Search

Aim: To test whether membrane fluidity and its changes are important for the sensitivity of cells to the action of perifosine (OPP), a new anticancer drug targeting cell membrane and not DNA., Method: Influence of OPP on the membrane structure of OPP-resistant MCF7, and OPP-sensitive MT3 breast cancer cell lines, as well as of mouse fibroblasts (L929) cell lines, and model cells (liposomes) was investigated by electron paramagnetic resonance, using spin labeled derivative of OPP (P5) and 5-doxylpalmitoyl methylester (MeFASL(10,3)) as spin probes., Results: OPP increased membrane fluidity of all cell lines at concentrations higher than 50 μM (on the level of P≤0.05, t test). In cells, the differences were observed only by P5 and not by MeFASL(10,3). Average order parameter Seff decreased for about 12% in MCF7 and L929 and only for 8% in OPP-sensitive MT3 cells, showing that there was no correlation between membrane fluidity changes and sensitivity of cells to OPP. The only correlation we found was between OPP sensitivity and the cell growth rate. In liposomes, both spin probes were sensitive to the action of OPP. Seff decreased with increasing concentration of OPP. For MeFASL(10,3) a significant decrease was observed at 4 mol% OPP, while for P5 it was observed at 8 mol%., Conclusion: Influence of OPP on plasma membrane fluidity of breast cancer cells is not the determining factor in the sensitivity of cells to OPP.

We studied the influence of sodium and calcium chloride on the global and local membrane properties of fluid palmitoyl-oleoyl phosphatidylcholine bilayers, applying synchrotron small-angle x-ray diffraction, spin-labeling electron paramagnetic resonance spectroscopy, and differential scanning calorimetry, as well as simultaneous density and acoustic measurements. The salt concentration was varied over a wide range from 0 to 5 M. We found that NaCl leads to a continuous swelling of the bilayers, whereas the behavior of the bilayer separation dW in the presence of CaCl2 is more complex, showing an initial large dW value, which decreased upon further addition of salt and finally increased again in the high concentration regime. This can be understood by a change of balance between electrostatic and van der Waals interactions. We were further able to show that both salts lead to a significant increase of order within the lipid bilayer, leading to a decrease of bilayer elasticity and shift of main phase transition temperature. This effect is more pronounced for Ca2+, and occurs mainly in the high salt-concentration regime. Thus, we were able to reconcile previous controversies between molecular dynamics simulations and x-ray diffraction experiments regarding the effect of salts on neutral lipid bilayers.

A novel membrane lateral domain approach was used to test whether the activity of the membrane-bound enzyme acetylcholinesterase (AChE) depends on the local properties (e.g. local lipid ordering) of bovine erythrocyte-ghost membrane. This issue has an additional aspect of interest due to an alternative mode of insertion of AChE molecules into the membrane by the glycosylphosphatidylinositol (GPI) anchor. In our experiments the lateral domain membrane structure was influenced by temperature and by the addition of n-butanol, and was quantitatively characterized using the method of EPR spectrum decomposition. The activity of AChE was determined by a colorimetric assay in the same samples. The results show that the membrane stabilizes the conformation of the membrane-bound AChE compared to the isolated AChE. In addition, a correlation was observed between the temperature dependence of order parameter of the most-ordered domain type and the activity of AChE. Therefore, our findings support the idea that the function of GPI proteins can be modulated by the lipid bilayer. Based on the assumption that the overall activity of AChE depends on the order parameters of particular domain types as well as their proportions, two models for AChE activity were introduced. In the first, a random distribution of enzyme molecules was proposed, and in the second, localization of enzyme molecules in a single (cholesterol-rich) domain type was assumed. Better agreement between measured and calculated activity values speaks in favor of the second model.

An electron paramagnetic resonance (EPR)-based membrane surface characterization method is presented to detect the properties of the carbohydrate-rich part of membrane surfaces as well as carbohydrate interaction with other membrane constituents and water-soluble molecules. The proposed method relies on the spin-labeling and spectral decomposition based on spectral simulation and optimization with EPRSIM software. In order to increase the sensitivity of characterization to the carbohydrate-rich part of the membrane surface, the sucrose-contrasting approach is introduced. With this method, which was established on model membranes with glycolipids and tested on erythrocyte membrane, we were able to characterize the surface and lipid bilayer lateral heterogeneity. Additionally, some properties of the interaction between glycocalyx and lipid bilayer as well as between glycocalyx and sucrose molecules were determined. The experiments also provided some information about the anchoring and aggregation of the glycosylated molecules. According to the results, some functions of the glycosylated surface are discussed.

Using EPR spectroscopy a typical lateral domain structure was detected in the membranes of spin-labeled bovine erythrocyte ghosts. The spectral parameters were determined by decomposing the EPR spectrum into three spectral components and tuned by a hybrid-evolutionary-optimization method. In our experiments the lateral domain structure and its properties were influenced by the variation in the temperature and by the addition of n-butanol. The specific responses of the particular domain types were detected. For the most-ordered domain type a break was seen in the temperature dependence of its order parameter, while the order parameters of the two less-ordered domain types exhibited a continuous decrease. Below the break-point temperature the alcohol-induced membrane fluidity variation is mainly a consequence of the change in the proportions of the least- and the most-ordered domain type and not the change of the domain-type ordering or dynamics (with n-butanol concentration). On the other hand, the fluidity variation above the break-point temperature arises from both types of changes. Interestingly, the proportion of the domain type that has its order parameter between that of the least- and the most-ordered domain type remains almost constant with concentration as well as with temperature, which implies its stability. Such characterization of the lateral membrane domain structure could be beneficial when considering the lipid-protein interactions, because it can be assumed that the activity of the membrane-bound enzyme depends on the properties of the particular domain type.