i(3)so3-galactosylceramide and fluorexon

The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells.

Topics: Animals; Carbon; Cattle; Computer Simulation; Cytotoxins; Fluoresceins; Lipids; Liposomes; Models, Molecular; Models, Statistical; Monte Carlo Method; Phosphatidylcholines; Phosphatidylserines; Protein Structure, Tertiary; Snakes; Sulfoglycosphingolipids; Thermodynamics; Water

The effect of sulfatide on membrane hydration of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles (SUVs) was investigated using steady-state and time-resolved fluorescence spectroscopy. The degree of hydration in the headgroup region of the bilayer lipids was assessed with the fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine along with the ratio of its fluorescence intensities measured in samples prepared either in D2O- or in H2O-based buffers. Similarly, hydration of acyl chains near the headgroup region and that close to the bilayer center were studied using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3, 5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine as probes. Increasing sulfatide concentration up to 30 mol% resulted in an increase in surface hydration and a decrease in interchain hydration. These were correlated with an increase in bilayer stability of the DOPE/sulfatide SUVs. Moreover, variation of pH was found to affect the hydration and stability of the bilayer vesicles. No further change in headgroup hydration and interchain hydration near the bilayer center was observed at sulfatide concentrations >/=30 mol%. At such high sulfatide concentrations, bilayer hydration and stability were no longer pH-sensitive. The effects of sulfatide on hydration and stability of DOPE bilayer vesicles are discussed by taking into account the electrostatic and geometrical properties of the sulfated galactosyl headgroups.

Topics: Dansyl Compounds; Diphenylhexatriene; Drug Stability; Fluoresceins; Hydrogen-Ion Concentration; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Structure-Activity Relationship; Sulfoglycosphingolipids; Water

The bilayer stabilization effect of sulfatide and the pH sensitivity of sulfatide-containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles were examined by light scattering and the release of entrapped calcein. At 30 mol% sulfatide, stable DOPE/sulfatide vesicles were formed at the physiological pH and their stability was preserved in the presence of human plasma. These vesicles were found to be pH-sensitive and became leaky at pH 6.0 or when there was a pH-gradient across the membrane bilayer. Under such conditions, the amount of calcein released after 24 h incubation at 37 degrees C was increased by one-fold compared to that found at pH 7.4. Our results suggest that the hydration and partial dehydration of the headgroup of sulfatide upon changing pH play an essential role in determining the pH sensitivity of DOPE/sulfatide vesicles, while the importance of the condensing effect of the glycolipid on membrane bilayer is less significant.

Topics: Fluoresceins; Fluorescence; Fluorescent Dyes; Hydrogen-Ion Concentration; Lipid Bilayers; Liposomes; Phosphatidylethanolamines; Sulfoglycosphingolipids