Conformational changes of phospholipid headgroups induced by a cationic integral membrane peptide as seen by deuterium magnetic resonance

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of a cationic amphiphilic peptide with pure DMPC membranes and with mixed bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). The choline and serine headgroups were selectively deuteriated at the alpha and beta positions. The amphiphilic peptide, with 20 leucine residues in the hydrophobic core and two cationic hydrophilic lysine residues at each end, spanned the lipid bilayer. Although 2H NMR experiments using DMPC with perdeuteriated fatty acyl chains showed that the average order parameter of the hydrophobic region was not significantly modified by the incorporation of the amphiphilic peptide, for either DMPC or DMPC/DMPS (5:1) bilayers, large perturbations of the quadrupolar splittings of the choline and serine headgroups were observed. The results obtained with the DMPC headgroup suggest that the incorporation of the cationic peptide in both DMPC and DMPC/DMPS (5:1) bilayers leads to a structural perturbation directly related to the net charge on the membrane surface. The magnitude of the observed effect seems to be similar to those observed previously with other cationic molecules [Seelig, J., MacDonald, P.M., & Scherer, P.G. (1987) Biochemistry 26, 7535-7541]. Two of the three quadrupolar splittings of the PS headgroup exhibited large variations in the presence of the amphiphilic peptide, while the third one remained unchanged. Our data have led us to propose a model describing the influence of membrane surface charges on headgroup conformation. In this model, the surface charge is represented as a uniform charge distribution. The electric field due to the charges produces a torque which rotates the polar headgroups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Phase and surface properties of lipid bilayers containing neoglycolipids

The physical properties conferred to DPPC bilayers by including neoglycolipids composed by two different trisaccharides: mannose-mannose-mannose (3M) and glucose-mannose-glucose (GMG) attached to a cholesterol (cho) and a distearylglycerol (diC18) lipid moiety by a spacer were evaluated by means of the measurement of the electrokinetic potential and interfacial fluorescent probes. The phase properties measured with diphenylhexatriene (DPH) were correlated with the surface properties measured with merocyanine 540, dansyl, and Laurdan probes. The results show that the surface properties of large unilamellar vesicles depend on the sugar exposure to the water phase and also on the hydrocarbon moiety by which it is anchored to the bilayer. The combination of the cholesterol moiety with the saccharide attenuates the cooperativity decrease induced by the cholesterol moiety without the sugar portion. The neoglycolipid GMG-diC18 promotes opposite effects affecting slightly the cooperativity at the hydrocarbon core of DPPC and displacing the phase transition temperature to higher values. The presence of neoglycolipid with diC18 introduces defects in the packing at the interface of the membrane in the gel state. It is concluded that a relatively low proportion of neoglycolipids affects significantly the interfacial properties of DPPC bilayers in large unilamellar vesicles in the absence of changes at the membrane bulk at 25 degrees C.     

Model study of interactions of high-molecular dextran sulfate with lipid monolayers and foam films

The interaction of high-molecular dextran sulfate (DS-5000) with dimyristoylphosphatidylcholine (DMPC) monolayers and foam films (FF) at the air–water interface in the presence of Ca²⁺ and Na⁺ ions was studied. DS-5000 was added in monolayer films (MF) and in FF as monomer molecules and in liposomal form. When added in liposomal form in FF, DS-5000 decreased the stability of DMPC common black films (CBF), and no formation of Newton black films (NBF) was observed. However, when included as monomer molecules in FF, DS-5000 caused film thinning, and drastically decreased the expansion rate of the black spots and transition of thick films to NBF, thus avoiding formation of CBF. The above effects were observed in both gel and liquid-crystalline phase states of DMPC in the presence of Ca²⁺ ions only, and not in the presence of Na⁺ ions. We postulate that the interaction of DMPC with DS-5000 in the plane of FF is mediated by Ca²⁺ bridges and results in dehydration of the DMPC polar heads. The interaction between DMPC and DS-5000 in monolayers resulted in slower adsorption and spreading of DMPC molecules at the interface, lower monolayer surface pressure, and penetration of DS-5000 molecules to DMPC monolayers when surface lipid density was higher than 50 Ų per DMPC molecule. The applicability of the FF model for studying the interactions of phospholipids with polysaccharides at interfaces surrounded by bulk solution, and for modeling such interactions in biological systems, e.g. LDL adhesion to the arterial walls, aggregation and fusion of liposomes, etc., is discussed.     

