Phosphatidylglycerol Monolayers Research Articles

Hydrolysis of three different head groups phospholipids by chicken group V phospholipase A2 using the monomolecular film technique

The kinetic aspects of lipolysis by pulmonary phospholipase A2 (ChPLA2-V), chicken intestinal phospholipase A2 (ChPLA2-IIA) and chicken pancreatic phospholipase A2 (ChPLA2-IB), from chicken have been compared using the monomolecular films technique, on short-chain phospholipids (with three different head groups) and on long-chain phospholipids.The main conclusions from our experimental data indicate that the maximum catalytic activities of ChPLA2-V on 1,2 phosphatidylcholine and 1,2 phosphatidylethanolamine reached 15.26 and 36.12 moles/cm2.min.mM, respectively, at a pressure of 15 and 35 dynes/cm, respectively. Whereas, those of ChPLA2-IB were 3.58 (at the pressure of 20 dynes/cm) and 4.9 moles/cm2.min.mM. However, hydrolysis of phosphatidylglycerol monolayers (C12PG), were very much higher compared with all the substrates tested with 122 moles/cm2.min. Surprisingly, the hydrolysis rate of ChPLA2-V on long-chain phosphatidylglycerol (C18PG) was very low (1.45 moles/cm2.min) compared with all tested substrates, even with the use of p-cyclodextrin. And thus, the fatty acid preference of ChPLA2-V was 2-decanoyl > 2-oleoyl with a PG head group.In order to gain significant correlations between enzyme’s structures and their relative functions, we tried to examine the surface electrostatic potentials of the various secreted phospholipase 2 (sPLA2) from chicken. In the present study, we detailed that the substrate affinity, specificity and the hydrolysis rates of sPLA2 at each interface is governed by the surface electrostatic potentials and hydrophobic interactions operative at this surface.

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with 2H- and 31P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.

Oriented Reconstitution of a Membrane Protein in a Giant Unilamellar Vesicle: Experimental Verification with the Potassium Channel KcsA

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.

Morphology modifications in negatively charged lipid monolayers upon mitochondrial creatine kinase binding

Mitochondrial creatine kinase (mtCK) may participate to membrane organization at the mitochondrial level by modulating lipid state and fluidity. The effect of the protein on lipid phase behaviour of different acyl chain length phosphatidylglycerol monolayers was analyzed from pressure-area isotherms and from the compressional modulus variation with respect to the surface pressure. Monolayer morphology was visualized by Brewster angle microscopy. No condensation effect was visible on dimyristoylphosphatidylglycerol (DMPG). For the other PG monolayers tested, dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG), mtCK facilitated the formation of a liquid condensed phase. The effect depended on the surface pressure at which transition phase occurred. The effect of mtCK was more pronounced for tetramyristoylcardiolipin (TMCL) monolayers, as liquid condensed regions appeared 10 mN/m below the transition phase of the pure TMCL monolayer. The observed domains were circular and rather uniform, indicating a stabilization of the condensed phase. The same effect, namely an overall condensation of the monolayer with formation of circular domains, was observed upon protein injection beneath TMCL monolayers in different condensation states at constant area. MtCK ability to induce and stabilize a LC phase on monolayers could have important consequences in membrane organization and emphasize its structural role at mitochondrial level.

Interaction between the antibacterial compound, oleuropein, and model membranes

Oleuropein, a secoiridoid glycoside extracted from the olive tree, Olea europaea L., has been described as showing antibacterial properties. However, the exact mechanism of these antimicrobial properties is not yet well understood. In the present study, we have studied the interaction of oleuropein with phosphatidylglycerol (PG) as a model membrane for Staphylococcus aureus (S. aureus) (Gram-positive bacteria) and phosphatidylethanolamine and Escherichia coli (E. coli) lipid extract as a model membrane for E. coli (Gram-negative bacteria). The study has been carried out using monolayers as model membranes and using kinetics at constant area and compression isotherms with Brewster angle microscopy (BAM) observations. The results show that oleuropein interacts in higher extent with PG monolayers, which is related with its stronger antibacterial effect against Gram-positive bacteria. The effects on the membrane are probably produced at the cell surface because oleuropein did not form stable mixed monolayers with the lipids assayed at the air/water interface.

