Cross-linking of Novikoff ascites hepatoma cytokeratin filaments.
Cross-linking of Novikoff ascites hepatoma cytokeratin filaments.
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Novikoff 腹水肝癌细胞角蛋白丝的交联。
DOI：
10.1021/bi00337a026
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Hnilica,LS
中科院分区：
文献类型：
--
作者：
Ward,WS;Schmidt,WN;Schmidt,CA;Hnilica,LS
生物化学和病理学系，A. B. Hancock, Jr.，范德比尔特实验室，大学癌症中心，范德比尔特大学医学院分子毒理学中心，纳什维尔，田纳西州 37232 收到 1984 年 10 月 5 日摘要：我们使用可裂解交联剂研究了来自 Novikoff 腹水肝癌的溶解细胞角蛋白的结构S((S)-二硫代双磺基琥珀酰亚胺基丙酸酯)在6M尿素存在下实现部分复合物熔化。通过二维凝胶电泳，其中蛋白质交联在二维上被破坏，我们鉴定了两个主要复合物，即p39-p56二聚体和(p39-p56)2四聚体，p39和p56是诺维科夫腹水肝癌中的两种主要细胞角蛋白。研究二聚体和四聚体之间可能关系的实验采用了免疫印迹和两种识别 p56 或 p39 细胞角蛋白的单克隆抗体。当交联的蛋白质浓度非常低时，二聚体是主要产物。随着蛋白质浓度的增加，我们注意到二聚体减少，四聚体相应增加，这表明二聚体可能是四聚体的前体。为了支持交联实验，在第一维中使用 4 M 尿素的二维凝胶电泳表明 Novikoff 腹水肝癌细胞角蛋白复合物中 p56 和 p39 的主要关联。细胞角蛋白是一组分子量范围为 40 000-70 000 的水不溶性蛋白质，包含上皮细胞的 10 nm 细胞结构中间丝（Lazarides，1980；Moll 等，1982）。上皮细胞类型可以根据细胞角蛋白组成进行分类（Moll 等，1982），最近的报告表明细胞角蛋白表达谱在各种肿瘤的病理诊断中可能很重要（Debus 等，1984；Franke 等，1982）。细胞角蛋白被分为两组：较大、碱性更强的 II 型蛋白和较小、酸性 I 型蛋白（Fuchs 等，1981）。迄今为止检查的每种细胞类型都至少含有一种来自每组的细胞角蛋白（Schiller 等人，1982）。
Departments of Biochemistry and Pathology, A. B. Hancock, Jr., Laboratory of the Vanderbilt, University Cancer Center, and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received October 5, 1984 abstract: We have investigated the structure of solubilized cytokeratins from Novikoff ascites hepatoma using the cleavable cross-linker S^'-dithiobisfsulfosuccinimidyl propionate) in the presence of 6 M urea to effect partial complex melting. By two-dimensional gel electrophoresis, in which the protein cross-links were broken in the second dimension, we have identified two major complexes as a p39-p56 dimer and a (p39—p56) 2 tetramer, p39 and p56 being two of the major cytokeratins in Novikoff ascites hepatoma. Experiments investigating possible relationships between the dimer and tetramer employed immunoblots and two monoclonal antibodies which recognized either p56 or p39 cytokeratins. When very low protein concentrations were cross-linked, the dimer was the predominant product. As protein concentration increased, we noted a decrease in dimers and a corresponding increase in tetramers, suggesting that the dimer may be a precursor to the tetramer. In support of the cross-linking experiments, two-dimensional gel electrophoresis using 4 M urea in the first dimension indicated a predominant association of p56 and p39 in the Novikoff ascites hepatoma cytokeratin complexes. e cytokeratins are a group of water-insoluble proteins in the molecular weight range of 40 000-70 000 that comprise the 10-nm, cytostructural intermediate filaments of epithelial cells (Lazarides, 1980; Moll et al., 1982). Epithelial celltypes can be classified on the basis of the cytokeratin composition (Moll et al., 1982), and recent reports suggest that profiles of cytokeratin expression may be important in the patho-diagnosis of various neoplasms (Debus et al., 1984; Franke et al., 1982). Cytokeratins have been divided into twogroups: the larger, more basic type II and the smaller, acidic type I proteins (Fuchs et al., 1981). Every cell type thus far examined contains at least one cytokeratin from each group (Schiller et al., 1982).