NOVEL PATHWAY OF ARGININE METABOLISM IN MACROPHAGES

巨噬细胞中精氨酸代谢的新途径

• 批准号: 3194867
• 负责人: MICHAEL A. MARLETTA
• 金额: $ 14.48万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3194867
• 关键词: NAD(H) phosphate; affinity chromatography; aminoacid metabolism; arginine; citrulline; dihydrolipoamide dehydrogenase; enzyme complex; enzyme mechanism; enzyme substrate; high performance liquid chromatography; macrophage; nitrates; nitroso compounds; nitrous acid; oxygen consumption; protein purification; tissue /cell culture; vasodilatation

The broad, long-term objectives of the research proposed is to fully characterize a novel metabolic pathway expressed in both macrophages and endothelial cells. The studies proposed here will be carried out exclusively with macrophages. This pathway, oxidation of L-arginine to N=O, NO2- and NO3-, plays an integral role in two important biological processes, namely macrophage cell- killing activity and vasodilation. This characterization will include the isolation and purification of the enzyme or enzymes involved in the pathway, kinetics of the various reactions and chemical characterization of the intermediaries in the reaction. The enzyme or enzymes involved the oxidation of L-arginine to NO2- and NO3- will be purified to homogeneity. The assay that is used presently measures NO2- and NO3- which are the end products of the reaction. Therefore, it will be consumption, and L-citrulline formation. The newly isolated enzymes will be characterized. This will include basic kinetic experiments to measure rates and substrate(s) affinity for any of the individual enzymes isolated. It may be that the reaction is carried out by a multienzyme complex, and if that is the case this protein will be characterized in similar manner. Substrate specificity intermediates in the reaction of L-arginine and NO2- and NO3- will be carried out. The intermediaries known at this time are N-hydroxy-L-arginine and N=O. Studies will involve stability of any intermediates isolated and the chemistry by which N=O is generated from L-arginine. Methods of protein purification, synthetic organic chemistry, an analytical biochemistry will be brought to bear on the overall problem. A complete understanding of the regulation and enzymology of this pathway should lead to national control over processes as diverse as immune system status and vasodilation.

提出的研究的广泛，长期目标是 完全表征了两者中表达的新型代谢途径 巨噬细胞和内皮细胞。 这里提出的研究将 仅用巨噬细胞进行。 这条路， l-精氨酸氧化至n = o，no2-和no3-扮演的氧化 在两个重要的生物过程中的作用，即巨噬细胞 - 杀死活动和血管舒张。 这种特征将 包括酶或酶的隔离和纯化 参与途径，各种反应的动力学和 反应中中介的化学表征。 酶或酶涉及将L-精氨酸氧化为No2- No3-将被纯化为同质性。 使用的测定 目前测量No2-和No3-，它们是最终产品 反应。 因此，它将是消费，L-Citrulline 形成。 新隔离的酶将被表征。 这 将包括基本的动力学实验，以衡量速率和 底物对任何分离的单个酶的亲和力。 可能是由多酶进行的反应 复杂，如果是这种情况，将会表征该蛋白质 以类似的方式。 底物特异性中间体 L-精氨酸和NO2和NO3-的反应将进行。 这 目前已知的中介机构是N-羟基-l-精氨酸和n =O。 研究将涉及任何分离的中间体的稳定性，并且 从L-精氨酸产生N = O的化学。 方法 蛋白质纯化，合成有机化学，一种分析 生物化学将在整体问题上承受。 一个 完全了解这一点的调节和酶学 途径应导致国家对流程的控制 作为免疫系统状态和血管舒张。