LIPID/LIPOXYGENASE INTERACTIONS

脂质/脂氧合酶相互作用

• 批准号: 2178228
• 负责人: BETTY J GAFFNEY
• 金额: $ 37.05万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178228
• 关键词: LIPID; LIPOXYGENASE; INTERACTIONS

Building on past progress in defining the structure and stability of the resting form of lipoxygenases and the EPR spectroscopy of the activated, ferric form, the overall aim of the next period is to obtain a molecular understanding of the lipoxygenase mechanism. EPR programs written in the last period will be expanded to include ferrous-nitric oxide complexes and dispersion and rapid passage spectra. Spectra at X- and Q-bands will be analyzed to determine the contributions of D and E to distributions of parameters in observed spectra. Polycrystalline and single crystal studies of lipoxygenase by EPR will continue using crystals of the enzyme in the ferric form and the nitric- oxide-complexed ferrous form. These studies are designed to determine the orientation of exchangeable ligands on iron within the lipoxygenase structure and how the principle axes of the magnetic tensors are related to the ligands. Pursuant to the goal of a molecular understanding of the fatty acid binding cavity and how positional specificity is achieved, new approaches will be developed for defining fatty acid binding, including using photoreactive fatty acid analogs and EPR studies. The importance of a novel pi-helical region for lipoxygenase function will be tested. Single site mutations and deletions, designed to vary the way the pi-helix contributes ligands to iron will be prepared and will be examined for activity, iron content, EPR spectra and thermal stability. Further studies of the mechanism of lipoxygenase unfolding will include studies to test whether iron binding is reversible, to use a soybean isoform to examine the reversible step in unfolding, and to use a spin labeled form of the resting enzyme to test the effects of glassing agents on cold denaturation. The crystallography effort will be supported in two ways. SBL-1 will be provided for continued experiments involving co-crystallization of ferric and ferrous enzyme with inhibitors. Human platelet 12-LO will continue to be produced in Sf9 insect cells, conditions for producing and storing this lipoxygenase will be improved and the protein will be supplied for x-ray crystallography. Fe levels will be measured in 12-LO.

以过去的进步来定义的结构和稳定性 脂氧酶的静息形式和激活的EPR光谱法， 铁形式，下一个时期的总体目的是获得分子 了解脂氧合酶机制。 在上一个时期编写的EPR程序将扩展到包括 铁氮氧化物复合物和分散和快速传递光谱。 将分析X和Q波段的光谱以确定贡献 D和E到观察到的光谱中参数的分布。 lipoxygogyasy by EPR的多晶和单晶研究将 继续使用铁形式的酶晶体和一氮 氧化杂色的亚铁形式。这些研究旨在确定 脂氧合酶内铁上可交换配体的方向 结构以及磁张量的原理轴如何相关 到配体。 根据对脂肪酸的分子理解的目标 绑定腔及其位置特异性如何实现新方法 将开发用于定义脂肪酸结合，包括使用 光电脂肪酸类似物和EPR研究。 新型的皮螺旋区对于脂氧合酶功能的重要性将 进行测试。单个站点突变和删除，旨在改变方式 Pi螺旋将准备配体配体，并将 检查了活性，铁含量，EPR光谱和热稳定性。 进一步研究脂氧合酶展开的机理将包括 测试铁结合是否可逆的研究，使用大豆 同工型检查展开的可逆步骤，并使用旋转 标记的静止酶的形式以测试玻璃剂的作用 关于冷变性。 晶体学的工作将通过两种方式支持。 SBL-1会 为涉及铁的共结晶而提供的持续实验 和用抑制剂的亚铁酶。人血小板12-lo将继续 在SF9昆虫细胞中生产，生产和存储的条件 脂氧合酶将得到改善，并将为X射线提供蛋白质 晶体学。 Fe水平将在12-LO中测量。