Biomembrane Systems Research Articles

The Modified Stalk Mechanism of Lamellar/Inverted Phase Transitions and Its Implications for Membrane Fusion

A model of the energetics of lipid assemblies (Siegel. 1993. Biophys. J. 65:2124–2140) is used to predict the relative free energy of intermediates in the transitions between lamellar (L α ) inverted hexagonal (H II), and inverted cubic (Q II) phases. The model was previously used to generate the modified stalk theory of membrane fusion. The modified stalk theory proposes that the lowest energy structures to form between apposed membranes are the stalk and the transmonolayer contact (TMC), respectively. The first steps in the L α /H II and L α /Q II phase transitions are also intermembrane events: bilayers of the L α phase must interact to form new topologies during these transitions. Hence the intermediates in these phase transitions should be similar to the intermediates in the modified stalk mechanism of fusion. The calculations here show that stalks and TMCs can mediate transitions between the L α , Q II, and H II phases. These predictions are supported by studies of the mechanism of these transitions via time-resolved cryoelectron microscopy (Siegel et al. 1994. Biophys. J. 66:402–414; Siegel and Epand. 1997. Biophys. J. 73:3089–3111), whereas the predictions of previously proposed transition mechanisms are not. The model also predicts that Q II phases should be thermodynamically stable in all thermotropic lipid systems. The profound hysteresis in L α /Q II transitions in some phospholipid systems may be due to lipid composition-dependent effects other than differences in lipid spontaneous curvature. The relevant composition-dependent properties are the Gaussian curvature modulus and the membrane rupture tension, which could change the stability of TMCs. TMC stability also influences the rate of membrane fusion of apposed bilayers, so these two properties may also affect the fusion rate in model membrane and biomembrane systems. One way proteins catalyze membrane fusion may be by making local changes in these lipid properties. Finally, although the model identifies stalks and TMCs as the lowest energy intermembrane intermediates in fusion and lamellar/inverted phase transitions, the stalk and TMC energies calculated by the present model are still large. This suggests that there are deficiencies in the current model for intermediates or intermediate energies. The possible nature of these deficiencies is discussed.

Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis.

Surfactin, a cyclic lipopeptide antibiotic and biosurfactant produced by Bacillus subtilis, is well-known for its interactions with artificial and biomembrane systems (e.g., bacterial protoplasts or enveloped viruses). To assess the applicability of this antiviral and antibacterial drug, we determined the cytotoxicity of surfactin with a 50% cytotoxic concentration of 30 to 64 microM for a variety of human and animal cell lines in vitro. Concomitantly, we observed an improvement in proliferation rates and changes in the morphology of mycoplasma-contaminated mammalian cells after treatment with this drug. A single treatment over one passage led to complete removal of viable Mycoplasma hyorhinis cells from various adherent cell lines, and Mycoplasma orale was removed from nonadherent human T-lymphoid cell lines by double treatment. This effect was monitored by a DNA fluorescence test, an enzyme-linked immunosorbent assay, and two different PCR methods. Disintegration of the mycoplasma membranes as observed by electron microscopy indicated the mode of action of surfactin. Disintegration is obviously due to a physicochemical interaction of the membrane-active surfactant with the outer part of the lipid membrane bilayer, which causes permeability changes and at higher concentrations leads finally to disintegration of the mycoplasma membrane system by a detergent effect. The low cytotoxicity of surfactin for mammalian cells permits specific inactivation of mycoplasmas without significant deleterious effects on cell metabolism and the proliferation rate in cell culture. These results were used to develop a fast and simple method for complete and permanent inactivation of mycoplasmas in mammalian monolayer and suspension cell cultures.

