The basic property of biomembranes

In order to explain the experimentally obtained results with biomembranes it has been assumed, that the diameter of all vesicles, defined by a lipid bilayer, form a perfect crystal. Later on it appeared that biomembranes are liquid crystals. Nevertheless already with biomembranes as crystals it was possible to calculate the diameters of all existing vesicles. As shown in the present paper, the results with liquid crystals were identical to those with a perfect crystal-model. Here I demonstrate that the geometric progression of the diameters of vesicles of biomembranes is based on the entropy of the lipids and it is shown that the diameters of biomembranes fit four geometric series. The surface areas of these series of biomembranes can be approximated by the two geometric series published previously. The experiments and the theoretical studies on which this work is based have been carried out at the Netherlands Cancer Institute, Amsterdam, The Netherlands.     

Interaction of peptides with biomembranes assessed by potential-sensitive fluorescent probes.

Peptide-membrane interaction is an important step to be evaluated in a study of the activity and mode of action of several bioactive peptides. A variety of methods are available; however, few of them satisfy the criteria of being sensitive, biocompatible, versatile, easy to perform, and allowing real-time monitoring as the use of potential-sensitive fluorescent probes. Here we review methods for detecting the effects of membrane-active peptides, even those that are not intrinsically fluorescent, on the different types of membrane potentials, with a special emphasis on studies conducted with living cells. FPE is a probe sensitive to surface potential and detects electrostatic interactions at the water-lipid interface. Di-8-ANEPPS is sensitive to dipole potential and detects membrane incorporations. Transmembrane potential changes reveal major membrane destabilizations, such as in pore formation. The combination of the information obtained from the three potential variations can lead to a more elucidative picture of the mechanisms of the interaction of relevant peptides with biomembranes.     

General Mathematical Frame for Open or Closed Biomembranes (Part I): Equilibrium Theory and Geometrically Constraint Equation

This paper aims at constructing a general mathematical frame for the equilibrium theory of open or closed biomembranes. Based on the generalized potential functional, the equilibrium differential equation for open biomembrane (with free edge) or closed one (without boundary) is derived. The boundary conditions for open biomembranes are obtained. Besides, the geometrically constraint equation for the existence, formation and disintegration of open or closed biomembranes is revealed. The physical and biological meanings of the equilibrium differential equation and the geometrically constraint equation are discussed. Numerical simulation results for axisymmetric open biomembranes show the effectiveness and convenience of the present theory.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Lipid phase transitions in model biomembranes: The effect of ions on phosphatidylcholine bilayers

Differential scanning calorimetry has been used to study the endothermic phase behaviour of some model biomembranes (i.e. phosphatidylcholine-water systems) in the presence of a wide range of alkaline, alkaline earth and heavy metal salts. Studies and comparisons were made of both cation and anion effects. Shifts occur in the temperatures of both the pre-transition and main transition endotherms. The observed shifts are smaller than those which have been reported for charged lipids, and no evidence has been found for the formation of specific complexes. Electron microscopic studies on freeze-fractured dispersions of phosphatidylcholine-water-salt systems show that with some salts the typical rippled surface observed with l-α-dimyristoyl phosphatidylcholine, when in the gel state, is replaced by a smooth surface.     

Combining molecular modeling with experimental methodologies: mechanism of membrane permeation and accumulation of ofloxacin

The interaction between ofloxacin, as a model drug of the fluoroquinolone class, and biomembranes was examined as the possible initial step in a transmembrane diffusion process. Dipalmitoylphosphatidylcholine was used for the preparation of biomembrane models. The influence of environmental conditions and protonation on molecular physicochemical behavior, and hence on the membrane interaction, was investigated by differential scanning calorimetry (DSC). This technique has been shown to be very effective in the interpretation of interactions of drug microspeciations with biomembranes. These findings suggest that the interaction occurred owing to ionic and hydrophobic forces showing how the passage through the membrane is mainly favored in the pH interval 6–7.4. It was demonstrated that a pH gradient through model membranes may be responsible for a poorly homogeneous distribution of ofloxacin (or other related fluoroquinolones), which justifies the in vivo accumulation properties of this drug. DSC experiments, which are in agreement with computational data, also showed that the complexing capability of ofloxacin with regard to Mg⁺⁺ or Ca⁺⁺ may govern the drug entrance into bacterial cells before the DNA Girase inhibition and could ensure the formation of hydrophobic and more fluid phospholipid domains on the surface of the model membrane. These regions are more permeable with regard to various solutes, as well as ofloxacin, allowing a so-called ‘self-promoted entrance pathway’. The combination of experimental methodologies with computational data allowed a further rationalization of the results and opened new perspectives into the mechanism of action of ofloxacin, namely its interaction with lipid bilayers and drug–divalent cation complex formation, which might be extended to the entire fluoroquinolone class. Ofloxacin accumulation within Escherichia coli ATCC 25922 was measured as a function of time. Also in this example, the environmental conditions influenced ofloxacin penetration and accumulation. The in vitro experiments, reported here, show that a suitable balance of hydrophilic and hydrophobic fluoroquinolone properties needs to occur for there to be increased drug permeation.     

