Association of diphtheria toxin with Vero cells. Demonstration of a receptor

Abstract

The interaction of radiolabeled diphtheria toxin with highly sensitive mammalian cell lines was studied. Toxin bound to (or was taken up by) Vero cells at 4 and 37 degrees C in a highly specific manner. At both temperatures, excess unlabeled toxin competed for up to 90% of the cell-associated label. The association at 37 degrees C was biphasic, increasing to a peak at 1 to 2 h and falling thereafter. At 4 degrees C, association increased with time to a steady state. Both fragment B and CRM-197 competed for the association of labeled toxin with cells. The magnitude of association correlated with the cytotoxic sensitivity of several cell lines. Both pH and exogenous nucleotides affected the association in a manner consistent with effects on cytotoxicity. The label associated with cells at 4 degrees C was largely intact toxin, while that at 37 degrees C was degraded. At 4 degrees C, the association was saturable (K = 9 X 10(8) liters/mol), was reversible, and indicated about 1 to 2 X 10(5) binding sites/cell.