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Biophys J, June 2001, p. 2761-2774, Vol. 80, No. 6
and
*CNR-ITC, Centro di Fisica degli Stati Aggregati, I-38050 Povo,
Italy, and Departamento de Bioquimica, Facultad de
Biologia, Universidad de la Habana, La Habana, Cuba
Sticholysin I and II (St I and St II), two basic
cytolysins purified from the Caribbean sea anemone Stichodactyla
helianthus, efficiently permeabilize lipid vesicles by forming
pores in their membranes. A general characteristic of these toxins is
their preference for membranes containing sphingomyelin (SM). As a
consequence, vesicles formed by equimolar mixtures of SM with
phosphatidylcholine (PC) are very good targets for St I and II. To
better characterize the lipid dependence of the cytolysin-membrane
interaction, we have now evaluated the effect of including different
lipids in the composition of the vesicles. We observed that at low
doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures,
permeabilization was optimal when the molar ratio of PA/SM was ~1.
The preference for membranes containing PA was confirmed by inhibition
experiments in which the hemolytic activity of St I was diminished by
pre-incubation with vesicles of different composition. The inclusion of
even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG),
phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also
elicited an increase in calcein release, the potency being in the order
CL 
PA 
PG PI PS. However, some boosting
effect was also obtained, including the zwitterionic lipid
phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the
positively charged lipid stearylamine (SA). This indicated that the
effect was not mediated by electrostatic interactions between the
cytolysin and the negative surface of the vesicles. In fact, increasing
the ionic strength of the medium had only a small inhibitory effect on
the interaction, but this was actually larger with uncharged vesicles
than with negatively charged vesicles. A study of the fluidity of the
different vesicles, probed by the environment-sensitive fluorescent dye
diphenylhexatriene (DPH), showed that toxin activity was also not
correlated to the average membrane fluidity. It is suggested that the
insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the
presence of lipids favoring a nonlamellar phase, in particular PA and
CL, strong inducers of negative curvature in the bilayer, could help in
the formation of the pore. This possibility is confirmed by the fact
that the formation of toxin pores strongly promotes the rate of
transbilayer movement of lipid molecules, which indicates local
disruption of the lamellar structure.
Biophys J, June 2001, p. 2761-2774, Vol. 80, No. 6
© 2001 by the Biophysical Society 0006-3495/01/06/2761/14 $2.00
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