Evaluation of membrane surface charge density: A discussion of some models

The theory of electrical double layer due to Gouy and Chapman, applied to the experimental data related to shifts of conductance-voltage curve along the voltage axis following changes in the external calcium concentration to derive values for the density of surface charges (σ) present on biomembranes, has been outlined. Similarly, the principal equations of resting membrane potential due to Kobatake and coworkers, also used for the first time by Lakshminarayanaiah to evaluate charges on biomembranes of several tissues, have been given. The values for σ derived by the first method have been considered to indicate the charge density at the channel region through which permeation of Na or K ion occurred. The values for σ derived by the second method are considered to represent the presence of charges smeared out uniformly over the membrane surface. Despite this difference in practical implementation of the concepts of the two theories which initially in their development are based on the assumption of charges covering the membrane surface uniformly, the ultimate values for σ derived by the two methods agree. The significance of this result in relation to different membrane models for surface charges is briefly discussed. Presented at the Society for Mathematical Biology Meeting, University of Pennsylvania, Philadelphia, August 19–21, 1976.     

Mathematical model of electrogenic transport through biomembranes via oligomeric channels capable of conformational transitions

A mathematical model of electrogenic ion transport across biomembranes by oligomeric channels liable to conformational transformations has been derived. The model describes changes with time of the membrane potential and near--membrane ion concentrations. Different types of the channel conductance regulation such as activation or inhibition by the permeating ions and membrane potential have been considered. It appears that in the presence of such regulations 1) the channel voltage-current curves have negative resistance regions; 2) the dependence of the quasisteady state (or resting) potential on the ion concentrations in the solution is of hysteresis nature; 3) the model may have multiple steady-state and oscillating solutions.     

Binding and antioxidant properties of therapeutically important plant flavonoids in biomembranes: insights from spectroscopic and quantum chemical studies

Plant flavonoids are emerging as novel therapeutic drugs for free radical mediated diseases, for which cell membranes mainly serve as targets for lipid peroxidation and related deleterious effects. Screening and characterization of these ubiquitous, therapeutically potent polyphenolic compounds require a clear understanding regarding their binding and possible locations in membranes, as well as quantitative estimates of relevant parameters such as partition coefficients, antioxidant and radical scavenging capacities. In this article we present perspectives emphasizing novel uses of the exquisitely sensitive 'two color' intrinsic fluorescence of plant flavonoids (which arise due to highly efficient photoinduced excited state intramolecular proton transfer (ESIPT) reactions) to explore their binding to model biomembranes consisting of phosphatidylcholine liposomes. Extension of such studies to natural biomembranes of relevant interest is also exemplified. Spectrophotometric assays reveal that typical mono- as well as poly-hydroxy substituted flavonoids have remarkable inhibitory actions on lipid peroxidation, and are significantly more potent antioxidants (2.5-4 times higher) compared to the reference compound Trolox (an water soluble derivative of vitamin E). The structure-activity relationships emerging from such studies are consistent with theoretical predictions based on quantum chemical computations.     

Protein-liposome interactions and their relevance to the structure and function of cell membranes

Summary Recent studies on the interactions of soluble proteins, membrane proteins and enzymes with phospholipid model membranes are reviewed. Similarities between the properties of such systems and the behavior of biomembranes, such as alterations in the redox potential of cytochromec after binding to membranes and effects of phospholipid fluidity on (Na + K) ATPase activity, are emphasized. The degree of correspondence between the behavior of model systems and natural membranes encourages the continuing use of model membranes in studies on protein-lipid interactions. However, some of the data on the increase of surface pressure of phospholipid monolayers by proteins and increases in the permeability of liposomes indicate that many soluble proteins also have a capability to interact hydrophobically with phospholipids. Thus a sharp distinction between both peripheral and integral membrane proteins and non-membrane proteins are not seen by these techniques. Cautious use of such studies, however, should lead to greater understanding of the molecular basis of cell membrane structure and function in normal and pathological states. Studies implicating protein-lipid interactions and (Na + K) ATPase activity in membrane alterations in disease states are also briefly discussed.An invited article.     

Interaction with membranes of the full C-terminal domain of protein NS4B from hepatitis C virus

Hepatitis C virus (HCV) NS4B protein is a transmembrane highly hydrophobic protein responsible for many key aspects of the viral replication process. The C-terminal part of NS4B is essential for replication and is a potential target for HCV replication inhibitors. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to the C-terminal domain of NS4B, NS4B(Cter). We show that NS4B(Cter) partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. NS4B(Cter) is located in a shallow position in the membrane but it is able to affect the lipid environment from the membrane surface down to the hydrophobic core. Our results identify the C-terminal region of the HCV NS4B protein as a membrane interacting domain, and therefore directly implicated in the HCV life cycle and possibly in the formation of the membranous web.     

