Egg Phosphatidylcholine Monolayers Research Articles

Interfacial Properties of NTAIL, an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure under physiological conditions in the absence of their biological partners and thus exist as dynamic ensembles of interconverting conformers, often highly soluble in water. However, in some cases, IDPs such as the ones involved in neurodegenerative diseases can form protein aggregates and their aggregation process may be triggered by the interaction with membranes. Although the interfacial behavior of globular proteins has been extensively studied, experimental data on IDPs at the air/water (A/W) and water/lipid interfaces are scarce. We studied here the intrinsically disordered C-terminal domain of the Hendra virus nucleoprotein (NTAIL) and compared its interfacial properties to those of lysozyme that is taken as a model globular protein of similar molecular mass. Adsorption of NTAIL at the A/W interface was studied in the absence and presence of phospholipids using Langmuir films, polarization modulated-infrared reflection-absorption spectroscopy, and an automated drop tensiometer for interfacial tension and elastic modulus determination with oscillating bubbles. NTAIL showed a significant surface activity, with a higher adsorption capacity at the A/W interface and penetration into egg phosphatidylcholine monolayer compared to lysozyme. Whereas lysozyme remains folded upon compression of the protein layer at the A/W interface and shows a quasi-pure elastic behavior, NTAIL shows a much higher molecular area and forms a highly viscoelastic film with a high dilational modulus. To our knowledge, a new disorder-to-order transition is thus observed for the NTAIL protein that folds into an antiparallel β-sheet at the A/W interface and presents strong intermolecular interactions.

An interfacial and comparative in vitro study of gastrointestinal lipases and Yarrowia lipolytica LIP2 lipase, a candidate for enzyme replacement therapy

Lipolytic activities of Yarrowia lipolytica LIP2 lipase (YLLIP2), human pancreatic (HPL) and dog gastric (DGL) lipases were first compared using lecithin-stabilized triacylglycerol (TAG) emulsions (Intralipid) at various pH and bile salt concentrations. Like DGL, YLLIP2 was able to hydrolyze TAG droplets covered by a lecithin monolayer, while HPL was not directly active on that substrate. These results were in good agreement with the respective kinetics of adsorption on phosphatidylcholine (PC) monomolecular films of the same three lipases, YLLIP2 being the most tensioactive lipase. YLLIP2 adsorption onto a PC monolayer spread at the air/water interface was influenced by pH-dependent changes in the enzyme/lipid interfacial association constant (KAds) which was optimum at pH 6.0 on long-chain egg PC monolayer, and at pH 5.0 on medium chain dilauroylphosphatidylcholine film. Using substrate monolayers (1,2-dicaprin, trioctanoin), YLLIP2 displayed the highest lipolytic activities on both substrates in the 25-35 mN m(-1) surface pressure range. YLLIP2 was active in a large pH range and displayed a pH-dependent activity profile combining DGL and HPL features at pH values found in the stomach (pH 3-5) and in the intestine (pH 6-7), respectively. The apparent maximum activity of YLLIP2 was observed at acidic pH 4-6 and was therefore well correlated with an efficient interfacial binding at these pH levels, whatever the type of interfaces (Intralipid emulsions, substrate or PC monolayers). All these findings support the use of YLLIP2 in enzyme replacement therapy for the treatment of pancreatic exocrine insufficiency, a pathological situation in which an acidification of intestinal contents occurs.

Mechanism of Electrochemical Detection of DNA Hybridization by Bilayer Lipid Membranes

Recent reports have suggested that bilayer lipid membranes (BLMs) and stabilized self-assembled bilayer lipid membranes (s-BLMs) can detect DNA hybridization, but have not explained the actual mechanism of signal generation. The electrochemical experiments have demonstrated that hybridization of complementary single-stranded DNA (ssDNA) 20mers, where one sequence is terminated by a C16 alkyl chain to assist incorporation into the lipid membrane, can cause reduction of ion current through membranes. The present work has explored egg-phosphatidylcholine monolayers at the air–water interface as a model of BLMs, and has investigated the effect of DNA on pressure–area relationships, electrostatic fields as measured using a noncontacting electrostatic voltmeter, and fluorescence microscopy of membranes that were loaded with fluorescein labelled DNA. Each of these experiments confirms that ssDNA partitions onto and changes the structural and electrostatic properties of lipid monolayers. On hybridization the DNA desorbs from the surface of the lipid membrane, causing a substantial alteration of surface properties.

