Rabbit or human erythrocytes lysed with Staphylococcus aureus a-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and a2-macroglobulin in gel chromatography . A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE . In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter . An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membranederived form of a-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of -3-nm effective diameter in toxin-treated membranes, the possibility is raised that native a-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel .
Native staphylococcus aureus a-toxin is secreted as a hydrophilic polypeptide chain of Mr 34,000. The presence of deoxycholate above the critical micellar concentration induced the toxin monomers to self-associate, forming ring or cylindrical oligomers. The oligomers were amphiphilic and bound detergent. In deoxycholate solution, the protein-detergent complexes exhibited a sedimentation coefficient of 10.4 S. A Mr of 238,700 was determined by ultracentrifugation analyses at sedimentation equilibrium. Because quantitative detergent-binding studies indicated a protein/detergent ratio of approximately 5:1 (wt/wt), the protein moiety in each protein-detergent complex was determined to be approximately Mr 200 000, corresponding to a hexamer of the native molecule. The amphiphilic toxin hexamers were ultrastructurally indistinguishable from the cytolytic, annular toxin complexes that form on and in biological target membranes. They bound lipid and could be incorporated into artificial lecithin lipid vesicles. The transition of toxin protein molecules from a hydrophilic monomer to an amphiphilic oligomer through self-association has thus been shown to be inducible solely through contact of the native protein molecules with an appropriate amphiphilic substrate.
The binding of Staphylococcus aureus a-toxin to rabbit and human erythrocytes was studied by hemolytic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. Hemolytic assays showed that toxin binding to 10% cell suspensions at neutral pH was very ineffective in the concentration range 3 x 10-8 to 3 x 10-7 M (1 to 10 ,ug/ml), and less than 5% of added toxin became cell bound. However, binding was augmented as toxin levels were raised, abruptly increasing to 50 to 60% at 2 x 10-6 to 3 x 10-6 M (60 to 100 ,ug/ml). When rabbit erythrocytes were lysed with 1 to 5 ,ug of toxin per ml, both monomeric and hexameric forms of the toxin could be detected on the membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. In contrast, human erythrocytes treated with 1 to 6 ,ug of toxin per ml did not lyse, and membrane-bound toxin was not detectable. When toxin concentrations were raised to 30 to 100 ,ug/ ml, human erythrocytes also lysed and toxin hexamers became membrane bound in comparable amounts as on rabbit cell membranes. Lowering the pH led to a marked increase in susceptibility of human, but not rabbit erythrocytes towards a-toxin. When human cells were lysed at pH 5.0 with 5 ,ug of toxin per ml, membranebound hexameric toxin became detectable. The demonstrated correlation between the presence of hexameric, cell-bound toxin and hemolytic activity supports the channel concept of toxin-mediated cytolysis. The results also show that toxin binding does not exhibit overall characteristics of a simple receptor-ligand interaction.
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