LABELED SYNTHETIC DPGS AND NOVEL CROSS-LINKERS FOR EM

本建议的重点有两个方面。 第一个是扩展 我们的膜蛋白标记研究，并作为其目标， 第一次开发了化学上定义明确，标记， 不对称取代的二磷脂酰甘油 脂质-蛋白质相互作用的生物化学和生物物理学研究， 生物膜 对这些相互作用的研究是一个重要的 长期目标的一部分是在分子水平上了解如何 细胞功能。 新的合成DPG将用于合作 Griffith和Jost博士的研究，以确定DPG结合位点， 细胞色素氧化酶通过光标记实验，并通过 掺入乙酰基氮氧自由基自旋标记，以确定 结合位点和相对平衡常数为每一类的 结合位点。 扩展固相合成的可能性 技术的复杂脂质的合成将探讨在 这项工作的过程。 对生物膜研究的补充 和其他细胞成分的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。 化学固定法是最广泛使用的保存方法， 用于透射EM的生物样品。 饱和脂质和碳水化合物 特别是难以使用现有的固定装置固定（即保持在适当位置 试剂 这项建议的第二个方面的目标是： 开发新的交联剂，专门设计用于固定这些 重要的细胞成分。 这些新的代理商将结合 以下反应性基团： 非特异性C-H的碳水化合物结合、光反应性卡宾前体 插入饱和脂质和其他残基，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

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.