Doherty J J, Kay D G, Lai W H, Posner B I, Bergeron J J
Department of Anatomy, McGill University, Montreal, Canada.
J Cell Biol. 1990 Jan;110(1):35-42. doi: 10.1083/jcb.110.1.35.
To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.
为了阐明内体在肝脏中胰岛素降解过程中的作用，我们采用了一种无细胞系统，在该系统中评估了内化的125I-胰岛素在分离的完整内体中的降解情况。在无细胞系统中孵育含有内化125I-胰岛素的内体，会迅速产生三氯乙酸（TCA）可溶性放射性标记产物（半衰期为6分钟）。孵育期间从内体中提取的放射性物质经葡聚糖G-50柱层析显示，洗脱为放射性碘酪氨酸的物质随时间增加。胰岛素降解活性的表观Vmax为4 ng胰岛素降解·分钟-1·毫克细胞组分蛋白-1，表观Km为60 ng胰岛素·毫克细胞组分蛋白-1。内体蛋白酶具有胰岛素特异性，因为通过TCA沉淀和葡聚糖G-50柱层析评估发现，内化的125I-表皮生长因子（EGF）和125I-催乳素在分离的内体中均未降解。将对氯汞苯甲酸（PCMB）、1,10-菲啰啉、杆菌肽或0.1% Triton X-100加入系统后，观察到降解受到显著抑制。最大程度的胰岛素降解需要向无细胞系统中添加ATP，添加ATP会导致吖啶橙积累所测量的酸化。在内体pH消散剂（如尼日利亚菌素、莫能菌素和氯喹）或质子转运酶抑制剂N-乙基马来酰亚胺（NEM）和二环己基碳二亚胺（DCCD）存在的情况下，内体胰岛素降解受到显著抑制。胰岛素-受体复合物的聚乙二醇（PEG）沉淀显示，内体降解增强了胰岛素与其受体的解离，并且解离的胰岛素作为内体蛋白酶的底物。结果表明，胰岛素内化后迅速但不完全与其受体解离。然后，解离的胰岛素被胰岛素特异性蛋白酶降解，导致进一步的解离和降解。
