title: Solubilization and partial characterization of inositol 1,4,5-trisphosphate receptor of bovine adrenal cortex reveal similarities with the receptor of rat cerebellum.
abstract: Inositol 1,4,5-trisphosphate (InsP3) is an intracellular messenger generated upon stimulation of a wide variety of cells by Ca2(+)-mobilizing stimuli. Specific binding sites for InsP3 have been identified in the adrenal cortex and many other tissues. The purpose of the present study was to solubilize and further characterize InsP3 receptors of bovine adrenal cortex. When adrenal cortex microsomes were incubated with Triton X-100 (1%) for 45 min and centrifuged at 100,000 x g for 1 hr, substantial InsP3-binding activity was recovered in the pellet fraction (82 +/- 46 fmol/mg of protein; Kd of 2.7 +/- 1.2 nM), suggesting a possible association with the cell skeleton. Similar results were also obtained with a microsomal preparation of rat cerebellum. On the other hand, the supernatant fraction also displayed important InsP3-binding activity (188 +/- 67 fmol/mg of protein; Kd of 10.4 +/- 2.2 nM). InsP3 binding in both fractions was inhibited by heparin and was increased upon pH elevations from 6 to 9. These are two characteristic properties of InsP3 receptors. Solubilized InsP3 receptors displayed a molecular size around 1,000,000, as estimated by gel filtration through Sepharose-4B column. Radiation inactivation analyses of the receptors of bovine adrenal cortex and rat cerebellum revealed unusual inactivation curves, indicating binding domains of Mr 65,000, much smaller than the smallest covalent structure (subunit) of Mr 260,000 estimated by gel electrophoresis. These results suggest that the binding domain of the receptor behaves independently from the rest of the molecule and that a direct hit on the domain is needed for inactivation. Our data show that the binding sites for InsP3 in the adrenal cortex have properties similar to those of sites recently purified from rat cerebellum, and they suggest that InsP3 receptors from both sources might be the same molecular entity.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Québec, Canada.
authors.forename: G
authors.initials: G
authors.lastname: Guillemette
authors.forename: I
authors.initials: I
authors.lastname: Favreau
authors.forename: G
authors.initials: G
authors.lastname: Boulay
authors.forename: M
authors.initials: M
authors.lastname: Potier
mesh_terms: D000302:Adrenal Cortex / Q000737:chemistry*; D000818:Animals; D015220:Calcium Channels; D002417:Cattle; D002531:Cerebellum / Q000737:chemistry*; D002850:Chromatography, Gel; D006493:Heparin / Q000494:pharmacology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins / Q000302:isolation & purification; D008861:Microsomes / Q000737:chemistry; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility
chemical_list: D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011092:Polyethylene Glycols; D015544:Inositol 1,4,5-Trisphosphate; D017830:Octoxynol; D006493:Heparin