THE INTERACTION OF ST I AND II, TWO PORE-FORMING TOXINS FROM THE SEA ANEMONE Stichodactyla helianthus WITH MEMBRANES

Alvarez C.(1), Lanio, M.E.(1), Nogueira, L.V.(2), Pazos, F.(1), Martinez, D.(1), Tejuca, M.(1), Casallanovo, F.(2), Shida, C.(2), Menestrina, G.(4), Lissi; E.(3), Schreier, S.(2)

(1)University of Havana, Cuba, (2)University of São Paulo, Brazil, (3)University of Santiago, Chile, (4)Center for Aggregate State Physics, Italy.

Sticholysins I (St I) and II (St II) are two highly hemolytic polypeptides that form oligomeric pores of radius around 1 nm in cell and lipid membranes containing sphingomyelin. The hemolytic activity of St II is ca. 5 times higher than that of St I. Binding to vesicles of phosphatidylcholine (PC): sphingomyelin (SM) produced an increase in both St I and II fluorescence intensity being larger for St I. This indicates a greater exposure of St I Trp to a less polar environment. Neither far- nor near-UV CD spectra revealed extensive conformational changes upon St I and St II binding to PC:SM vesicles. The inclusion of small proportions of a third lipid, particularly phosphatidic acid (PA) and cardiolipin (CL) into PC:SM vesicles led to a marked increase in calcein release caused by St I and St II. Probably, the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules. Lipid-protein interaction and the degree of penetration of both toxins into PC:SM membranes was monitored by EPR. The proteins interact with the bilayer conducing to a more immobilized lipid population. St I and St II had essentially no effect on the spectra of probes labeled at C-16. We have also examined fluorescence quenching by PC-spin labeled. The smallest effect was observed for 16-PCSL, both for St I and St II, corroborating the EPR findings and could be explained considering the existence of toroidallipid pore.

CORRESPONDENCE TO:

Carlos Alvarez, Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Calle 25, entre J e I, Vedado, Ciudad Habana, Cuba, Email: calvarez@infomed.sld.cu