CHEMISTRY OF TUBULIN GUANINE NUCLEOTIDE INTERACTIONS

微管蛋白鸟嘌呤核苷酸相互作用的化学性质

• 批准号: 3289724
• 负责人: Daniel Lee Purich
• 金额: $ 11.1万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289724
• 关键词: brain; cell biology; conformation; guanosine triphosphate; hydrolase; ligands; mass spectrometry; polymerization; radiotracer; stereochemistry

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.

在此应用中，我们概述了一系列旨在提供 更深入地了解控制结合和的因素 水解步骤共同构成微管蛋白 GTP 酶反应和 它们与微管自组装的关系：（1）检查 中和中间氧 18 交换反应与 组装诱导的 GTPase（此处的目标是了解可逆性 水解机制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 使用标记有氧-16、-17 和 -17 的 GTP Lambda S 进行水解的过程 -18 立体定向地存在于 Lambda-磷酸盐中（这将有助于区分 在涉及单个串联攻击或两个反转的反应之间， 磷酰蛋白中间体的后一个特征）； (3) 鸟嘌呤核苷酸结合机制的表征（The 目的是了解更多有关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 复合物定义微管蛋白结合的立体化学 金属-核苷酸复合物（该方法利用了反转性 铬-核苷酸复合物与配体交换以帮助衍生 否则几乎无法获取有关短期绑定的信息 MgGTP2 结合。）； (5) 水解过程的研究 单伸长步骤（这将有助于区分提示和 延迟 DTP 水解，与微管蛋白结合延伸 微管。）； (6) 5'-鸟苷基过氧二磷酸盐的用途 改变 GTP 酶作用的化学过程或抑制 GTP 结合 微管蛋白（这将代表首次尝试利用另一种 GTP 在可交换核苷酸位点的可水解类似物）； (7) 复审32Pi小于大于GTP和32Pi小于 大于猪微管蛋白的GDP交换反应（这 研究有望解决牛和牛之间相互冲突的交换特性 猪脑微管蛋白）； (8) 等温线的开发 聚合方法以消除潜在的误导性冷稳定形式 微管蛋白（这种快速等温浓缩的新方法 可能有助于研究集会的规定。）。 总体目标 当这些研究和其他研究的结果出来时，该项目的目标就会实现 融入全球机制。