Kinetics of adenosinediphosphoribosylation of elongation factor 2 in cells exposed to diphtheria toxin.

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RESUMO

When susceptible cells are exposed to diphtheria toxin (Mr, 62,000) the N-terminal 21,150-dalton A fragment of toxin reaches the cytoplasm, where it catalyzes the transfer of adenosinediphosphoribose from nicotinamide adenine dinucleotide to elongation factor 2 (EF2). Adenosinediphosphoribose-EF2 is inactive, so that protein synthesis is blocked. Using a simple, rapid assay for the amount of adenosinediphosphoribosylatable EF2 in unfractionated lysates of cultured cells we have followed the kinetics of inactivation of EF2 in CV-1 and BHK cells exposed to diphtheria toxin. With both cell lines a lag was observed between the addition of toxin to the cells and the adenosinediphosphoribosylation of EF2. The lag decreased with increasing toxin concentration until a limiting value of about 12 min was reached. The rate of adenosinediphosphoribosylation of EF2 after the lag was 10 to 20 times more rapid in CV-1 cells than in BHK cells exposed to the same toxin concentration. The concentration of fragment A active in the cytoplasm of toxin-treated cells was estimated from the rate of adenosinediphosphoribosylation observed. Comparison of these estimates with data from studies of binding of 125I-toxin to cells suggests that the fragment A of only a minor fraction of toxin molecules bound to cell surface receptors reaches the cytoplasm and participates in the inactivation of EF2. A model summarizing our current views on the process by which fragment A enters cells is presented.

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