Wileman T, Boshans R, Stahl P
J Biol Chem. 1985 Jun 25;260(12):7387-93.
During endocytosis, mannosylated ligands enter vesicles which have a density intermediate between that of the plasma membrane and secondary lysosomes. Mannosylated ligands are transferred from these vesicles to lysosomes. A solubilization-precipitation assay was used to study the dissociation of mannosylated ligands from their receptor. In whole cells dissociation was rapid (t 1/2 (37 degrees C) = 8 min) and took place before delivery of the ligand to lysosomes. Receptor-ligand dissociation within membrane vesicles, washed free of cytosol, could be induced by addition of ATP and GTP but not ADP. Receptor-ligand dissociation caused by manipulating the pH of the vesicles suggested that the pH within endosomes was lowered to 5.5 by addition of ATP. Dissociation was blocked by proton ionophores and Zn2+, but was unaffected by inhibitors of the F1, Fo-ATPase or the Na+,K+-ATPase. Dissociation did not require Na+ or K+ and was blocked by anion transport inhibitors. Dissociation was slowed in the absence of permeant anions (Cl-). Receptor-ligand complexes within vesicles isolated as early as 2 min following ligand internalization responded to addition of ATP. The results suggest that receptor-ligand dissociation in endosomes requires ATP, possibly to power endosomal acidification via an ATP-dependent proton pump. Dissociation is enhanced in the presence of permeant anions, suggesting the involvement of an anion channel or carrier.
在胞吞作用过程中，甘露糖基化配体进入密度介于质膜和次级溶酶体之间的囊泡。甘露糖基化配体从这些囊泡转移至溶酶体。采用溶解-沉淀分析法研究甘露糖基化配体与其受体的解离。在完整细胞中，解离迅速（37℃时的半衰期t1/2 = 8分钟），且在配体转运至溶酶体之前就已发生。在洗去胞质溶胶的膜囊泡内，添加ATP和GTP可诱导受体-配体解离，但添加ADP则不能。通过调节囊泡pH值引起的受体-配体解离表明，添加ATP可使内体的pH值降至5.5。质子离子载体和Zn2+可阻断解离，但F1、F0-ATP酶或Na+、K+-ATP酶的抑制剂对其无影响。解离不需要Na+或K+，但可被阴离子转运抑制剂阻断。在没有渗透性阴离子（Cl-）的情况下，解离速度减慢。早在配体内化后2分钟分离得到的囊泡内的受体-配体复合物对添加ATP有反应。结果表明，内体中的受体-配体解离需要ATP，可能是通过ATP依赖的质子泵为内体酸化提供能量。在存在渗透性阴离子的情况下，解离增强，提示存在阴离子通道或载体参与其中。
