Hudson T H, Neville D M
J Biol Chem. 1985 Mar 10;260(5):2675-80.
The rate-limiting step in diphtheria toxin (DT) intoxication of Vero cells has been determined utilizing cycloheximide as an inhibitor of the intoxication process. Cycloheximide is shown to inhibit the toxin catalyzed ADP-ribosylation of elongation factor 2 (EF-2). The inhibition is blocked by puromycin thus establishing the ribosome as the location of cycloheximide protection. Washing cells free of cycloheximide rapidly reverses the protective effect. The initial rates of protein synthesis inhibition observed after removal of cycloheximide from DT-intoxicated cells are 5 to 12-fold greater than rates observed in unprotected cells and are shown to reflect ADP-ribosylation of EF-2 by cytosolic DT. Ten to thirty minutes after cycloheximide removal, the rate of protein synthesis inhibition abruptly changes to values identical to those of unprotected cells. Both the initial rates and extent of the initial rapid inactivation are directly related to toxin concentration and time of incubation with DT in the presence of cycloheximide. We concluded that: the rate-limiting step in protein synthesis inhibition by DT is not the ADP-ribosylation of EF-2 by cytosolic toxin but rather the earlier entry step of DT into the cytosol. DT enters the cytosol as a bolus of sufficient size to rapidly inactivate all EF-2 in that cell. It is inferred from 1 and 2 that the first order inactivation rate exhibited by DT is the result of the probability of the release of a bolus of toxin to the cytosol of any cell in the population per unit time. Autoradiographic analysis of intoxicated cell populations support this two-population state model. The size of a single bolus or quantum of DT is calculated from data over the range of 10(-11) to 10(-9) M DT and is found to remain constant. We suggest that the cytosolic entry mechanism of DT results from a unique ability of the internalized toxin molecules to destabilize the vesicular membrane resulting in a random release of a bolus of toxin into the cytosol. Because the bolus size remains constant over a 50-fold change in receptor occupancy the possibility is raised that DT undergoes a post-receptor packaging process, package size remaining a constant and package number increasing with receptor occupancy.
利用环己酰亚胺作为中毒过程的抑制剂，已确定了Vero细胞白喉毒素（DT）中毒的限速步骤。结果表明，环己酰亚胺可抑制毒素催化的延伸因子2（EF-2）的ADP核糖基化。嘌呤霉素可阻断这种抑制作用，从而确定核糖体为环己酰亚胺的保护位点。将细胞洗去环己酰亚胺可迅速逆转这种保护作用。从DT中毒细胞中去除环己酰亚胺后观察到的蛋白质合成抑制的初始速率比未受保护细胞中观察到的速率高5至12倍，并且表明这反映了胞质DT对EF-2的ADP核糖基化作用。去除环己酰亚胺后10至30分钟，蛋白质合成抑制速率突然变为与未受保护细胞相同的值。初始速率和初始快速失活的程度均与毒素浓度以及在环己酰亚胺存在下与DT孵育的时间直接相关。我们得出结论：DT抑制蛋白质合成的限速步骤不是胞质毒素对EF-2的ADP核糖基化作用，而是DT进入胞质溶胶的较早进入步骤。DT以足够大的团块形式进入胞质溶胶，从而迅速使该细胞中的所有EF-2失活。从1和2可以推断，DT表现出的一级失活速率是每单位时间向群体中任何细胞的胞质溶胶释放一团毒素的概率的结果。对中毒细胞群体的放射自显影分析支持这种双群体状态模型。根据10（-11）至10（-9）M DT范围内的数据计算出单个DT团块或量子的大小，发现其保持恒定。我们认为，DT的胞质进入机制源于内化毒素分子使囊泡膜不稳定的独特能力，从而导致一团毒素随机释放到胞质溶胶中。由于在受体占有率变化50倍的情况下团块大小保持恒定，因此提出了DT经历受体后包装过程的可能性，包装大小保持恒定，包装数量随受体占有率增加。
