area of 13.5 nm2 which agrees well with the molecular area obtained from the monolayer surface. Although no drastic changes in secondary structure were detected in the course of this study, some conformational changes are indicated by solvent-dependent differences in the surface pressure versus area isotherms.     

Modulation of tetracycline-phospholipid interactions by tuning of pH at the water-air interface

This paper is part of a systematic study of the interactions of tetracycline antibiotics with phospholipid monolayers at the water-air interface. Tetracyclines are widespread antibiotics that undergo a series of protonation equilibria in solution, depending on the pH. The surface activity of tetracyclines was determined by means of surface tension measurements for three different systems, i.e. water, TRIS and McIlvaine-EDTA buffer. Surface pressure-molecular area and surface potential-molecular area isotherms were acquired for dipalmitoylphosphatidic acid monolayers on TRIS buffer (pH=7.0) and McIlvaine-EDTA buffer (pH=4.0) solution as a function of tetracycline concentration in the subphase. Comparative analysis of surface potential data, with the molecular dipole moment of tetracycline obtained from semiempirical calculations, provided information on the orientation of tetracycline at the interface. Surface pressure measurements as a function of monolayer compression were described, applying either a continuous partition model or Langmuir adsorption isotherms. The results obtained in the case of buffer solutions were compared to those obtained for tetracycline in water subphases. The analysis of the results indicated that electrostatic interactions dictate the migration of tetracycline to the monolayer interface. Penetration of the molecule to the lipophilic portion of the monolayer was unlikely, especially at high surface pressures. The results showed that stronger interactions are established between the zwitterionic tetracycline and the deprotonated phosphatidic group in TRIS buffer solution; in this case, tetracycline binds at the monolayer interface following a Langmuir type adsorption. In the case of water, where the monodeprotonated acid and the tetracycline zwitterions are the only species involved, the data can be described by continuous partition of tetracycline between interfacial and bulk phases. The same holds for McIlvaine-EDTA buffer subphases, although the high concentrations of citrate ions in this buffer competitively interfere with tetracycline association at the monolayer interface.     

Interfacial properties of the M1 segment of the nicotinic acetylcholine receptor

We have studied the thermodynamic, surface, and structural properties of alphaM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of -7.2 kcal/mol. A cross-sectional molecular area of 210 A(2)/molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl(4), respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoyl-phosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyl-oleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.     

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.     

Lateral lipid distribution is a major regulator of lipase activity. Implications for lipid-mediated signal transduction.

Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lateral proton conduction in mixed monolayers of phosphatidylethanolamine and cetyltrimethylammonium bromide.

Proton conduction is known to be facilitated along phospholipid monolayers spread on aqueous phases. This property was monitored with mixed cetyltrimethylammonium bromide/phosphatidyl-ethanolamine monolayers. The film was shown to be metastable by surface pressure and fluorescence measurements. The detergent was leaving the interface for the bulk phase. Nevertheless, a fraction of the detergent remained in the lipid matrix, as shown by the binding of the fluorescent probe 8-anilino-1-naphthalensulfonate. Its dissociation constant decreased, and the nature of its binding site was affected, as shown by a shift of its emission spectrum. Apart from film expansion, the properties of the film were affected only at the water/membrane interface. Proton conduction was prevented only when the surface concentration of the detergent was larger than a critical value. Such an effect could be due either to the disruption in the continuity of the conducting hydrogen-bond network or to an electrostatic repulsion of the protons by the interface.     

Influence of temperature on phytohormone interactions with monolayers obtained from phospholipids of wheat calli

The effect of temperatures (15 and 5 degrees C) on adsorption parameters of phytohormones at monolayers prepared from a mixture of phospholipids extracted from non-embryogenic (NE) and embryogenic (E) winter wheat calli initiated from inflorescences (inf) and embryos (emb) was studied. The surface parameter values, i.e. limiting area and collapse pressure, were determined using the Langmuir method. Phytohormones 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), kinetin, zeatin and zearalenone were investigated. The phytohormones, at a concentration of 0.2 microg/ml dissolved in water, were injected into the subphase. Phospholipids, at the concentration of 2 mg/ml, were spread at the water surface and the monolayer was compressed. The anomalous temperature effect was observed, especially, in non-embryogenic systems. In monolayers obtained from E phospholipids, the temperature effect was dependent on the kind of tissue from which the callus was initiated. Among all the examined phytohormones, the greatest changes (monolayer expansion) were found for IAA and zearalenone. However, this activity depended strongly on the kind of tissue from which the phospholipid mixture was extracted.     

Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.     

