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Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

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Summary

The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.

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Abbreviations

EqT-II:

Actinia equina equinatoxin II

SM:

sphingomyelin

PC:

phosphatidylcholine

PE:

phosphatidylethanolamine

POPC:

palmitoyl-oleoyl-phosphatidylcholine

SUV:

small unilamellar vesicles

HRBC:

human red blood cells

PLM:

planar lipid membranes

TLC:

thin layer chromatography

SDS:

sodium dodecyl sulfate

LDS:

lithium dodecyl sulfate

Tween-20:

polyoxyethylene sorbitan monolaurate

Brij-35:

polyoxyethylene(23)lauryl ether

Triton X-100:

octylphenoxy polyethoxy ethanol

LubrolPX:

polyethylenglycol(9)dodecyl ether

PLP:

pyridoxal-5′-phosphate

EDC:

1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide

DM5:

dimethyl suberimidate

PEG:

polyethyleneglycol

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Authors and Affiliations

  1. Dipartimento di Fisica, Universitá di Trento, I-38050, Povo (TN), Italy

    Giovanna Belmonte, Cecilia Pederzolli & Gianfranco Menestrina

  2. Istituto per la Ricerca Scientifica e Tecnologica, I-38050, Povo (TN), Italy

    Cecilia Pederzolli

  3. Department of Biology, Biotechnical Faculty, University of Ljubljana, SLO-61000, Ljubljana, Slovenia

    Peter Maček

  4. CNR Centro di Fisica degli Stati Aggregati, I-38050, Povo (TN), Italy

    Gianfranco Menestrina

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  1. Giovanna Belmonte
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  2. Cecilia Pederzolli
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  3. Peter Maček
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  4. Gianfranco Menestrina
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Belmonte, G., Pederzolli, C., Maček, P. et al. Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes. J. Membrain Biol. 131, 11–22 (1993). https://doi.org/10.1007/BF02258530

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  • DOI: https://doi.org/10.1007/BF02258530

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