Ohnishi T, Tan C, Yokoyama S
Department of Medicine, University of Alberta, Edmonton, Canada.
Biochemistry. 1994 Apr 19;33(15):4533-42. doi: 10.1021/bi00181a014.
The substrate-specific rate of the human plasma lipid transfer protein (LTP) reaction was studied using pyrene-labeled substrate lipid analogues as probes for various lipids, by monitoring the ratio of the fluorescence intensities of their excimers to those of their monomers as an indicator of pyrene concentration in the microenvironment. Transfer of cholesteryl ester (CE) and triglyceride (TG) was demonstrated between human high-density lipoproteins, between low-density lipoproteins, and between these two lipoprotein, and the specific fractional transfer rate of CE was always higher than that of TG by a factor of 2.4-7.9. On the other hand, the transfer by LTP of CE, TG, and phosphatidylcholine (PC) was also demonstrated between lipid microemulsions having an average diameter of 25-26 nm using the same probes, but only when the emulsions were activated by apolipoproteins A-I, A-II, E, or C-III. The maximally activated rates of the transfer of CE and TG were the same when measured between the emulsions with cores composed exclusively of either lipid. The specific fractional transfer rate of pyrene-CE, however, was inversely proportional to the percentage of CE in the TG core of the emulsions, and the initial transfer of TG was almost completely inhibited by the presence of small percentages of CE in the TG core. Thus, the transfer of CE between the emulsions is highly selective over that of TG by orders of magnitude, much more selective than the reaction between any natural plasma lipoproteins, but this selectivity is not a rate-limiting step of the overall LTP reaction. The maximally activated LTP-catalyzed transfer rate of PC between the emulsions was somewhat higher than that of CE or TG and was not affected by the composition of the core lipids of the emulsion, TG or CE. When an excess amount of LTP was incubated with emulsion containing a small percentage of pyrene-CE in the TG core in the absence of the acceptor particles, excimer fluorescence rapidly decreased to the base line, and this change was suppressed when pyrene-CE was diluted with CE in the core. This result may indicate that LTP selectively disrupts pyrene-CE excimer formation on the basis of its selective interaction with the CE molecule over TG in the emulsion system as a putative background mechanism for the selective transfer of CE.
利用芘标记的底物脂质类似物作为各种脂质的探针，通过监测其准分子荧光强度与单体荧光强度的比值作为微环境中芘浓度的指标，研究了人血浆脂质转运蛋白（LTP）反应的底物特异性速率。已证实在人高密度脂蛋白之间、低密度脂蛋白之间以及这两种脂蛋白之间存在胆固醇酯（CE）和甘油三酯（TG）的转运，且CE的特异性分数转运率总是比TG高2.4至7.9倍。另一方面，使用相同的探针也证实在平均直径为25至26nm的脂质微乳液之间存在LTP介导的CE、TG和磷脂酰胆碱（PC）的转运，但前提是这些微乳液由载脂蛋白A-I、A-II、E或C-III激活。当在仅由其中一种脂质构成核心的微乳液之间测量时，CE和TG转运的最大激活速率相同。然而，芘-CE的特异性分数转运率与微乳液TG核心中CE的百分比成反比，并且TG核心中少量CE的存在几乎完全抑制了TG的初始转运。因此，微乳液之间CE的转运在数量级上比TG的转运具有高度选择性，比任何天然血浆脂蛋白之间的反应选择性高得多，但这种选择性不是整个LTP反应的限速步骤。微乳液之间LTP催化的PC最大激活转运率略高于CE或TG，且不受微乳液核心脂质（TG或CE）组成的影响。当在没有受体颗粒的情况下，将过量的LTP与TG核心中含有少量芘-CE的微乳液一起孵育时，准分子荧光迅速降至基线，并且当核心中的芘-CE用CE稀释时，这种变化受到抑制。这一结果可能表明，作为CE选择性转运的一种假定背景机制，LTP在乳液系统中基于其与CE分子而非TG的选择性相互作用，选择性地破坏芘-CE准分子的形成。
