Bilayers, artificial

The question is How much can one infer from Dr. Thomas diffusion experiments on the diffusion mechanism in large (bulk) artificial bilayer membranes about mechanisms on bilayer membranes of phospholipids with proteins inclusion Some part of the formulation may break down because we pass from bulk to surface only, from macro- to microdescription. 

I would like to extend Prof. Simon s characterizations of these beautiful new molecules to include a description of the effects on lipid bilayers of his Na+ selective compound number 11, which my post-doctoral student, Kun-Hung Kuo, and I have found to induce an Na+ selective permeation across lipid bilayer membranes [K.-H. Kuo and G. Eisenman, Naf Selective Permeation of Lipid Bilayers, mediated by a Neutral Ionophore, Abstracts 21st Nat. Biophysical Society meeting (Biophys. J., 17, 212a (1977))]. This is the first example, to my knowledge, of the successful reconstitution of an Na+ selective permeation in an artificial bilayer system. (Presumably the previous failure of such well known lipophilic, Na+ complexing molecules as antamanide, perhydroan-tamanide, or Lehn s cryptates to render bilayers selectively permeable to Na+ is due to kinetic limitations on their rate of complexation and decomplexation). 

Peracchia C, Shen L Gap junction channel reconstitution in artificial bilayers and evidence for calmodulin binding sites in MIP26 and connexins from rat heart, liver and xenopus embryo in Hall JE, Zampighi GA, Davis RM (eds) Gap Junctions. Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 163-170. 

On the other hand, as we have already seen, cholesterol tends to reduce the mobility of molecules in membranes and causes phospholipid molecules to occupy a smaller area than they would otherwise. Myelin is especially rich in long-chain sphingolipids and cholesterol, both of which tend to stabilize artificial bilayers. Within our bodies, the bilayers of myelin tend to be almost solid. Bilayers of some gram-positive bacteria growing at elevated temperatures are stiffened by biosynthesis of bifunctional fatty acids with covalently joined "tails" that link the opposite sides of a bilayer.149... 

Tab. 1.8 The physical characteristics of artificial bilayers and biological membranes. (Reprinted from Table 2.3 of ref. 2, with permission from Macmillan)...

However, as pointed out earlier, the artificial bilayer membrane, the BLM, can be made to serve as a model for the reality of biomembranes. One forms the BLM itself (Section 14.2) and then introduces into it various entities in order to examine their chemical and electrochemical effects. The appropriate membrane can be assembled by the use of a Langmuir-Blodgett trough in which long lipid molecules (those that make up the bilayer) are floated on the surface of water and then gently pushed together by a plastic slider (Rejou-Michel and Habib, 1986). A sensitive mechanism measures the force of this pushing and then when the force necessary increases suddenly, one knows the molecules have all been pushed into contact and a monolayer formed. 

Artificial bilayer lipid membranes (BLM) have an electrical conductivity of k = 10-14-10-12 S cm-1, less than cell membranes by a factor of 106. The conductivity is increased to physiological levels with the introduction of electron acceptors or proteins in the artificial membranes that form charge transfer complexes with the lipids, as for solid state lipids11. 

In summary, the incorporation of Ceo into artificial bilayer membranes, despite being successful in principle, gives rise to a number of unexpected complications. In consideration of the strong aggregation forces among fullerene cores, it is imperative to separate the individual fullerene moieties. Only the adequate hydrophilic-hydrophobic balance of the host matrix is an appropriate means to hinder the spontaneous cluster formation [88]. 

As in the case of the ATP synthase, the most convincing evidence for the function of the respiratory chain as an autonomous proton pump comes from the ability to purify energy-conserving segments of the chain and reconstitute them into artificial bilayers with the recovery of their proton translocating capacity [21,22]. 

Annexins isolated from chondroblast matrix vesicles may be reconstituted with phospholipids to form calcium ion channels in the complete absence of Ca2+ ions. Indeed, annexin V has domains that directly bind calcium ions glutamate and aspartate residues provide the ion binding site (EF-hand domains). Figure 9.6b illustrates putative annexin V channels that mediate an influx of Ca2+ ions into artificial bilayers and liposomes (detectable with a calcium-sensitive fluorescent dye). These in vitro annexin Ca2+ channels, and also the Ca2+ influx into matrix vesicles in cell culture and in vivo, are blocked by Zn2+ ions, or a derivative of 1,4-benzothiazepine (inhibitor K201). 

