Quantitative analysis of the binding and oligomerization of staphylococcal alpha-toxin in target erythrocyte membranes.
The binding of staphylococcal alpha-toxin to rabbit and human erythrocytes was quantitated over a wide range of toxin concentrations (3 x 10(-11) to 3 x 10(-6) M) with the use of an enzyme-linked immunosorbent assay that permitted simultaneous quantitation of monomeric and oligomeric toxin forms. Three basic observations were made. First, in no range of concentrations did the binding of alpha-toxin to rabbit erythrocytes display characteristics of a receptor-ligand interaction. Net binding to rabbit cells was nil at sublytic concentrations (10(-10) M or 3 ng/ml). The onset of binding occurred at around 10 ng/ml and remained fairly constant and ineffective (5 to 8% of toxin offered) over a wide concentration range (up to 10 micrograms/ml). Second, hemolysis of rabbit and human erythrocytes at 37 degrees C was always accompanied by the formation of toxin oligomers in the membrane. Third, overall toxin binding at 0 degree C followed a pattern similar to that at 37 degrees C. However, oligomer formation and cell lysis were retarded (but not totally inhibited) at 0 degree C. When rabbit erythrocytes were incubated with low levels of toxin at 0 degree C (0.5 microgram/ml) for 30 min, the toxin became bound exclusively in monomer form, and no lysis occurred. When cells thus treated were washed and suspended at 37 degrees C, lysis rapidly ensued, and native monomeric toxin was replaced by oligomeric toxin. The collective results directly support the oligomer pore concept of toxin action and also indicate that toxin oligomers form by lateral aggregation of bound monomers in the bilayer. They speak against the existence of specific binding sites for alpha-toxin on rabbit erythrocytes.
D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis* / Q000633:toxicity; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism; D006460:Hemolysin Proteins; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels; D046911:Macromolecular Substances; D011485:Protein Binding; D011817:Rabbits; D013696:Temperature
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D007473:Ion Channels; D046911:Macromolecular Substances; C021091:staphylococcal alpha-toxin