Evidence for the presence of a specific ganglioside GM1/valinomycin complex in mixed monolayers

The effect of a negatively charged mono-sialoglyco-sphingolipid (GM1-ganglioside) on the molecular organization and on physiochemical properties of lipid/peptide (valinomycin) systems was investigated in monolayers at the air/water interface. At a high molar fraction of GM1, the surface pressure/area isotherms of the two-component films of the system GM1/valinomycin and the isotherm of the pure ganglioside monolayer are identical concerning the space requirement of the molecules and thereby the packing of the monolayer. Using space-filling molecular models, a simple calculation gives the theoretical amount of 4.5 ganglioside molecules associated with one molecule of the depsipeptide valinomycin. The average surface potential indicates, that valinomycin, interacting with the polar head group of GM1, becomes partly embedded within the lipid interface. For GM1/eicosanol and valinomycin/eicosanol mixtures, the agreement between theory and experimental data strongly supports the model of ideal mixing without any molecular interactions between the different components. The results suggest the formation of a ganglioside/valinomycin complex with simultaneous alteration of the surface potential and molecular structure of the single components.     

Modification of ion transport in lipid bilayer membranes in the presence of 2,4-dichlorophenoxyacetic acid. II. Suppression of tetraphenylborate conductance and changes of interfacial potentials.

It has been shown that the blocking of negatively charged tetraphenylborate ion transport in phosphatidylcholine (PC)-cholesterol membranes by the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is dominated by suppression of TPhB- diffusion across the membrane interior, rather than by the decrease of adsorption of TPhB- ions at the membrane surface. The blocking effect can be associated with the decrease of electric potential inside the membrane with respect to that of the aqueous medium, this decreases being proportional to the concentration of 2,4-D in the aqueous solution. It has been estimated that 25 - 30% of the total 2,4-D-induced change of the potential difference is between the plane of absorption of TPhB- and the aqueous solution, and the remaining fraction is between the membrane interior and the absorption plane. The results of this study support the dipolar hypothesis of 2,4-D action in lipid membranes. These conclusions are further supported by measurements changes of electric potential difference across air/water and air/lipid monolayer/water interfaces. It has been found that the electric potential of the nonpolar side of the interface decreases in the presence of neutral molecules of 2,4-D and that this effect becomes more prominent in presence of electrolyte. We have confirmed that PC-cholesterol monolayer cannot be considered as a model for half of the bilayer membrane because of the disagreement between the changes of the interfacial potential difference of PC-cholesterol monolayers and those determined from studied of transport of positive and negative ions across bilayer membranes. In contract, we have found close agreement between the 2,4-D-induced changes of electric potential of the lipid hydrocarbon region in glycerolmonooleate (GMO) membranes and GMO monolayers. We suggest that the action of 2,4-D in lipid membranes is not associated with the changes of orientation of dipoles of lipids constituting the membranes, but rather with a layer of 2,4-D molecules absorbed at the nonpolar/polar membrane boundary.     

The liquid condensed diffusional transition of dipalmitoylphosphoglycerocholine in monolayers

The fine details of the phase transition of dipalmitoylphosphoglycerocholine (DPPC) monolayers at air/NaCl solution interfaces were investigated at 21 +/- 1 degrees C by using the fluorescence after photobleaching technique employing 12-(9-anthroyloxy)stearic acid as fluorescent probe. The mode of compression of the monolayer (i.e., continuous compression or successive additions of the lipid at fixed area) together with the ionic strength of the subphase (0.1 or 1.0 M NaCl) were particularly studied. The photobleaching results show that the lateral diffusion coefficient of the probe molecules decreases drastically within the liquid-condensed phase, i.e., from the end of the liquid-expanded-liquid-condensed phase transition to the beginning of the solid-condensed phase. The molecular areas at which the phase transition occurs under the various experimental conditions, together with a parallel analysis of the hydration states and related molecular areas of the DPPC molecules in multilayers, strongly suggest that the steric hindrance associated with the hydration water of the polar head of DPPC molecules in the monolayer is responsible for the drastic decrease in diffusion coefficient in the liquid-condensed phase. Furthermore, the fluorescence characteristics of the probe molecules also show that, together with the aforementioned reorganization of the polar head, a structural reorganization of the aliphatic chains of the lipid molecules also takes place in the liquid-condensed phase. The liquid-condensed phase therefore appears as a transition region from liquid to solid phases in which the lipid molecules present a significant decrease in their lateral diffusion related to a structural reorganization of both their polar and aliphatic components.     

