A RADIORECEPTOR ASSAY FOR MASS MEASUREMENT OF INOSITOL (1,4,5)-TRISPHOSPHATE USING SAPONIN-PERMEABILIZED OUTDATED HUMAN PLATELETS

The binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] to saponin -permeabilized blood-bank-outdated human platelets, 6 days old, has be en characterized (K-d = 3.8 nM; B-max = 1.7 pmol/mg protein) and used to develop a novel radioreceptor assay which allows the measurement of the Ins(1,4,5)P-3 content in resting or agonist-stimulated cells. Thi s assay is as sensitive (0.25 pmol in a 0.25 ml volume), specific, and reproducible as previously proposed methods. In addition, obtaining l arge batches of the Zns(1,4,5)P-3 binding protein by treating outdated platelets with saponin is simple and quick and uses otherwise discard ed material. Moreover, the assay is considerably cheaper than commerci ally available kits. Using this method we confirmed that thrombin evok es a rapid, transient, and dose-dependent increase in the platelet con centration of Ins(1,4,5)P-3. (C) 1998 Academic Press.