ESR DETECTION OF NITRIC OXIDE PRODUCED IN VIVO BY PROKARYOTIC CELLS

原核细胞体内一氧化氮的 ESR 检测

• 批准号: 7723985
• 负责人: Rosemary Loria
• 金额: $ 0.13万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723985
• 关键词: Appearance; Arginine; Bacillus anthracis; Binding; Biology; Characteristics; Chemistry; Citrulline; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Deinococcus radiodurans; Detection; Diffuse; Doctor of Philosophy; Electrons; Funding; Gram-Positive Bacteria; Grant; Heme; Homologous Gene; Institution; Iron; Mammals; N-terminal; Nitric Oxide; Nitric Oxide Synthase; Organism; Oxidoreductase; Oxygenases; Paper; Production; Prokaryotic Cells; Proteins; Research; Research Personnel; Resources; Rosemary; Sampling; Sarcosine; Signal Transduction; Source; Spin Trapping; Staphylococcus aureus; Students; United States National Institutes of Health; Work; in vivo; interest; mutant; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Prof Rosemary Loria is interested in detection, via ESR, of nitric oxide produced in vivo by prokaryotic cells. Nitric oxide synthases (NOSs) in mammals are dimeric proteins containing N-terminal oxygenase domains that convert arginine into citrulline and NO, and a reductase domain that shuttles electrons to the heme of the oxygenase domain. Homologs to NOSs exist in prokaryots, but direct evidence for NO production by these proteins has been lacking. The aim of current work was to demonstrate that NOS also produces diffusible NO. To demonstrate NO production, the spin trap dithiocarboxy-sarcosine (DTCS) was used. DTCS binds iron to form the active spin trap Fe(DTCS)2 that lacks an ESR signal. After NO binds to the Fe(DTCS)2 complex, the spin trap produces a characteristic 3-peak signal. Incubation of Fe(DTCS)2 with the wild type culture of S. turgidiscabies resulted in the appearance of the characteristic 3-peak signal of NO-Fe(DTCS)2 identical to that produced in control experiments by addition of artificial NO-donor. This NO-specific 3-peak signal was not observed in samples from NOS-deletion mutant cultures. These results confirmed that NOS-dependent free-diffusing NO is released by thaxtomin-producing streptomycetes and were the first data to document in vivo production of NO by prokaryotic NOSs. Structural conservation of bacterial NOSs predicts NO production by other Gram-positive bacteria including medically and environmentally important organisms such as Bacillus anthracis, Staphylococcus aureus, and Deinococcus radiodurans. A paper on this work was submitted to Chemistry & Biology. This work also represents a substantial part of the PhD thesis of a graduate student of Prof. Loria: Evan Johnson.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Rosemary Loria 教授对通过 ESR 检测原核细胞体内产生的一氧化氮感兴趣。哺乳动物中的一氧化氮合酶 (NOS) 是二聚体蛋白，含有将精氨酸转化为瓜氨酸和 NO 的 N 末端加氧酶结构域，以及将电子传送到加氧酶结构域的血红素的还原酶结构域。原核生物中存在 NOS 的同源物，但缺乏这些蛋白质产生 NO 的直接证据。当前工作的目的是证明 NOS 也产生扩散性 NO。 为了证明 NO 的产生，使用了自旋陷阱二硫代羧基肌氨酸 (DTCS)。 DTCS 与铁结合形成缺乏 ESR 信号的活性自旋陷阱 Fe(DTCS)2。 NO 与 Fe(DTCS)2 复合物结合后，自旋陷阱产生特征性 3 峰信号。 Fe(DTCS)2 与 S. turgidiscabies 野生型培养物一起孵育，导致出现 NO-Fe(DTCS)2 特征性 3 峰信号，与添加人工 NO 供体的对照实验中产生的信号相同。 在 NOS 缺失突变体培养物的样品中未观察到这种 NO 特异性 3 峰信号。 这些结果证实，产thax​​tomin的链霉菌可以释放依赖于NOS的自由扩散NO，并且是记录原核NOS在体内产生NO的第一个数据。 细菌 NOS 的结构保守性预示着其他革兰氏阳性菌（包括医学和环境上重要的生物体，如炭疽杆菌、金黄色葡萄球菌和耐辐射球菌）会产生 NO。 关于这项工作的论文已提交给Chemistry & Biology。 这项工作也是 Loria 教授的研究生 Evan Johnson 博士论文的重要组成部分。