The conformational analysis of peptides using fourier transform IR spectroscopy

Fourier transform infrared spectroscopy (FTIR) can be used for conformational analysis of peptides in a wide range of environments. Measurements can be performed in aqueous solution, organic solvents, detergent micelles as well as in phospholipid membranes. Information on the secondary structure of peptides can be derived from the analysis of the strong amide I band. Orientation of secondary structural elements within a lipid bilayer matrix can be determined by means of polarized attenuated total reflectance-FTIR spectroscopy. Hydrogen-deuterium exchange can be monitored by the analysis of the amide II band. This review gives some example of peptide systems studied by FTIR spectroscopy. Studies on alamethicin and alpha-aminoisobutyric acid containing peptides have shown that FTIR spectroscopy is a sensitive tool for identifying 3(10)-helical structures. Changes in the structure of the magainins upon interaction with charged lipids were detected using FTIR spectroscopy. Tachyplesin is an example of a beta-sheet containing membrane active peptide. Polarized ir spectroscopy reveals that the antiparallel beta-sheet structures of tachyplesin are oriented parallel to the membrane surface. Synthesis of peptides corresponding to functionally/structurally important regions of large proteins is becoming increasingly popular. FTIR spectroscopy has been used to analyze the structure of synthetic peptides corresponding to the ion-selective pore of the voltage-gated potassium channel. In biomembrane systems these peptides adopt a highly helical structure. Under conditions, where these peptides are aggregated the presence of some intermolecular beta-sheet structure can also be detected.

Triply-periodic nanostructures in surfactant, block copolymer, and biomembrane systems, and simulation of TEMs

First discovered in surfactant-water liquid crystalline systems, so-called ‘bicontinuous cubic phases’ have the property that hydropnilic and lipophilic microdomains form interpenetrating networks conforming to cubic lattices on the scale of nanometers. Later these same structures were found in star diblock copolymers, where the simultaneous continuity of elastomeric and glassy domains gives rise to unique physical properties. Today it is well-established that the symmetry and topology of such a morphology are accurately described by one of several triply-periodic minimal surfaces, and that the interface between hydrophilic and hydrophobic, or immiscible polymer, domains is described by a triply-periodic surface of constant, nonzero mean curvature. One example of such a dividing surface is shown in figure 5.The study of these structures has become of increasing importance in the past five years for two reasons:1)Bicontinuous cubic phase liquid crystals are now being polymerized to create microporous materials with monodispersed pores and readily functionalizable porewalls; figure 3 shows a TEM from a polymerized surfactant / methylmethacrylate / water cubic phase; and2)Compelling evidence has been found that these same morphologies describe biomembrane systems in a wide range of cells.

Quenching of fluorescein-conjugated lipids by antibodies. Quantitative recognition and binding of lipid-bound haptens in biomembrane models, formation of two-dimensional protein domains and molecular dynamics simulations.

Three model biomembrane systems, monolayers, micelles, and vesicles, have been used to study the influence of chemical and physical variables of hapten presentation at membrane interfaces on antibody binding. Hapten recognition and binding were monitored for the anti-fluorescein monoclonal antibody 4-4-20 generated against the hapten, fluorescein, in these membrane models as a function of fluorescein-conjugated lipid architecture. Specific recognition and binding in this system are conveniently monitored by quenching of fluorescein emission upon penetration of fluorescein into the antibody's active site. Lipid structure was shown to play a large role in affecting antibody quenching. Interestingly, the observed degrees of quenching were nearly independent of the lipid membrane model studied, but directly correlated with the chemical structure of the lipids. In all cases, the antibody recognized and quenched most efficiently a lipid based on dioctadecylamine where fluorescein is attached to the headgroup via a long, flexible hydrophilic spacer. Dipalmitoyl phosphatidylethanolamine containing a fluorescein headgroup demonstrated only partial binding/quenching. Egg phosphatidylethanolamine with a fluorescein headgroup showed no susceptibility to antibody recognition, binding, or quenching. Formation of two-dimensional protein domains upon antibody binding to the fluorescein-lipids in monolayers is also presented. Chemical and physical requirements for these antibody-hapten complexes at membrane surfaces have been discussed in terms of molecular dynamics simulations based on recent crystallographic models for this antibody-hapten complex (Herron et al., 1989. Proteins Struct. Funct. Genet. 5:271-280).