The influence of cholesterol on interactions and dynamics of ibuprofen in a lipid bilayer

In this work, molecular dynamics (MD) simulations with atomistic details were performed to examine the influence of the cholesterol on the interactions and the partitioning of the hydrophobic drug ibuprofen in a fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer. Analysis of MD simulations indicated that ibuprofen molecules prefer to be located in the hydrophobic acyl chain region of DMPC/cholesterol bilayers. This distribution decreases the lateral motion of lipid molecules. The presence of ibuprofen molecules in the bilayers with 0 and 25 mol% cholesterol increases the ordering of hydrocarbon tails of lipids whereas for the bilayers with 50 mol% cholesterol, ibuprofen molecules perturb the flexible chains of DMPC lipids which leads to the reduction of the acyl chain order parameter. The potential of the mean force (PMF) method was used to calculate the free energy profile for the transferring of an ibuprofen molecule from the bulk water into the DMPC/cholesterol membranes. The PMF studies indicated that the presence of 50 mol% cholesterol in the bilayers increases the free energy barrier and slows down the permeation of the ibuprofen drug across the DMPC bilayer. This can be due to the condensing and ordering effects of the cholesterol on the bilayer.     

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.     

Solutes in small amounts provide for lipid-bilayer softness: cholesterol,short-chain lipids,and bola lipids

The effect of the incorporation of small amounts (∼1 mole%) of amphiphilic solutes, such as cholesterol, a short-chain lipid (DC₁₀PC), and a bola lipid, into multilamellar DMPC bilayers is studied by small-angle neutron scattering and differential-scanning calorimetry. The anomalous swelling behavior observed in the transition region of pure DMPC bilayers is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Small amounts of the solutes are found to maintain or even enhance the bilayer softness. In the case of cholesterol, a systematic study shows that the well-known rigidification effect is observed only for cholesterol concentrations above 3–4 mole%. The results are discussed in relation to the physical properties of internal cell membranes. Received: 28 May 1996 / Accepted: 27 July 1996     

Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers

Anionic lipids are key components in the cell membranes. Many cell-regulatory and signaling mechanisms depend upon a complicated interplay between them and membrane-bound proteins. Phospholipid bilayers are commonly used as model systems in experimental or theoretical studies to gain insight into the structure and dynamics of biological membranes. We report here 200-ns-long MD simulations of pure (DMPC and DMPG) and mixed equimolar (DMPC/DMPG, DMPC/DMPS, and DMPC/DMPA) bilayers that each contain 256 lipids. The intra- and intermolecular interaction patterns in pure and mixed bilayers are analyzed and compared. The effect of monovalent ions (Na⁺) on the formation of salt-bridges is investigated. In particular, the number of Na⁺-mediated clusters in the presence of DMPS is higher than with DMPG and DMPA. We observe a preferential clustering of DMPS (and to some extent DMPA) lipids together rather than with DMPC molecules, which can explain the phase separation observed experimentally for DMPC/DMPS and DMPC/DMPA bilayers.     

Spin-labelled vacuolar-ATPase inhibitors in lipid membranes

Two spin-labelled derivatives of the 5-(2-indolyl)-2,4-pentadienoyl class of inhibitors of the vacuolar ATPase have been synthesised and their EPR properties characterised in phospholipid membranes. One spin-labelled inhibitor is the amide derivative of pentadienic acid and 4-amino-TEMPO (INDOL6), and the other is the 3-hydroxymethyl-PROXYL ester (INDOL5). The response of the EPR spectra to the chain-melting transition of dimyristoyl phosphatidylcholine (DMPC) bilayers demonstrates that both derivatives incorporate in phospholipid membranes. The axially anisotropic EPR spectra of INDOL6 in fluid DMPC membranes indicate that the indolyl-pentadienoyl inhibitors intercalate between the lipid chains, in the membrane. INDOL5, designed to possess additional internal segmental mobility, exhibits more nearly isotropic motion of the spin-label moiety in fluid membranes than does INDOL6. The EPR characteristics of INDOL5 are therefore well suited to detecting specific ligand-protein interactions. Progressive saturation EPR experiments with polar and hydrophobic relaxation agents (aqueous Ni2+ and oxygen) show that the nitroxide group is buried in the membrane, with the indole moiety providing the anchor at the membrane polar-apolar interface. Rates of spin-label reduction by externally added ascorbate confirm this assignment. These two spin-labelled derivatives provide complementary EPR probes of the lipid environment (INDOL6), and of ligand-protein interactions (INDOL5), for this class of V-ATPase inhibitor.     

