Gonatas J, Stieber A, Olsnes S, Gonatas N K
J Cell Biol. 1980 Dec;87(3 Pt 1):579-88. doi: 10.1083/jcb.87.3.579.
The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques. The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population. Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP. In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.
利用形态学和生物化学技术研究了小鼠神经母细胞瘤单层培养物对蓖麻毒素、辣根过氧化物酶（HRP）以及蓖麻毒素-HRP偶联物的内吞作用。在4℃下，（125）I-蓖麻毒素和（125）I-蓖麻毒素-HRP与细胞的结合，作为配体浓度的函数，是一个可饱和的过程。在平衡时测定的表观亲和常数，蓖麻毒素为2.8×10（6）M（-1），蓖麻毒素-HRP为1×10（6）M（-1）。每个细胞的结合位点数，凝集素为8×10（7），偶联物为3×10（7）。（125）I-蓖麻毒素与单层的结合与细胞密度不成比例。我们发现在较高细胞浓度下结合减少，这表明随着细胞数量增加，配体与受体位点的可及性降低或位点减少。神经母细胞瘤细胞在高尔基体（GERL）附近有一个酸性磷酸酶阳性的池和囊泡网络。蓖麻毒素-HRP在与GERL对应的囊泡和池中以及残余体（致密体）中发生内吞作用。蓖麻毒素-HRP的细胞摄取量比游离HRP大100-200倍，并且蓖麻毒素不会刺激液相内吞作用。当单层暴露于天然HRP浓度为偶联物100倍时，过氧化物酶的细胞摄取量相当，但HRP仅定位于残余体中，从未定位于GERL成分中。这些结果支持以下结论：GERL参与蓖麻毒素-HRP的吸附性内吞作用，而残余体参与HRP的大量摄取。此外，通过定量电子显微镜放射自显影检查了蓖麻毒素的（125）I-亚基B（结合亚基）和（125）I-蓖麻毒素的结合、摄取和可能的再循环。在4℃下，蓖麻毒素及其结合亚基显示出相似的放射自显影颗粒分布，并且在2小时的内吞作用期间没有证据表明它们会分解或再循环到质膜。
