Nakamura Makiko, Mie Masayasu, Funabashi Hisakage, Yamamoto Kimiko, Ando Joji, Kobatake Eiry
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Anal Biochem. 2006 May 1;352(1):61-7. doi: 10.1016/j.ab.2006.02.019. Epub 2006 Mar 9.
We demonstrate the application of an assay technique for the detection of ATP efflux from the cell surface. Until recently, the firefly luciferase assay has conventionally been used to detect ATP release because of its high sensitivity and wide detection ability. However, since this technique can be used only to infer the amount of diffused ATP in bulk solutions, it does not accurately reflect the dynamic ATP flux at the cell membrane. We therefore constructed luciferase fusion proteins that could be immobilized onto the cell surface. However, the low activities and limited application ranges of these proteins prompted us to use biotinylated luciferase given its attributes of strong and stable localization. Furthermore, luciferase can be immobilized strongly onto the biotinylated cell surface via streptavidin-biotin binding and can thus be used to determine the dynamic release of ATP near the cell surface.
我们展示了一种用于检测细胞表面ATP流出的检测技术的应用。直到最近，萤火虫荧光素酶检测法因其高灵敏度和广泛的检测能力，一直被传统地用于检测ATP释放。然而，由于该技术仅能用于推断大量溶液中扩散的ATP量，它并不能准确反映细胞膜处的动态ATP通量。因此，我们构建了可固定在细胞表面的荧光素酶融合蛋白。然而，这些蛋白的低活性和有限的应用范围促使我们使用生物素化荧光素酶，因为其具有强而稳定的定位特性。此外，荧光素酶可通过链霉亲和素 - 生物素结合牢固地固定在生物素化的细胞表面，因此可用于确定细胞表面附近ATP的动态释放。