Modeling membrane targeting: interaction and recognition of proteins with model biomembrane systems

Model biomembrane systems are useful for understanding fundamental biochemical and biophysical barriers that confront drug targeting strategies at the cellular level. Membrane receptor-ligand recognition and binding functions, mimicking aspects of physiological membrane phenomena, demonstrate the effects of membrane organization and lipid architecture on protein binding. Phospholipase A 2 binding and hydrolysis of phospholipid monolayers produces phase-separated domains of hydrolysis products that induce enzyme adsorption and organization at the interface. Fluorescein, presented as a model hapten to a monoclonal antifluorescein antibody, is recognized at lipid vesicle, micelle, and monolayer interfaces to widely varying degrees, depending on lipid-hapten chemical architecture. Molecular dynamics simulations based on the known crystallographic coordinates for proteins in these systems are used in tandem to provide additional insight into binding requirements at interfaces on a molecular level. These examples demonstrate the utility of simplistic membrane models in understanding protein recognition and receptor-ligand interactions on membrane surfaces.

The Influence of the Local Anaesthetic Tetracaine on the Temperature and Pressure Dependent Phase Behaviour of Model Biomembranes

AbstractBy using differential scanning calorimetry and neutron small‐angle scattering, the effect of the local anaesthetic tetracaine on the temperature and pressure dependent phase behaviour of model biomembrane systems, such as pure and cholesterol—containing 1, 2‐dimyristoyl‐phosphatidylcholine bilayers, has been investigated. The results provide insight into the thermodynamic and structural consequences of the incorporation of the anaesthetic into the membranes and might thus help in understanding the molecular mechanisms underlying the pharmacological action of local anaesthetics and the antagonistic effect of pressure against anaesthesia, which has been observed in vivo. Furthermore, as tetracaine can be viewed as a model system for amphiphilic molecules, knowledge of its concentration, temperature and pressure dependent phase behaviour helps in understanding the general physico‐chemical action of amphiphilic molecules on membranes.

Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radical by reduced ubiquinone-10 in solution

Kinetic study of the reaction between tocopheroxyl (vitamin E radical) and reduced ubiquinone, n = 10) has been performed. The rates of reaction of ubiquinol with α-tocopheroxyl 1 and seven kinds of alkyl substituted tocopheroxyl radicals 2–8 in solution have been determined spectrophotometrically, using a stopped-flow technique. The result shows that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of tocopheroxyls increases. For the tocopheroxyls with two alkyl substituents at ortho positions (C-5 and C-7), the second-order rate constants, k 1, obtained vary i n the order of 10 2, and decrease predominantly, as the size of two ortho-alkyl groups (methyl, ethyl, isopropyl and tert-buty) in tocopheroxyl increases. On the other hand, the reaction between tocopheroxyl and ubiquinone-10 (oxidized ubiquinone) has not been observed. The result indicates that ubiquinol-10 regenerates tocopherol by donating a hydrogen atom of the 1-OH and/or 4-OH group to the tocopheroxyl radical. For instance, the k 1 values obtained for α-tocopheroxyl are 3.74 · 10 5 M −1 · s −1 and 2.15 · 5 M −1 · s −1 in benzene and ethanol solution at 25°C, respectively. The above reaction rates, k 1, obtained were compared with those of vitamin C with α-tocopheroxyl reported by Packer et al. ( k 2 = 1.55 · 10 6 M −1 · s −1) and Scarpa et al. ( K 2 = 2 · 10 5 10 5 M −1 · s −1), which is well known as a usual regeneration reaction of tocopheroxyl in biomembrane systems. The result suggests that ubiquinol-10 also regenerates the tocopheroxyl to tocopherol and prevents lipid peroxidation in various tissues and mitochondria.

High pressure neutron-scattering experiments on phospholipid model biomembranes

Abstract By use of neutron small-angle scattering (SANS) the structural properties of saturated and unsaturated phosphatidylcholine model biomembrane systems have been investigated in the temperature range from 0°C to 70°C and at pressures up to 3.5 kbar. The effect of chemical constitution of the hydrocarbon chains of the lipids and the effect of cholesterol on the thermotropic and barotropic phase behaviour is discussed.

