Bulinski J C, Bossler A
Department of Anatomy and Cell Biology, Columbia University, College of Physicians & Surgeons, New York, NY 10032.
J Cell Sci. 1994 Oct;107 ( Pt 10):2839-49. doi: 10.1242/jcs.107.10.2839.
In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.
在之前的研究中（布林斯基和博里西（1979年）。《美国国家科学院院刊》76卷，293 - 297页；韦瑟比等人（1980年）。《生物化学》19卷，4116 - 4123页），一种相对分子质量（M(r)）约为125,000的微管相关蛋白（MAP）被鉴定为HeLa细胞中一种主要的MAP。我们着手对这种蛋白质进行生化特性分析，并确定其体外功能和体内分布。我们确定，与促进组装的MAPs，即tau、MAP2和MAP4一样，125 kDa的MAP对蛋白酶敏感且具有热稳定性。这种MAP的另一个特性，即在紫杉醇存在的情况下，它与对钙不敏感的微管群体异常紧密结合，被用于设计一种高效的纯化策略。由于该MAP与稳定的微管群体紧密结合，且在体外似乎有助于该微管群体的稳定性，我们将这种蛋白质命名为ensconsin。我们检测了纯化的ensconsin与微管的结合；ensconsin表现出结合，其微管结合位点在大约1:6（ensconsin:微管蛋白）的摩尔比下饱和。与迄今已鉴定的其他MAPs不同，ensconsin与微管的结合对中等盐浓度（≤0.6 M）不敏感。我们使用小鼠多克隆抗ensconsin抗体以及与先前描述的MAPs（如高分子量tau亚型、发动蛋白、STOP、CLIP - 170和驱动蛋白）反应的抗体，在免疫印迹实验中进一步对ensconsin进行了特性分析。这些实验表明，ensconsin与可能与微管相关的其他类似相对分子质量的蛋白质不同。用抗ensconsin抗体进行免疫荧光检测表明，在几种人上皮细胞、成纤维细胞和肌肉细胞系的大多数或所有微管中都能检测到ensconsin；发现其体内特性和分布，特别是对细胞的药物或其他处理的反应，与这些细胞类型中主要的MAP4不同。我们得出结论，ensconsin是一种存在于多种人类细胞中的MAP，在生化方面——也许在功能方面——与非神经元细胞中存在的其他MAPs不同。
