Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.
Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.
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发表时间：
1992-02
影响因子：
4
通讯作者：
D. Cole;W. Cande;R. Baskin;D. Skoufias;AN CHRISTOPHERJ.HOG;J. Scholey
中科院分区：
文献类型：
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作者：
D. Cole;W. Cande;R. Baskin;D. Skoufias;AN CHRISTOPHERJ.HOG;J. Scholey
为了了解Kinesin及其近亲在细胞分裂中的作用，有必要从有丝分裂细胞中鉴定和鉴定Kinesin超家族的多个成员。为此，我们提出了与运动蛋白超家族中几个已知成员的运动域的高度保守区域相对应的多肽的抗血清。正如预期的那样，这些多肽抗体与运动蛋白和NCD蛋白的运动域发生特异性反应，它们还与几种多肽(包括运动蛋白重链)反应，这些多肽与从AMPPNP处理的海胆卵胞浆中沉淀的微管(MTS)共形成。随后对这些MT的ATP洗脱物进行分级，得到相对分子质量为330×10(3)的蛋白质，其表现为三种不同于传统动蛋白亚基或其片段的多肽的复合体。该复合体包含85 kDa和95 kDa的多肽，与我们的多肽抗体发生反应，以及115 kDa的多肽，不与我们的多肽抗体反应。这种三聚体多肽，我们称之为Krp(85/95)，以AMPPNP增强的、ATP敏感的方式与纯化的海胆卵管蛋白结合，并诱导微管束的形成。因此，我们认为该三联体对应于一种新的海胆卵动蛋白相关蛋白。
To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 x 10(3) that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.