HIGH DENSITY LIPOPROTEIN STRUCTURE FUNCTION CORRELATIONS

这项研究的总体目标是从分子的角度阐明， 人体高密度的组成-结构-功能关系 脂蛋白(Hdl)。使用包含正常的重组高密度脂蛋白(RHDL) 载脂蛋白A-I(apo A-I)、apo A-I片段或突变的apo A-I 结合纯脂，我们建议研究三种- 这个载脂蛋白的空间折叠，以及它在定义 盘状和球状重组高密度脂蛋白的亚类及其与 卵磷脂胆固醇酰基转移酶。此外，我们计划分离出一种 只含载脂蛋白A-I的天然HDL3的均一组分，并制备 用洗涤剂增溶方法从载脂蛋白A-I中分离出rHDL， 目的比较“天然”载脂蛋白A-I和“天然”载脂蛋白A-I的结构和功能 其常规隔离形式的性质(通过有机溶剂 提取)。 高密度脂蛋白颗粒及其载脂蛋白A-I和脂类成分将 以各种生化和生物物理方法为特征的 包括用超速离心法和FPLC法分离颗粒， 非变性梯度凝胶电泳法的研究 通过荧光、圆二色谱和傅立叶变换红外光谱测定蛋白质结构， 单抗结合，限制性蛋白水解酶和多肽 测序。高密度脂蛋白中脂类成分的有序性和动态性 将通过荧光和傅里叶变换红外光谱方法进行研究，并对生物 所有高密度脂蛋白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 酶分析中的放射性标记脂类。

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.