Shimizu T, Toyoshima Y Y, Edamatsu M, Vale R D
National Institute of Bioscience and Human-Technology, Ibaraki, Japan.
Biochemistry. 1995 Feb 7;34(5):1575-82. doi: 10.1021/bi00005a013.
Cytoplasmic dynein and ncd, a kinesin-related protein from Drosophila, are motor proteins that move toward the minus ends of microtubules, while kinesin moves to the microtubule plus end. In previous work, we examined the nucleotide dependence of motility and enzymatic activity by kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F., & Vale, R. D., (1991) J. Cell Biol. 112, 1189-1197]. In this study, we examined these activities of the cytoplasmic dynein from bovine brain and ncd in order to explore what enzymatic features might be shared by these two minus-end-directed motors. Both ncd and cytoplasmic dynein demonstrated an activation of ATPase activity upon the addition of microtubules (30-fold and 6-fold, respectively). A significant difference between ncd and cytoplasmic dynein was their relative sensitivity to vanadate and to aluminum fluoride. In contrast to cytoplasmic dynein, ncd polypeptide was not cleaved by UV-vanadate treatment, and its ATPase and motility were unaffected by vanadate (up to 0.1 mM). When the nucleotide requirement for movement as examined using a battery of 20 nucleotides and nucleotide analogues, cytoplasmic dynein was found to exhibit a specificity very similar to that of axonemal dyneins from Tetrahymena. Surprisingly, however, the nucleotide specificities of in vitro motility produced by ncd or its construct, GST/MC1 (a fusion protein of glutathione S-transferase and 210-700 of the predicted ncd amino acid sequence), were quite distinct from that of kinesin. Thus, the nucleotide specificity profiles of members of the kinesin motor superfamily do not appear to be identical.
胞质动力蛋白和果蝇中的一种与驱动蛋白相关的蛋白质ncd是向微管负端移动的运动蛋白，而驱动蛋白则向微管正端移动。在之前的工作中，我们研究了驱动蛋白运动性和酶活性的核苷酸依赖性[Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F., & Vale, R. D., (1991) J. Cell Biol. 112, 1189 - 1197]。在本研究中，我们检测了牛脑胞质动力蛋白和ncd的这些活性，以探究这两种向负端移动的运动蛋白可能共有的酶学特征。ncd和胞质动力蛋白在添加微管后均表现出ATP酶活性的激活（分别为30倍和6倍）。ncd和胞质动力蛋白之间的一个显著差异是它们对钒酸盐和氟化铝的相对敏感性。与胞质动力蛋白不同，ncd多肽在紫外线 - 钒酸盐处理下不会被切割，其ATP酶活性和运动性不受钒酸盐影响（高达0.1 mM）。当使用一系列20种核苷酸和核苷酸类似物检测运动所需的核苷酸时，发现胞质动力蛋白表现出与嗜热四膜虫轴丝动力蛋白非常相似的特异性。然而，令人惊讶的是，ncd或其构建体GST/MC1（谷胱甘肽S - 转移酶与预测的ncd氨基酸序列210 - 700的融合蛋白）产生的体外运动性的核苷酸特异性与驱动蛋白的截然不同。因此，驱动蛋白运动超家族成员的核苷酸特异性谱似乎并不相同。
