Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes.
Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes.
复制标题
DOI：
--
复制
发表时间：
1990-11
期刊：
影响因子：
--
通讯作者：
E. Feener;Wei-Chiang Shen;Hugues J.-P. Ryser
中科院分区：
文献类型：
--
作者：
E. Feener;Wei-Chiang Shen;Hugues J.-P. Ryser
虽然有生物学证据表明，二硫键的还原裂解对于内吞的大分子如毒素、免疫毒素和其他药物载体的活化至关重要，但实际上对这种裂解的具体情况一无所知。为了研究这一过程，合成了一种模型化合物，其中放射性碘化酪胺通过二硫键连接到不可降解的载体聚（D-赖氨酸），已知其被有效内吞。用该探针脉冲标记培养的中国仓鼠卵巢细胞，并在不同追踪时间测定二硫键裂解释放的酸溶性放射性。脉冲标记的细胞也进行亚细胞分级分离，以确定与二硫键裂解相关的细胞内结构。裂解开始时无延迟，在第一个小时内约为初始细胞结合放射性的7%，并持续6小时以上。在N-乙基马来酰亚胺存在下，其被消除。当细胞表面存在的巯基被细胞不渗透的巯基试剂阻断时，二硫键裂解的初始阶段被抑制，表明裂解开始于细胞表面。一个持久的细胞内阶段的二硫键裂解后开始约30分钟的追逐。亚细胞分级分离和动力学分析表明，无论是溶酶体，也没有内体参与该阶段，离开高尔基体作为最可能的网站内吞二硫键裂解。
Whereas there is biological evidence that the reductive cleavage of disulfide bonds is critical for the activation of endocytosed macromolecules such as toxins, immunotoxins, and other drug carriers, virtually nothing is known about the specifics of this cleavage. To study this process, a model compound was synthesized in which a radioiodinated tyramine was linked through a disulfide bond to an undegradable carrier, poly(D-lysine), known to be efficiently endocytosed. Cultured Chinese hamster ovary cells were pulse-labeled with this probe, and the disulfide cleavage was measured as released acid-soluble radioactivity at different times of chase. Pulse-labeled cells were also subjected to subcellular fractionation to identify intracellular structures associated with disulfide cleavage. Cleavage began without lag, amounted to about approximately 7% of the initial cell-bound radioactivity in the first hour and continued for more than 6 h. It was abolished in the presence of N-ethylmaleimide. When sulfhydryl groups present at the cell surface were blocked with cell-impermeant sulfhydryl reagent, the initial phase of disulfide cleavage was inhibited, indicating that cleavage began at the cell surface. A long-lasting intracellular phase of disulfide cleavage began after about approximately 30 min of chase. Subcellular fractionation and kinetic analysis indicated that neither lysosomes nor endosomes were participating in that phase, leaving the Golgi apparatus as the most probable site of endocytic disulfide cleavage.