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A novel, specific binding protein assay for quantitation of intracellular inositol 1,3,4,5‐tetrakisphosphate (InsP 4 ) using a high‐affinity InsP 4 receptor from cerebellum
Author(s) -
Donié Frédéric,
Reiser Georg
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81029-4
Subject(s) - inositol , intracellular , chemistry , biochemistry , receptor , cerebellum , biology , neuroscience
A membrane preparation from porcine cerebellum displays high‐affinity binding sites for [ 3 H]inositol 1,3,4,5‐tetrakisphosphate ([ 3 H]InsP 4 ) 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 InsP 4 , since Ins(1,3,4,5,6)P 5 and Ins(1,4,5,6)P 4 displaced binding of InsP 4 with EC 50 values of 0.2 and 0.3 μM, respectively. Ins(1,4,5)P 3 and other inositol phosphates were less effective. Using this InsP 4 receptor, an assay for measuring tissue content of InsP 4 was developed. The detection limit of the assay was 0.1 pmol. In the same tissue samples the amount of Ins(1,4,5)P 3 was determined in parallel with a similar assay using a binding protein preparation from beef liver.