In summary, incorporation of [60]flillerene into artificial bilayer membranes, despite being successful in principle, nevertheless, disclosed a number of unexpected complications. The most dominant parameter, in this view, is the strong aggregation forces among the fullerene cores. The lack of appropriately structured domains within the vesicular hosts, which could assist in keeping the fullerene units apart, is believed to be the reason for the instaneous cluster formation. The incorporation of a number of suitably functionalized derivatives, which on their own bear hydrophobic and hydrophilic substructures, will be discussed further below. 

Note that clarification of the spatial localization of the electron-transfer chain components inside the artificial bilayer membranes is of a key value for the development of biomimetic systems modeling natural photosynthesis. The direct methods of identification of localization specificity of the functional molecules are usually quite laborious. For this reason, in practice, in this particular research, some studies commonly make use of certain analogs of molecular electron relays or of special molecules such as, e.g., paramagnetic spin labeled ones [2,5,6]. 

All these effects have an important, hitherto mostly neglected influence on drug activity [214]. The differences in inhibitory activities of trimethoprim (TMP) analogs in cell-free and whole cell systems of Escherichia coli strains being sensitive and resistant to TMP, the interaction of amphiphilic benzylamines with artificial bilayers, the interaction of neuroleptics with bilayer membranes (measured by NMR techniques), and the reversal of multidrug resistance by amphiphilic agents could quantitatively be described in relation to these effects [214]. 

The changes in phase transition of artificial bilayers for example upon addition of drugs is only a parameter which allows an interpretation of strength and type of interaction with the studied phospholipid and is useful for the derivation of quantitative structure-interaction relationships. 

Unit conductances were seen only when the polyenes were added to both sides of the artificial bilayer, reminiscent again of the proposed mechanism for gramicidin pore formation. Many of the properties of single channels formed by the polyenes are similar to those for gramicidin, as for example, complex IjV relations. In this instance, however, the channels are anion selective (for membranes bathed in KCl the transport numbers for K and Cl were 0.15 and 0.85, respectively) in contrast to those formed by gramicidin. The structure of the single channels produced by polyenes is unknown, but it seems likely that they consist of polymeric assemblies of polyenes associated with sterols [27]. 

There seems little doubt therefore that permeant molecules which possess suitable ion binding sites and which are perhaps water-lined provide a means for ion translocation across artificial bilayers. 

In a further example of pore formation in artificial bilayers, the properties of alamethicin appear to be rather more complex than those for gramicidin and polyenes. 

The physical description of sodium currents in excitable membranes, together with evidence cited earlier [39], makes it certain that sodium moves through the membrane by some sort of channel. When the temperature is lowered, nerve membranes show no abrupt changes in ionic conductance as is found in artificial bilayers incorporating carrier substances [69]. 

One of the greatest obstacles in the successful modeling of the cytochrome P-450 system is the choice and control of electrons for the reductive activation of dioxygen. In our laboratory we began by designing a system that closely matched the environment of the native enzyme. Artificial bilayers in the form of polymerizied vesicles of 9 were utilized in an attempt to compartmentalize and separate the various components of the artificial enzyme system (Figure 7).2i... 

Several interesting review articles have been recently published focusing on the use of NMR methods to study peptide-lipid and small molecular weight molecule interactions in model and natural membranes. Maler as well as Kang and Li highlighted the unique possibilities of solution-state NMR to investigate the structure, dynamics and location of proteins and peptides in artificial bilayers and peptide-lipid interactions. On the other hand, Renault et reviewed recent advances in cellular solid-state nuclear magnetic resonance spectroscopy (SSNMR) to follow the structure, function, and molecular interactions of protein-lipid complexes in their cellular context and at atomic resolution. 

For a lecithin with chain containing 18 carbon atoms, a value of d = 70 A is found, which is nearly equal to two times the length of a fully extended lecithin molecule. This and other findings support the conclusion that the membrane consists essentially of a bimolecular layer (bilayer) of oriented lipid molecules. The polar head groups of the lipid molecules point toward the aqueous medium, whereas the fatty acid chain forms the interior of the membrane. Some hydrocarbon solvent remains dissolved in the film, but otherwise the structure of the artificial bilayer closely resembles the arrangement of the lipid molecules in biological membranes. Experiments with artificial lipid membranes have indicated some of the basic mechanisms by which ions may cross biological membranes [323]. 

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