CHEMISTRY OF TUBULIN GUANINE NUCLEOTIDE INTERACTIONS

在本申请中，我们概述了一系列旨在提供 更深入地了解控制约束力的因素， - 水解步骤，其共同构成微管蛋白GTdR反应， 它们与微管自组装的关系：（1）检测微管的 与氧-18交换反应有关的中等和中等氧-18交换反应 汇编诱导的GTcloud（这里的目标是了解 水解机理中的某些步骤和速率常数 定义它们的速率和通量。）(2)立体化学的测定 使用用氧-16、-17和-18标记的GTP λ S的水解过程 -18立体特异性地在λ-磷酸盐中（这将有助于区分 在涉及一次串联攻击或两次反转的反应之间， 后者是磷酰基蛋白质中间体的特征）;（3） 鸟嘌呤核苷酸结合机制的表征（The 目的是了解微管蛋白的构象变化 在配体结合之前或参与配体结合。(4)铬（III）的应用 GTP复合物来定义微管蛋白结合的立体化学， 金属-核苷酸复合物（该方法利用了 铬-核苷酸复合物的配体交换， 否则几乎无法访问的信息绑定的短命 MgGTP 2结合）; (5)研究了水解过程中的时间过程， 单伸长步骤（这将有助于区分快速和 延迟的DTP水解与微管蛋白结合到延长的 微管）; (6)5 '-鸟苷酰过氧二磷酸的用途， 改变GTP作用的化学过程或抑制GTP比宁， 微管蛋白（这将是首次尝试利用另一种 在可交换的核苷酸位点处的GTP的可水解类似物）;（7） 复查32 Pi小于大于GTP和32 Pi小于 大于猪微管蛋白的GDP交换反应（这 这项研究有望解决牛和牛之间相互矛盾的交换特性， 猪脑微管蛋白）;（8）等温 消除潜在误导的冷稳定形式的聚合方法 微管蛋白的快速等温浓缩新方法 可能有助于研究组装的调节）。 总目标 当这些研究和其他研究的结果出来时， 纳入全球机制。

In this application, we outline a series of experiments designid to provide a more in-depth understanding of the factors controlling the binding and hydrolytic steps which together constitute the tubulin GTPase reaction and their relationship to microtubule self-assembly: (1) Examination of the medium and intermediate oxygen-18 exchange reactions associated with the assembly-induced GTPase (The goal here is to learn about the reversibility of certain steps in the hydrolytic mechanism and the rate constants which define their rates and fluxes.); (2) determination of the stereochemical course of the hydrolysis using GTP Lambda S labeled with oxygen-16, -17 and -18 stereospecifically in the Lambda-phosphate (This will help discriminate between reactions involving a single in-line attack or two inversions, the latter characteristic of a phosphoryl-protein intermediates); (3) Characterization of the binding mechanism for guanine nucleotides (The objective is to learn more about the tubulin conformational changes preceding or attending ligand binding.); (4) Application of chromium (III) GTP complexes to define the stereochemistry of tubulin binding of metal-neucleotide complexes (The approach taken advantage of the invertness of chromium-nucleotide complexes to ligand exchange to help derive otherwise nearly inaccessible information about the binding of short-lived MgGTP2-binding.); (5) Studies of the time-course of hydrolysis during a single elongation step (This will help to distinguish between prompt and delayed DTP hydrolysis with repsect to tubulin binding to an elongating microtubule.); (6) Use of 5'-guanylyl peroxy-diphosphate to potentially modify the chemical course of GTPase action or to inhibit GTP bining to tubulin (This would represent the first attempt to utilize another hydrolyzable analogue of GTP at the exchangeable nucleotide site); (7) Reexamination of the 32Pi less than greater than GTP and 32Pi less than greater than GDP exchange reactions of porcine microtubule protein (This study promises to resolve the conflicting exchange properties of bovine and porcine brain microtubule proteins); (8) Development of an isothermal polymerization method to eliminate potentially misleading cold-stable forma of microtubule protein (This new approach of rapid isothermal concentration may be helpful in studying the regulation of assembly.). The overall goals of the project will be realized when results of these and other studies integrate into a global mechanism.