Monolayers of long chain lecithins at the air/water interface and their hydrolysis by phospholipase A2

The behavior of phosphatidylcholine monolayers at the air/water interface was studied by measuring their surface isotherm, surface potential, surface viscosity, and rate of hydrolysis by the dimeric phospholipase A2 from the venom of Crotalus atrox. The monolayers showed typical liquid-expanded behavior. In this phase, the surface potential was linearly dependent on surface concentration and extrapolated at zero concentration to a value characteristic of a liquid hydrocarbon/water interface. The rate of the reaction was measured by monitoring changes in area at constant surface pressure for 1,2-dioctanoyl- and 1,2-didecanoyl-3-sn-phosphatidylcholines, and by monitoring changes in surface potential for 1,2-dimyristoyl-, 1,2-dipalmitoyl-, 1-palmitoyl-2-oleoyl-, and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholines. The enzymatic hydrolysis is first order with respect to the enzyme-calcium complex which forms with a Kd = 1.5 mM. A mechanism is proposed to account for the dependency of the reaction rates on the surface concentration of the substrate. We postulate that the rate-limiting step is the decomposition of a quaternary complex formed from two phospholipid molecules, one calcium ion and one dimeric enzyme. The rate is independent of the surface pressure per se; addition of inert lipids to a monolayer at constant area, and hence constant surface concentration of the substrate, increases the surface pressure without changing the surface density of the substrate yielding maximal enzymatic rate. The enzyme is specific for loosely packed substrate molecules in the liquid-expanded state: transition into the liquid-condensed state or compression of the liquid-expanded layer beyond 80 A2/phospholipid strongly inhibits the enzymatic reaction. Our results show that surface recognition is a direct consequence of a bifunctional active site since it is only at a phospholipid surface that the distance between two substrate molecules is optimal for forming a catalytically competent enzyme-Ca2+-(substrate)2 complex.     

Conformation of poly(l-glutamic acid) at the air/water interface

The conformation of the monolayer of poly(l-glutamic acid) on subsolutions of different pH values was studied by the film-balance technique, obtaining surface pressure measurements, together with polarized infrared spectroscopy and Raman spectroscopy. The monolayers of poly(l-glutamic acid) gave different surface pressure-area curves on subsolutions of various pH values. It was found that the conformation of poly(l-glutamic acid) monolayer spread at the air/water interface differs from that in solution. It can be presumed that poly(l-glutamic acid) in a monolayer is in the form of an α-helix at pH 2.0, in the β-form at pH 3.5 and in an ‘intramolecular’ heterogeneous conformation (consisting of a random coil and an α-helix) at pH 4.0.     

Influence of levofloxacin and clarithromycin on the structure of DPPC monolayers

Research on lipid/drug interactions at the nanoscale underpins the emergence of synergistic mechanisms for topical drug administration. The structural understanding of bio-mimetic systems employing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a lung surfactant model mixed with antibiotics, as well as their biophysical properties, is of critical importance to modulate the effectiveness of therapeutic agents released directly to the airways. In this paper, we investigate the structural details of the interaction between Levofloxacin, ‘a respiratory quinolone’, and the macrolide Clarithromycin, with DPPC monolayers at the air-water interface, using a combination of Brewster angle microscopy, polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS), surface pressure isotherms and neutron reflectometry (NR) to describe the structural details of this interaction. The results allowed association of changes in the π-A isotherm profile with changes in the molecular organization and the co-localization of the antibiotics within the lipid monolayer by NR measurements. Overall, both antibiotics are able to increase the thickness of the acyl tails in DPPC monolayers with a corresponding reduction in tail tilt as well as to interact with the phospholipid headgroups as shown by PM-IRRAS experiments. The effects on the DPPC monolayers are correlated with the physical-chemical properties of each antibiotic and dependent on its concentration.     

Association of protein kinase C with phospholipid monolayers: two-stage irreversible binding

The association of protein kinase C (PKC) with phospholipid (PL) monolayers spread at the air-water interface was examined. PKC-PL binding induced surface pressure changes that were dependent on the amount of PKC, the phospholipid composition of the monolayers, the presence of Ca2+, and the initial surface pressure of the monolayer (pi 0). Examination of surface pressure increases induced by PKC as a function of phospholipid surface pressure, pi 0, revealed that PKC-phosphatidylserine (PS) association had a critical pressure of 43 dyn/cm. Above this surface pressure, PKC cannot cause further surface pressure changes. This high critical pressure indicated that PKC should be able to penetrate many biological membranes which appear to have surface pressures of about 30 dyn/cm. PKC-induced surface pressure changes were Ca2+ dependent only for PL monolayers spread at a pi 0 greater than 26 dyn/cm. PKC alone (in the absence of PL) formed a film at the air-water interface with a surface pressure of about 26 dyn/cm. Calcium-dependent binding was studied at the higher surface pressures which effectively excluded PKC from the air-water interface. Subphase depletion measurements suggested that association of PKC with PS monolayers consisted of two stages: a rapid Ca2+-dependent interaction followed by a slower process that resulted in irreversible binding of PKC to the monolayer. The second stage appeared to involve penetration of PKC into the hydrocarbon region of the phospholipid. The commonly used in vitro substrates for PKC, histone and protamine sulfate, also associated with and penetrated PS monolayers with critical pressures of 50 and 60 dyn/cm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)