Ritter M C, Scanus A M
J Biol Chem. 1977 Feb 25;252(4):1208-16.
For a better definition of the role of human serum apolipoprotein A-I (apo A-I) in high density lipoprotein structure, a systematic investigation was carried out on factors influencing the in vitro association of this apoprotein with lipids obtained from the parent high density lipoprotein (HDL); these lipids include phospholipids, free cholesterol, cholesteryl esters, and triglycerides. Following equilibration, mixtures of apo A-I and lipids in varying stoichiometric amounts were fractionated by sequential flotation, CsCl density gradient ultracentrifugation, or gel-permeation chromatography, and the isolated complexes were characterized by physicochemical means. As defined by operational criteria (flotation at density 1,063 to 1.21 g/ml), only two types of HDL complexes were reassembled; one, reconstituted HDLS, small with a radius of 31 A, and the other, reconstituted HDLL, large with a radius of 39 A. The two types incorporated all of the lipid constituents of native HDL and contained 2 and 3 mol of apo A-I, respectively. A maximal yield of reconstituted HDL (R-HDL) was observed at an initial protein concentration of 0.1 muM, where apo A-I is predominantly monomeric. At increasing protein concentrations, the amount of apo A-I recovered in R-HDL was found to be proportional to the initial concentration of monomer and dimer in solution. The composition and yield of the complexes were independent of ionic strength and pH within the ranges studied. Both simple incubation and cosonication of apo A-I with HDL phospholipids produced complexes of identical composition, although the yeild of complexes was higher with co-sonication. When the comparison of the same methods was extended to mixtures of apo A-I and whole HDL lipids, the results confirmed previous observations that co-sonication is essential for the incorporation of the neutral lipid into the R-HDL complexes. The results indicate that (a) in vitro complexation of apo A-I with lipids is under kinetic control; (b) apo A-I can generate a lipid-protein complex with properties similar to those of the parent lipoprotein; (c) the process requires well defined experimental conditions and, most importantly, the presence in solution of monomers and dimers of apo A-I; (d) the number of apo A-I molecules incorporated into R-HDL determines the size and structure of the reassembled particle. All of these observations strongly support the essential role of apo A-I in the structure of human HDL.
为了更明确人血清载脂蛋白A-I（apo A-I）在高密度脂蛋白结构中的作用，我们对影响该载脂蛋白与源自母体高密度脂蛋白（HDL）的脂质进行体外结合的因素展开了系统研究；这些脂质包括磷脂、游离胆固醇、胆固醇酯和甘油三酯。平衡后，通过连续浮选、CsCl密度梯度超速离心或凝胶渗透色谱法对不同化学计量比的apo A-I与脂质混合物进行分级分离，并用物理化学方法对分离出的复合物进行表征。根据操作标准（在密度1.063至1.21 g/ml下浮选）定义，仅重新组装了两种类型的HDL复合物；一种是重组HDLS，半径为31 Å的小颗粒，另一种是重组HDLL，半径为39 Å的大颗粒。这两种类型包含了天然HDL的所有脂质成分，分别含有2摩尔和3摩尔的apo A-I。在初始蛋白质浓度为0.1 μM时观察到重组HDL（R-HDL）的最大产量，此时apo A-I主要为单体形式。随着蛋白质浓度增加，发现R-HDL中回收的apo A-I量与溶液中单体和二聚体的初始浓度成正比。在所研究的离子强度和pH范围内，复合物的组成和产量与它们无关。apo A-I与HDL磷脂的简单孵育和共超声处理均产生了组成相同的复合物，尽管共超声处理时复合物的产量更高。当将相同方法的比较扩展到apo A-I与整个HDL脂质的混合物时，结果证实了先前的观察结果，即共超声处理对于将中性脂质掺入R-HDL复合物至关重要。结果表明：（a）apo A-I与脂质的体外复合受动力学控制；（b）apo A-I可以产生一种性质与母体脂蛋白相似的脂蛋白复合物；（c）该过程需要明确的实验条件，最重要的是溶液中存在apo A-I的单体和二聚体；（d）掺入R-HDL中的apo A-I分子数量决定了重组颗粒的大小和结构。所有这些观察结果都有力地支持了apo A-I在人HDL结构中的重要作用。
