ISOLATION AND CHARACTERIZATION OF THE APICAL SURFACE OF POLARIZED MADIN-DARBY CANINE KIDNEY EPITHELIAL-CELLS
ISOLATION AND CHARACTERIZATION OF THE APICAL SURFACE OF POLARIZED MADIN-DARBY CANINE KIDNEY EPITHELIAL-CELLS
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DOI：
10.1073/pnas.85.5.1529
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发表时间：
1988-03-01
影响因子：
11.1
通讯作者：
RODRIGUEZBOULAN, E
中科院分区：
文献类型：
--
作者：
SAMBUY, Y;RODRIGUEZBOULAN, E
我们已经开发了一种快速有效的方法来分离Madin-Darby犬肾上皮细胞的顶面。汇合后的细胞单层涂有交替层的阳离子化的胶体二氧化硅和聚阴离子，60%的顶端表面被回收作为大膜片通过应用程序的聚赖氨酸涂层的玻璃表面。顶端表面的细胞质方面的扫描电子显微镜显示了蜂窝状图案，由细胞边界断裂或以上的紧密连接的水平。通过透射电子显微镜，顶端准备似乎是由质膜和细胞质薄层。酶分析和免疫印迹证明了6至7倍的顶端标记物的富集和细胞质和基底侧标记物的低水平污染。通过碱提取去除细胞溶质污染物和外周膜蛋白后，通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（NaDodSO 4/PAGE）表征顶端整合膜蛋白。凝集素印迹和[3 H]葡糖胺标记确定了两个主要的唾液酸糖蛋白的表观先生200，000和100，000。这里描述的顶端膜片提供了一个有用的模型，用于膜的分子组成的系统表征，在无细胞系统中的脂质和蛋白质运输的重建，并为研究膜下细胞骨架蛋白与顶端质膜结构域的相互作用。
We have developed a fast and efficient method to isolate the apical surface of Madin-Darby canine kidney epithelial cells. After confluent cell monolayers were coated with alternate layers of cationized colloidal silica and a polyanion, 60% of the apical surface was recovered as large membrane sheets through the application of a polylysine-coated glass surface. Scanning electron microscopy of the cytoplasmic aspect of the apical surface revealed a honeycomb pattern given by the cell borders fractured at or above the level of the tight junctions. By transmission electron microscopy, the apical preparation appeared to be composed of plasma membrane and a thin layer of cytoplasm. Enzyme assays and immunoblots demonstrated a 6- to 7-fold enrichment of an apical marker and a low level of contamination by cytoplasmic and basolateral markers. After removal of cytosolic contaminants and peripheral membrane proteins by alkaline extraction, apical integral membrane proteins were characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Lectin blotting and [3H]glucosamine labeling identified two major sialoglycoproteins of apparent Mr 200,000 and 100,000. The apical membrane sheets here described provide a useful model for systematic characterization of the molecular components of the membrane, for reconstitution of lipid and protein transport in cell-free system, and for study of the interactions of submembranous cytoskeletal proteins with the apical plasma membrane domain.