CYTOCHROME B AND NEUTROPHIL SUPEROXIDE PRODUCTION

细胞色素 B 和中性粒细胞过氧化物的产生

• 批准号: 2063498
• 负责人: ALGIRDAS JOSEPH JESAITIS
• 金额: $ 19.62万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-03-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2063498
• 关键词: NAD(P)H dehydrogenase; antibody; conformation; cytochrome b; electron transport; electrospray ionization mass spectrometry; epitope mapping; fluorescent dye /probe; genetic library; hemoprotein structure; high performance liquid chromatography; human tissue; laboratory rabbit; molecular biology; monoclonal antibody; neutrophil; nuclear magnetic resonance spectroscopy; phosphorylation; physical model; protein sequence; protein structure function; stoichiometry; superoxides; synthetic peptide

The broad long term objective of this proposal is to determine the molecular basis of superoxide production in human neutrophils. The proposed investigation will focus on the structure of human neutrophil cytochrome b and how it changes upon activation of the superoxide generating system. The fundamental assumption made in this proposal is that this cytochrome is the central electron transferase of superoxide production and that modifications of its structure will regulate the flow of electrons across the plasma membrane. Understanding the molecular mechanisms of regulation of this electron flow will provide crucial information necessary for a molecular understanding of microbicidal killing and misdirected tissue injury functions of human neutrophils. Hence, understanding the structure of this cytochrome may lead to the development of rationally designed drugs that could ameliorate neutrophil mediated tissue damage and enhance neutrophil mediated microbicidal killing. More specifically, this proposal outlines strategies for making monoclonal antibodies recognizing native cytochrome b and defining the binding epitopes of these antibodies using a random sequence phage library. It describes a plan to covalently modify cytochrome b in order to mark different sites on its surface with fluorescent probes, identify possible phosphorylation sites, and identify the locations of the heme prosthetic groups. To precisely identify these modifications, it proposes to use HPLC combined with electrospray mass spectrometry and peptide sequencing of proteolytic digests of the modified proteins. Using this information and covalent modification of the cytochrome with fluorescent probes, it then should be possible to map the cytochrome surface relative to intrinsic hemes using fluorescence energy transfer as a spectroscopic ruler. Lastly the proposal describes a strategy to use multidimensional NMR techniques employing TOCSY, ROSEY, and Transferred NOESY to determine the bound structures of interfacial sites of the cytochrome that interact with its functional partners m the superoxide-generating NADPH oxidase. Successful completion of this work will permit the development of a structural model of the cytochrome incorporating sequence information, transmembrane topology, locations of specific sites, and high resolution maps of interfacial regions.

该提案的广泛长期目标是确定 人类中性粒细胞超氧化物产生的分子基础。 这 拟议的研究将集中于人类中性粒细胞的结构 细胞色素 b 及其在超氧化物激活后如何变化 生成系统。该提案的基本假设是 该细胞色素是超氧化物的中心电子转移酶 生产及其结构的修改将调节流量 电子穿过质膜。了解分子 这种电子流的调节机制将提供至关重要的 分子了解杀菌剂所需的信息 杀伤和误导人类中性粒细胞的组织损伤功能。 因此，了解这种细胞色素的结构可能会导致 开发合理设计的可以改善中性粒细胞的药物 介导的组织损伤并增强中性粒细胞介导的杀菌作用 杀戮。更具体地说，该提案概述了制定战略 单克隆抗体识别天然细胞色素 b 并定义 使用随机序列噬菌体结合这些抗体的表位 图书馆。它描述了共价修饰细胞色素 b 的计划，以便 用荧光探针在其表面标记不同位点，识别 可能的磷酸化位点，并确定血红素的位置 假肢组。为了准确识别这些修改，它建议 使用 HPLC 结合电喷雾质谱和肽 对修饰蛋白的蛋白水解消化物进行测序。 使用这个 荧光细胞色素的信息和共价修饰 探针，那么应该可以绘制相对于细胞色素表面的图 使用荧光能量转移作为光谱 统治者。最后，该提案描述了使用多维的策略 采用 TOCSY、ROSEY 和 Transferred NOESY 的 NMR 技术来确定 相互作用的细胞色素界面位点的结合结构 及其功能伙伴是产生超氧化物的 NADPH 氧化酶。 成功完成这项工作将有助于开发 结合序列信息的细胞色素的结构模型， 跨膜拓扑、特定位点的位置和高分辨率 界面区域图。