Cleavage of cell surface proteins by thrombin.
Cleavage of cell surface proteins by thrombin.
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凝血酶裂解细胞表面蛋白。
DOI：
10.1002/jsscb.1981.380150106
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
Cunningham,DD
中科院分区：
文献类型：
--
作者：
Moss,M;Cunningham,DD
这项研究是基于我们以前的发现，凝血酶对培养的成纤维细胞的有丝分裂作用是在没有内化的情况下凝血酶与细胞表面相互作用的结果，并且凝血酶的蛋白分解活性是刺激细胞分裂所必需的。这促使我们使用标记细胞表面蛋白的二维凝胶电泳法寻找凝血酶介导的裂解。用3种方法标记表面膜组分：(1)用~(125)I-−用乳过氧化酶催化碘化标记蛋白质；(2)用半乳糖氧化酶氧化糖蛋白的半乳糖和氨基半乳糖残基，用~3H-BH4还原；(3)用~3H-岩藻糖孵育细胞代谢性标记糖蛋白。前两个步骤的标记是在凝血酶处理后进行的；相反，用~3H-岩藻糖代谢标记的细胞随后用凝血酶处理以寻找蛋白水解性裂解。总而言之，这些研究表明，只有大约5种细胞表面蛋白是凝血酶敏感的，这与该酶的高度特异性一致。每一种标记程序都显示了一种凝血酶敏感的细胞表面糖蛋白，通过免疫沉淀实验鉴定为纤维连接蛋白。此外，约140K和55K道尔顿的细胞表面蛋白对凝血酶敏感。但经凝血酶处理后，约45K道尔顿和130K~150K道尔顿的细胞表面蛋白增加。这些实验是在已建立的中国仓鼠肺细胞系上进行的，最终目标是研究凝血酶对凝血酶促有丝分裂作用有反应或无反应的大量克隆群体中凝血酶介导的细胞表面蛋白的切割。这种方法应该能够识别凝血酶刺激的细胞分裂所必需的蛋白水解性切割。
This study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin‐mediated cleavages using 2‐dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: (1) proteins were labeled by lactoperoxidase‐catalyzed iodination using125I−; (2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with3H‐NaBH4; and (3) glycoproteins were metabolically labeled by incubating cells with3H‐fucose. Labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with3H‐fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin‐sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin‐sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin‐sensitive. However, cell surface proteins of about 45K daltons and 130K to 1 50K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin‐mediated cleavages of cell surface proteins in a large number of cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages that are necessary for thrombin‐stimulated cell division.