Naiki H, Nakakuki K
Department of Pathology, Fukui Medical School, Japan.
Lab Invest. 1996 Feb;74(2):374-83.
Recently, several studies have proposed models describing the mechanisms of Alzheimer's beta-amyloid fibril formation in vitro. However, these models are somewhat controversial and no exact kinetic analyses measuring the polymerization velocity as an indicator of the reaction, have thus far been available. We first formed beta-amyloid fibrils from a synthetic peptide, beta-amyloid(1-40), and determined the optimum conditions for quantitative fluorometry of these beta-amyloid fibrils with thioflavine T. Optimum fluorescence measurements of beta-amyloid fibrils were obtained at the excitation and emission wavelengths of 446 and 490 nm, respectively, with the reaction mixture containing 5 microM thioflavine T and 50 mM of glycine-NaOH buffer, pH 8.5. We then focused our study on the extension phase of beta-amyloid fibril formation in vitro. When beta-amyloid fibrils were incubated with monomeric beta-amyloid(1-40) in conditions where de novo seed formation does not occur, the extension of beta-amyloid fibrils was observed with electron microscopy. Quantitative fluorometry revealed that: (a) extension of amyloid fibrils proceeded by a pseudo-first-order exponential increase as measured by the fluorescence of thioflavine T; (b) the rate of extension was maximum around pH 7.5, and was dependent on the incubation temperature. Between 20 and 37 degrees C, good linearity was observed between the common logarithm of the initial rate and the reciprocal of the absolute temperature; (c) the rate of polymerization was found to be proportional to the product of beta-amyloid fibrils number concentration and the beta-amyloid(1-40) concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization. These results show that beta-amyloid fibril formation can be explained by a first-order kinetic model: i.e., the extension of beta-amyloid fibrils proceeds via the consecutive association of beta-amyloid(1-40) onto the ends of existing fibrils.
最近，几项研究提出了描述体外阿尔茨海默病β-淀粉样蛋白原纤维形成机制的模型。然而，这些模型存在一定争议，并且目前还没有精确的动力学分析以测量聚合速度作为反应指标。我们首先从合成肽β-淀粉样蛋白（1-40）形成β-淀粉样蛋白原纤维，并确定了用硫黄素T对这些β-淀粉样蛋白原纤维进行定量荧光测定的最佳条件。β-淀粉样蛋白原纤维的最佳荧光测量分别在激发波长446nm和发射波长490nm下获得，反应混合物含有5μM硫黄素T和50mM甘氨酸-NaOH缓冲液，pH8.5。然后我们将研究重点放在体外β-淀粉样蛋白原纤维形成的延伸阶段。当β-淀粉样蛋白原纤维在不发生从头种子形成的条件下与单体β-淀粉样蛋白（1-40）一起孵育时，通过电子显微镜观察到β-淀粉样蛋白原纤维的延伸。定量荧光测定显示：（a）淀粉样蛋白原纤维的延伸通过硫黄素T荧光测量的准一级指数增加进行；（b）延伸速率在pH7.5左右最大，并且取决于孵育温度。在20至37摄氏度之间，初始速率的常用对数与绝对温度的倒数之间观察到良好的线性关系；（c）发现聚合速率与β-淀粉样蛋白原纤维数浓度和β-淀粉样蛋白（1-40）浓度的乘积成比例；（d）净延伸速率是聚合和解聚速率之和。这些结果表明，β-淀粉样蛋白原纤维的形成可以用一级动力学模型来解释：即β-淀粉样蛋白原纤维的延伸是通过β-淀粉样蛋白（1-40）连续结合到现有纤维的末端进行的。
