Binding and mobility of anti-dinitrophenyl monoclonal antibodies on fluid-like, Langmuir-Blodgett phospholipid monolayers containing dinitrophenyl-conjugated phospholipids

The association of a fluorescently labelled anti-dinitrophenyl monoclonal antibody (ANO2) with Langmuir-Blodgett monolayers composed of three different binary mixtures of phosphatidylcholine and dinitrophenyl-conjugated phosphatidylethanolamine has been characterized. Quantitative fluorescence microscopy measurements demonstrated that measurable amounts of antibodies bound to the monolayers only at high molar fractions of dinitrophenyl-conjugated lipid (greater than or equal to 5 mol%). Fluorescence pattern photobleaching recovery measurements showed that the apparent translational diffusion coefficients and mobile fractions of a fluorescent lipid were high for all monolayer compositions and that the antibody translational mobility was measurable but slow and depended on the two-dimensional antibody density. The results demonstrate that the ANO2-binding characteristics of Langmuir-Blodgett monolayers containing dinitrophenyl-conjugated phospholipids are substantially different from those of similar model systems but that the ANO2 antibodies, when bound, display similar diffusive behavior.     

Interaction between a growth-hormone releasing hexapeptide and phospholipids spread as monolayers at the air/water interface

The interaction between a growth-hormone releasing hexapeptide and phospholipids was studied on mixed monolayers models by means of surface fluorescence. When in a monolayer this hexapeptide which contains two tryptophan molecules was observed to fluoresce. Isothermal compression experiments showed that the complex was destroyed upon compression in the case of phosphatidylethanolamine. With phosphatidylglycerol it was observed to be stable but a dramatic reversible decrease in emission was observed at high surface pressure. This is indicative of a reversible change in the organization of the peptide-phospholipid complex. These observations indicate that, in the complex, hydrophobic interactions were weak but electrostatic ones, when present, were strong enough to maintain the GHRP attached to the monolayer and not to destabilize it. The integrity of the lipid monolayer appeared not to be affected by the peptide.     

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir-Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein-lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion.     

Hepatic lipase hydrolysis of lipid monolayers. Regulation by apolipoproteins

A monolayer technique was used to study the substrate specificity of hepatic lipase (HL) and the effect of surface pressure and apolipoproteins on hydrolysis of lipid monolayers by this enzyme. HL hydrolyzed readily phosphatidylethanolamine monolayers. Pure trioctanoylglycerol was found to be a poor substrate but when progressively diluted with nonhydrolyzable 1,2-didodecanoylphosphatidylcholine hydrolysis of triacylglycerol by HL reached maximum at a molar ratio of 1:1 triacylglycerol to phosphatidylcholine. The activation of triacylglycerol hydrolysis was not due to altered penetration of HL. The surface pressure optimum of HL for the hydrolysis of phosphatidylethanolamine monolayers was broad between 12.5 and 25 mN/m. When apolipoprotein E was injected beneath the monolayer of phosphatidylethanolamine prior to enzyme addition, a 3-fold activation of HL was observed at surface pressures equal to or below 15 mN/m. Below surface pressures of 20 mN/m apolipoprotein E did not affect the penetration of HL into the lipid-water interface. Apolipoprotein E slightly activated the hydrolysis of triacylglycerol by HL at 10 mN/m. At a high surface pressure of 25 mN/m all apolipoproteins tested (apolipoproteins A-I, A-II, C-I, C-II, C-III, and E) inhibited the penetration into and HL activity on phosphatidylethanolamine At 18.5 mN/m all apolipoproteins except apolipoprotein E inhibited the hydrolysis of triacylglycerol in the triacylglycerol:phosphatidylcholine mixed film. Based on these results we present a hypothesis that phospholipid present in apolipoprotein E-rich high density lipoprotein-1 and triacylglycerol in intermediate density lipoprotein would be preferred substrates for HL.     

Effect of polar head groups on the activity of aspartyl protease adsorbed to lipid membranes

The proteolytic activity of an aspartyl protease of Mucor miehei was correlated with the adsorption of the protease to lipid vesicles. It was observed that the presence of phosphatidylethanolamines (PE's) in the membrane increased the enzyme activity in a 20% in the gel phase and 10% in the fluid phase. The effects of protease on the surface pressure of monolayers composed by dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dimyristoyl phosphatidylethanolamine (DMPE) were measured at constant temperature as a function of the surface pressure. At low surface pressures, the major changes were induced by protease on DOPC and DMPC monolayers. However, the effect were much lower when the monolayer was composed by DMPE. The low hydration and strong head-head interaction between the phosphates and the amine groups of adjacent PE's would result in an area per molecule much lower in PE than in phosphatidylcholine (PC) in concordance with the lower penetration in PE. Protease adsorption on PE membranes increases the proteolytic activity in which condition is less susceptible to inhibition by pepstatin. However, PC's do not alter the enzyme activity being the action of inhibitor unaffected.     

