Tarone G, Ferracini R, Galetto G, Comoglio P
J Cell Biol. 1984 Aug;99(2):512-9. doi: 10.1083/jcb.99.2.512.
The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction.
幼仓鼠肾BHK细胞中不溶于Triton X-100的骨架由细胞核、中等大小的细丝和肌动蛋白纤维组成。通过透射电子显微镜观察，发现膜碎片与这些不溶性结构相关。当用0.5% Triton提取放射性碘化或[3H]葡糖胺标记的细胞时，除了一种分子量为85,000的糖蛋白（gp85）仍与不溶性骨架相关外，大多数质膜糖蛋白都被溶解。用特异性抗血清进行免疫沉淀表明，gp85不是纤连蛋白（一种不溶于去污剂的细胞外基质糖蛋白）的蛋白水解降解产物。制备了一种对BHK细胞gp85特异的单克隆抗体。用该单克隆抗体进行免疫荧光分析表明，gp85不与细胞外基质相关，而是局限于细胞膜。在固定和未固定的完整细胞中，荧光都集中在细胞表面优先排列成条纹的点上。发现gp85表现为一种与脂质双层疏水核心相互作用的整合膜蛋白，因为它可被SDS等离子去污剂从膜制剂中提取出来，但在没有去污剂的情况下不能被0.1 N NaOH（pH 12）提取，已知这种条件可释放外周分子。将Triton浓度提高到5%对gp85与细胞骨架的结合没有影响，但当肌动蛋白丝解离或在Triton存在下使用蛋白质变性剂（6 M尿素）时，gp85与细胞骨架的结合受到影响，这表明蛋白质-蛋白质相互作用参与了gp85与细胞骨架的结合。我们得出结论，gp85是一种整合的质膜糖蛋白，可能在细胞表面-细胞骨架相互作用中起作用。
