BIOCHEMISTRY OF MACROPHAGE NITRIC OXIDE SYNTHESIS

巨噬细胞一氧化氮合成的生物化学

基本信息

批准号：
2095582
负责人：
DENNIS J STUEHR
金额：
$ 10.06万
依托单位：
CLEVELAND CLINIC FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-01-01 至 1995-12-31
项目状态：
已结题

项目摘要

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.

哺乳动物从 L-精氨酸 (L-Arg) 合成一氧化氮 (NO) 的新方法被发现并且机制新颖。 NO 的合成方式多种多样细胞和组织中，它充当信号或效应分子一些生理和病理过程。这些包括控制血压和血流、神经元信号传导、趋化性、细胞增殖、血栓形成和脓毒症的肝脏反应。理解 NO 合成的生物化学将极大地帮助研究该代谢途径的功能、分布和控制。初步的部分纯化的巨噬细胞 NO- 的生化特征产生活性表明它需要 NADPH、四氢生物蝶呤、FAD 和除L-Arg外还有硫醇；并且可以是多组分的。此举的目标建议纯化、表征和克隆小鼠巨噬细胞酶。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。酶可能的解离色谱过程中的复合物将在每个色谱中解决步骤通过混合实验。二亚苯基碘鎓 (DPI)，一种不可逆的 NO 生成抑制剂，将使用 [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。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。他们包括确定 L-Arg 的底物特异性和米氏常数（及其类似物）、NADPH 和 DPI；之间的化学计量关系 L-Arg依赖的NADPH氧化、NO产生和O2消耗；和产品中氧的来源（NO 和脲基氧） L-瓜氨酸）。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，反应中间体将被分离并得到它们的结构通过NMR和气相色谱-质谱法鉴定。

项目成果

期刊论文数量（99）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

High-level expression of mouse inducible nitric oxide synthase in Escherichia coli requires coexpression with calmodulin.

小鼠诱导型一氧化氮合酶在大肠杆菌中的高水平表达需要与钙调蛋白共表达。

DOI：
10.1006/bbrc.1996.0763
发表时间：
1996
期刊：
Biochemical and biophysical research communications.
影响因子：
0
作者：
Wu,C;Zhang,J;Abu-Soud,H;Ghosh,DK;Stuehr,DJ
通讯作者：
Stuehr,DJ

Isoform-specific effects of salts on nitric oxide synthase activity.

盐对一氧化氮合酶活性的异构体特异性影响。

DOI：
10.1016/s0167-4838(98)00138-1
发表时间：
1998
期刊：
Biochimica et biophysica acta
影响因子：
0
作者：
Schrammel,A;Gorren,AC;Stuehr,DJ;Schmidt,K;Mayer,B
通讯作者：
Mayer,B

Nitric oxide blocks cellular heme insertion into a broad range of heme proteins.

一氧化氮阻止细胞血红素插入多种血红素蛋白中。

DOI：
10.1016/j.freeradbiomed.2010.02.038
发表时间：
2010
期刊：
Free radical biology & medicine
影响因子：
7.4
作者：
Waheed,SyedMohsin;Ghosh,Arnab;Chakravarti,Ritu;Biswas,Ashis;Haque,MohammadMahfuzul;Panda,Koustubh;Stuehr,DennisJ
通讯作者：
Stuehr,DennisJ

Fast ferrous heme-NO oxidation in nitric oxide synthases.