Kinetics of protein synthesis inactivation by diphtheria toxin in toxin-resistant L cells. Evidence for a low efficiency receptor-mediated transport system

Abstract

In an attempt to gain an understanding of the mechanism by which toxins enter cells, we have analyzed the intoxication of diphtheria toxin in the toxin-resistant L cell and compared these results with those previously reported for toxin-sensitive HeLa and KB cells (Uchida, T., Pappenheimer, A. M., Jr., and Harper, A. A. (1973) J. Biol. Chem. 248, 3845-3850; Moehring, J. M., and Moehring, T. J. (1976) Infect. Immun. 13, 221-228). The inactivation of protein synthesis in diphtheria toxin-treated L cells is characterized by first order inactivation kinetics and a concentration-dependent lag phase. The log rate of inactivation of protein synthesis when plotted versus toxin concentration yields a complex hyperbola with a saturable component detected in the range of 5 to 10 X 10(-5) M. We conclude that the intoxication of L cells by diphtheria toxin in the range of 10(-8) to 5 X 10(-6) M occurs via a saturable process and that the saturable component probably represents a plasma membrane toxin receptor with an affinity 200-fold lower than that found in sensitive HeLa cells. Since L cells require a 10(4)- to 10(5)-fold higher toxin dose than HeLa cells, a 200-fold affinity difference cannot fully explain the resistance of L cells to diphtheria toxin. In addition, L cells must have either fewer toxin receptors or a deficiency in the transport step(s). Because toxin-exposed L cells in contrast to sensitive cells exhibit different behavior with respect to pH and pH reversal of NH4Cl protection, a deficiency in the transport step is considered the more likely case.