生理活性物質の生体膜系への移行と活性発現

This review article deals with i) the relation between chemical structures and partition coefficients of bioactive compounds, ii) the possible mode of interaction of ionizable compounds in biomembranes, iii) phase transition of phospholipid membranes induced by bioactive compounds, iv) the affinity of bioactive compounds for liposomes, v) the process of transfer of chemical compounds into membranes and vi) the action mechanism of membrane-targeting bioactive compounds in biomembrane systems. The biological activities of organic compounds is mostly dependent on their hydrophobicities determined by their partition coefficients between octanol and water [P (oct) ]. Similarly, the partition of organic compounds between liposomes and water is wall correlated with P (oct) . On transfer of compounds into liposome membranes, change of the phase transition temperature (Tm) takes place. The mechanism of change in Tm induced by incorporation of compounds into phospholipid membranes is discussed in terms of the structure of biocative compounds and phospholipids.Next, the process of transfer of bioactive compounds into phospholipid membranes is described. There are two possible models of the transfer : a monomer transfer model and a transient collision model. The former process is regarded as available for various compounds. Finally, the mechanism of action of membrane-targeting bioactive compounds is discussed. There are two kinds of mechanism : a sitespecific mechanism and a non-site-specific mechanism. The site-specific mechanism consists of the direct interaction of bioactive compounds with membrane proteins. This specific interaction leads to specific biological activity. In the non-site-specific mechanism, the interaction of compounds with the phospholipid part of the membrane leads to modification of the state of a membrane protein that responds specifically to a certain change of phospholipid membrane property, inducing specific biological activity.

Freezing of isolated thylakoid membranes in complex media

Chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw treatment in a buffered medium containing 70 mM KCl, 30 mM NaNO3 and 20 mM K2SO4 in different combinations. In the presence of the three predominant inorganic electrolytes, inactivation of photophosphorylation was mainly caused by a decrease in the capacity of the photosynthetic electron transport; release of proteins from the membranes was not manifest and light-induced H(+) gradient and proton permeability were largely unaffected. Omission of nitrate from the medium had little effect. When either sulfate or chloride or both were omitted prior to freezing, inactivation of photophosphorylation was correlated with stimulation of the phosphorylating electron flow, marked increase in H(+) permeability and loss of the ability of the thylakoids to accumulate protons in the light. In the absence of sulfate, uncoupling was mainly a consequence of the dissociation of chloroplast coupling factor (CF1). Partial restoration of proton impermeability and pH gradient occurred upon the addition of N,N'-dicyclohexylcarbodiimide (DCCD). When sulfate was present but chloride omitted, CF1 remained attached to the membranes and the addition of DCCD had no effect, indicating that the increase in proton efflux was caused by a different mechanism. It is concluded that sulfate stabilizes the CF1 and prevents its release from the membranes, but KCl is also necessary for maintaining the low permeability of the membranes to protons. The importance of complex media for investigations on isolated biomembrane systems is stressed.

Phospholipid monolayers at the mercury / water interface

This paper describes the formation and properties of stable phospolipid monolayers on mercury electrodic surfaces. Various voltammetric techniques are used to investigate their behavior in the presence of an electric field. The permeability of the films to faradaic reactions is evaluated using Cd(II) and Zn(II) as probes. Between −0.2V and −0.7 (vs. Ag/AgCl) the lipids are oriented with their hydrocarbon moiety next to the mercury. Experimental evidence indicates the lipid layers are fluid and there is coiling and overlap of the hydrocarbon chains. At more positive and more negative potentials the membrane undergoes thinning and becomes permeable to faradaic reactions. At more negative potentials a voltammetric peak is consistent with a ferroelectric type switching process which appears to involve the P—N dipoles. These adsorbed phospholipid films can be used in future studies of model biomembrane systems.

Derivative Raman spectroscopy applied to biomembrane systems

AbstractDerivative Stokes‐Raman spectroscopy has been applied to biomembrane systems containing the fluorescent antibiotic amphotericin B. The membranes studied included weakly scattering solutions of dimyristoylphosphatidylcholine (DMPC) and multilamellar liposomes composed of DMPC and cholesterol. In this techqnique, the wavelength of an Ar ion‐laser pumped, tunable CW Rhodamine 6G dye laser was sinusoidally modulated with an electro‐optic birefringent tuner. The wavelength‐modulated Raman signal becomes amplitude‐modulated at the exit slit of a scanning monochromator. The signal, demodulated by a phase‐sensitive detector, was proportional to the first derivative of the Raman spectrum under appropriate conditions. The results indicate that this modulation technique can offer improvements in Raman signal‐to‐noise ratios of up to a factor of two for the conformationally sensitive phospholipid acyl‐chain vibrations of the biomembranes.

