Gong E L, Nichols A V, Forte T M, Blanche P J, Shore V G
Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Biochim Biophys Acta. 1988 Jul 1;961(1):73-85. doi: 10.1016/0005-2760(88)90132-4.
Using a cholate-dialysis recombination procedure, complexes of apolipoprotein A-I and synthetic phosphatidylcholine (1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC] were prepared in mixtures at a relatively high molar ratio of 150:1 phosphatidylcholine/apolipoprotein A-I. Particle size distribution analysis by gradient gel electrophoresis of the recombinant mixtures indicated the presence of a series of discrete complexes that included species migrating at RF values observed for discoidal particles in nascent high-density lipoproteins (HDL) in plasma of lecithin-cholesterol acyltransferase-deficient subjects. One of these complex species, designated complex class 6, formed with either phosphatidylcholine, was isolated by gel filtration and characterized at follows: discoidal shape (mean diameter 20.8 nm (POPC) and 19.0 nm (DOPC]; molar ratio, phosphatidylcholine/apolipoprotein A-I, 155:1 (POPC) and 130:1 (DOPC); and both containing 4 molecules of apolipoprotein A-I per particle. Incubation of class 6 complexes with lecithin-cholesterol acyltransferase (EC 2.3.1.43) and a source of unesterified cholesterol (low-density lipoprotein (LDL] was shown by electron microscopy to result in a progressive transformation of the discoidal particles (0 h) to deformable (2.5 h) and to spherical particles (24 h). The spherical particles (diameter 13.6 nm (POPC) and 12.5 nm (DOPC) exhibit sizes at the upper boundary of the interval defining the human plasma (HDL2b)gge (12.9-9.8 nm). The spherical particles contain a cholesteryl ester core that reaches a limiting molar ratio of approx. 50-55:1 cholesteryl ester/apolipoprotein A-I. The deformable particles assume a rectangular shape under negative staining and, relative to the 24-h spherical product, are enriched in phosphatidylcholine. Chemical crosslinking (by dimethyl suberimidate) of the isolated transformation products shows the 24-h spherical particle to contain predominantly 4 apolipoprotein A-I molecules; products produced after intermediate periods of time appear to contain species with 3 and 4 apolipoproteins per particle. Our in vitro studies indicate a potential pathway in the origins of large, apolipoprotein A-I-containing plasma HDL particles. The deformable species observed during transformation were similar in size and shape to particles observed in interstitial fluid.
采用胆酸盐透析重组方法，以相对较高的150:1磷脂酰胆碱/载脂蛋白A-I摩尔比在混合物中制备载脂蛋白A-I与合成磷脂酰胆碱（1-棕榈酰-2-油酰磷脂酰胆碱（POPC）或二油酰磷脂酰胆碱（DOPC））的复合物。通过对重组混合物进行梯度凝胶电泳分析颗粒大小分布，结果表明存在一系列离散的复合物，其中包括一些在卵磷脂-胆固醇酰基转移酶缺乏受试者血浆中新生高密度脂蛋白（HDL）的盘状颗粒所观察到的迁移率（RF值）下迁移的物质。这些复合物种之一，命名为复合物6类，由任何一种磷脂酰胆碱形成，通过凝胶过滤分离并进行如下表征：盘状形状（平均直径20.8nm（POPC）和19.0nm（DOPC））；摩尔比，磷脂酰胆碱/载脂蛋白A-I，155:1（POPC）和130:1（DOPC）；且每个颗粒均含有4个载脂蛋白A-I分子。电子显微镜显示，将6类复合物与卵磷脂-胆固醇酰基转移酶（EC 2.3.1.43）和未酯化胆固醇来源（低密度脂蛋白（LDL））一起孵育，会导致盘状颗粒（0小时）逐渐转变为可变形颗粒（2.5小时）并最终转变为球形颗粒（24小时）。球形颗粒（直径13.6nm（POPC）和12.5nm（DOPC））的大小处于定义人血浆（HDL₂b）亚类（12.9 - 9.8nm）区间的上限。球形颗粒含有一个胆固醇酯核心，其胆固醇酯/载脂蛋白A-I的摩尔比达到约50 - 55:1的极限值。可变形颗粒在负染下呈矩形，相对于24小时的球形产物，其磷脂酰胆碱含量更高。对分离出的转化产物进行化学交联（通过辛二酸二甲酯）显示，24小时的球形颗粒主要含有4个载脂蛋白A-I分子；在中间时间段产生的产物似乎含有每个颗粒具有3个和4个载脂蛋白的物质。我们的体外研究表明了在含载脂蛋白A-I的大血浆HDL颗粒起源中的一条潜在途径。在转化过程中观察到的可变形物质在大小和形状上与在组织液中观察到的颗粒相似。
