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.

在此应用程序中，我们概述了一系列实验DesignID，以提供 对控制结合和的因素有更深入的了解 构成微管蛋白GTPase反应的水解步骤和 它们与微管自组装的关系：（1）检查 培养基和中间氧18交换反应 组装引起的GTPase（这里的目标是了解可逆性 水解机理的某些步骤和速率常数 定义其速率和通量。）； （2）确定立体化学 使用GTP Lambda S标记的氧气16，-17和 -18在lambda-磷酸盐中立体特定（这将有助于区分 在涉及一次或两次反转的反应之间 磷酸蛋白中间体的后一种特征）； （3） 表征鸟嘌呤核苷酸的结合机制（ 目的是了解有关微管蛋白构象变化的更多信息 之前或参加配体结合。）; （4）铬的应用（III） GTP复合物来定义小管蛋白结合的立体化学 金属加核苷酸复合物（该方法利用了反转的优势 核苷酸复合物与配体兑换以帮助得出 否则，几乎无法访问有关短暂寿命的结合的信息 MGGTP2结合。）; （5）研究在 单个伸长步骤（这将有助于区分提示和 延迟的DTP水解延迟，重复到小管蛋白与拉长结合 微管。）; （6）使用5'-甘尼氏二磷酸盐 修改GTPase动作的化学过程或抑制GTP 3 微管蛋白（这将代表第一次使用另一种尝试 GTP在可兑换核苷酸位点的可水解类似物）； （7） 32PI的重新审查小于GTP，而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 猪脑微管蛋白）； （8）等温的发展 聚合方法以消除潜在误导性冷稳定式的形式 微管蛋白（这种快速热浓度的新方法 可能有助于研究组装的调节。）。 总体目标 当这些研究结果和其他研究的结果时，将实现该项目的 集成到全球机制中。