ASSEMBLY AND DISASSEMBLY OF CYTOPLASMIC MICROTUBULES

拟议研究的长期目标是了解 微管蛋白对GTP的水解、结合与细胞功能的关系 微管相关蛋白(MAP)，以及微管相关蛋白的几何和动力学 微管的组装。五个相互关联的具体项目是 建议。(1)了解GTP和GDP与微管蛋白的结合 亚基将被扩展到对分子基础的研究 微管组装和拆卸对镁离子的依赖性。(2) 在无MAP的微管蛋白组装过程中，从 将研究从带状中间体到微管的过程。角色 GTP水解、MAP和聚合核在生产圆柱形中的作用 我们将研究微管蛋白聚集体的形状。(3)非共价络合物 由孤立但完整的地图组成，可能由超过 一种类型的MAP和微管蛋白将根据它们的大小来表征， 组成，以及它们的化学计量比和互换性 蛋白质。(4)MAP与Main绑定之间的潜在差异 GDP-微管蛋白在微管中心的晶格和到 GTP-微管蛋白在“帽”的末尾将被检查。两者的差异 将寻求结合蛋白的种类和数量。(5)自发形成 由微管在溶液中的近乎平行排列的区域 调查以了解浓度和长度依赖关系有多好 这一过程符合现有的理论。建议的方法主要是生物物理方法。 和生物化学。GTP的水解度将通过放射性同位素进行评估 微管和片状模板的测量和组装 浊度和小角光散射的测量。地图复合体 并通过层析和层析获得它们的蛋白质含量 电泳法。它们的大小和形状将通过凝胶排斥来测量 层析和分析超速离心法。微管和微管蛋白的交换 MAP将通过快速凝胶过滤和离心法(Airfuge)进行评估 借助放射性同位素和荧光标记。这项工作将提供 对微管形成机制的基本洞察 既能动态组装/拆卸，又能相对稳定 将有助于为研究它们在病理上的故障提供基础 情况，例如，外周轴突运输中断 神经病、细胞内运动障碍和控制障碍 有丝分裂，如在癌症中。

Long-term objectives of the proposed research are to understand the relationships between GTP hydrolysis by tubulin, binding and function of microtubule-associated proteines (MAPs), and the geometry and kinetics of assembly of microtubules. Five interrelated specific projects are proposed. (1) Understanding of the binding of GTP and GDP to tubulin subunits will be extended to an investigation of the molecular basis of the Mg2+-dependence of assemby and disassembly of microtubules. (2) The kinetics of the transition, during assembly of MAP-free tubulin, from ribbon-like intermediates to microtubules will be investigated. The roles of GTP hydrolysis, MAPs, and polymerization nuclei in producing cylindrical shape in tubulin aggregates will be studied. (3) The noncovalent complexes formed by isolated but unfractionated MAPs, probably composed of more than one type of MAP and tubulin , will be characterized as to their size, composition, and the stoichiometric ratios and exchangeability of their proteins. (4) Potential differences between binding of MAPs to the main lattice of GDP-tubulin in the center of the microtubule and to the GTP-tubulin in the "cap" at the end will be examined. Differences in both kind and amount of bound protein will be sought. (5) Spontaneous formation by microtubules in solution of regions of nearly parallel alignment will be investigated to learn how well the concentration- and length-dependence of the process fit existing theory. Proposed methods are chiefly biophysical and biochemical. GTP hydrolysis will be assessed by radioisotope measurements, and assembly of microtubules and sheetlike forms by measurement of turbidity and small-angle light-scattering. MAP complexes will be isolated and their protein content obtained by chromatography and electrophoresis. Their size and shape will be measured by gel exclusion chromatography and analytical ultracentrifugation. Exchange of tubulin and MAPs will be assessed by rapid gel-filtration and centrifugation (Airfuge) aided by radioisotope and fluorescent labeling. The work will provide fundamental insight into the mechanisms that allow microtubules to be capable both of dynamic assembly/disassembly and of relative stability, and will help provide a basis for studies of their malfunction in pathological conditions, e.g., interruption of axonal transport in peripheral neuropathies, failures of intracellular movement, and disruption of control of mitosis, as in cancer.