Modification of a competitive protein binding assay for determination of inositol 1,4,5-trisphosphate.

A modification of a competitive protein-binding method for the determination of inositol 1,4,5-triphosphate (Ins(1,4,5)P3), facilitated by the use of a cell harvester, is described. The method is based on the competition of [3H]Ins(1,4,5)P3 with Ins(1,4,5)P3 in the sample for binding to a binding protein prepared from bovine adrenal cortex. The assay is carried out in 96-well microtiter plates in a final volume of 100 microliters and free [3H]Ins(1,4,5)P3 is separated from bound by filtration using a cell harvester. This allows the rapid measurement of large numbers of samples, with high reproducibility. Ins(1,4,5)P3 bound to a single class of high-affinity receptors with a KD of 2.3 +/- 0.2 nM and a Bmax of 289 +/- 7 fmol/mg of protein. The binding was rapid, reaching equilibrium after 20 min, and reversible. The sensitivity of the assay allows the determination of amounts of Ins(1,4,5)P3 as small as 0.1 pmol, which corresponds to a concentration of 5 nM in the sample. The application of the method to measure the formation of Ins(1,4,5)P3 in DDT1 MF-2 smooth muscle cells activated with ATP or bradykinin is described. The modification is a rapid, sensitive, convenient, and relatively inexpensive method for the determination of Ins(1,4,5)P3 in large numbers of samples.