Intrinsic protein-lipid interactions: Infrared spectroscopic studies of gramicidin A, bacteriorhodopsin and Ca 2+-ATPase in biomembranes and reconstituted systems

Infrared spectroscopy has been applied to the study of a number of aqueous systems of model and natural biomembranes. The absorption bands arising from water and buffer solutions were eliminated by means of an infrared spectrometer data station. Spectra were examined using H 2O and 2H 2O aqueous buffer systems. Pure lecithin-water systems, and various model biomembranes containing cholesterol, gramicidin A, bacteriorhodopsin or Ca 2+-ATPase were examined. The infrared spectra of the reconstituted biomembranes were compared with those of the corresponding natural biomembranes, i.e. the purple membrane of Halobacterium halobium and also sarcoplasmic reticulum membranes, respectively. Changes in lipid chain conformation caused by the various intrinsic molecules incorporated within the model lipid bilayer structures were monitored by studying the shifts in frequency (cm −1) of the CH 2 symmetric and asymmetric absorption bands arising from the lipid chains. The effect of gramicidin A and also the intrinsic proteins, as indicated by the shift of band frequencies, are quite different from that of cholesterol at temperatures above the main lipid transition temperature t c. Cholesterol causes a reduction in gauche isomers which increases with concentration of cholesterol within the lipid bilayer. Whilst gramicidin A and the intrinsic proteins at low concentration cause a reduction of gauche isomers, at higher concentrations of these molecules, however, there is little difference in gauche isomer content when the intrinsic molecule is present compared with that of the fluid lipid alone. These results are considered and compared with previously published studies using deuterium nuclear magnetic resonance spectroscopy on similar model biomembrane systems. Below the lipid t c value, all the intrinsic molecules produce an increase in gauche isomers presumably by disturbing the lipid chain packing in the crystalline lipid arrangement. Information about the polypeptide structure within gramicidin A. the reconstituted proteins and also the proteins in the natural biomembranes was obtained by examining the region of the infrared spectrum between 1600 and 1700 cm −1 associated with the amide I and amide II bands. An examination of the infrared band frequencies of the different systems in this region leads to the conclusions: (1) that gramicidin A within a phospholipid bilayer structure probably has a single helix rather than a double helix structure; (2) that there are differences in band widths of the reconstituted Ca 2+-ATPase and bacteriorhodopsin compared with the spectra of the corresponding sarcoplasmic reticulum and purple membrane; (3) different membrane proteins adopt different conformations as evinced by a comparison of the spectra of the sarcoplasmic reticulum and purple membrane; (4) the polypeptide arrangement in the purple membrane is mainly helical but the abnormal frequency of the amide I band suggests that some distortion of the helix occurs: and (5) the sarcoplasmic reticulum membrane contains unordered as well as α-helix polypeptide arrangements.

A rapid controller of temperature for use in determining Arrhenius profiles in biomembrane systems.

To minimize artifacts in temperature-velocity (Arrhenius) profiles due to aging of preparations of biological membranes, a rapid controller of temperature was developed for spectrophotometric or polarographic (02 electrode) measurements. The reaction mixture is cooled or heated through contact with Peltier elements. One Pt temperature sensor in the cuvette or electrode holder controls current flow into the Peltier units, and another Pt temperature sensor in the reaction mixture is used to read out the sample temperature on a meter or recorder, and to provide feedback control. The sample temperature can be reproducibly set to within 0.1 degree C, with a noise level of 0.04 degrees C or less; a change of 4 degrees C takes 1 min.

