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

Abstract

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 (Kd) 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 microM, 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.