Effect of the Arrangement of Tandem Repeating Units of Class A Amphipathic α-Helixes on Lipid Interaction

Exchangeable apolipoproteins possess tandem repeating units of class A amphipathic helical segments and many of them are linked together by proline residues. To understand the optimal arrangement of the amphipathic helixes for lipid association, we have studied the interactions of three model class A amphipathic helical peptides with lipids. The three peptides are: 37pA, a dimer of 18A (DWLKAFYDKVAEKLKEAF) linked together by a Pro (18A-Pro-18A); 37aA, a dimer of 18A linked together by an Ala (18A-Ala-18A); and 36A, a dimer of 18A without any linker residue (18A-18A). Circular dichroism (CD) spectra showed that the peptides are predominantly alpha-helical in aqueous and lipid environments. Temperature dependent CD studies indicated that in buffer helix stability decreases in the order 36A > 37aA > 37pA; however, in the presence of dimyristoyl phosphatidylcholine (DMPC), the above order is reversed. The retention times of the peptides on a C18 reversed-phase high performance liquid chromatography column decreased in the order 36A > 37aA > 37pA, consistent with the lengths of the nonpolar faces of the alpha-helixes being in the same order; the retention time of the parent 18A was shorter than 37pA. While 37pA adsorbed to egg phosphatidylcholine monolayers most strongly, the degree and rate of association of 36A were significantly lower. Differential scanning calorimetry indicated that, while 37pA was most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of DMPC multilamellar vesicles, 36A was least effective; 36A was even less effective than 18A. Fluorescence quenching experiments with iodide and acrylamide indicated that, in the presence of DMPC, Trp residues in 36A are most exposed to the quenchers while in 37pA they are least exposed. In the presence of DMPC, shielding of Trp in 18A from the quenchers was more than that observed with Trp residues in 36A. The results of this study suggest that the arrangement of tandem repeating amphipathic helical units which results in the formation of a class A amphipathic helix with a nonpolar face longer than five or six turns reduces the ability of the helix to associate with phospholipid.

Interfacial properties of recombinant human cholesterol ester transfer protein.

We investigated the interfacial behavior of recombinant human cholesterol ester transfer protein (rCETP) using monolayer and surface balance techniques. rCETP bound to egg phosphatidylcholine monolayers spread at the air/water interface with a maximum surface pressure of 23 millinewtons (mN)/m at subphase concentrations between 3 and 5 x 10(-5) g/dl; the estimated dissociation constant was 7.5 x 10(-6) g/dl or 1 nM. The binding of rCETP to the lipid interface decreased linearly with increasing initial surface pressure; rCETP was excluded at pressures greater than 31 mN/m. rCETP catalyzed the desorption of [14C]cholesterol oleate from mixed lipid monolayers in a concentration dependent fashion. Similar studies with apolipoproteins A-I and A-IV established that cholesterol ester desorption was not caused by changes in surface pressure or cholesterol ester solubility. The desorption rate was proportional to subphase rCETP concentration, but at all concentrations surface radioactivity remained constant until surface pressure reached a plateau. The calculated binding stochiometry was one molecule of cholesterol ester desorbed for every 1000 molecules of rCETP in the subphase. We conclude that rCETP is surface active, binds to phospholipid monolayers with an affinity equivalent to that of the plasma apolipoproteins, and effects the desorption of cholesterol ester molecules from phospholipid monolayers by a carrier mechanism. Moreover, the relatively low equilibrium surface pressure of rCETP suggests that when bound to lipid the entire rCETP molecule may not penetrate the interface.

An investigation into the effects of surfactants on phospholipid monolayers using a Langmuir-Blodgett film balance

The interactions between egg phosphatidylcholine monolayers and two surface-active agents, sodium lauryl sulphate (SLS) and cetyltrimethylammonium bromide (CTAB), have been studied using a Langmuir-Blodgett film balance using a range of surfactant concentrations below the critical micelle concentration. The presence of SLS in the subphase was shown to result in an increase in the surface pressure (using equivalent surfactant solutions for comparison) while retaining the same basic shape and transitional behaviour of the phospholipid profile. In contrast, the presence of CTAB resulted in a concentration dependent inhibition of film formation. It is concluded that these two surfactants interact with the phospholipid monolayers by different mechanisms over the concentration ranges studied and that surface pressure measurements represent an effective means of characterising such interactions.

