Artificial lipid bilayer membranes

The lipid molecule is the main constituent of biological cell membranes. In aqueous solutions amphiphilic lipid molecules form self-assembled structures such as bilayer vesicles, inverse hexagonal and multi-lamellar patterns, and so on. Among these lipid assemblies, construction of the lipid bilayer on a solid substrate has long attracted much attention due to the many possibilities it presents for scientific and practical applications [4]. Use of an artificial lipid bilayer often gives insight into important aspects ofbiological cell membranes [5-7]. The wealth of functionality of this artificial structure is the result of its own chemical and physical properties, for example, two-dimensional fluidity, bio-compatibility, elasticity, and rich chemical composition. 

A second approach with respect to anisotropic flavin (photo-)chemistry has been described by Trissl 18°) and Frehland and Trissl61). These authors anchored flavins in artificial lipid bilayers by means of C18-hydrocarbon chains at various positions of the chromophore. From fluorescence polarization analysis and model calculations they conclude, that the rotational relaxation time of the chromophore within the membrane is small compared to the fluorescence lifetime (about 2 ns74)). They further obtain the surprising result that the chromophore is localized within the water/lipid interface, with a tilt angle of about 30° (long axis of the chromophore against the normal of the membrane), irrespective of the position where the hydrocarbon chain is bound to the flavin nucleus. They estimate an upper limit of the microviscosity of the membrane of 1 Poise. 

A parallel development came from studies on artificial lipid bilayer membranes. Hladky and Hay don (1984) found that when very small amounts of the antibiotic gramicidin were introduced into such a membrane, its conductance to electrical current flow fluctuated in a stepwise fashion. It looked as though each gramicidin molecule contained an aqueous pore that would permit the flow of monovalent cations through it. Could the ion channels of natural cell membranes act in a similar way To answer this question, it was first necessary to solve the difficult technical problem of how to record the tiny currents that must pass through single channels. 

The frequency response of various chemical constituents of nerve membrane was studied. Biological membranes in general consist of lipids and proteins. Firstly, impedance characteristics of artificial lipid bilayer membranes are examined using lecithin-hexadecane preparations. It was observed that the capacitance of plain lipid membranes was independent of frequency between 100 Hz and 20 KHz. Moreover, application of external voltages has no effect up to 200 mV. Secondly, membrane capacitance and conductance of nerve axon were investigated. There are three components in nerve membranes, i.e., conductance, capaci-... 

Channel activity is best studied electrochemically as charged species cross a cell membrane or artificial lipid bilayer. There is a difference in electrical potential between the interior and exterior of a cell leading to the membrane itself having a resting potential between -50 and -100 mV. This can be determined by placing a microelectrode inside the cell and measuring the potential difference between it and a reference electrode placed in the extracellular solution. Subsequent changes in electrical current or capacitance are indicative of a transmembrane flux of ions. 

Ions and small molecules may be transported across cell membranes or lipid bilayers by artificial methods that employ either a carrier or channel mechanism. The former mechanism is worthy of brief investigation as it has several ramifications in the design of selectivity filters in artificial transmembrane channels. To date there are few examples where transmembrane studies have been carried out on artificial transporters. The channel mechanism is much more amenable to analysis by traditional biological techniques, such as planar bilayer and patch clamp methods, so perhaps it is not surprising that more work has been done to model transmembrane channels. 

For this purpose liposomes are used as lipid phase. Unilamellar liposomes are artificial lipid bilayer vesicles. They can be considered as real model bilayer membranes as they ideally consist of a circular bilayer membrane. The hydrophobic acyl chains are assembled in the hydrophobic core of the liposome whereas the hydrophilic head groups point to the water in the inside and outside of the vesicle. Liposomes can be produced from a variety of lipids and from mixtures of lipids. This possibility allows studying the influence of membrane constituents on the partition of solutes. Kramer et al. (1997) studied the influence of the presence of free fatty acids in membranes on the partition behaviour of propranolol. The influence on a-Tocopherol in membranes on the partition behaviour of desipramine has been reported recently (Marenchino et al. 2004) using a liposome model. 

Information Conversion and Amplification - Signal Transduction When an extracellular signal is recognized by a receptor on a cell membrane, the G-protein activates the enzyme inside the cell. The activation of an enzyme by external chemicals can be mimicked using a system consisting of an artificial receptor and an enzyme immobilized on an artificial lipid bilayer membrane. 

Clegg RM, Vaz WLC. Translational diffusion of proteins and lipids in artificial lipid bilayer membranes. A comparison of experiment with theory. In Progress in Protein-Lipid Interactions, Vol. I. Watts A, De Pont JJHHM, eds. 1985. Elsevier, Amsterdam, The Netherlands, pp. 173-229... 

