DEVELOPMENT OF A SCINTILLATION PROXIMITY ASSAY FOR CALCINEURIN PHOSPHATASE-ACTIVITY

We have developed a scintillation proximity assay (SPA) that allows th e Ca2+/calmodulin (CaM)-dependent Serine/threoine (Ser/Thr) phosphopro tein phosphatase 2B (calcineurin) activity to be analyzed. A [P-33] la beled and biotinylated peptide containing a partial sequence of the re gulatory subunit (R-II) of the cyclic adenosine monophosphate (cAMP)-d ependent protein kinase was synthesized and used as a synthetic substr ate for calcineurin, Following incubation of the peptide with calcineu rin, which removes the [P-33] label, streptavidin-coated SPA beads wer e added to capture the biotinylated peptide (the level of the signal d etected is inversely proportional to that of the calcineurin activity) , Sensitivity is increased in this system by settling or centrifuging the streptavidin-coated SPA beads after binding has occurred, This met hod allows calcineurin phosphatase assays to be carried out in a 96-we ll format that is amenable to screening large numbers of compounds.