Evans W H, Flint N
Biochem J. 1985 Nov 15;232(1):25-32. doi: 10.1042/bj2320025.
The complexity of rat liver endosome fractions containing internalized radioiodinated asialotransferrin, asialo-(alkaline phosphatase), insulin and prolactin was investigated by using free-flow electrophoresis and isopycnic centrifugation in Nycodenz gradients. Two subfractions were separated by free-flow electrophoresis. Both subfractions contained receptors for asialoglycoprotein and insulin. Glycosyltransferase activities were associated with the more electronegative vesicles, whereas 5'-nucleotidase and alkaline phosphodiesterase activities were associated with the less electronegative vesicles. Three subfractions were separated on Nycodenz gradients. Two subfractions, previously shown to become acidified in vitro, contained the ligands. At short intervals after uptake (1-2 min), ligands were mainly in subfraction DN-2 (density 1.115 g/cm3), but movement into subfraction DN-1 (density 1.090 g/cm3) had occurred 10-15 min after internalization. Low amounts of glycosyltransferase activities were associated with subfraction DN-2, and 5'-nucleotidase and alkaline phosphodiesterase activities were mainly located in subfraction DN-1. The binding sites for asialoglycoproteins and insulin were distributed towards the higher density range in the Nycodenz gradients, thus indicating a segregation of receptor-enriched vesicles and those vesicles containing the various ligands 10-15 min after internalization. Electron microscopy of the subfractions separated on Nycodenz gradients indicated that whereas the ligand-transporting fractions consisted mainly of empty vesicles (average diameter 100-150 nm), the receptor-enriched component was more granular and smaller (average diameter 70-95 nm). The properties of the endosome subfraction are used to assign their origin to the regions of the endocytic compartment where ligand-receptor dissociation and separation occur.
利用自由流动电泳和在Nycodenz梯度中的等密度离心法，研究了含有内化放射性碘化去唾液酸转铁蛋白、去唾液酸（碱性磷酸酶）、胰岛素和催乳素的大鼠肝内体组分的复杂性。通过自由流动电泳分离出两个亚组分。两个亚组分都含有去唾液酸糖蛋白和胰岛素的受体。糖基转移酶活性与电负性更强的囊泡相关，而5'-核苷酸酶和碱性磷酸二酯酶活性与电负性较弱的囊泡相关。在Nycodenz梯度上分离出三个亚组分。先前显示在体外会酸化的两个亚组分含有配体。摄取后短时间间隔（1-2分钟）时，配体主要在亚组分DN-2（密度1.115 g/cm³）中，但内化10-15分钟后已转移到亚组分DN-1（密度1.090 g/cm³）中。亚组分DN-2中糖基转移酶活性较低，5'-核苷酸酶和碱性磷酸二酯酶活性主要位于亚组分DN-1中。去唾液酸糖蛋白和胰岛素的结合位点在Nycodenz梯度中向更高密度范围分布，这表明内化10-15分钟后富含受体的囊泡与含有各种配体的囊泡发生了分离。对在Nycodenz梯度上分离的亚组分进行电子显微镜观察表明，转运配体的组分主要由空囊泡组成（平均直径100-150 nm），而富含受体的组分更具颗粒性且更小（平均直径70-95 nm）。内体亚组分的特性用于确定其起源于内吞区室中发生配体-受体解离和分离的区域。
