A rapid ion-exchange assay for detergent-solubilized inositol 1,4,5-trisphosphate receptors

S. R. Hingorani, William S. Agnew

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)204-213
Number of pages10
JournalAnalytical biochemistry
Volume194
Issue number1
DOIs
StatePublished - Apr 1991

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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