BINDING OF ESCHERICHIA-COLI VEROTOXINS TO CELL-SURFACE PROTEIN ON WILD-TYPE AND GLOBOTRIAOSYLCERAMIDE-DEFICIENT VERO CELLS

We have examined verotoxin (VT) binding to cell surface proteins, When Vero or globotriaosylceramide (Gb(3)) deficient Vero (VRP) cells were incubated with I-125-labelled verotoxin 2 (VT2) and disuccinimidyl su berate cross-linker, SDS-PAGE of cell lysates showed radiolabelled ban ds at 44, 50, 60, 86, 102, and 138 kDa, When I-125-labelled verotoxin 1 (VT1) was cross-linked, radioactive bands occurred at 51, 67, 101, 1 60, 188, and 232 kDa. In contrast, I-125-labelled VT1 B subunit produc ed a single radioactive band migrating at 50 kDa. CHO cells did not bi nd labelled VT. VT2, binding to VRP cells fit a rectangular hyperbola suggesting a single class of binding sites. In contrast, VT1 and VT1 B subunit binding to VRP cells was best fit by sigmoidal curves suggest ing the presence of positive cooperativity between at least two bindin g sites. Scatchard analysis of VT2 binding data yielded 3.5 x 10(9) mo lecules bound/mu g of cell protein with an equilibrium dissociation co nstant (K-D) of 13 nM. The apparent K-D was 9.7 nM for VT1 and 73.2 nM for VT1 B subunit. These results indicate that VT binds to a protein, or proteins, on the surface of susceptible cells and that there appea r to be differences between VT1 and VT2 binding. Interactions between VT1 or VT2 and the proteins demonstrated here may be important in the biological activity of VT.