CYTOCHROME B AND NEUTROPHIL SUPEROXIDE PRODUCTION

这项建议的广泛长期目标是确定 人中性粒细胞产生超氧化物的分子基础。这个 拟议的调查将集中在人中性粒细胞的结构上 细胞色素b及其在超氧化物歧化时的变化 发电系统。这项提案所做的基本假设是 这种细胞色素是超氧化物的中心电子转移酶 生产及其结构的改变将调节流动 穿过质膜的电子。理解分子 这种电子流动的调节机制将提供至关重要的 了解杀微生物剂分子所需的信息 人中性粒细胞的杀伤和误导组织损伤功能。 因此，了解这种细胞色素的结构可能会导致 改善中性粒细胞的合理设计药物的开发 介导的组织损伤和增强中性粒细胞介导的杀菌作用 杀戮。更具体地说，这项提案概述了制定 识别天然细胞色素b的单抗和定义 利用随机序列噬菌体结合这些抗体的表位 图书馆。它描述了一种共价修饰细胞色素b的计划，以便 用荧光探针在其表面标记不同的位置，识别 可能的磷酸化位点，并确定血红素的位置 假肢小组。为了准确识别这些修改，它提出了 高效液相色谱-电喷雾-质谱法联用 对修饰蛋白的蛋白水解酶进行测序。使用这个 细胞色素的荧光信息及其共价修饰 探针，然后应该可以映射细胞色素表面的相对 利用荧光能量转移作为光谱对本征血红素的影响 尺子。最后，该提案描述了使用多维 使用TOCSY、Rosey和Transfer NOESY的核磁共振技术来确定 细胞色素相互作用的界面位置的结合结构 和它的功能伙伴是产生超氧化物的NADPH氧化酶。 这项工作的成功完成将允许开发一个 结合了序列信息的细胞色素的结构模型， 跨膜拓扑、特定部位的位置和高分辨率 界面区域的地图。

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.