KINETIC-ANALYSIS OF INOSITOL TRISPHOSPHATE BINDING TO PURE INOSITOL TRISPHOSPHATE RECEPTORS USING SCINTILLATION PROXIMITY ASSAY

Inositol 1,4,5-triphosphate (InsP(3)) receptors are regulated by many intracellular signals including proteins and small messengers. By link ing purified cerebellar InsP(3) receptors to scintillation proximity a ssay beads, binding of radioligands can be measured without separation of bound from free ligand. InsP(3) receptors assayed by scintillation proximity assay bound heparin with high affinity and stereoselectivel y bound InsP(3) with similar affinity to cerebellar membranes. By rapi dly freezing scintillation proximity assay reactions and then counting the frozen samples, both fast and slow components of [H-3] InsP(3) as sociation and dissociation were identified. Our novel freeze-quench me thod in combination with conventional stopped-quench equipment and sci ntillation proximity assay allows the rapid kinetics of the interactio ns of pure receptors with their ligands to be resolved. (C) 1996 Acade mic Press, Inc.