BIOCHEMISTRY OF MACROPHAGE NITRIC OXIDE SYNTHESIS

哺乳动物由L-精氨酸（L-Arg）合成一氧化氮（NO）是近年来发现的新的生物学过程。 发现和机械新颖。NO的合成方式多种多样 在细胞和组织中，它作为一种信号或效应分子， 几种生理和病理过程。其中包括控制 血压和血流，神经元信号，趋化性，细胞 增殖、血栓形成和对脓毒症的肝反应。理解 NO合成的生物化学将大大有助于研究 功能，分布和控制这一代谢途径。初步 部分纯化的巨噬细胞NO- 产生活性表明它需要NADPH，四氢生物蝶呤，FAD， 巯基以及L-Arg;并且可以是多组分的。这个目标 一项旨在纯化、鉴定和克隆小鼠巨噬细胞的提案 酶。纯化将使用常规色谱树脂（凝胶 过滤、阴离子交换）和基于结合的亲和树脂 L-Arg、NADPH和FAD的位点。一种酶可能的解离 色谱过程中的复杂性将在每个色谱 混合实验的步骤。二苯基碘鎓（DPI），一种不可逆的 NO生成的抑制剂，将使用[125 I]NaI合成并使用 鉴定和纯化具有NADPH/FAD结合的酶（或亚基 网站.将对酶进行测序和克隆。表征研究 将在纯化期间和之后进行。它们包括 测定L-Arg的底物特异性和米氏常数 (and其类似物）、NADPH和DPI; L-Arg依赖性NADPH氧化、NO产生和O2消耗;以及 产物中氧的来源（NO和脲基氧 L-瓜氨酸）。为了帮助确定反应机理，反应 将寻求导致中间体积累的条件，如果 发现，反应中间体将被分离和它们的结构 通过NMR和气相色谱-质谱鉴定。

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.