Antiprogestin ZK-98.299 and progesterone display differential binding characteristics in the human myometrial cytosol

To investigate whether the synthetic progesterone antagonist ZK-98.299 binds to progesterone receptor or also has distinct binding sites, the binding characteristics of ZK-98.299 were compared with those of progesterone in the human myometrial cytosol. [3H]ZK-98.299 and [3H]progesterone showed specific binding in the myometrial cytosol and the binding of each radiolabelled ligand could be displaced with the respective ligand in a dose-response manner. However, while the binding of [3H]progesterone could be completely blocked with progesterone or ZK 98.299, the binding of [3H]ZK-98.299 could not be displaced more than 50%. The non-specific binding of [3H]ZK-98.299 was very high as compared to that of [3H]progesterone. Using [3H]progesterone, the relative binding affinity (RBA) of progesterone was more than that of ZK 98.299, whereas using [3H]ZK-98.299 the RBA of ZK 98.299 exceeded that of progesterone. Treatment of myometrial cytosol with increasing concentrations of -SH-modifying agents (iodoacetamide (IA) 0-10 mM or N-ethylmaleimide (NEM) 0-1000 nM) decreased the binding of progesterone by over 80%, whereas similar treatment did not have appreciable effect on the binding of [3H]ZK-98.299. Although both preformed ligand-receptor complexes were relatively stable in the presence of IA and NEM, the [3H]progesterone-receptor complex was more sensitive as compared to the [3H]ZK-98.299-receptor complex. The addition of 20 mM molybdate in the cytosol had a protective effect against the -SH-modifying agents. [3H]ZK-98.299 and [3H]progesterone-receptor complexes also showed differential stability when incubated at elevated temperatures (25 degrees C and 37 degrees C), [3H]ZK-98.299-binding sites being more thermolabile as compared to [3H]progesterone binding sites. Prior occupation of the receptor by the two ligands gave the complexes the ability to resist an elevated temperature of 25 degrees C. Moreover, molybdate stabilized both the liganded and unoccupied receptors at 25 degrees C. When the ligand-receptor complexes were applied onto a prefocused polyacrylamide gel, the progesterone and ZK-98.299-receptor complexes were resolved and focused at pH 7.2 and 8.4, respectively. The results of this study suggest that although progesterone and ZK-98.299 are mutually competitive for binding to progesterone receptor, ZK-98.299 also has distinct binding sites.