Thin liquid films and monolayers of DMPC mixed with PEG and phospholipid linked PEG

In this work thin liquid films (TLFs) and monolayers at the air/water interface formed by dimyristoylphosphatidylcholine (DMPC) and by DMPC mixed with poly ethylene glycols (PEGs) and dimyristoylphosphatidylethanolamine (DMPE) linked PEGs were studied. Film forming dispersions were composed of two types of particles: liposomes and micelles. TLFs stability, threshold concentration C _t (i.e., the minimum one for stable film formation), and hydrodynamic behavior were measured. At equivalent conditions, DMPC films were Newton black films (real bilayers), while DMPE-PEGs films were much thicker with free water between the monolayers. DMPE-PEG addition to DMPC films caused both C _t decrease (depending on PEG moiety length and Mw) and change of TLF formation mechanism. TLFs’ hydrodynamic behavior also strongly depended on DMPE-PEG content and Mw. It was observed that thinning of the DMPC and DMPE-PEGs films continued to different film types and thickness, being much thicker for the latter films. Addition of free PEGs (PEG-200/6000) did not alter TLF type or stability, but changed TLF thinning time, confirming that free PEGs with Mw<8000 could not penetrate in the membrane and alter “near-membrane” water layer viscosity. Monolayer studies showed improved formation kinetics of both adsorbed and spread films, decrease of surface tension (equilibrium and dynamic), and of film compression/decompression histeresis area in DMPE-PEGs monolayers compared with DMPC pure films. Our study shows that combining the models of phospholipid TLFs and monolayers provide the opportunity to investigate the properties of membrane surface and to clarify some mechanisms of its interactions with membrane-active agents.     

Solid state 13C NMR of unlabeled phosphatidylcholine bilayers: spectral assignments and measurement of carbon-phosphorus dipolar couplings and 13C chemical shift anisotropies.

The direct measurement of 13C chemical shift anisotropies (CSA) and 31P-13C dipolar splitting in random dispersions of unlabeled L alpha-phase phosphatidylcholine (PC) has traditionally been difficult because of extreme spectral boradening due to anisotropy. In this study, mixtures of dimyristoyl phosphatidylcholine (DMPC) with three different detergents known to promote the magnetic orientation of DMPC were employed to eliminate the powder-pattern nature of signals without totally averaging out spectral anisotropy. The detergents utilized were CHAPSO, Triton X-100, and dihexanoylphosphatidylcholine (DHPC). Using such mixtures, many of the individual 13C resonances from DMPC were resolved and a number of 13C-31P dipolar couplings were evident. In addition, differing line widths were observed for the components of some dipolar doublets, suggestive of dipolar/chemical shift anisotropy (CSA) relaxation interference effects. Oriented sample resonance assignments were made by varying the CHAPSO or DHPC to DMPC ratio to systematically scale overall bilayer order towards the isotropic limit. In this manner, peaks could be identified based upon extrapolation to their isotropic positions, for which assignments have previously been made (Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355-358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821-3849). It was observed that the plots of CSA or dipolar coupling versus overall bilayer order obtained from DHPC and CHAPSO titrations were linear. Estimates of the intrinsic dipolar couplings and chemical shift anisotropies for pure DMPC bilayers were made by extrapolating shifts and couplings from the detergent titrations to zero detergent. Both detergent titrations led to similar "intrinsic" CSAs and dipolar couplings. Results extracted from an oriented Triton-DMPC mixture also led to similar estimates for the detergent-free DMPC shifts and couplings. The results from these experiments were found to compare favorably with limited measurements made from pure L alpha PC. This detergent-based method for assigning spectra and for determining dipolar couplings and CSA in detergent-free systems should be extendable to other lipid systems. The resulting data set from this study may prove useful in future modeling of the structure and dynamics of DMPC bilayers. In addition, the fact that experiments utilizing each of the three detergents led to similar estimates for the spectral parameters of pure DMPC, and the fact that spectral parameter versus bilayer order plots were linear, indicate that the averaged conformation and dynamics of DMPC in the presence of the three detergents are very similar to those of pure L alpha DMPC.     

