Analysis of progesterone receptor binding in the ovine uterus

The appropriate conditions for the measurement of ovine uterine cytoplasmic progesterone receptors (PR) have been determined to be 20 nM 3H-progesterone (3H-P4) with and without a 100-fold excess of non-radioactive progesterone (P4) 0-4 degrees C and 4 h of incubation. Under these conditions PR readily exchanged bound progesterone for progesterone added during the assay. This exchange occurred even when saturating concentrations of P4 were present. The progestins, R5020 and P4, effectively competed for the ovine uterine PR binding while non-progestin steroids and diethylstilbestrol failed to compete for the PR binding. The dissociation constant (Kd) measured for the 3H-P4 binding was 1.60 x 10-9 M indicating that the 3H-P4 binding was of high affinity. The levels of PR and the dissociation constant measured using 3H-R5020 in place of 3H-P4 were similar indicating a lack of corticosteroid binding globulin (CBG)-like binding in the ovine uterus.