MEMBRANE-GLYCOPROTEINS INVOLVED IN CELL-SUBSTRATUM ADHESION
MEMBRANE-GLYCOPROTEINS INVOLVED IN CELL-SUBSTRATUM ADHESION
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DOI：
10.1073/pnas.78.10.6071
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
BUCK, CA
中科院分区：
文献类型：
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作者：
KNUDSEN, KA;RAO, PE;BUCK, CA
结合免疫学和生化方法来鉴定参与细胞-基质粘附的表面膜成分。针对仓鼠细胞（幼仓鼠肾成纤维细胞 BHK-21/C-13 细胞和劳斯肉瘤转化的 C-13/B-4 细胞）表面膜制备的广谱抗血清，在体外诱导仓鼠成纤维细胞可逆性变圆和从培养基中脱离。这种现象被仓鼠细胞的 Nonidet P-40 提取物抑制。开发了抗体中和测定法来检测细胞 Nonidet P-40 提取物分级分离过程中抗原的存在。经过 2 个差异沉淀步骤、阴离子交换层析和连续凝集素亲和层析后，分离出具有阻断抗血清诱导的细胞粘附和外观变化的能力的极大富集的级分。通过 NaDodSO4[十二烷基硫酸钠]/聚丙烯酰胺凝胶电泳分析该级分揭示了一组高度限制的糖蛋白，其分子量约为 100。 140,000。凝集素纯化的糖蛋白级分用于产生更高滴度的抗血清，该抗血清能够诱导细胞可逆地变圆和从培养基上脱离，并且当固定在抗体亲和柱上时，能够结合并释放能够阻止抗血清诱导的细胞变圆的物质。这些方法使人们的注意力集中在一组有限的糖蛋白上，它们是表面膜的组成部分，并且在细胞-基质粘附过程中发挥一些尚未确定的作用。
A combination of immunological and biochemical methods was used to identify surface membrane components involved in cell-substratum adhesion. Broad-spectrum antiserum, prepared against surface membranes from hamster cells [baby hamster kidney fibroblast BHK-21/C-13 cell and Rous sarcoma transformed C-13/B-4 cell], induced reversible rounding and detachment of hamster fibroblasts from a substratum in vitro. This phenomenon was inhibited by Nonidet P-40 extracts of hamster cells. An antibody neutralization assay was developed to detect the presence of antigen during the fractionation of Nonidet P-40 extracts of cells. After 2 differential precipitation steps, anion exchange chromatography and sequential lectin affinity chromatography, a fraction greatly enriched in ability to block antiserum-induced changes in cell adhesion and appearance was isolated. Analysis of this fraction by NaDodSO4[sodium dodecylsulfate]/polyacrylamide gel electrophoresis revealed a highly restricted group of glycoproteins with MW .apprxeq. 140,000. A lectin-purified glycoprotein fraction was used to raise a higher titer antiserum that was able to induce reversible rounding and detachment of cells from a substratum and, when immobilized on an antibody affinity column, was able to bind and release material capable of blocking antiserum-induced cell rounding. These methods have allowed attention to be focused on a restricted group of glycoproteins that are integral constituents of the surface membrane and which play some as yet undetermined role in the process of cell-substratum adhesion.