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

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

Modulation of the interbilayer hydration pressure by the addition of dipoles at the hydrocarbon/water interface

The effects of the cholesterol analog 5 alpha-cholestan-3 beta-ol-6-one (6-ketocholestanol) on bilayer structure, bilayer cohesive properties, and interbilayer repulsive pressures have been studied by a combination of x-ray diffraction, pipette aspiration, and dipole potential experiments. It is found that 6-ketocholestanol, which has a similar structure to cholesterol except with a keto moiety at the 6 position of the B ring, has quite different effects than cholesterol on bilayer organization and cohesive properties. Unlike cholesterol, 6-ketocholestanol does not appreciably modify the thickness of liquid-crystalline egg phosphatidylcholine (EPC) bilayers, and causes a much smaller increase in bilayer compressibility modulus than does cholesterol. These data imply that 6-ketocholestanol has both its hydroxyl and keto moieties situated near the water-hydrocarbon interface, thus making its orientation in the bilayer different from cholesterol's. The addition of equimolar 6-ketocholestanol into EPC bilayers increases the magnitude, but not the decay length, of the exponentially decaying repulsive hydration pressure between adjacent bilayers. Incorporation of equimolar 6-ketocholestanol into EPC monolayers increases the dipole potential by approximately 300 mV. These data are consistent with our previous observation that the magnitude of the hydration pressure is proportional to the square of the dipole potential. These results mean that 6-ketocholestanol, despite its location in the bilayer hydrocarbon region, approximately 10 A from the physical edge of the bilayer, modifies the organization of interlamellar water. We argue that the incorporation of 6-ketocholestanol into EPC bilayers increases the hydration pressure, at least in part, by increasing the electric field strength in the polar head group region.

Cholesteryl-pullulan and cholesteryl-amylopectin interactions with egg phosphatidylcholine monolayers

The extent of adsorption of cholesteryl-pullulan and cholesteryl-amylopectin at air-water interface was assessed from the surface pressure measurements at constant area. It was found that areas per adsorbed cholesterol derivative of pullulan and amylopectin are 0.40 and 0.19 nm 2, respectively. These small areas indicate that sugar moieties of both polysaccharide derivatives are completely immersed in the aqueous phase. The surface potential data strongly suggest that the cholesteryl moieties of adsorbed cholesteryl derivative of pullulan are stretched toward the air phase, but lay flat, exposing lateral CH 3 groups to the interface, in the case of a cholesteryl derivative of amylopectin. Surface pressure and surface potential isotherms of egg-phosphatidylcholine monolayers were shown to be greatly modified in the presence of cholesterol-substituted polysaccharides in the aqueous subphase. The results reveal the ability of both polysaccharide derivatives to penetrate the lipid monolayer. However, this effect is superior for cholesteryl-amylopectin, which interacts strongly with the lipid even at very high surface coverages. cholesteryl-amylopectin also compensates lipid surface potential to a higher extent than cholesteryl-pullulan. This would explain why liposomes coated with cholesteryl-amylopectin exhibit lower stability relative to those coated with a cholesterol derivative of pullulan.

ヒト血漿脂質転送蛋白の反応機構について

Human blood plasma contains the catalytic activity of transfer cholesteryl ester among the plasma lipoproteins. [14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate to investigate the cholesteryl ester transfer reaction of the plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of a density>1.006 at 37°C. When the distribution of the radiolabeled cholesteryl ester was equilibrated for the liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose. The LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (ajusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. The radioactivity in this preparation was present exclusively in the cholesteryl ester. The apolipoprotein B-100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high density lipoproteins (HDL) by using this labeled LDL, the kinetics were consistent with the equilibrium transfer model. However, the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.The lipid-transfer protein (LTP) was partially purified about 1, 600 times from human plasma using this new radiolabeled LDL in the LTP assay in order to monitor the activity. Using this LTP preparation we studied the transfer of lipids between LDL and triolein particles coated with an egg phosphatidylcholine monolayer, having a diameter of 27±4nm. The lipid particles were unstable and seemed to attach themselves to the LDL when incubated with LDL either in the presence or absence of bocine serum albumin. The human apolipoproteins, A-I, A-II, C-II, C-III, and E stabilized the lipid particles and completely prevented this process. Cholesterol rapidly appeared in the lipid particles reaching a homogeneous distribution among the phospholipid surfaces of LDL and the lipid particles, both when the apolipoproteins were present and when they were absent. Cholesteryl ester spontaneously appeared in the lipid particles to some extent in the abscence of the apolipoproteins, and human plasma lipid transfer protein enhanced this reaction only to a very limited extent. When the lipid particles were stabilized with the apolipoproteins. The spontaneous cholesteryl ester transfer was minimized and the lipid-transfer protein markedly catalyzed the transfer of cholesteryl ester. There were no specific differences among the apolipoproteins in stabilizing the particles or enhancing the transfer reaction. A reciprocal decrease in the volume of triglyceride was observed at the same time in the lipid particles until the relative content of cholesteryl ester in the cores of the LDL was the same as in the lipid particles. The kinetics of the cholesteryl ester and triglyceride transfer was consistent with the model, showing that the reaction is bidirectional in equilibrium and takes both non-polar lipids as substrate in a single pool.

Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes.

Pressure versus distance relationships have been obtained for egg phosphatidylcholine bilayers containing a range of cholesterol concentrations. Water was removed from between adjacent bilayers by the application of osmotic pressures in the range of 0.4-2600 atm (4 x 10(5)-2.6 x 10(9) dyn/cm2), and the distance between adjacent bilayers was obtained by Fourier analysis of X-ray diffraction data. For applied pressures up to about 50 atm and bilayer surface separations of 15-5 A, the incorporation of up to equimolar cholesterol has little influence on plots of pressure versus bilayer separation. However, for the higher applied pressures, cholesterol reduces the interbilayer separation distance by an amount that depends on the cholesterol concentration in the bilayer. For example, the incorporation of equimolar cholesterol reduces the distance between bilayers by as much as 6 A at an applied pressure of 2600 atm. At this applied pressure, electron density profiles show that the high-density head-group peaks from apposing bilayers have merged. This indicates that equimolar concentrations of cholesterol spread the lipid molecules apart in the plane of the bilayer enough to allow the phosphatidylcholine head groups from apposing bilayers to interpenetrate as the bilayers are squeezed together. All of these X-ray and pressure-distance data indicate that, by reducing the volume fraction of phospholipid head groups, cholesterol markedly reduces the steric repulsion between apposing bilayers but has a much smaller effect on the sum of the longer ranged repulsive hydration and fluctuation pressures. Increasing concentrations of cholesterol monotonically increase the dipole potential of egg phosphatidylcholine monolayers, from 415 mV with no cholesterol to 493 mV with equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of non-polar lipid transfer reaction through stabilization of substrate lipid particles with apolipoproteins.

Transfer of lipids was studied between human plasma low density lipoproteins (LDL) and triolein particles coated with an egg phosphatidylcholine monolayer, with diameter of 27 +/- 4 nm. The lipid particles were unstable and seemed to aggregate to LDL when incubated with LDL either in the presence or the absence of bovine serum albumin. Human apolipoproteins A-I, A-II, C-II, C-III, and E stabilized the lipid particles and completely prevented this process. Cholesterol rapidly appeared in the lipid particles to reach homogeneous distribution among the phospholipid surfaces of LDL and the lipid particles regardless of whether apolipoproteins were present or absent. Cholesteryl ester spontaneously appeared in the lipid particles to some extent in the absence of the apolipoproteins, and human plasma lipid transfer protein enhanced this reaction only to a very limited extend. When the lipid particles were stabilized with the apolipoproteins, spontaneous cholesteryl ester transfer was minimized and the lipid transfer protein catalyzed the transfer of cholesteryl ester markedly. There was no specific difference among the apolipoproteins in stabilizing the particles and enhancing the transfer reaction. Reciprocal decrease in volume of triglyceride was observed at the same time in the lipid particles until the relative content of cholesteryl ester in the cores of LDL was the same as in the lipid particles. The kinetics of the cholesteryl ester and triglyceride transfer was consistent with the model that the reaction is bidirectional in equilibrium and takes both non-polar lipids as substrate in a single pool.

A comparison of the surface activities of rat plasma apolipoproteins C-II, C-III-0, C-III-3

Rat apolipoproteins C-II, C-III-0 and C-III-3 give similar surface pressure (π)— molecular area isotherms when spread at the air/water interface. When allowed to adsorb to the clean air/water interface, the intrinsic surface activity of apolipoprotein C-II is somewhat higher than that of apolipoprotein C-III. All three apolipoprotein C molecules can penetrate an egg phosphatidylcholine monolayer spread at the air/water interface causing an increase (Δπ) in surface pressure. Increasing the initial surface pressure (π i) of the lipid monolayer decreases Δπ, and Δπ = 0 when π i ⩽ 32 ± 2 mN· m −1 for all three apolipoprotein C proteins. This implies that apolipoproteins C-II, C-III-0 and C-III-3 would adsorb and desorb similarly from the surfaces of lipoprotein particles during metabolism.

Interactions of cytochrome c and [14C

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.