Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination

被引：9

作者：

Oura, Tai ^{[1
]}

Murata, Kaori ^{[2
]}

Morita, Takao ^{[3
]}

Nezu, Akihiro ^{[3
]}

Arisawa, Mitsuhiro ^{[1
]}

Shuto, Satoshi ^{[1
]}

Tanimura, Akihiko ^{[3
]}

机构：

[1] Hokkaido Univ, Fac Pharmaceut Sci, Kita Ku, Kita 12,Kita 12Nishi 6, Sapporo, Hokkaido 0600812, Japan

[2] Hlth Sci Univ Hokkaido, Dept Pediat Dent, Sch Dent, Kanazawa Ku, Kita 121757, Ishikari, Hokkaido 0610293, Japan

[3] Hlth Sci Univ Hokkaido, Dept Pharmacol, Sch Dent, Kanazawa Ku, Kita 121757,Kita 12, Ishikari, Hokkaido 0610293, Japan

来源：

CHEMBIOCHEM | 2016年 / 17卷 / 16期

基金：

日本学术振兴会;

关键词：

fluorescent probes; FRET; inositol; 1; 4; 5-trisphosphate; ligand-binding domain; sensors; IP3 RECEPTOR LIGANDS; ADENOPHOSTIN-A; PENICILLIUM-BREVICOMPACTUM; POTENT AGONISTS; TRISPHOSPHATE; PHOSPHATES; CHANNELS; ANALOGS; NUCLEOSIDES; DESIGN;

D O I：

10.1002/cbic.201600096

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Based on the results of our previous adenophostinA 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 IP3 sensor in protein-expressing cells caused FRET. This principle was extended to a cell-free assay system by using the fluorescent IP3 sensor bound to agarose beads. The effect of FLL on the FRET signal was reduced by subsequent addition of IP3. The IC50 values of IP3 on the FRET signals were 139.7 and 352.1nm for 30 and 100nm FLL, respectively. This method allowed quantitative measurement of IP3 concentrations below 10nm and was applied to measure cytosolic IP3 concentrations in COS-7 cells and to examine the potency of synthesized adenophostinA analogues.

引用

页码：1509 / 1512

页数：4

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