Olsnes S, Sandvig K, Refsnes K, Pihl A
J Biol Chem. 1976 Jul 10;251(13):3985-92.
The kinetics of protein synthesis inhibition in a cell-free system from rabbit reticulocyte lysate was studied after addition of abrin and ricin and the isolated A chains. The toxin A chains inhibited protein synthesis at a rate proportional to the amount added. When intact toxins were added to the reticulocyte lysate, the kinetics of protein synthesis inhibition indicated that the A chains must be liberated before ribosome inactivation can take place. The splitting of the toxin in the lysate was directly demonstrated by the use of labeled toxins. The amount of abrin and ricin bound to HeLa cells under different experimental conditions was correlated to the concomitant inhibition of cellular protein synthesis. In the presence of lactose, which inhibits toxin binding, much higher concentrations of toxins were required to inhibit protein synthesis than in the absence of lactose. A linear relationship was found between the lactose concentration in the medium and the toxin concentration required to give 50% reduction in protein synthesis after 3 hours. The amount of toxin bound to the cell surfaces in the presence of lactose was either determined directly or calculated from the apparent association constant between toxins and surface receptors at the various lactose concentrations. Under different conditions involving a 300-fold variation in the concentration of toxin required to reduce protein synthesis by 50% after 3 hours, the amount of toxin bound to the cell surface was found to be the same. The toxicity thus appears to be determined by the number of toxin molecules bound to the cell surface. Purified ricin B chain was used to compete with the toxins for the receptor sites. Only after addition of high amounts of B chain was the toxicity of abrin and ricin appreciably reduced. The data do not support the view that receptors with especially high affinity are involved in the uptake of the toxins. When the time required for 50% inhibition was plotted versus the inverse value of the square root of the number of toxin molecules bound per cell, a straight line was obtained, intercepting at about 30 min. The data indicate that the observed lag time cannot be due entirely to the fact that the A chains must be liberated before they can act.
在添加相思子毒素、蓖麻毒素及其分离出的A链后，研究了兔网织红细胞裂解液无细胞体系中蛋白质合成抑制的动力学。毒素A链以与添加量成比例的速率抑制蛋白质合成。当完整毒素添加到网织红细胞裂解液中时，蛋白质合成抑制的动力学表明，在核糖体失活发生之前，A链必须被释放出来。通过使用标记毒素直接证明了毒素在裂解液中的裂解。在不同实验条件下，相思子毒素和蓖麻毒素与HeLa细胞的结合量与细胞蛋白质合成的同步抑制相关。在存在抑制毒素结合的乳糖的情况下，与不存在乳糖时相比，需要更高浓度的毒素来抑制蛋白质合成。发现培养基中乳糖浓度与3小时后使蛋白质合成减少50%所需的毒素浓度之间存在线性关系。在存在乳糖的情况下，与细胞表面结合的毒素量要么直接测定，要么根据不同乳糖浓度下毒素与表面受体之间的表观缔合常数计算得出。在不同条件下，3小时后使蛋白质合成减少50%所需的毒素浓度变化了300倍，发现与细胞表面结合的毒素量是相同的。因此，毒性似乎由与细胞表面结合的毒素分子数量决定。使用纯化的蓖麻毒素B链与毒素竞争受体位点。只有在添加大量B链后，相思子毒素和蓖麻毒素的毒性才会明显降低。这些数据不支持存在具有特别高亲和力的受体参与毒素摄取的观点。当将50%抑制所需的时间与每个细胞结合的毒素分子数量的平方根的倒数作图时，得到一条直线，截距约为30分钟。数据表明，观察到的延迟时间不能完全归因于A链在发挥作用之前必须被释放这一事实。
