Purification and characterization of native conventional kinesin, HSET, and CENP-E from mitotic HeLa cells
Purification and characterization of native conventional kinesin, HSET, and CENP-E from mitotic HeLa cells
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DOI：
10.1074/jbc.m102801200
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发表时间：
2001-07-27
影响因子：
4.8
通讯作者：
Wilson, L
中科院分区：
文献类型：
--
作者：
DeLuca, JG;Newton, CN;Wilson, L
我们已经开发了一种从有丝分裂的HeLa细胞中纯化天然微管运动蛋白的策略，并在这里描述了人类传统运动蛋白和两种人类运动蛋白相关蛋白HSET和CENP-E的纯化和表征。我们发现，120 kda的HeLa细胞常规运动蛋白是一个主动马达，在室温下以类似于30妈妈/分钟的速度诱导微管滑动。这种活性形式的HeLa细胞激酶不含轻链，尽管在其他组分中检测到轻链。HSET是c端激酶亚家族的一员，也是首次以天然形式纯化，该蛋白在约75 kDa处以单带形式迁移。纯化后的HSET是一种主动马达，可诱导微管以接近5妈/分钟的速度滑动，微管在停止运动前平均滑动3妈。最后，我们纯化了原生的CENP-E，这是一种与有丝分裂期间染色体聚集有关的运动蛋白，我们发现这种形式的CENP-E不会诱导微管滑动，但能够与微管结合。
We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at similar to 30 mum/min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at similar to 75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of similar to5 mum/min, and microtubules glide for an average of 3 mum before ceasing movement. Finally, we Purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.