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Eur J Biochem. 1996 Sep 15;240(3):646-54.

HlyA hemolysin of Vibrio cholerae O1 biotype E1 Tor. Identification of the hemolytic complex and evidence for the formation of anion-selective ion-permeable channels.

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Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum), Universität Würzburg, Germany.


Hemolysin (HlyA) was concentrated from supernatants of different Vibrio cholerae O1 biotype E1 Tor strains by ammonium sulfate precipitation. The concentration of the toxin in the supernatants and in the precipitates was quantified using its hemolytic activity. The toxin formed a high molecular-mass band (about 220 kDa) on SDS/PAGE while the toxin monomer had a molecular mass of 60 kDa when it was heated. The addition of the E1 Tor hemolysin oligomers, but not that of the monomers, to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels, which had long lifetimes at small voltages. The hemolysin channel had a single-channel conductance of 350 pS in 1 M KCl. These results defined hemolysin (HlyA) from V. cholerae as a channel-forming component with properties similar to other cytolytic toxins. The long lifetime of the channel suggested that the channel-forming oligomer did not show a rapid association/dissociation reaction. At voltages larger than 50 mV, the hemolysin channel was voltage dependent in an asymmetric fashion dependent on the side of its addition. The single-channel conductance of the hemolysin (HlyA) from V. cholerae O1 biotype E1 Tor channel was a linear function of the bulk aqueous conductance, which suggested that the toxin forms aqueous channels with an estimated minimum diameter of about 0.7 nm. The hemolysin channel of V. cholerae was found to be moderately anion-selective. The pore-forming properties of hemolysin (HlyA) from V. cholerae O1 biotype E1 Tor were compared with those of aerolysin of Aeromonas sobria and alpha-toxin from Staphylococcus aureus. All these cytolytic toxins must probably oligomerize for activity in biological and artificial membranes and form anion-selective channels.

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