ALTERED PORE-FORMING PROPERTIES OF PROTEOLYTICALLY NICKED STAPHYLOCOCCAL ALPHA-TOXIN

Staphylococcal alpha-toxin is a single-chain polypeptide with a molecu lar weight of 34,000 that hexamerizes in lipid bilayers to form pores of 1-1.5 nm effective diameter in membranes. We demonstrate that limit ed proteolysis of purified alpha-toxin with proteinase K generates a h emolytically active product that yields one major protein band of 17-1 8 kDa in SDS-polyacrylamide gel electrophoresis. The 17-18-kDa protein band harbors two major fragments of similar size representing the N- and C-terminal halves, which remain associated with each other in non- denaturing buffers but dissociate in 6 M urea. Dissociation in urea le ads to loss of hemolytic activity. In contrast, unnicked alpha-toxin i s not inactivated by urea. Nicked, hemolytically active alpha-toxin fo rms hexamers on erythrocyte membranes and on lymphocytes and monocytes . However, the nicked toxin can only lyse erythrocytes and fails to pe rmeabilize nucleated cells. Osmotic protection experiments indicate th at the size of pores generated by the nicked toxin is considerably sma ller (0.6-0.9 nm effective diameter) than that generated by native tox in. The collective results do not support a previous proposal that dif ferent functions of alpha-toxin are contained in separate domains of t he molecule.