CHEMISTRY OF TUBULIN GUANINE NUCLEOTIDE INTERACTIONS

在本应用程序中，我们概述了一系列旨在提供 更深入地了解控制约束和 共同构成微管蛋白GTP酶反应和 它们与微管自组装的关系：(1)检查微管 与之相关的中等和中间氧-18交换反应 装配诱导的GTP酶(这里的目标是了解可逆性 水解机理中的某些步骤和反应速率常数 确定它们的速率和通量。)；(2)立体化学物质的测定 氧-16，-17和氧标记的GTP Lambda S的水解过程 -18在磷酸二氢叶酸中的立体专一性(这将有助于区分 在涉及一次内联攻击或两次反转的反应之间， 后者具有磷酸化-蛋白质中间体的特性)； 鸟嘌呤核苷酸结合机制的表征 目的是了解更多关于微管蛋白构象变化的信息 在配体结合之前或之后。)；(4)铬(III)的应用 GTP络合物用于确定微管蛋白结合的立体化学 金属-核苷酸络合物(利用反转性的方法 铬-核苷酸络合物的配体交换，以帮助衍生 否则几乎无法访问有关短期的绑定的信息 MgGTP2结合。)；(5)研究在 单一延伸步骤(这将有助于区分提示和 延迟型DTP水解物与微管蛋白结合 微管)；(6)使用5‘-鸟氧基过氧二磷酸潜在地 改变GTP酶作用的化学过程或抑制GTP结合 微管蛋白(这将是首次尝试利用另一种 (7)GTP的可交换核苷酸位置上的可水解类似物)； 32PI小于GTP和32PI小于GTP的复审 猪微管蛋白的GDP交换反应(这是 研究承诺解决牛和牛之间相互冲突的交换属性 猪脑微管蛋白)；(8)等温线的建立 消除潜在误导性冷稳型的聚合方法 微管蛋白(这种快速等温浓缩的新方法 可能有助于研究组装的调节。)。总体目标 当这些研究和其他研究的结果时，将实现该项目的 融入全球机制。

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.