BINDING OF ANTIBODIES TO FUNCTIONAL EPITOPES ON THE PORE FORMED BY ESCHERICHIA-COLI HEMOLYSIN IN CELLS AND MODEL MEMBRANES

Escherichia coli hemolysin (HlyA) inserts into target membranes produc ing a cation-selective pore. We approached the problem of determining which portions of this protein remain exposed on the side of attack by applying specific antibodies. Results obtained with resealed erythroc yte ghosts and planar phospholipid membranes were compared. The effect s of one polyclonal and four monoclonal anti-hemolysin antibodies (mAb s) were studied. Using ghosts we found one mAb which strongly reduced the ion-permeability through the preinserted HlyA channels and one whi ch clearly increased it. Experiments with planar bilayers corroborated these results by showing that the former mAb effectively promoted the closed state of the channel whereas the latter forced the HlyA channe l into an open configuration. Anti-hemolysin polyclonal antibodies ini tially stimulated but then prevented channel opening, indicating they contained clones able to act on both these channel determinants. They were effective only when applied on the same side as the hemolysin ind icating that the epitopes were exposed to that side. Finally, the anti genic epitopes of three of the mAbs were localised on the HlyA molecul e by using different mutants (amber and frame shift mutants and hemoly sin gene hybrids).