Effects of chain packing and chain mobility on the raman spectra of biomembranes

The degree of lateral crystal-like order between hydrocarbon chains in biomembrane systems can be estimated from Raman measurements in the C-H stretching region. Observations on the temperature dependence of the Raman spectra of crystalline n- C 16 H 34 and the urea clathrate of n- C 16 H 34 have enabled us to separate to some extent the overlapping effects of chain packing and chain mobility, effects that are normally not distinguished in considering lateral order. The mobility is associated with the freedom of an extended chain to rotate and twist about its long axis. A high degree of such motion must be ascribed to n- C 16 H 34 in a urea clathrate in order to explain the unusual temperature behavior observed for Raman bands at 2885 and 1174 cm −1. Comparison of the temperature behavior of the Raman spectra of the clathrate with that of crystalline n- C 16 H 34 permits the effects due to packing and to mobility to be distinguished. The same effects can be expected to be present in the Raman spectra of biomembranes.

Difference infrared spectroscopy of aqueous model and biological membranes using an infrared data station

A Perkin-Elmer infrared Data Station associated with a simple IR spectrometer (model 298) is shown to give excellent results with aqueous model and biomembrane systems. Examples are presented of difference spectra obtained with lipid--water systems, reconstituted lipid--protein systems and a natural biomembrane. The spectra of the lipid after water subtraction and of the intrinsic protein after lipid subtraction from a model reconstituted Ca2+-ATPase membrane system are shown. The potential for studying intrinsic protein conformations is emphasised.

Significance of multiple forms of brain monoamine oxidase in situ as probed by electron spin resonance.

Spin-labeled hydroxyamphetamine, a competitive reversible inhibitor of brain monoamine oxidase, has been shown to be useful as an electron spin resonance (ESR) probe of the microenvironment of the active sites of the possible monoamine oxidase multiple forms. The ESR spectrum of spin-labeled hydroxyamphetamine was strongly quenched upon binding to the enzyme. The conformation of the active site of rat brain monoamine oxidase existing in various physical states, i.e. monoamine oxidase in situ (intact brain mitochondria), crude solubilized monoamine oxidase (MAOS) and isolated monoamine oxidase fractions (MAOa and MAOb) were critically and systematically examined. Nonlinear least squares regression analyses have been used to fit the binding data (obtained at room temperature with varying spin-labeled hydroxyamphetamine concentrations) to three groups of independent noninteracting ligand-binding models. A Gibbs-Helmholtz relationship was applied to the interpretation of the measured apparent association constant K as a function of temperature ranging from 4-50 degrees with increments of 2 degreesmfrom the extracted intensive parameters, k (intrinsic association constant) and deltaF (intrinsic free energy), as well as the apparent heat, deltaH, it was clear that the microenvironment of the binding sites existing in the more purified enzyme fractions MAOa and MAOb were similar to those found in the crude solubilized enzyme. More importantly, they correlated well with the conformation of the sites characterized in situ. The data suggested that the microenvironment of this multienzyme system was unperturbed in spite of the treatment due to the isolation process. In terms of the composition of binding sites, MAOa appeared to be heterogeneous while MAOb appeared to be more homogeneous. Since the isolated fractions MAOa and MAOb possessed marked different substrate specificities, these observations directly implied that monoamine oxidase multiple forms do exist in situ. The extracted extensive parameters, n (specific binding activity, nanomoles/mg of protein), as well as the measured characteristic transition temperatures, indicated that the relative abundance of the sites which directly affected substrate specificities was indeed altered. The consistency of the characteristic transition temperatures of 21 degrees and 38 degrees for the case of intact membrane preparations was particularly significant. A tenable hypothesis is that the manipulation in the composition of the monoamine oxidase binding forms through intimate lipid-protein interactions, which has been amply demonstrated in many biomembrane systems to be functionally important might be the underlying regulatory mechanism in vivo.

Fluorescence studies of benzo-[a]-pyrene in liposome membrane systems

This investigation concerns the change in the fluorescence properties of benzo-[a]-pyrene when added to membrane systems. The feasibility of the fluorescence quenching technique using metal ions which have specific transport properties to study diffusion processes of carcinogenic benzo-[a]-pyrene molecules in biomembrane systems is demonstrated.