BIOCHEMISTRY OF MACROPHAGE NITRIC OXIDE SYNTHESIS

哺乳动物从L-精氨酸(L-精氨酸)合成一氧化氮(NO)是新发现的 被发现并且在机械上是新颖的。一氧化氮的合成种类繁多 在细胞和组织中，它充当信号或效应器分子 几个生理和病理过程。这些措施包括控制 血压和血流，神经元信号，趋化，细胞 脓毒症的增殖、血栓形成和肝脏反应。理解 NO合成的生物化学将极大地帮助研究 这种代谢途径的功能、分布和控制。初步 一株部分纯化的巨噬细胞NO-1的生化特性 产生活性表明它需要NADPH、四氢生物蝶呤、FAD和 硫醇除L-精氨酸外，还可以是多组分。这样做的目的是 建议对小鼠巨噬细胞进行纯化、鉴定和克隆 酶(S)。纯化将使用常规的层析树脂(凝胶 过滤、阴离子交换)和基于结合的亲和树脂 L的网站-arg，nadph，和fad。一种酶的可能解离 层析过程中的复合体将在每一层析中处理 一步一步地混合实验。二苯基碘(DPI)，一种不可逆的 NO生成的抑制剂，将用[125I]NaI合成并使用 鉴定和纯化具有NADPH/FAD结合的酶(或亚基) 网站。该酶(S)将被测序和克隆。特性研究 将在净化过程中和之后进行。它们包括 L-精氨酸底物专一性和米氏常数的测定 (及其类似物)、NADPH和DPI；化学计量关系 L依赖精氨酸的NADPH氧化、NO产生和O2消耗； 产品中的氧的来源(NO和 L-瓜氨酸)。以协助确定反应机理、反应 将寻求导致中间体积累的条件，如果 发现，反应中间产物会被分离出来，它们的结构 经核磁共振和气相色谱-质谱联用鉴定。

Mammalian synthesis of nitric oxide (NO) from L-arginine (L-Arg) is newly discovered and mechanistically novel. NO is synthesized in a wide variety of cells and tissues, where it acts as a signal or effector molecule in several physiological and pathological processes. These include control of blood pressure and blood flow, neuron signaling, chemotaxis, cell proliferation, thrombosis, and hepatic response to sepsis. Understanding the biochemistry of NO synthesis will greatly aid investigations into the function, distribution, and control of this metabolic pathway. Preliminary biochemical characterization of a partially-purified macrophage NO- generating activity shows it requires NADPH, tetrahydrobiopterin, FAD, and thiol in addition to L-Arg; and may be multi-component. The goal of this proposal is to purify, characterize, and clone the mouse macrophage enzyme(s). Purification will use conventional chromatographic resins (gel filtration, anion exchange) and affinity resins that are based on binding sites for L-Arg, NADPH, and FAD. The possible dissociation of an enzyme complex during chromatography will be addressed in each chromatographic step by mixing experiments. Diphenyleneiodonium (DPI), an irreversible inhibitor of NO production, will be synthesized using [125I]NaI and used to identify and purify the enzyme (or subunit) possessing NADPH/FAD binding sites. The enzyme(s) will be sequenced and cloned. Characterization studies will be done both during and following purification. They include determining the substrate specificity and Michaelis constants for L-Arg (and its analogs), NADPH, and DPI; the stoichiometric relationships between L-Arg-dependent NADPH oxidation, NO production and O2 consumption; and the source of the oxygens in the products (NO and the ureido oxygen of L-citrulline). To assist in determining the reaction mechanism, reaction conditions that lead to a buildup of intermediates will be sought and, if found, reaction intermediates will be isolated and their structure identified by NMR and gas chromatography-mass spectroscopy.