HIGH DENSITY LIPOPROTEIN STRUCTURE-FUNCTION CORRELATIONS

高密度脂蛋白结构-功能相关性

• 批准号: 3335118
• 负责人: ANA JONAS
• 金额: $ 11.08万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3335118
• 关键词: apolipoproteins; blood lipoprotein metabolism; circular dichroism; crystallization; detergents; electron microscopy; fluorescence spectrometry; gel electrophoresis; high density lipoproteins; high performance liquid chromatography; human tissue; lipid solubility; lipid structure; monoclonal antibody; phosphatidylcholine sterol acyltransferase; protein folding; protein structure function; radiotracer; ultracentrifugation; ultraviolet spectrometry

The overall objective of this research is to elucidate, in molecular terms, the composition-structure-function relationship in human high density lipoproteins (HDL). Using reconstituted HDL (rHDL) containing normal apolipoprotein A-I (apo A-I), apo A-I fragments, or a mutant apo A-I, in conjunction with pure lipids, we propose to investigate the three- dimensional folding of this apolipoprotein, and its role in defining the subclasses of discoidal and spherical rHDL, and their reactivity with lecithin cholesterol acyltransferase. Furthermore, we plan to isolate a homogeneous fraction of native HDL3 containing only apo A-I, and to prepare rHDL from apo A-I isolated by detergent solubilization methods, with the objective of comparing the structure and function of "native" apo A-I with its properties in the conventionally isolated form (by organic solvent extraction). The HDL particles and their apo A-I and lipid components will be characterized by a variety of biochemical and biophysical approaches including isolation of the particles by ultracentrifugal and FPLC methods, analysis by non-denaturing gradient gel electrophoresis, investigation of the protein structure by fluorescence, CD, and FT-TR spectroscopy, monoclonal antibody binding, and limited proteolysis and polypeptide sequencing. The order and dynamics of the lipid constituents of the HDL will be studied by fluorescence and FT-TR methods, and the biological function of all the HDL particles will be assessed by their ability to activate the lecithin cholesterol acyltransferase reaction, using radiolabeled lipids in the enzymatic assays.

这项研究的总体目标是从分子角度阐明， 人体高密度物质组成-结构-功能关系 脂蛋白（HDL）。 使用含有正常HDL的重构HDL（rHDL）， 载脂蛋白A-I（apo A-I）、apo A-I片段或突变体apo A-I， 结合纯脂质，我们建议调查三个- 这种载脂蛋白的三维折叠，以及它在定义 盘状和球形rHDL的亚类，以及它们与 卵磷脂胆固醇酰基转移酶。 此外，我们计划分离出一种 仅含有apo A-I的天然HDL 3的均匀级分，并制备 通过去污剂增溶方法从apo A-I分离的rHDL， 目的是比较“天然”载脂蛋白A-I的结构和功能， 其在常规分离形式（通过有机溶剂）中的性质 提取）。 HDL颗粒及其载脂蛋白A-I和脂质组分将被 以多种生化和生物物理方法为特征 包括通过超离心和FPLC方法分离颗粒， 非变性梯度凝胶电泳分析 通过荧光、CD和FT-TR光谱分析蛋白质结构， 单克隆抗体结合，有限的蛋白水解和多肽 测序 高密度脂蛋白脂质组分的顺序和动力学 将通过荧光和FT-TR方法进行研究， 所有HDL颗粒的功能将通过它们的能力来评估， 激活卵磷脂胆固醇酰基转移酶反应，使用 放射性标记的脂质在酶测定中。