No abstract

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.

Amphotericin B (AmB) is a polyene antibiotic used to treat deep-seated mycoses. Both the therapeutic action and the toxic side effects of this drug are dependent on its molecular organization. AmB appears as a zwitterion at neutral pH owing to -NH 3 + and -COO -groups.The results obtained with electronic absorption, fluorescence, resonance light scattering and infrared absorption spectroscopic analyses show that in the aqueous medium at pH above 10 AmB appears in the monomeric form owing to the negative net electric charge of the molecule. On the contrary, anomalously high aggregation level has been observed at pH below 2, despite the positive net electric charge. The effect is interpreted in terms of the permanent polarization of the polyene chain at low pH, associated with relative rotational freedom of the charged mycosamine fragment of the molecule. The pH-dependent aggregation of AmB is discussed in aspect of pharmacological action of the drug.

Amphotericin B (AmB) is a life-saving polyene antibiotic used to treat deep-seated mycotic infections. Both the mode of therapeutic action as well as toxic side effects are directly dependent on molecular organization of the drug. Binding of AmB to lipid monolayers formed with dipalmitoylphosphatidylcholine, pure and containing 40 mol% cholesterol or ergosterol, the sterols of human and fungi respectively, has been examined by means of