| Abstract: | The receptor-mediated internalization and degradation of radiolabeled diphtheria toxin by cultured monkey kidney cells was studied. The ability of a number of enzymes and chemicals to remove cell surface-bound toxin was tested; the combination of pronase and inositol hexaphosphate (PIHP) proved most effective. Using PIHP, the kinetics of toxin-cell association at 37 degrees C was resolved into two compounds: surface binding and internalization. The PIHP assay also allowed estimation of the half-time of toxin internalization (about 25 min). An assay involving precipitation of culture supernatants with trichloroacetic acid was developed and used to measure the rate of degradation and excretion of cell-associated toxin. Agents which markedly inhibited toxin internalization similarly prevented degradation, implying an intracellular location for the degradative process. The primary radioactive product excreted by Vero cells was monoiodotyrosine. The extent and rate of toxin degradation indicated lysosomal involvement. Finally, agents which blocked internalization or degradation, or both, (e.g. antibody and concanavalin A), protected cells from the cytotoxin action of diphtheria toxin, suggesting that these processes are necessary for expression of biological effect. |