Relationships between Equilibrium Spreading Pressure and Phase Equilibria of Phospholipid Bilayers and Monolayers at the Air−Water Interface

The intricate interplay between the bilayer and monolayer properties of phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) phospholipids, in relation to their polar headgroup properties, and the effects of chain permutations on those polar headgroup properties have been demonstrated for the first time with a set of time-independent bilayer-monolayer equilibria studies. Bilayer and monolayer phase behavior for PE is quite different than that observed for PC and PG. This difference is attributed to the characteristic biophysical PE polar headgroup property of favorable intermolecular hydrogen-bonding and electrostatic interactions in both the bilayer and monolayer states. This characteristic hydrogen-bonding ability of the PE polar headgroup is reflected in the condensed nature of PE monolayers and a decrease in equilibrium monolayer collapse pressure at temperatures below the monolayer critical temperature, T(c) (whether above or below the monolayer triple point temperature, T(t)). This interesting phenomena is compared to equilibrated PC and PG monolayers which collapse to form bilayers at 45 mN/m at temperatures both above and below monolayer T(c). Additionally, it has been demonstrated by measurements of the equilibrium spreading pressure, pie, that at temperatures above the bilayer main gel-to-liquid-crystalline phase-transition temperature, T(m), all liquid-crystalline phospholipid bilayers spread to form monolayers with pie around 45 mN/m, and spread liquid-expanded equilibrated monolayers collapse at 45 mN/m to form their respective thermodynamically stable liquid-crystalline bilayers. At temperatures below bilayer T(m), PC and PG gel bilayers exhibit a drop in bilayer pi(e) values < or =0.2 mN/m forming gaseous monolayers, whereas the value of pic of spread monolayers remains around 45 mN/m. This suggests that spread equilibrated PC and PG monolayers collapse to a metastable liquid-crystalline bilayer structure at temperatures below bilayer T(m) (where the thermodynamically stable bilayer liquid-crystalline phase does not exist) and with a surface pressure of 45 mN/m, a surface chemical property characteristically observed at temperatures above bilayer T(m) (monolayer T(c)). In contrast, PE gel bilayers, which exist at temperatures below bilayer T(m) but above bilayer T(s) (bilayer crystal-to-gel phase-transition temperature), exhibit gel bilayer spreading to form equilibrated monolayers with intermediate pie values in the range of 30-40 mN/m; however, bilayer pie and monolayer pic values remain equal in value to one another. Contrastingly, at temperatures below bilayer T(s), PE crystalline bilayers exhibit bilayer pie values < or =0.2 mN/m forming equilibrated gaseous monolayers, whereas spread monolayers collapse at a value of pic remaining around 30 mN/m, indicative of metastable gel bilayer formation.

Effect of phloretin on the dipole potential of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol monolayers.

The effect of phloretin on the potential of phosphatidylcholine (PC), phosphatidylethanolamine (PE,) and phosphatidylglycerol (PG) monolayers below and above the phase transition in mixtures of different PC/PE ratios with and without cholesterol of ester and ether phospholipids have been determined. The effectiveness of phloretin to decrease the dipole potential of monolayers in the fluid state is lessened by the moieties esterified to the phosphate group in the sequence choline > ethanolamine > glycerol. These effects on the dipole potential of monolayers are independent of the presence of carbonyls. In addition, in the gel state phloretin does not affect the dipole potential on dimyristoylphosphatidylethanolamine, although it is very pronounced in dimyristoylphosphatidylcholine. The changes of the dipole potential induced by phloretin were correlated with the packing of the lipids and with the formation of intermolecular hydrogen bonds between adjacent phospholipid molecules. These results may be indicative of the different distribution of polarized water around the phosphate groups imposed by the surrounding environment.

Surface dilational behaviour of spread dipalmitoyl phosphatidyl glycerol monolayers

The stress relaxation process in spread dipalmitoyl phosphatidyl glycerol (DPPG) monolayers was investigated by using the pendant drop technique as a microfilm balance in combination with the axisymmetric drop shape analysis. The stress relaxation caused by a transient drop volume change was analysed by a new model, which takes into account material inhomogeneity by means of a distribution of relaxation γ. The surface pressure decay in the DPPG monolayers at 35 and 40 °C is governed by 2 main relaxation γ. One is of the order of 20 s, the other one is in the range of 200-600 s. The pi/A isotherms, the surface dilational modulus, and the higher relaxation γ are influenced by temperature and by the presence of electrolyte in the subphase; 0.15 mol dm-3 NaCl yields a smaller area demand per molecule in the monolayer. Harmonic oscillation experiments were carried out and the dilational elasticities and viscosities were determined for different frequencies in the range between the two main relaxation γ. Both, elasticities and viscosities, depend on frequency. The surface rheological behaviour is structural viscous, i.e. high viscosities at small frequencies, which decrease at faster deformations. The results are related to the surface dilational rheological behaviour of DPPC monolayers

Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme.

