Gnaccarini Claudio, Ben-Tahar Wajih, Lubell William D, Pelletier Joelle N, Keillor Jeffrey W
Département de chimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada H3C 3J7.
Bioorg Med Chem. 2009 Sep 1;17(17):6354-9. doi: 10.1016/j.bmc.2009.07.031. Epub 2009 Jul 18.
Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity gamma-glutamyl donor substrate and a biotinylated amine as a gamma-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.
在此，我们报告了一种用于测定组织转谷氨酰胺酶（TG2）活性的直接间断荧光转酰胺化测定方法的开发。在测定反应中，TG2使用荧光、高亲和力的γ-谷氨酰供体底物和生物素化胺作为γ-谷氨酰受体底物，催化生物素-荧光团共轭物的形成。反应后，共轭物固定在链霉亲和素包被的珠子上，洗去过量的底物，通过荧光测量对转酰胺化活性进行定量。该方法用于检测低至0.6 mU的纯化TG2的活性，并且可用于检测粗细胞裂解物中的活性。此外，该测定可用于筛选潜在的抑制剂和合成底物，本文展示了后者。
