Baenziger J U, Fiete D
J Biol Chem. 1986 Jun 5;261(16):7445-54.
Rat hepatocytes incubated in high K+ buffer (all Na+ is replaced by K+) internalize glycoproteins bearing terminal galactose moieties but are not able to deliver them to lysosomes (Baenziger, J. U., and Fiete, D. (1982) J. Biol. Chem. 257, 6007-6009). Instead, internalized ligand accumulates in a prelysosomal compartment(s) with a density similar to that of plasma membrane. We have separated two populations of prelysosomal endocytic vesicles from hepatocytes incubated in high K+ buffer. The vesicle population VR.L has a mean density of 1.14 by sucrose gradient centrifugation and contains functionally active Gal/GalNAc-specific receptor which is able to bind intravesicular ligand. The vesicle population VL has a mean density of 1.19. It contains ligand, but is deficient in Gal/GalNAc-specific receptor when compared to VR.L. These two vesicle populations appear to arise from intracellular organelles which participate in receptor-ligand segregation in rat hepatocytes. Pulse-chase experiments indicate that ligand passes from VR.L to VL. VR.L and VL are also detected in hepatocytes incubated in buffers containing physiologic amounts of Na+; however, the proportion of ligand found in VL is less than in cells incubated in K+-containing buffer. The primary effect of high K+ buffer is to prevent exit of ligand from VL whereas the accumulation of ligand in VR.L is likely secondary to the effect on VL. Membrane protein constituents of VR.L and VL were identified by vectorial lactoperoxidase labeling using a galactosyl conjugate of lactoperoxidase. Vesicles containing Gal-lactoperoxidase were isolated and labeling initiated by addition of 125I, glucose, and glucose oxidase. The labeling patterns for VR.L and VL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinct from the more complex labeling pattern obtained at the cell surface. Analysis by two-dimensional electrophoresis demonstrated a highly selective labeling pattern with only a small number of differences between VR.L and VL. This suggests that the major membrane components of the compartments prior to and following receptor-ligand segregation are the same. Thus, receptors may be selectively removed from these membranes during the process of receptor-ligand segregation.
在高钾缓冲液（所有钠离子被钾离子取代）中孵育的大鼠肝细胞会内化带有末端半乳糖部分的糖蛋白，但无法将它们转运至溶酶体（贝恩齐格，J. U.，和菲耶特，D.（1982年）《生物化学杂志》257卷，6007 - 6009页）。相反，内化的配体聚集在一个密度与质膜相似的溶酶体前区室中。我们从在高钾缓冲液中孵育的肝细胞中分离出了两群溶酶体前内吞小泡。通过蔗糖梯度离心，小泡群体VR.L的平均密度为1.14，并且含有功能活跃的半乳糖/ N - 乙酰半乳糖胺特异性受体，该受体能够结合小泡内的配体。小泡群体VL的平均密度为1.19。它含有配体，但与VR.L相比，缺乏半乳糖/ N - 乙酰半乳糖胺特异性受体。这两群小泡似乎源自参与大鼠肝细胞中受体 - 配体分离的细胞内细胞器。脉冲追踪实验表明配体从VR.L传递至VL。在含有生理量钠离子的缓冲液中孵育的肝细胞中也检测到了VR.L和VL；然而，在VL中发现的配体比例低于在含钾缓冲液中孵育的细胞。高钾缓冲液的主要作用是阻止配体从VL中排出，而配体在VR.L中的积累可能是对VL影响的继发结果。通过使用乳过氧化物酶的半乳糖基共轭物进行向量性乳过氧化物酶标记，鉴定了VR.L和VL的膜蛋白成分。分离出含有半乳糖 - 乳过氧化物酶的小泡，并通过添加125I、葡萄糖和葡萄糖氧化酶启动标记。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳得到的VR.L和VL的标记模式与在细胞表面获得的更复杂的标记模式不同。二维电泳分析显示出高度选择性的标记模式，VR.L和VL之间只有少数差异。这表明在受体 - 配体分离之前和之后的区室的主要膜成分是相同的。因此，在受体 - 配体分离过程中，受体可能会从这些膜中被选择性去除。
