title: ZK98299--a new antiprogesterone: biochemical characterization of steroid binding parameters in the calf uterine cytosol.
abstract: We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11 beta-(4-dimethylaminophenyl)-17 alpha-hydroxy-17 beta-(3-hydroxypropyl)- 13 alpha-methyl-4,9-gonadien-3-one], in the calf uterus cytosol and compared the nature of this interaction with the binding of progesterone receptor (PR) agonist R5020 [promegestone, 17,21-dimethylpregna-4,9-diene-3,20-dione]. In the freshly prepared cytosol, [3H]ZK98299 interacted specifically with a macromolecule: the binding was abolished in the presence of excess progestins (R5020 and progesterone) and the antiprogesterone ZK98299. The high affinity (Kd = 2.5 nM) interaction between [3H]ZK98299 and PR was temperature- and time-dependent, reaching an optimum by 2-3 h at 0 degrees C, and was facilitated by 20 mM Na2MoO4. Under nontransforming conditions, [3H]ZK98299-receptor complexes sedimented as 8 S species in 8-30% linear glycerol gradients. Upon salt or thermal transformation, there was a loss of the 8 S form, with only a small fraction of total complexes (5-7%) binding to DNA-cellulose. In contrast, transformed [3H]R5020-receptor complexes exhibited a greater extent of binding (25-55%) to DNA-cellulose. [3H]ZK98299-receptor complexes could be resolved into two ionic species over DEAE-Sephacel following incubation of the complexes at 0 or 23 degrees C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as beta-mercaptoethanol increased the extent of steroid binding. Although treatment with iodoacetamide (IA) abolished [3H]R5020 binding, there was a significant (nearly twofold) increase in the [3H]ZK98299 binding. The results of this study point to similarities and differences between the steroid binding properties of the uterine PR occupied by R5020 and ZK98299: both steroids appear to bind the same 8 S receptor but exhibit differential DNA binding and sensitivity to IA. The reported antagonist properties of ZK98299 may, therefore, be explained on the basis of a distinct receptor conformation induced by the antisteroid.
authors.affiliation: Department of Biological Sciences, Oakland University, Rochester, Michigan 48309-4401.
authors.forename: R
authors.initials: R
authors.lastname: Nath
authors.forename: A
authors.initials: A
authors.lastname: Bhakta
authors.forename: V K
authors.initials: VK
authors.lastname: Moudgil
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D003600:Cytosol / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D005260:Female; D005300:Fertility Agents, Female / Q000737:chemistry*; D006067:Gonanes / Q000737:chemistry*; D007700:Kinetics; D011374:Progesterone / Q000037:antagonists & inhibitors*; D011987:Receptors, Steroid / Q000032:analysis*; D014599:Uterus / Q000737:chemistry*
chemical_list: D005300:Fertility Agents, Female; D006067:Gonanes; D011987:Receptors, Steroid; D011374:Progesterone; D004247:DNA; C053238:onapristone