Membrane-associated inositol hexakisphosphate binding in bovine retina

Abstract

We investigated the InsP₆ binding proteins in bovine retinal membranes and rod outer segments (ROS) by radioligand binding assay and western blotting. The relative affinity of InsP₆ for the binding protein was determined by competitive binding of [³H]-InsP₆ with increasing concentrations of the unlabeled InsP₆ or other isomers. InsP₆ specifically binds to both bovine retinal membranes and ROS; maximum binding was achieved after one-hour incubation at 4°C and was unchanged up to 2 h. Tris-HCl or acetate buffer was equally suitable for the binding assay over a broad range of pH, although specific binding was slightly increased at acidic pH. The order of potencies of displacement was InsP₆ > Ins(1,3,4,5,6)P₅ > Ins(1,3,4,6)P₄ = Ins(1,3,4,5)P₄, whereas Ins(1,4,5)P₃, Ins(1,4)P₂, Ins(4,5)P₂, and Ins(1)P were not effective displacers. Scatchard analyses of the binding data were consistent with an equilibrium dissociation constant (K_d) of 2.5 ± 0.2 μM and maximal binding capacity (B_max) of 123.7 ± 25.0 pmol/mg at pH 7.4. Western blotting was used to detect whether AP-2 (an InsP₆ binding protein) is present in the retina. Immunoreactivity to AP-2 α and β subunits was found in retinal membranes and ROS. Thus, bovine retinal membranes and ROS contain membrane-associated InsP₆ binding protein(s) which is distinct from proteins that bind InsP₅, InsP₄, or InsP₃.

Key Words:

• inositol polyphosphates
• arrestin
• phosphatidylinositols
• phytic acid
• retina
• cattle (bovine)