Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells.
Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells.
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猴肾细胞受体介导的白喉毒素内化和降解。
DOI：
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发表时间：
1979
影响因子：
4.8
通讯作者：
Stephen H. Leppla
中科院分区：
文献类型：
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作者：
R. Dorland;J. Middlebrook;Stephen H. Leppla
研究了培养的猴肾细胞对放射性标记白喉毒素的受体介导的内化和降解。测试了多种酶和化学物质去除细胞表面结合毒素的能力；链霉蛋白酶和肌醇六磷酸 (PIHP) 的组合被证明是最有效的。使用 PIHP，37 摄氏度下毒素-细胞结合的动力学被解析为两种化合物：表面结合和内化。 PIHP 测定还可以估计毒素内化的半衰期（约 25 分钟）。开发了一种涉及用三氯乙酸沉淀培养物上清液的测定法，并用于测量细胞相关毒素的降解和排泄率。显着抑制毒素内化的药物同样可以防止降解，这意味着降解过程位于细胞内。 Vero 细胞分泌的主要放射性产物是单碘酪氨酸。毒素降解的程度和速率表明溶酶体参与。最后，阻断内化或降解或两者的试剂（例如抗体和刀豆球蛋白A）保护细胞免受白喉毒素的细胞毒素作用，表明这些过程对于表达生物效应是必要的。
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.