Actin-binding and dimerization domains of HeLa cell filamin.
Actin-binding and dimerization domains of HeLa cell filamin.
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DOI：
10.1021/bi00406a011
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发表时间：
1988-03
期刊：
影响因子：
2.9
通讯作者：
R. Weihing
中科院分区：
文献类型：
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作者：
R. Weihing
HeLa 细胞细丝蛋白是一种线性、二价、高分子量亚基同型二聚体（Mr 250,000），可以在体内将肌动蛋白丝交联成超分子结构，例如网络和束。我们使用来自鸡胸肌的毫摩尔 Ca 蛋白酶将亚基切割成更小的片段，我们根据二聚体的整体结构绘制这些片段。该酶将 HeLa 细丝蛋白切割成更大的（Mr 250,000）。 192,000) 和一个较小的 (Mr 104,000) 片段；较小的片段是一个更小的 (Mr 92,000) 片段的前体，在共沉淀测试中只有较大的片段与肌动蛋白结合，表明它含有亚基的肌动蛋白结合区域。 192,000 片段，但 Mr 104,000/92,000 片段的产率很低，因为天然细丝蛋白是头对头二聚体，预计交联最容易在二聚化位点进行，因此，Mr 192,000 片段似乎从交联细丝蛋白中裂解，因为它位于二聚化区域的远端，而 Mr 192,000 片段似乎从交联细丝蛋白中裂解出来。 104,000/92,000 个片段不存在，因为它们位于二聚化区域，并且交联成十二烷基硫酸钠电泳无法识别的形式。
HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.