Transbilayer and interbilayer phospholipid exchange in dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine large unilamellar vesicles

The rates of spontaneous interbilayer and transbilayer exchange of [3H]dimyristoylphosphatidylcholine ([3H]DMPC) were examined in DMPC and DMPC/dimyristoylphosphatidylethanolamine (DMPE) large unilamellar vesicles in the liquid-crystalline-, gel-, and mixed-phase states. DMPC desorption rates from either gel or liquid-crystalline phases containing DMPE are very similar to the corresponding rates from pure DMPC gel or liquid-crystalline phases. This is not the case for DMPC desorption from distearoylphosphatidylcholine (DSPC)-containing gel phases, where the desorption rates are significantly faster than from a pure DMPC gel phase [Wimley, W. C., & Thompson, T. E. (1990) Biochemistry 29, 1296-1303]. We proposed that the DMPC/DSPC behavior results from packing defects in gel phases composed of both DMPC and DSPC molecules because of the four-carbon difference in the acyl chain lengths of the two species. The present results strongly support this hypothesis because no such anomalous behavior is observed in DMPC/DMPE, which is similar to DMPC/DSPC in phase behavior but does not have the chain length difference. The inclusion of 10-30 mol % DMPE in DMPC bilayers was also found to have a significant effect on the rate of transbilayer movement (flip-flop) of [3H]DMPC in the liquid-crystalline phase. Between 10 and 30 mol % DMPE, flip-flop of DMPC is slowed by at least 10-fold relative to flip-flop in DMPC bilayers, and the entropy and enthalpy of flip-flop activation are both substantially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solvent effect on phosphatidylcholine headgroup dynamics as revealed by the energetics and dynamics of two gel-state bilayer headgroup structures at subzero temperatures.

The packing and dynamics of lipid bilayers at the phosphocholine headgroup region within the temperature range of -40 to -110 degrees C have been investigated by solid-state nuclear magnetic resonance (NMR) measurements of selectively deuterium-labeled H2O/dimyristoylphosphatidylcholine (DMPC) bilayers. Two coexisting signals with 2H NMR quadrupolar, splittings of 36.1 and 9.3 (or smaller) kHz were detected from the -CD3 of choline methyl group. These two signals have been assigned to two coexisting gel-state headgroup structures with fast rotational motion of -CD3 and -N(CD3)3 group, respectively, with a threefold symmetry. The largest quadrupolar splitting of the NMR signal detected from the -CD2 of C alpha and C beta methylene segment was found to be 115.2 kHz, which is 10% lower than its static value of 128.2 kHz. Thus, there are extensive motions of the entire choline group of gel-state phosphatidylcholine bilayers even at a subzero temperature of -110 degrees C. These results strongly support the previous suggestion (E. J. Dufourc, C. Mayer, J. Stohrer, G. Althoff, and G. Kothe, 1992, Biophys. J. 61:42-57) that 31P chemical shift tensor elements of DMPC determined under similar conditions are not the rigid static values. The free energy difference between the two gel-state headgroup structures was determined to be 26.3 +/- 0.9 kJ/mol for fully hydrated bilayers. Furthermore, two structures with similar free energy difference were also detected for "frozen" phosphorylcholine chloride solution in a control experiment, leading to the conclusion that the two structures may be governed solely by the energetics of fully hydrated phosphocholine headgroup.(ABSTRACT TRUNCATED AT 250 WORDS)     

New insights into the influence of monofluorination on dimyristoylphosphatidylcholine membrane properties: A solid-state NMR study

Solid-state ¹⁹F NMR spectroscopy is a method of choice to study the interactions between lipid membranes and other molecules such as peptides, proteins or drugs. Numerous fluorine-labeled NMR probes have been developed over the last few years, especially fluorine-labeled peptides. In order to develop a new kind of NMR reporter molecule and a complementary approach to fluorine-labeling of peptides, we synthesized six monofluorinated derivatives of the lipid dimyristoylphosphatidylcholine (F-DMPC), with the fluorine atom located along the acyl chain linked to the central glycerol position. To better understand the behavior of these fluorine-labeled lipids, we report here the investigation of F-DMPC membrane properties using solid-state ²H, ¹⁵N, ¹⁹F‐ and ³¹P‐NMR spectroscopy. This study was carried out on pure F-DMPC bilayers as well as F-DMPC/DMPC mixtures at various ratios. Slight perturbations were observed for pure F-DMPC multilamellar vesicles (MLVs), most noticeable for lipids with the fluorine atom located at the extremities of the acyl chain. On the other hand, no significant perturbations were observed for F-DMPC/DMPC MLVs containing up to 25% F-DMPC, nor for any fluorine-labeled bilayers that were prepared as macroscopically oriented samples. To test the interaction with some representative peptides, ¹⁵N-labeled α-helical antimicrobial peptides (AMPs) were incorporated into F-DMPC/DMPC (1/3) bilayers. ¹⁵N SS-NMR analyses confirmed that the known orientation of each AMP in pure DMPC was preserved in the presence of 25% monofluorinated DMPC, irrespective of the position of the ¹⁹F-label. In summary, F-DMPC/DMPC (1/3) model membranes can be used as NMR reporter to study membrane interactions with other molecules.     

