LABELED SYNTHETIC DPGS AND NOVEL CROSS-LINKERS FOR EM

标记的合成 DPGS 和新型 EM 交联剂

• 批准号: 3286792
• 负责人: JOHN F KEANA
• 金额: $ 10.31万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286792
• 关键词: carbene; cardiolipins; crosslink; cytochrome oxidase; electron microscopy; membrane lipids; nitrous oxide; reagent /indicator

Two areas form the focus of this proposal. The first is an extension of our membrane protein labeling studies and has as its objective the development for the first time of chemically well defined, labeled, unsymmetrically substituted diphosphatidylglycerols of immediate importance to biochemical and biophysical studies of lipid-protein interactions in biological membranes. The study of these interactions is an important segment of the long range goal of understanding at the molecular level how cells function. The new synthetic DPGs will be used in collaborative studies with Drs. Griffith and Jost to determine the DPG binding site(s) on cytochrome oxidase through photolabeling experiments, and, through incorporation of a proxyl nitroxide spin label, to determine the number of binding sites and the relative equilibrium constants for each class of binding sites. The possibility of extending solid phase synthesis techniques to the synthesis of complex lipids will be explored during the course of this work. Complementary to the study of biological membranes and other cellular components by biophysical techniques has been the direct visualization of cellular ultrastructure through electron microscopy. Chemical fixation is the most widely used method of preservation of biological samples for transmission EM. Saturated lipids and carbohydrates in particular are difficult to fix (i.e. hold in place) using existing reagents. The second area of this proposal has as its objective the development of new cross-linking agents specifically designed to fix these important cellular constituents. These new agents incorporate combinations of the following reactive groups: p-nitrophenylboronic acid for carbohydrate binding, photoreactive carbene precursors for non-specific C-H insertions into saturated lipids and other residues, and a glutaraldehyde group that should mimic the reactivity of the widely used fixative agent glutaraldehyde.

这项提案的重点是两个方面。第一个是对 我们的膜蛋白标记研究并以此为目标 第一次开发化学上定义明确的，标记的， 立即重要的不对称取代二磷脂酰甘油 脂肪-蛋白质相互作用的生化和生物物理研究 生物膜。对这些相互作用的研究是一个重要的 在分子水平上了解长期目标的部分内容 细胞的功能。新的合成DPG将用于协作 与Griffith和Jost博士一起研究确定DPG结合位点(S) 细胞色素氧化酶通过光标记实验，并通过 掺入丙氧基氮氧化物自旋标记，以确定 每类化合物的结合位点数和相对平衡常数 结合部位。扩大固相合成的可能性 在此期间，我们将探索合成复杂脂质的技术。 这项工作的过程。对生物膜研究的补充 和其他细胞成分通过生物物理技术已经直接 通过电子显微镜观察细胞超微结构。 化学固定是使用最广泛的保存方法 用于传播EM的生物样本。饱和脂肪和碳水化合物 尤其是难以使用现有设备固定(即固定到位) 试剂。这项提案的第二个领域的目标是 开发专门为解决这些问题而设计的新型交联剂 重要的细胞成分。这些新的药剂结合了多种药物 以下反应基团：对硝基苯基硼酸 非特异性C-H的碳水化合物结合、光反应卡宾前体 插入饱和脂肪和其他残留物，以及戊二醛 应模拟广泛使用的固定剂的反应性的基团 戊二醛。