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.     

Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.     

Hydrolysis of lipid monolayers and the substrate specificity of hepatic lipase

The substrate specificities of the phospholipase and triglyceridase activities of purified rat liver hepatic lipase were compared using lipid monolayers so that the substrates were presented to the enzyme in a controlled physical state. The rate of hydrolysis of 14C-labeled lipid at constant surface pressure in the presence of hepatic lipase and fatty acid-free bovine serum albumin at 33 degrees C was determined by monitoring the decrease of surface radioactivity. In monolayers of sphingomyelin/cholesterol (2:1, mol/mol) containing either 1 mol% triacylglycerol, 1 mol% phosphatidylethanolamine, or 10 and 20 mol% phosphatidylcholine, hepatic lipase clearly showed a preference for unsaturated over saturated lipids. In addition, with a sphingomyelin/cholesterol (2:1) monolayer containing 1 mol% of lipid substrate, hepatic lipase showed the following preference: triolein = dioleoylphosphatidylethanolamine much greater than dioleoylphosphatidylcholine; the respective rates of hydrolysis were 15.3 +/- 1.2, 14.9 +/- 0.8, and 0.5 +/- 0.1 mumol fatty acid produced/h per mg hepatic lipase. Overall, it appears that when comparing rates of hydrolysis of molecules within a given lipid class, hydrocarbon chain interactions are important. However, when comparing different lipid classes such as phosphatidylcholines and phosphatidylethanolamines, it is apparent that the polar group has a significant influence on the rate of hydrolysis. The rate of [14C]triolein hydrolysis, when mixed at surface concentrations of up to 2 mol% in a sphingomyelin/cholesterol (2:1) monolayer, was significantly faster than when triolein was present in a 1-oleyl-2-palmitylphosphatidylcholine monolayer; the rates of hydrolysis were 47.7 +/- 5.4 and 8.9 +/- 0.8 mumol fatty acid produced/h per mg hepatic lipase, respectively. The monolayer physical state and the miscibility of the substrate in the inert matrix influence the presentation of the substrate to the enzyme, thereby affecting the hydrolysis rate.     

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.     

Lipid headgroup discrimination by antimicrobial peptide LL-37: insight into mechanism of action

Interaction of the human antimicrobial peptide LL-37 with lipid monolayers has been investigated by a range of complementary techniques including pressure-area isotherms, insertion assay, epifluorescence microscopy, and synchrotron x-ray scattering, to analyze its mechanism of action. Lipid monolayers were formed at the air-liquid interface to mimic the surface of the bacterial cell wall and the outer leaflet of erythrocyte cell membrane by using phosphatidylglycerol (DPPG), phosphatidylcholine (DPPC), and phosphatidylethanolamine (DPPE) lipids. LL-37 is found to readily insert into DPPG monolayers, disrupting their structure and thus indicating bactericidal action. In contrast, DPPC and DPPE monolayers remained virtually unaffected by LL-37, demonstrating its nonhemolytic activity and lipid discrimination. Specular x-ray reflectivity data yielded considerable differences in layer thickness and electron-density profile after addition of the peptide to DPPG monolayers, but little change was seen after peptide injection when probing monolayers composed of DPPC and DPPE. Grazing incidence x-ray diffraction demonstrated significant peptide insertion and lateral packing order disruption of the DPPG monolayer by LL-37 insertion. Epifluorescence microscopy data support these findings.     

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and beta-casein with phospholipids.

The conformations adopted by beta-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being alpha-helical and the beta-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the beta-casein is in a compressed state whereas the apoprotein is extended as alpha-helices in the plane of the interface. The chain-length depedences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and β-casein with phospholipids

The conformations adopted by β-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being α-helical and the β-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the β-casein is in a compressed state whereas the apoprotein is extended as α-helices in the plane of the interface. The chain-length dependences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B(max)) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in deltapi change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The deltapi change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a "raft-like" mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.     

Eutectic mixed monolayers in equilibrium with phospholipid-bilayers and triolein-liquid phase.

