A novel, specific binding protein assay for quantitation of intracellular inositol 1,3,4,5‐tetrakisphosphate (InsP4) using a high‐affinity InsP4 receptor from cerebellum

FEBS Letters

Donié, Frédéric¹  Reiser, Georg¹ 

[1] Physiologisch-Chemisches Institut der Universität Tübingen, Hoppe-Seyler-Str. 4, 7400 Tübingen, FRG

关键词: Inositol 1;    3;    4;    5-tetrakisphosphate;    Inositol 1;    4;    5-trisphosphate;    Receptor;    inositol phosphate;    Bradykinin;    InsP4;    D-myo-inositol 1;    3;    4;    5-P4;    InsP3;    D-myo-inositol 1;    4;    5-P3;   

DOI  :  10.1016/0014-5793(89)81029-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A membrane preparation from porcine cerebellum displays high-affinity binding sites for [³H]inositol 1,3,4,5-tetrakisphosphate ([³H]InsP₄) with a dissociation constant (K _d) of 1.0 nM and a density of 220 fmol/mg protein. Specific binding was maximal in the presence of 25 mM phosphate and at pH 5.0. The receptor site was specific for InsP₄, since Ins(1,3,4,5,6)P₅ and Ins(1,4,5,6)P₄ displaced binding of InsP₄ with EC₅₀ values of 0.2 and 0.3 μM, respectively. Ins(1,4,5)P₃ and other inositol phosphates were less effective. Using this InsP₄ receptor, an assay for measuring tissue content of InsP₄ was developed. The detection limit of the assay was 0.1 pmol. In the same tissue samples the amount of Ins(1,4,5)P₃ was determined in parallel with a similar assay using a binding protein preparation from beef liver.

【 授权许可】

Unknown