Structural homologies between PFTs and other toxins have not been identified. However, the process of membrane permeabilization may be operative in many cases where proteins have to escape from an intracellular compartment. Well known examples are diphtheria toxin, the neurotoxins and anthrax toxin. Specific domains in many intracel-lularly active toxins have in fact been shown to produce pores in artificial lipid bilayers, and membrane permeabilization is thought to form the basis for translocation of the active moieties from the late endo-some to the cytoplasm (reviewed in Montecucco et ai, 1994). The molecular mechanism of this translocation remains obscure. In the... 

J.W. Morzycki and co-workers described the synthesis of dimeric steroids to be used as components of artificial lipid bilayer membranes. The key coupling of two steroid derivatives was achieved by the Wurtz reaction. The steroid primary alkyl iodide was dissolved in anhydrous toluene and treated with an excess of sodium metal. After 20h of reflux, the desired homocoupled product was obtained in moderate yield along with a considerable amount (36%) of the reduced compound. 

As a consequence, researchers from different disciplines of the life sciences ask for efficient and sensitive techniques to characterize protein binding to and release from natural and artificial membranes. Native biological membranes are often substituted by artificial lipid bilayers bearing only a limifed number of components and rendering the experiment more simple, which permits the extraction of real quantitative information from binding experiments. Adsorption and desorption are characterized by rate constants that reflect the interaction potential between the protein and the membrane interface. Rate constants of adsorption and desorption can be quantified by means of sensitive optical techniques such as surface plasmon resonance spectroscopy (SPR), ellipsometry (ELL), reflection interference spectroscopy (RIfS), and total internal reflection fluorescence microscopy (TIRE), as well as acoustic/mechanical devices such as the quartz crystal microbalance (QCM)... 

The lateral mobility of proteins and lipids in natural and artificial lipid bilayer membranes was determined by different methods. For long-range mobility, fluorescence recovery after photobleaching (13-15) and electrophoresis of membrane components (16) were employed. We employed the electrophoresis method for determination of the eletrophoretic and diffusional mobilities of PSI in the plane of hypotonically inflated, spherical thylakoid vesicles. To monitor the redistribution of PSI particles, we made use of the spatial characteristics of the contribution of PSI particles to electrophotoluminescence (EPL) (17, 18). The contribution of PSII to EPL was eliminated by heat treatment of the chloroplasts (19). The EPL originates from the PSI particles at the hemisphere of the vesicles at which the induced electrical field destabilizes the photoinduced charge separation (18). The electrophoretic and diffusional mobilities were measured in vesicular suspensions to avoid immobilization for microscopic visualization (20). The photosynthetic membranes are devoid of cytoskeletal elements that might interfere with the lateral mobility. 

Solute permeability is also a function of membrane composition. The rate of permeation of doxorubicin measured through artificial lipid bilayers composed of cholesterol, PC, PS, PE, and SM (abbreviations defined in Table 5.1) changes substantially with composition (Figure 5.7). This is an important finding, since cells have distinct membrane compositions. In addition, the composition of the individual leaflets of a membrane can vary (Table 5.1), suggesting the possibility of asymmetric passive transport (i.e., permeabilities that differ depending on the direction of transport, out-to-in or in-to-out). 

Fig. 6. Partition coefficients and permeability coefficients of amides as a function of carbon chain length. Dashed hues partition coefficients for (oUve oil + fatty acid)/water system (OOFA) or ether. Solid lines permeabihties across red cell membranes of dog or man, as indicated data from (11 ] or across artificial lipid bilayer membranes, data from Poznansky et al. [25].

Artificial lipid bilayer membranes can be made [22,23] either by coating an orifice separating two compartments with a thin layer of dissolved lipid (which afterwards drains to form a bilayered structure—the so-called black film ) or by merely shaking a suspension of phospholipid in water until an emulsion of submicroscopic particles is obtained—the so-called liposome . Treatment of such an emulsion by sonication can convert it from a collection of concentric multilayers to single-walled bilayers. Bilayers may also be blown at the end of a capillary tube. Such bilayer preparations have been very heavily studied as models for cell membranes. They have the advantage that their composition can be controlled and the effect of various phospholipid components and of cholesterol on membrane properties can be examined. Such preparations focus attention on the lipid components of the membrane for investigation, without the complication of protein carriers or pore-forming molecules. Finally, the solutions at the two membrane interfaces can readily be manipulated. Many, but not all, of the studies on artificial membranes support the view developed in the previous sections of this chapter that membranes behave in terms of their permeability properties as fairly structured and by no means extremely non-polar sheets of barrier molecules. 

Sahl H-G, Kordel M, Benz R. Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin- Arch Microbiol 1987 149 12 -124. 

Electroporation has been studied in different systems as artificial lipid bilayers, lipid vesicles, and animal and plant cells, and the effect has been considered at the level of plasma membranes. During the last two decades nanoelectroporation has been developed. The extremely short pulses applied in this approach allow the poration to occur at the level of subcellular structures. 

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