Model membrane experiments

As an illustrative example for the successful realization of this concept, here the interplay between three disciplines, organic synthesis, biophysics and cell biology in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in precise molecular detail is described. The interplay is highlighted using the Ras protein as a representative example. Included herein is the development of methods for the synthesis of Ras-derived peptides and fully functional Ras proteins, the determination of the biophysical properties, in particular the ability to bind to model membranes, and finally the use of synthetic Ras peptides and Ras proteins in cell biological experiments. 

Natural biological membranes consist of lipid bilayers, which typically comprise a complex mixture of phospholipids and sterol, along with embedded or surface associated proteins. The sterol cholesterol is an important component of animal cell membranes, which may consist of up to 50 mol% cholesterol. As cholesterol can significantly modify the bilayer physical properties, such as acyl-chain orientational order, model membranes containing cholesterol have been studied extensively. Spectroscopic and diffraction experiments reveal that cholesterol in a lipid-crystalline bilayer increases the orientational order of the lipid acyl-chains without substantially restricting the mobility of the lipid molecules. Cholesterol thickens a liquid-crystalline bilayer and increases the packing density of lipid acyl-chains in the plane of the bilayer in a way that has been referred to as a condensing effect. 

Model Membranes and Their Characteristics Liposome preparation and size characterization, 171, 193 preparation of microcapsules from human erythrocytes use in transport experiments of glutathione and its S-conjugate, 171, 217 planar lipid-protein membranes strategies of formation and of detecting dependencies of ion transport functions on membrane conditions, 171,... 

As expected, system 13 did in fact bind and transport zwitterionic a-amino acids through a model membrane barrier with good selectivity under conditions where the porphyrin-derived control system (14), lacking the carboxylate anion chelation ability inherent in 13, would not. Specifically, it was found that at neutral pH compound 13 acts as a very efficient carrier for the through model membrane (H2O-CH2CI2-H2O) transport of phenylalanine and tryptophan. Further, in direct competition experiments, L-phenylalanine was found to be transported four times faster than L-tryptophan and 1000 times faster than L-tyrosine. As implied above, little or no transport was observed when a porphyrin control (14) was used. Nor was significant transport observed when a mixture of sapphyrin and lasalocid was used. 

A very brief description of biological membrane models, and model membranes, is given. Studies of lateral diffusion in model membranes (phospholipid bilayers) and biological membranes are described, emphasizing magnetic resonance methods. The relationship of the rates of lateral diffusion to lipid phase equilibria is discussed. Experiments are reported in which a membrane-dependent immunochemical reaction, complement fixation, is shown to depend on the rates of diffusion of membrane-bound molecules. It is pointed out that the lateral mobilities and distributions of membrane-bound molecules may be important for cell surface recognition. 

Fig. 4. Schematic representation of transient method employed by Devaux and McConnell9 to measure the rates of lateral diffusion of phospholipids in model membranes. The upper diagram represents a concentrated patch of labels at the beginning of the experiment, time f = 0. At later times f>0, the molecules diffuse laterally, as shown in the lower two drawings. The paramagnetic resonance spectra depend on the spin-label concentration in the plane of the membrane, and an analysis of the time dependence of these spectra yielded the diffusion constant. [Reprinted with permission from P. Devaux and H. M. McConnell, J. Am. Chem. Soc., 94, 4475 (1972). Copyright by American Chemical Society.]...

Fig. 11. Evidence that a membrane-associated immunochemical reaction (complement fixation) depends on the mobility of the target hapten (IX) in the plane of a model membrane. The extent of the immunochemical reaction, complement fixation, is measured by A Absorbance at 413 nm. Temperature is always 32°C, which is above the chainmelting temperature (23°C) of dimyristoylphosphatidylcholine used for the data given in A and below the chain-melting transition temperature (42°C) of dipalmitoylphosphatidyl-choline used for the data in B. Thus A refers to a fluid membrane and B refers to a solid membrane. The numbers by each curve are equal to c, the mole % of spin-label hapten IX in the plane of the lipid membrane. It will be seen that complement fixation, as measured by A Absorbance at 413 nm is far more effective in the fluid membrane than in the solid membrane at low hapten concentrations (i.e., c 0.3 mo e%). In C the lipid membrane host is a 50 50 mole ratio mixture of cholesterol and dipalmitoylphosphatidylcholine. The immunochemical data suggest that this membrane is in a state of intermediate fluidity. Specific affinity-purified IgG molecules were used in these experiments. (For further details, see Ref. 5.)... 

Porcine skin is used as a model membrane in all the screening experiments performed with INSIGHT. Porcine skin closely resembles human skin and serves as an excellent model membrane (95-98). 

Selectivity sequences in solvents such as water, methanol and ethanol do not guarantee a similar behaviour in the lipid membrane. Experiments have been carried out in attempts to investigate the selective transfer of cations across model membranes, and these are exemplified here by reference to an investigation concerning the cryptands [2.2.2], [3.2.2], [3.3.3] and [2.2.C8]. Two aqueous phases (IN and OUT) were bridged by a chloroform layer into which the carrier can be dissolved. Alkali metal picrate was dissolved in two aqueous layers such that the IN layer was 1000 times more concentrated than the OUT layer. All layers were stirred and the transport monitored via increase in picrate in the OUT layer (UV) and increase in potassium in the OUT layer (atomic absorption). The membrane phase was also analyzed at the end of the experiment.497... 

Numerous surface-active molecules have been studied as GI absorption promoters in a wide variety of testing conditions, including model membranes, everted intestinal sacs, tissue cultures, intestinal epithelia in diffusion chambers, intact animals, and humans. The physical properties of a chemical enhancer may be strongly dependent on the interactions with the endogenous GI components such as bile salts, pH, and bacteria. Thus the in vitro experiments on enhancing GI absorption are not necessarily predictive of the behavior of the promoter in animals or humans, and we will mainly focus on summarizing results from in vivo studies. 

The interactions of TDZ (6) with model membranes composed of different phospholipids were also studied by the same group [78]. Calorimetric studies demonstrated that TDZ (6) altered the thermotropic properties of negatively charged DMPC membranes to a larger extent than of zwitterionic phospholipids (PC and PE). The character of the drug-induced changes of the transition parameters of all studied lipids indicated that TDZ (6), similarly to other phenothiazine derivatives, was likely to be localized close to the po-lar/apolar interface of the bilayers. Experiments in which fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was employed revealed that TDZ (6) reduced the mobility of lipid molecules in a concentration-dependent manner and thus decreased membrane fluidity. The influence of TDZ (6) on isolated... 

In the model membrane studies discussed here, results from experiments using bovine brain ceramides differed from those using epidermal ceramides in the presence of a solid phase in the former, i.e. a lipid organization in which molecular motion is less than in a gel, and similar to that observed in an anhydrous powder. Model membranes composed of epidermal ceramides are clearly an advantage in studying the behavior of SC intercellular membranes, and appropriate NMR studies to detect such a phase will resolve this issue. One possible explanation is found by considering the kinetics of the formation of the solid phase In the bovine brain ceramide mixtures it was very slow to form, on the order of days to weeks, and was even more sluggish at pH 7.4... 

Model membranes have been used, such as multilamellar vesicles composed of dipalmitoyl phosphatidylcholine. It is appreciated that there are no phospholipids in the skin, but the types of interactions expected are similar, and interpretation of the results is much easier, when the lipid is pure. In order to simplify interpretation further, experiments with monolayers have been conducted and the results interpreted in light of published information. 

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