Binding of quinacrine to phospholipids and porcine pancreatic phospholipase A2 (PLA2) was investigated using fluorescence resonance energy transfer, Langmuir films, assay for the enzymatic activity, and molecular modeling. No significant binding of this drug to the zwitterionic phosphatidylcholine was observed whereas a high affinity for acidic phospholipids was revealed by quenching of pyrene-labeled phospholipid analogues. Partial reversal of this binding was observed due to the addition of 4 mM CaCl2. Quinacrine efficiently and independently of the lipid surface pressure penetrated into monolayers of phosphatidylglycerol while only a weak penetration into phosphatidylcholine films was evident. Quinacrine also bound to eosin-labeled PLA2, and the addition of 4 mM CaCl2 reversed this interaction almost completely. In the presence of acidic phospholipids both the drug and the enzyme were attached to the lipid surface. Studies on the influence of quinacrine on the activity of PLA2 toward pyrene-labeled phospholipid analogues revealed that the hydrolysis of phosphatidylcholine was progressively reduced as a function of increasing [quinacrine]. At low [CaCl2] and low quinacrine:lipid molar ratios (<1:5) quinacrine enhanced slightly the rate of hydrolysis of acidic phospholipids whereas at higher drug:lipid molar ratios (>1:2) an inhibition was observed. In the presence of 1 mM CaCl2 quinacrine inhibited PLA2-catalyzed hydrolysis of phosphatidylglycerol only when the drug:lipid molar ratio exceeded 1:1. The presence of 4 mM CaCl2 abolished nearly completely the inhibition with all the substrate analogues used. Our data suggest that the inhibition of PLA2 by quinacrine is due to its binding to the enzyme. This is supported also by molecular modeling which suggested a binding site for quinacrine close to the active site and Ca2+ binding site of the enzyme. Importantly, our data indicate that quinacrine binds avidly to acidic phospholipids and their presence may influence the drug-enzyme interaction and the inhibition of the enzyme action. Accordingly, presence of quinacrine may interfere also with other processes that require the presence of acidic lipids and/or Ca2+, such as the function of the nicotinic acetylcholine receptor.

Hydrolysis of supported pyrenephospholipid monolayers by phospholipase A 2

Hydrolysis by pancreatic and snake venom ( Crotalus atrox) phospholipase A 2 of fluorescent monolayers of pyrene-labelled phosphatidylglycerol on solid support was studied. We used a fluorescence microscope equipped with video camera, video recorder and an image analyzer to monitor changes in fluorescence. Decrease in pyrene excimer emission was evident when pyrene phosphatidylglycerol monolayers transferred onto quartz glass slides (at a surface pressure of 15 mN m −1) were subjected to enzymatic hydrolysis. Snake venom phospholipase A 2 could hydrolyze the monolayers almost completely while pancreatic phospholipase A 2 could cause only 50% decrease in fluorescence intensity. EDTA totally inhibited the action of both A 2 phospholipases. When monolayers were transferred onto solid supports at a surface pressure of 31 mN m −1 C. atrox phospholipase A 2 could still exert activity whereas porcine pancreatic phospholipase A 2 was inactive.

Modulation by subphase pH and ions, of 12-(9-anthroyloxy)stearic acid mobility in phospholipid monolayers. A fluorescence recovery after photobleaching study

The mobility of acidic phospholipid monolayers was investigated by means of fluorescence recovery after photobleaching of the 12-(9-anthroyloxy)stearic acid embedded in it. It was shown that for a given fatty acyl chain composition, the mobility in the liquid expanded state is modulated both by the nature and the concentration of the aqueous subphase cations. On NaCl, where dilauroylphosphatidylglycerol and phosphatidylglycerol with methyl-branched fatty acyl chains are in the liquid expanded state, the probe mobility is slower for the branched phospholipid. It is also shown that the probe mobility in monolayers of phosphatidylglycerol extracted from the bacterium Micrococcus luteus membranes, is modulated by cations in the increasing order 1 mM Ca 2+ > 1 M Mg 2+ > 100 mM Na +. On 0.1 M NaCl subphase the mobility of membrane isolated phospholipids varies in the order phosphatidylglycerol < cardiolipin < phosphatidylinositol. The results are interpreted with reference to the “free volume” model of diffusion which also includes a drag force that acts on the head group of the lipid to modulate its mobility.