Phase behavior of fully hydrated DMPC-amphiphilic cyclodextrin systems

With the aim of exploring relationships between the chemical structure and the physico-chemical properties of amphiphilic beta-cyclodextrin, a reappraisal of the obtaining of pure heptakis (2,3-di-O-hexanoyl)-beta-cyclodextrin (beta-CDC(6)) was undertaken. In this paper the chemical characterization of the newly synthesized beta-CDC(6) and its ability to form mixed structures with dimyristoylphosphatidylcholine (DMPC) are reported. Miscibility of the two amphiphiles is examined: (i) in monolayers formed at the air-water interface by analyzing the surface pressure-area isotherms; and (ii) in fully hydrated mixtures by differential scanning calorimetry (DSC) and X-ray diffraction at small and wide angles. Results demonstrate that the beta-cyclodextrin derivative is partially miscible to the phospholipid: intimate mixing occurs at beta-CDC(6) molar ratios smaller than 7-15 mol%, depending on the dimensional scale considered, while beyond these compositions phase separation is observed. At the air-water interface, the miscibility region of the two compounds shows non-ideal behavior characterized by the non-additivity of the molecular areas in the mixed monolayers. At the three-dimension level, the formation of a beta-CDC(6)/DMPC mixed lamellar phase occurs except at beta-CDC(6) molar ratios close to 5 mol% at which a highly ordered structure is depicted below the solid-to-liquid state transition of the DMPC hydrocarbon chains. At beta-CDC(6) contents higher than 7 mol%, the mixed assemblies coexist with excess amphiphilic cyclodextrin which then forms a separated hexagonal structure.     

In vitro effects of the antitumor drug miltefosine on human erythrocytes and molecular models of its membrane

This study was aimed at elucidating the molecular mechanisms of the interaction of the antitumor alkylphospholipid drug miltefosine with human erythrocytes (RBC) and molecular models of its membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray results showed that the drug interacted with DMPC multilayers; however, no effects on DMPE were detected. The experimental findings obtained by differential scanning calorimetry (DSC) indicated that miltefosine altered the thermotropic behavior of both DMPC and DMPE vesicles. Fluorescence spectroscopy evidenced an increase in the fluidity of DMPC vesicles and human erythrocyte membranes. Scanning electron microscopy (SEM) observations on human erythrocytes showed that miltefosine induced morphological alterations to RBC from its normal biconcave to an echinocyte type of shape. These results confirm that miltefosine interacts with the outer moiety of the human erythrocyte membrane affecting the cell morphology.     

Dibucaine effects on structural and elastic properties of lipid bilayers

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPC) were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes.     

Location and dynamics of anthracyclines bound to unilamellar phosphatidylcholine vesicles

We have exploited the intrinsic fluorescence properties of the anthracycline antitumor antibiotics to study the dependence on drug structure of relative drug location and dynamics when the anthracyclines were bound to sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Iodide quenching experiments at constant ionic strength were used to evaluate the relative accessibilities of the bound fluorophores to membrane-impermeable iodide. Iodide was found to quench the fluorescence of anthracyclines in free solution by both static and dynamic mechanisms, whereas quenching of membrane-bound fluorophores was predominantly due to the dynamic mechanism. Modified Stern-Volmer plots of anthracyclines bound to fluid-phase DMPC bilayers were linear, and the biomolecular rate constant (kq) values ranged from 0.6 X 10(9) to 1.3 X 10(9) M-1 s-1. Modified Stern-Volmer plots of anthracyclines bound to solid-phase DPPC bilayers were curved, indicative of a heterogeneous-bound drug population. A strong correlation between drug hydrophobicity and penetration of the fluorophore into the bilayer was observed for the daunosamine-containing anthracyclines. Steady-state fluorescence anisotropy measurements under iodide quenching conditions were used to investigate the diffusive motions of anthracyclines in isotropic solvent and in fluid-phase DMPC bilayers. Anthracycline derivatives free in solution exhibited limiting anisotropy (alpha infinity) values which decayed to zero at times long compared to the excited-state lifetime, in contrast to anthracyclines bound to fluid-phase DMPC bilayers, which showed nonzero alpha infinity values. Steady-state anisotropies of membrane-bound anthracyclines were found to be governed principally by alpha infinity and not by the mean rotational rate (R).(ABSTRACT TRUNCATED AT 250 WORDS)