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、结合和功能之间的关系 微管相关蛋白（MAPs），和几何形状和动力学 微管的组装。 五个相互关联的具体项目是 提出了 (1)对GTP和GDP与微管蛋白结合的理解 亚基将扩展到研究的分子基础， Mg ~（2+）对微管组装和拆卸的依赖性。 (2)的 在无MAP的微管蛋白组装期间， 将研究微管的带状中间体。 的角色 GTP水解，MAPs和聚合核在生产圆柱形 将研究微管蛋白聚集体的形状。 (3)非共价复合物 由分离但未分级的MAP形成，可能由超过 一种类型的MAP和微管蛋白，将表征它们的大小， 化学计量比和交换率 proteins. (4)MAPs与主要药物结合之间的潜在差异 在微管中心的GDP-微管蛋白的晶格中， GTP-微管蛋白在“帽”的最后将被检查。 性差异 将寻求结合蛋白质种类和量。 (5)自发形成 通过微管在溶液中几乎平行排列的区域， 调查以了解浓度和长度的依赖性如何 该工艺符合现有理论。 提出的方法主要是生物物理的 和生物化学。 将通过放射性同位素评估GTP水解 测量和组装微管和片状形式， 浊度和小角度光散射的测量。 MAP复合物 将被分离，并通过色谱法获得其蛋白质含量， 电泳 它们的大小和形状将通过凝胶排阻法测量 色谱和分析超离心。 微管蛋白交换， 将通过快速凝胶过滤和离心（Airfuge）评估MAP 通过放射性同位素和荧光标记辅助。 这项工作将提供 对微管的基本机制的深入了解， 能够动态组装/拆卸和相对稳定，以及 将有助于为研究其在病理学上的功能障碍提供依据。 条件，例如，外周神经轴突运输中断 神经病变、细胞内运动失败和控制中断 有丝分裂，如癌症。

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.