BIOCHEMICAL-ANALYSIS OF THE MOVEMENT OF A MAJOR LYSOSOMAL MEMBRANE GLYCOPROTEIN IN THE ENDOCYTIC MEMBRANE SYSTEM
BIOCHEMICAL-ANALYSIS OF THE MOVEMENT OF A MAJOR LYSOSOMAL MEMBRANE GLYCOPROTEIN IN THE ENDOCYTIC MEMBRANE SYSTEM
复制标题
DOI：
10.1093/oxfordjournals.jbchem.a122922
复制
发表时间：
1989-10-01
影响因子：
2.7
通讯作者：
KATO, K
中科院分区：
文献类型：
--
作者：
FURUNO, K;YANO, S;KATO, K
以HRP-抗LGP107 Fab‘’和125I-抗LGP107-Ig G为探针，研究了LGP107在原代培养大鼠肝细胞胞膜转运系统中的运动。在培养液中加入HRP-抗LGP107 Fab‘’后，通过Percoll密度梯度的细胞分级检测抗体结合物从细胞表面到溶酶体的转移。HRP示踪剂在质膜和溶酶体部分呈双峰型亚细胞分布。随着细胞孵育时间的延长，溶酶体组分中的HRP含量增加。HRP在溶酶体中的蓄积率为每小时给药负荷的0.13%/106细胞。当给予细胞125I-抗LGP107抗体时，抗体不储存，但在溶酶体中迅速降解。通过测量释放到培养液中的TCA可溶放射性标记降解产物来评估细胞对125I-Ig G的摄取，随着给予抗体的浓度和孵育时间的增加而成比例地增加。多价~(125)I-Ig G的摄取速率与单价HRP-Fab‘’的摄取速率相当，即使在细胞长期暴露于饱和浓度的多价~(125)I-Ig G后仍保持不变。这种摄取过程在暴露于蛋白质合成抑制剂放线菌亚胺的细胞中持续了数小时。这些结果表明，LGP107在肝细胞表面和溶酶体之间存在持续循环。
HRP-anti LGP107 Fab'' and 125I-anti LGP107 IgG were used as probes to study the movement of LGP107 in the endocytic membrane transport system in primary cultured hepatocytes of rats. Following the addition of HRP-anti LGP107 Fab'' to the culture medium, the transfer of the antibody conjugate from the cell surface to lysosomes was examined by cell fractionation on Percoll density gradients. The HRP tracer showed a bimodal subcellular distribution, in plasma membrane and lysosomal fractions. The amount of HRP found in the lysosomal fractions became larger as the period of cell incubation was increased. The rate of HRP accumulation in lysosomes was 0.13% of the administered load per hour per 106 cells. When cells were given 125I-anti LGP107 IgG, the antibody was not stored but was rapidly degraded in the lysosomes. The uptake of 125I-IgG by the cells, which was assessed by measuring the TCA-soluble radiolabeled degradation products released into the medium, increased proportionally to the administered concentration of the antibody and to the incubation time. The rate of uptake of the polyvalent 125I-IgG was comparable to that for the uptake of the monovalent HRP-Fab'', and remained unchanged even after long exposure of the cells to a saturating concentration of the polyvalent IgG. This uptake process continued for many hours in the cells exposed to the protein synthesis inhibitor, cycloheximide. These results suggest that there is a continuous circulation of LGP107 between the cell surface and lysosomes in hepatocytes.