Singh T K, Scraba D G, Ryan R O
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1992 May 5;267(13):9275-80.
The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.
在体外脂质转移试验中，利用高密度脂蛋白和人低密度脂蛋白（LDL）作为供体/受体底物，检测了烟草天蛾脂质转移颗粒（LTP）的脂质底物特异性。发现未酯化胆固醇在这些底物脂蛋白之间自发交换，且转移/交换程度不受LTP影响。相比之下，标记的磷脂酰胆碱和胆固醇酯的转移以浓度依赖方式依赖于LTP。促进的磷脂酰胆碱转移比促进的胆固醇酯转移发生得更快；这一观察结果表明，要么LTP可能对极性脂质有内在偏好，要么供体底物颗粒中特定脂质的可及性影响它们的转移速率。通过在转移试验中增加高密度脂蛋白与LDL的比例，研究了LDL从脂蛋白接受外源脂质的能力。在存在LTP的情况下，当比例为3:1（蛋白质）时，LDL变得浑浊（通过电子显微镜观察到聚集的LDL），表明LDL在维持整体稳定结构的同时，接受外源脂质的能力有限。当在转移实验中加入分离的人非B型极低密度脂蛋白（VLDL）载脂蛋白或昆虫载脂蛋白III（apoLp - III）时，样品没有变得浑浊，尽管脂质转移程度与未添加载脂蛋白时相同。外源载脂蛋白引起的样品浊度降低以浓度依赖方式发生，表明这些蛋白质与LDL表面结合，并稳定了由LTP介导的净脂质转移产生的脂质/水界面的增加。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析证实了载脂蛋白与修饰LDL表面的结合，扫描密度测定显示apoLp - III以1:14的apoB:apoLp - III摩尔比与LDL表面结合。电子显微镜显示，apoLp - III稳定的修饰LDL颗粒的直径（29.2±2.6 nm）大于对照LDL（22.7±1.9 nm），这与观察到的颗粒密度、脂质和载脂蛋白含量的变化一致。因此，LTP催化的矢量脂质转移可用于对分离的LDL颗粒进行显著修饰，并提供了一种研究VLDL - LDL相互关系的新机制。