Incorporation of a synthetic mitochondrial signal peptide into charged and uncharged phospholipid monolayers

The interaction of the chemically synthesized 25-residue signal peptide of subunit IV of yeast cytochrome c oxidase with synthetic and natural phospholipids was studied by using a monolayer technique. Incorporation of the peptide into phospholipid monolayers was measured as surface area increase at constant surface pressure. The peptide was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into phospholipid monolayers up to limiting pressures of 30-40 mN/m. The incorporation of the positively charged peptide was strongly enhanced by the presence of negatively charged phospholipids. The molecular area of the signal peptide in monolayers was determined with a 14C-labeled signal peptide and was 560 +/- 170 A2. This is consistent with a 25-residue alpha-helical peptide incorporating with its long axis parallel to the plane of the monolayer. Incorporation isotherms into synthetic phosphatidylcholine and phosphatidylglycerol monolayers at different charge densities were analyzed in terms of a simple incorporation/binding model, involving partitioning of the peptide into the monolayer and an in-plane binding reaction of the negatively charged phospholipids to the partitioned peptide.

Hydrolysis of phospholipid monolayers by human spermatozoa. Inhibition by male contraceptive gossypol

Monomolecular films of phospholipid were used to study the interaction of intact human spermatozoa with model membranes. Exclusively with negatively charged phosphatidylglycerol monolayers rapid penetration of spermatozoa into the monolayer with subsequent hydrolysis of the lipid was triggered by the addition of 5 mM calcium into the medium. The results suggest the localization of a calcium-dependent phospholipase A 2 at the outer acrosomal or plasma membrane of human spermatozoa with its active site exposed to the external environment. Preincubation of the cells with 100μM gossypol completely abolished the ability of human spermatozoa to hydrolyze or penetrate monolayers of phosphatidylglycerol. The inhibition of the phospholipase activity by gossypol may contribute to the unknown contraceptive mechanisms of this non-steroidal male antifertility agent.

The effect of a phase transition on penetration of phospholipid monolayers by melittin and glucagon

The penetration of melittin and glucagon into phospholipid monolayers was studied by measuring compression isotherms of phospholipids in the absence and presence of various concentrations of protein in the subphase. Differences in molecular area were calculated as a function of protein concentration at constant pressure. Area change as a function of surface pressure at constant protein concentration was also calculated. Melittin showed greater affinity for penetration into phosphatidylglycerol (PG) than into phosphatidylcholine (PC) monolayers. The cutoff pressure for melittin penetration was 45 mN/m with PC and 60 mN/m (extrapolated) with PG. Dipalmitoyl PC and PG monolayers show phase transitions upon compression at 25 °C. Both melittin and glucagon showed increased penetration as measured by area change within the region of the phase transition with both lipids. Glucagon showed a cutoff pressure of 25 mN/m for penetration into dimyristoyl PC. The preference of glucagon for interaction with lipid bilayers in the gel phase is discussed with respect to monolayer penetration as a function of surface pressure.

Influence of monovalent and divalent cations on the surface area of phosphatidylglycerol monolayers

A theory is presented on the electrostatic properties of the surface area of phosphatidyl-glycerol monolayers spreading at an air-water interface in the presence of monovalent and divalent cations. In the present theory, the adsorption of monovalent and divalent cations to the membranes is taken into account, besides the dissociation of protons, as a possible cause of the change of surface charge density with the variation of pH or ion concentrations. It is also pointed out that, in the presence of structure-making ions such as Li + and Na +, the nearest-neighbour interactions between proton dissociation sites become important for the monolayers in the gel state to yield a sharp expansion of the surface area with the increase of pH. The present theory shows quantitative agreements with previously-observed data.

Comparative studies of lipase and phospholipase A2 acting on substrate monolayers.

The kinetic aspects of lipolysis by pancreatic lipase and phospholipase A2 from different sources have been compared using monomolecular films of short chain lipids as the substrates. Phosphatidylcholine monolayers, in contrast to phosphatidylethanolamine and phosphatidylglycerol monolayers, were resistant to hydrolysis by pancreatic lipase. The induction time, measured during pre-steady state conditions, increased abruptly for a given value of the surface pressure. This appears to be due to a degree of lipid packing above which the enzyme no longer can penetrate the lipid film. The existence of an optimum in the velocity versus surface pressure profile is the result of at least two counterbalancing factors. As the surface pressure increases, the amount of enzyme present in the interface decreases, whereas the minimal specific activity of the enzyme increases. From this study with monolayers we can conclude that activity of lipolytic enzymes used as tools for probing biological membranes will be greatly influenced by the physiochemical nature of the membrane-water interface. Thus, studies such as this one which can measure the penetrating ability of various lipolytic enzymes can be useful in deriving a better understanding of biological membrane structure.