Cole D G, Cande W Z, Baskin R J, Skoufias D A, Hogan C J, Scholey J M
Department of Zoology, University of California, Davis 95616.
J Cell Sci. 1992 Feb;101 ( Pt 2):291-301. doi: 10.1242/jcs.101.2.291.
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.
为了解驱动蛋白及其相关蛋白在细胞分裂中的作用，有必要从有丝分裂细胞中鉴定并表征驱动蛋白超家族的多个成员。为此，我们针对与驱动蛋白超家族几个已知成员的马达结构域高度保守区域相对应的肽段制备了抗血清。如预期的那样，这些肽抗体与驱动蛋白和ncd蛋白的马达结构域特异性反应，并且它们还与几种与从经AMPPNP处理的海胆卵胞质溶胶中沉淀的微管（MT）共沉降的多肽（包括驱动蛋白重链）发生反应。随后对这些MT的ATP洗脱液进行分级分离，得到一种相对分子质量为330×10³的蛋白质，其表现为三种多肽的复合物，这三种多肽不同于传统的驱动蛋白亚基或其片段。该复合物包含与我们的肽抗体发生反应的85 kDa和95 kDa多肽，以及不发生反应的115 kDa多肽。我们将这种三联体多肽称为KRP(85/95)，它以AMPPNP增强的、ATP敏感的方式与纯化的海胆卵微管蛋白结合，并诱导微管束的形成。因此，我们提出该三联体对应于一种新的海胆卵驱动蛋白相关蛋白。
