Furuno K, Ishikawa T, Kato K
J Biochem. 1982 May;91(5):1485-94. doi: 10.1093/oxfordjournals.jbchem.a133840.
The administration to rats of leupeptin produced prominent numbers of enlarged and irregularly shaped autolysosomes in hepatocytes. Percoll density equilibration of crude lysosomal fractions from rat livers showed that most lysosomal enzyme migrated form normal lysosomal density fractions toward higher density fractions within 30 min after leupeptin injection. The denser particles were ultrastructurally identified with the autolysosomes. In analysis by differential centrifugation of the liver homogenates, lysosomal enzymes became sedimentable with particles of greater sedimentation rate within 30 min after leupeptin injection. These changes in physical properties of lysosomes reverted to normal after 24 h, and concomitant disappearance of autolysosomes in hepatocytes was observed by electron microscopy. When the time course of distribution of leupeptin in subcellular fractions was analyzed, particle-bound leupeptin shifted with time to larger particle fractions in parallel with the shift of lysosomal enzyme distribution. Upon injection with leupeptin, cathepsin B activity was inhibited by more than 80% for about 3 h and was gradually restored to a normal level after 24 h. Leupeptin treatment caused marked delay of the degradation of endocytosed FITC-labeled asialofetuin in hepatic lysosomes, and its inhibitory effect lasted for over 9 h after the injection. It was suggested that autophagy is probably a normal process of protein degradation in hepatocytes, and because of a retarded digestion of sequestered materials, autolysosomes persisted for a long period and made up the majority of the lysosome population in the leupeptin-treated cells.
给大鼠注射亮抑酶肽后，肝细胞中出现大量增大且形状不规则的自噬溶酶体。对大鼠肝脏粗溶酶体组分进行Percoll密度平衡分析表明，注射亮抑酶肽后30分钟内，大多数溶酶体酶从正常溶酶体密度组分向更高密度组分迁移。超微结构鉴定显示，密度较高的颗粒为自噬溶酶体。通过对肝脏匀浆进行差速离心分析，注射亮抑酶肽后30分钟内，溶酶体酶可与沉降速率更高的颗粒一起沉降。溶酶体这些物理性质的变化在24小时后恢复正常，并且通过电子显微镜观察到肝细胞中的自噬溶酶体同时消失。当分析亮抑酶肽在亚细胞组分中的分布时间进程时，与溶酶体酶分布的变化平行，与颗粒结合的亮抑酶肽随时间转移到更大颗粒组分中。注射亮抑酶肽后，组织蛋白酶B活性在约3小时内被抑制超过80%，24小时后逐渐恢复到正常水平。亮抑酶肽处理导致肝溶酶体内内化的异硫氰酸荧光素标记的去唾液酸胎球蛋白降解明显延迟，其抑制作用在注射后持续超过9小时。这表明自噬可能是肝细胞中蛋白质降解的正常过程，并且由于被隔离物质的消化受阻，自噬溶酶体长期存在，并在亮抑酶肽处理的细胞中构成溶酶体群体的大部分。
