Solubilization and Characterization of PGE 2 Receptor in Porcine Ciliary Epithelium

Abstract

ABSTRACT

PGE₂ binding sites or receptors of porcine ciliary nonpigmented epithelial (NPE) and pigmented epithelial (PE) membranes were solubilized with detergents (CHAPS and Triton X100). From the Scatchard plots of PGE₂ binding to CHAPS-solubilized proteins, the Kd and Bmax values were calculated to be 35 nM and 470 fmol/mg protein for NPE protein and 65 nM and 430 fmol/mg protein for PE protein, respectively. On the basis of the Kd and Bmax values, the solubilized receptor proteins correspond to PGE₂ binding sites of the membranes which have previously been shown to be coupled to adenylate cyclase inhibition. For both NPE and PE proteins, the order of binding potency was PGE₂>PGF_2α>PGD₂. By gel filtration chromatography of NPE and PE proteins, the molecular mass of the major PGE₂ binding peak was estimated to be about 150 KDa when solubilized in CHAPS and 46 KDa for Triton X100 extracts. The Bmax values of membrane-associated binding proteins were increased by GTP, indicating a close association of the PGE₂ binding sites with a GTP-binding protein. However, GTP did not affect the Bmax values of detergent-solubilized receptor proteins.