HIGH DENSITY LIPOPROTEIN STRUCTURE FUNCTION CORRELATIONS

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 以各种生物化学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。

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.