Inhibitory effects of apolipoprotein E on Alzheimer's beta-amyloid fibril formation in vitro.

最近，许多研究小组研究了载脂蛋白E(ApoE)在体外对β-淀粉样原纤维(BetaAf)形成的影响。然而，他们的数据有些争议，而且到目前为止还没有对载脂蛋白E作用的准确动力学评估。我们从不同浓度的新制备的β-淀粉样蛋白(1-40)(β1-40)开始，利用硫代黄素T的荧光光谱，在体外检测了人载脂蛋白E对β-Af形成的影响。当50 mM的β-1-40与1：1000至1：100摩尔比的载脂蛋白E孵育时，观察到载脂蛋白E的剂量依赖性抑制作用。ApoE可抑制βAf的体外成核期和延伸期，而当300um的β1-40与1：100摩尔比的apoE孵育时，apoE的抑制作用完全消失。然后，我们利用最近建立的BetaAf体外延伸的一级动力学模型[Naki，H.，&Nakakuki，K.(1996)Lab.Invest.74,374-383]，重点研究了apoE对BetaAf体外延伸阶段的抑制作用的动力学。对数据的数学处理表明，apoE通过与Beta1-40形成络合物，从而从反应混合物中消除游离的Beta1-40，从而抑制BetaAf在体外的延伸。在三种主要的重组apoE亚型中，与β1-40的平衡结合常数基本相同。这些结果表明，载脂蛋白E在体外对βAf形成的影响是不同的，可以解决一些关于体外β-淀粉样蛋白-载脂蛋白相互作用的争议。

Recently, many research groups have examined the effect of apolipoprotein E (apoE) on beta-amyloid fibril (betaAf) formation in vitro. However, their data were somewhat controversial and no exact kinetic assessment of the role of apoE has thus far been available. We examined the effect of human apoE on betaAf formation in vitro, starting with various concentrations of freshly-prepared beta-amyloid (1-40) (beta1-40) and using fluorescence spectroscopy with thioflavine T.When 50 muM of beta-1-40 was incubated with an 1 : 1000 to 1 : 100 molar ratio of apoE,a dose dependent inhibitory effect of apoE was observed. Both the nucleation and extension phases of betaAf formation in vitro were inhibited by apoE.On the other hand, when 300 muM of beta1-40 was incubated with an 1 : 100 molar ratio of apoE,the inhibitory effect of apoE was completely abolished. We then focused our study on the kinetics of the inhibitory effect of apoE on the extension phase of betaAf formation in vitro, utilizing the recently established first-order kinetic model of betaAf extension in vitro [Naiki, H., & Nakakuki, K.(1996) Lab.Invest.74,374-383]. The mathematical treatment of the data suggests that apoE inhibits the extension of betaAf in vitro, by making a complex with beta1-40, thus eliminating free beta1-40 from the reaction mixture. The equilibrium association constant with beta1-40 was practically the same among the three major recombinant apoE isoforms. These results indicate that the effects of apoE on betaAf formation in vitro is differential, and could settle some of the controversy about beta-amyloid-apoE interaction in vitro.