TY - JOUR
T1 - ZK98299-A new antiprogesterone
T2 - Biochemical characterization of steroid binding parameters in the calf uterine cytosol
AU - Nath, Rathna
AU - Bhakta, Amrita
AU - Moudgil, V. K.
N1 - Funding Information:
i Supported by NIH Grant DK-20893. * To whom correspondence should be addressed. 3 Abbreviations used: DMSO, dimethyl sulfoxide; E, estradiol; ethylenediaminetetraacetate; acetamide; P-mercaptoethanol; MTG, N-ethylmaleimide; P, progesterone; PMSF, oride; PR, progesterone receptor; R5020, thylpregna-4,9-diene-3,20-dione; RU486, 11~-[4-(dimethyl)aminophenyl]-l7or(prop-l-ynyl)estra-4,9-dien-3-one; SH, sulfhydryl; Tris, tris(hydroxymethyl)aminomethane; antigestagen, onapristone, 116-(4-dimethylaminophenyl)-17a-hydroxy-17P-(3-hydroxypropyl)-l3~-methyl-4,9-gona~en-3-one; ticoid receptor; PRE, progesterone response tropin-releasing hormone; LH, luteinizing
PY - 1992/1
Y1 - 1992/1
N2 - We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11β-(4-dimethylaminophenyl)-17α-hydroxy-17β-(3-hydroxypropyl)-13α-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 °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 °C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as β-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.
AB - We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11β-(4-dimethylaminophenyl)-17α-hydroxy-17β-(3-hydroxypropyl)-13α-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 °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 °C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as β-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.
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U2 - 10.1016/0003-9861(92)90083-9
DO - 10.1016/0003-9861(92)90083-9
M3 - Article
C2 - 1727646
AN - SCOPUS:0026537010
SN - 0003-9861
VL - 292
SP - 303
EP - 310
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -