Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination
Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination
复制标题
使用新型荧光配体和配体结合域组合对肌醇 1,4,5-三磷酸进行高灵敏度测量
作者：
Oura;T.; Murata;K.; Morita;T.; Nezu;A.; Arisawa;M.; Shuto;S.; Tanimura;A.
在前期腺磷蛋白 A构效关系研究的基础上，设计了两种荧光1，4，5-三磷酸(IP_3)受体配体，即荧光腺磷蛋白A(FADA)和荧光低亲和力配体(Fll)。在表达蛋白质的细胞中，荧光配体与荧光IP3传感器的结合引起了FRET。这一原理通过使用结合在琼脂糖珠上的荧光IP3传感器扩展到无细胞检测系统。FLL对FRET信号的影响通过随后添加IP3而减小。IP3对FRET信号的IC50值分别为139.7 nm和352.1 nm。该方法可定量测量10 以下的IP3浓度，并可用于测定COS7细胞胞浆IP3浓度，并检测合成的腺磷蛋白 A类似物的效价。
Based on the results of our previous adenophostin A structure–activity relationship studies, two fluorescent inositol 1,4,5‐trisphosphate (IP3) receptor ligands, fluorescent adenophostin A (FADA) and fluorescent low‐affinity ligand (FLL), were designed. Binding of the fluorescent ligands to the fluorescent IP3sensor in protein‐expressing cells caused FRET. This principle was extended to a cell‐free assay system by using the fluorescent IP3sensor bound to agarose beads. The effect of FLL on the FRET signal was reduced by subsequent addition of IP3. The IC50values of IP3on the FRET signals were 139.7 and 352.1 nmfor 30 and 100 nmFLL, respectively. This method allowed quantitative measurement of IP3concentrations below 10 nmand was applied to measure cytosolic IP3concentrations in COS‐7 cells and to examine the potency of synthesized adenophostin A analogues.