Kinetics of amyloid β-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays
Kinetics of amyloid β-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays
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DOI：
10.1074/jbc.m305627200
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发表时间：
2003-09-26
影响因子：
4.8
通讯作者：
Selkoe, DJ
中科院分区：
文献类型：
--
作者：
Leissring, MA;Lu, A;Selkoe, DJ
降解β - 淀粉样蛋白（Aβ）的蛋白酶是健康状态和阿尔茨海默病中大脑Aβ水平的重要调节因子，但用于研究其详细动力学的实用方法却很少。在此，我们描述了基于新型底物荧光素 - Aβ -（1 - 40）- 赖氨酸 - 生物素（FAbetaB）的可靠且定量的Aβ降解检测方法。液相色谱/质谱分析表明，FAbetaB被脑啡肽酶和胰岛素降解酶（两种被广泛研究的Aβ降解蛋白酶）在与野生型Aβ极为相似的位点水解。衍生化的肽是一种亲和性底物，适用于生物样本以及高通量化合物筛选。我们开发的检测方法易于实施，对于生成定量动力学数据特别有用，正如我们通过测定几种Aβ降解蛋白酶（包括此前未被表征的纤溶酶）对FAbetaB降解的动力学参数所证明的那样。这些检测方法的使用应该会对Aβ降解蛋白酶的生物学特性产生更多新的见解，并有助于识别此类酶的激活剂和抑制剂。
Proteases that degrade the amyloid beta-protein (Abeta) are important regulators of brain Abeta levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Abeta degradation assays based on the novel substrate, fluorescein-Abeta- (1-40)-Lys-biotin (FAbetaB). Liquid chromatography/ mass spectrometric analysis shows that FAbetaB is hydrolyzed at closely similar sites as wild-type Abeta by neprilysin and insulin-degrading enzyme, the two most widely studied Abeta-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAbetaB degradation by several Abeta-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Abeta-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.