Identification and Characterization of High-Affinity Binding Sites for Inositol Trisphosphate in Red Beet

Inositol 1,4,5-trisphosphate (InsP3) is thought to play a primary role in intracellular Ca2+ mobilization during signal transduction in plant cells. Although InsP3-elicited Ca2+ release across the vacuolar membrane has been demonstrated in a variety of species, little is known of the properties of the putative InsP3 receptor. Using a 3H-InsP3 ligand-displacement assay with detergent-solubilized microsomes from the storage root of red beet, we determined that InsP3 binds specifically to a single class of high-affinity binding sites (dissociation constant [Kd] = 121 [plus or minus] 10 nM) with an estimated receptor density of 0.84 pmol/mg. Binding of InsP3 is selective, because other inositol phosphates exhibited only supramicromolar affinities for the binding site. Low molecular weight heparin was a potent competitive inhibitor of InsP3 binding (Kd = 301 [plus or minus] 72 nM). High concentrations of ATP also displaced 3H-InsP3 (Kd = 0.66 mM). Preincubation of microsomes with sulfhydryl reagents reduced InsP3-specific binding in an InsP3-protectable manner. Density gradient centrifugation of microsomes led to copurification of InsP3-specific binding with a fraction enriched in vacuolar membrane. Despite a probable difference in cellular location, the putative InsP3 receptor of red beet has characteristics that are very similar to those of animal InsP3 receptors. These studies provide direct evidence of InsP3-specific binding in plant tissue and strengthen the argument that InsP3-induced Ca2+ release is a component in plant cell signal transduction.