A rapid ion-exchange assay for detergent-solubilized inositol 1,4,5-trisphosphate receptors.
We describe a rapid ion-exchange syringe assay for [3H]inositol 1,4,5-trisphosphate binding to detergent-solubilized receptors. In extracts of rat cerebellar membranes, the assay resolves rapidly dissociating ligand complexes, detecting two to three times higher receptor abundance than conventional gel filtration spun column assays, and provides evidence for two classes of IP3-binding sites, representing 0.5-1.0% of total cerebellar membrane protein. Receptors purified from bovine and rat cerebellum exhibit a single class of high-affinity sites, with equilibrium dissociation constants (Kd = 4-8 nM) reflecting 20 to 25-fold higher affinity than reported in studies with spun-column methods.
Hingorani
S R
SR
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Agnew
W S
WS
D000818:Animals; D015220:Calcium Channels; D002531:Cerebellum / Q000378:metabolism / Q000648:ultrastructure; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D003902:Detergents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility; D013594:Syringes; D014316:Tritium
D015220:Calcium Channels; D003902:Detergents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D014316:Tritium