Theorie des catastrophes et fonction physiologique membranaire

This comunication is based on a preliminary work which emphasized a topological model of biomembranes from Thom's Catastrophe Theory. In this model called swallowtail bifurcation set, the structural state of a biomembrane was within the control of two structural attractors. Then, the physiological act of this biomembrane resulted in a sudden transfer of weight from the hydrophilic attractor to the hydrophobic attractor.In this consecutive work, the physiological act appears to be one of the four stages which permit to describe the larger notion of cyclic membrane function. Two of these stages unfold in the structural axis of the swallowtail model. They prepare the two others (physiological act and refractory stages) which expand in the functional direction.This conceiving of cyclic membrane function is applied to physiological examples such as the action potential and the endocytosis. Then, changes in this function are discussed on the basis of pathological data.

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Theorie des catastrophes et fonction physiologique membranaire

     

Electrical oscillation and fluctuation in phospholipid membranes. Phospholipids can form a channel without protein

Fluctuations and/or step-wise changes in membrane potential and electrical current were observed in bilayer membranes of dioleoylphosphatidylcholine (DOPC) in the absence of any channel protein. The DOPC membranes consisted of three types: black lipid membranes, pipette-clamp membranes and lipid membranes transferred to porous filter paper by conventional Langmuir-Blodgett techniques. This finding is significant since phospholipids are the main constituents of biomembranes. Lipid molecules with a cis double bond in their carbon skeleton are suggested to be important in the gating or excitation of biomembranes.     

Dynamics of pore growth in membranes and membrane stability.

Pores can form and grow in biomembranes because of factors such as thermal fluctuation, transmembrane electrical potential, and cellular environment. We propose a new statistical physics model of the pore growth treated as a non-Markovian stochastic process, with a free energy barrier and memory friction from the membrane matrix treated as a quasi-two-dimensional viscoelastic and dielectric fluid continuum. On the basis of the modern theory of activated barrier crossing, an analytical expression for membrane lifetime and the phase diagram for membrane stability are obtained. The memory effect due to membrane viscoelasticity and the elasticity due to cytoskeletal network are found to induce sharp transitions to membrane stability against pore growth and compete with other factors to manifest rich dynamic transitions over the membrane lifetime.     

Intrinsic protein-lipid interactions. Infrared spectroscopic studies of gramicidin A, bacteriorhodopsin and Ca2+-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 H₂O and ²H₂O aqueous buffer systems. Pure lecithin-water systems, and various model biomembranes containing cholesterol, gramicidin A, bacteriorhodopsin or Ca²⁺-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 CH₂ 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 Ca²⁺-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.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.     

First-Leaflet Phase Effect on Properties of Phospholipid Bilayer Formed Through Vesicle Adsorption on LB Monolayer

Phospholipid bilayers were formed on mica using the Langmuir–Blodgett technique and liposome fusion, as a model system for biomembranes. Nanometer-scale surface physical properties of the bilayers were quantitatively characterized upon the different phases of the first leaflets. Lower hydration/steric forces on the bilayers were observed at the liquid phase of the first leaflet than at the solid phase. The forces appear to be related to the low mechanical stability of the lipid bilayer, which was affected by the first leaflet phase. The first leaflet phase also influenced the long-range repulsive forces over the second leaflet. Surface forces, measured using a modified probe with an atomic force microscope, showed that lower long-range repulsive forces were also found at the liquid phase of the first leaflet. Force measurements were performed at 300 mM sodium chloride solution so that the effect of the phase on the long-range repulsive forces could be investigated by reducing the effect of the repulsion between the second-leaflet lipid headgroups on the long-range repulsive forces. Forces were analyzed using the Derjaguin–Landau–Verwey–Overbeek theory so that the surface potential and surface charge density of the lipid bilayers were quantitatively acquired for each phase of the first leaflet.     

Structural and functional features of membranes with different cholesterol content

The paper deals with the present-day ideas of the structure and functions of membranes with different cholesterol content and relative sterols. The data on the molecular bases of lipids and sterols interaction in model systems, monolayers and liposomes, are obtained using 1H-, 13C-, 31P-NMR, EPR-investigations, methods of differential scanning calorimetry and X-ray diffraction. The methods for obtaining biomembranes with the different content of cholesterol and its effect on the membrane structure and functions are discussed. Reasons are analyzed of principal differences in behaviour of biomembranes with different content of cholesterol and respective model systems. The nature of cholesterol binding with phospholipids, proteins and its asymmetric distribution in biomembranes is considered. The cholesterol effect on the erythrocyte membrane charge density and possible changes in structural and functional properties of the membranes are demonstrated through the author's own experimental data. It is concluded that the cholesterol effect on structure and functions of the membranes is determined to a considerable extent by their phospholipid composition, localization and phase behaviour of phospholipids and sometimes depends on the membrane charge density.