Triolein (TO) and phospholipids (egg yolk phosphatidylcholine, egg yolk phosphatidylethanolamine, and bovine brain phosphatidylserine) had low mutual solubilities and separated into the TO-liquid phase and phospholipid-bilayers. Spreading pressures of the TO-phospholipid mixture (i.e., surface pressures of the mixed monolayer in equilibrium with the phase-separating lipid mixture) at the air/saline interface were independent of the lipid composition. On the other hand, collapse pressures of the mixed monolayer of TO and phospholipid (i.e., surface pressures of the mixed monolayer in equilibrium with the TO-liquid phase) at the interface changed with the monolayer composition and were lower than the spreading pressure. The experimental data indicated the spreading and collapse pressures as offering a phase diagram for the presence of equilibrium between the mixed monolayer, the phospholipid-bilayers and the TO-liquid phase. The diagram showed that TO and the phospholipids were miscible in the mixed monolayer, forming an eutectic mixed monolayer. When the mixed monolayer initially had the eutectic composition, no collapse of the monolayer was detected until the surface pressure reached the value of the spreading pressure. No specific complex between TO and the phospholipid is required to explain the stability and collapse of the mixed monolayers. The bulk immiscibility of the lipids elucidated by the spreading pressure-measurements, immediately leads to the phase behaviors observed.     

Interaction of proteins with lipid monolayers at the air-solution interface studied by reflection spectroscopy

The interaction of a fluorescein-labelled insulin and of cytochrome C with the air-solution interface and with lipid monolayers at the air-solution interface has been studied by measuring the change in surface pressure at constant area and by reflection spectroscopy. Chromophores at the interface only give rise to enhanced light reflection without contribution to the signal from chromophores in the bulk. The accumulation of labelled insulin at the solution surface is very weak as concluded from the shape of the spectrum and reflection intensity. No interaction with a monolayer of dipalmitoyl-phosphatidylcholine at initial surface pressure of 5mN/m was detected. In contrast, the interaction with monolayers of dioctadecyl-dimethyl-ammonium bromide at initial surface pressures between 5 and 40 mN/m is much stronger, leading to a remarkable increase of surface pressure at constant area and strong reflection signal. The technique was also used to detect cytochrome C at the air-solution interface.     

Interaction of methionine–enkephalins with raft-forming lipids: monolayers and BAM experiments

Enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are opioid peptides with proven antinociceptive action in organism. They interact with opioid receptors belonging to G-protein coupled receptor superfamily. It is known that these receptors are located preferably in membrane rafts composed mainly of sphingomyelin (Sm), cholesterol (Cho), and phosphatidylcholine. In the present work, using Langmuir’s monolayer technique in combination with Wilhelmy’s method for measuring the surface pressure, the interaction of synthetic methionine–enkephalin and its amidated derivative with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sm, and Cho, as well as with their double and triple mixtures, was studied. From the pressure/area isotherms measured, the compressional moduli of the lipids and lipid–peptide monolayers were determined. Our results showed that the addition of the synthetic enkephalins to the monolayers studied led to change in the lipid monolayers characteristics, which was more evident in enkephalinamide case. In addition, using Brewster angle microscopy (BAM), the surface morphology of the lipid monolayers, before and after the injection of both enkephalins, was determined. The BAM images showed an increase in surface density of the mixed surface lipids/enkephalins films, especially with double and triple component lipid mixtures. This effect was more pronounced for the enkephalinamide as well. These observations showed that there was an interaction between the peptides and the raft-forming lipids, which was stronger for the amidated peptide, suggesting a difference in folding of both enkephalins. Our research demonstrates the potential of lipid monolayers for elegant and simple membrane models to study lipid–peptide interactions at the plane of biomembranes.     

Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes

The phospholipid composition of ram spermatozoa plasma membranes has been investigated. An exclusively high participation of the choline- and ethanolamine-plasmalogens in the phosphatidylcholine and phosphatidylethanolamine fractions has been established. Phosphatidylcholine of ram spermatozoa plasma membranes contains a great amount of polyunsaturated fatty acids. The phospholipid distribution in spermatozoa plasma membrane was investigated. It was established that the choline containing phospholipids are situated mainly in the outer membrane lipid monolayer, whereas diphosphatidylglycerol and phosphatidylserine are localized predominantly in the inner monolayer. The rest of the phospholipids are evenly distributed among the two monolayers. Ram spermal plasma membranes exhibit high phospholipase A2 activity.     

Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 microM and 19 microM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 microM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (<5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other "single-chain" lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

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

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

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

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