Characterization of Prostaglandin Receptors in the Bovine Corpus Luteum Cell Membranes

Abstract

Abstract [3H]Prostaglandin (PG) E1 bound to the cell membrane fraction of bovine corpus luteum with high affinity and specificity. Unlabeled PGE1 and E2 inhibited [3H]PGE1 binding to the membranes in a dose-dependent manner with complete inhibition occurring at 1.4 x 10-7 m. Other PGs (PGF1α, F2α, B1, A1, and A2) inhibited 80 to 100% of [3H]PGE1 binding only at 100-fold greater concentrations than that of PGEs. Membrane-bound and free [3H]PGE1 were found to be essentially unchanged under the present incubation conditions. The Scatchard plot analysis of the equilibrium binding data suggested the presence of high and low affinity binding sites having apparent dissociation constants (Kd) of 1.3 x 10-9 m and 1.0 x 10-8 m, respectively. Free energy changes of +12.6 and +11.4 Cal per mole were calculated for the dissociation of [3H]PGE1 from high and low affinity binding sites, respectively. The rate constants for association and dissociation, 1.9 x 105 m-1 s-1 and 2.8 x 10-3 s-1, were measured at 38°. The apparent Kd calculated from the ratio of rate constants at 22° and 38° (2.8 x 10-8 m versus 1.5 x 10-8 m) agreed very well with each other. Maximum specific binding of [3H]PGE1 occurred at pH 6.2 from 30 to 38°. For concentrations of various cations, up to 1 x 10-1 m, only Ca2+ and Mg2+ moderately increased [3H]PGE1 binding. When the concentrations were increased above 1 x 10-1 m, both mono- and divalent cations significantly decreased [3H]PGE1 binding. Incubation of membranes with various enzymes revealed that PGE receptor molecules are protein in nature which require lipids and specific phospholipids, but not sialic acid residues, of membranes and/or receptor in binding of [3H]-PGE1. Treatment of membranes with several protein-modifying reagents suggested that tyrosyl residues but not sulfhydryl groups were involved in the binding interaction.