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149. Synthesis and decay of estrogen-induced progesterone receptors in the hamster uterus
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Abstracts microsomal progesterone binders are distinctly different from the known cytosolic progesterone receptors of rat myometrium in regard to binding affinity and steroid specificity.
5B 2. Progestogen receptors-11 147. Characterization and purification of the progesterone receptor from human uterus SMITH, R. G., IRAMAIN, C. A. and BUTTRAM,V. C., Departments of Cell Biology and Obstetrics and Gynaecology, Baylor College of Medicine, Houston, Texas, U.S.A. For the first time a specific high affinity progesterone receptor has been substantially purified (> 10,000 fold) from human uterus. The progesterone receptor was initially isolated by ammonium sulphate fractionation of human uterine cytosoi. The receptor was characterized as a macromolecule using sucrose density gradient analysis ( _ 3.7 S) and Sephadex gel filtration. Its high specificity and the absence of CBG contamination was confirmed using a charcoal adsorption assay in the presence of estradiol, dihydrotestosterone, corticosterone and cortisol. The receptor has an association constant for progesterone of 1.78 x lo9 M and a binding site concentration of 4.34 x 10 -9 M/mg protein. The receptor was further purified by affinity chromatography using deoxycorticosterone hemisuccinatecoupled to Sepharose-albumin. Scatchard analyses of the material which was not retained by the affinity column showed that all the receptor was tightly bound by the column; elution was accomplished by displacement with excess 3H-progesterone. Sephadex chromatography and sucrose density gradient analysis in the presence of competing steroids showed that the purified human receptor had retained its native physical-chemical characteristics. 148. Progesterone receptor in calf uterus cytosol BEST-BELPOMME, M., Laboratoire d’Enzymologie, 91190, Gif sur Yvette, France; Present address: Unite 33 INSERM, Lab Hormones, 94270, Bicetre, France The presence of a proteic receptor which binds specifically progesterone is demonstrated in calf uterus cytosol by the method of adsorption on small hydroxylapatite columns. The progesterone receptor does not,bind corticosteroids. It is equally distributed between the endometrium and the myometrium and exhibits an identicai affinity constant in both cases. In crude extracts (at 4”C), the progesterone receptor is unstable (half degradation in 48 h). Stabilization is obtained by precipitating with 35% ammonium sulfate. Its activity decreases with increasing KC1 concentration (half inactivation obtained at -0.25 M KCI). After sucrose gradient ultracentrifugation (low ionic strength), analysis by the hydroxylapatite method permits the distinction between the CBG-like-protein (~“4s”) and the non-specific sites (majority in the “45 region”) and the progesterone receptor which is clearly present in two active conformations: “8 s” and “aggregates”. Several parameters of the interaction of the receptoiand itsligand have been measured at 0°C: (a) Intrinsic affinity constant Ka = lOaM-‘. (b) Second order association rate constant k, = I.8 x lo* M-’ see- i. (c) Respective dissociation rate constants
(dissociation of the complex progesterone-receptor proceeds by two steps) kdl = 1.6 x 10e4 set-’ and kd2 = 8 x low6 see-‘. These kinetic and equiiib~um data and others obtainedwith calfuterus oestradiol receptor can be explained by a model which implicates a change in the information of the receptor induced by binding of steroids. 149. Synthesis and decay of estrogen-induced progesterone receptors in the hamster uterus REEL, J. R. and SHIH, Y., Department of Pharmacology, Parke-Davis Research Laboratories, Ann Arbor, Michigan 48106, U.S.A. Using sucrose-glycerol density gradient analysis, we present here data concerned with the kinetics of synthesis and decay of estrogen-induced uterine progesterone (P) receptors in the ovariectomized (ovx) immature hamster. Hamsters ovx 2 weeks prior were injected S.C.with 100 pg of 17~~-estradiol (E-1 oer ke bw at 6. 12. 16. 20. 24. 24. 72. 96 and 12Oh . before autopsy. A 7 S P receptor in the uterine cytosol was elevated 6h and continued to rise until a maximum was attained at 20 h. This new steady state level persisted for at least another 52 h; thereafter, the 7 S binder steadily declined whereas a 4.5 S binder. which was a minor component during the induction phase, gradually increased becoming the predominant species during the decay phase. Actinomycin D and cycloheximide blocked Ez induction of the 7 S receptor, suggesting that RNA and protein synthesis is involved in this response. Ancillary results suggest that the 4.5 S component is derived from the 7S species. Incubation of the cytosol at 25 “C or exposure to O-l 5-0.4M KC1 dissociated the 7 S species into the 4.5 S form. In summary, induction of P receptors is one of the early uterine responses to estrogen. During the induction and decay phases there are quantitative and qualitative changes in the molecular forms of the receptors, raising further questions as to their interrelationship and physiological significance. *a
.
150. Multiple forms of the progesterone receptor in the guinea pig uterus CEIL.P. D.. ACKER. G. and BARDIN. C. W.. The Milton S. Hershey Medical Center. The Pennsylvania State University, Hershey, Pennsylvania, U.S.A. Studies of the progesterone receptor in the guinea pig uterus indicated that the cytoplasmic (CR) and the nuclear (NR) forms of this intracellular binder were similar when prepared in high-salt buffers. It was, therefore, pertinent to distinguish CR from NR as a function of ionic strength. The CR complex sedimented as a 6.9 f 0.2 S (f SD) molecule in low-salt sucrose-glycerol gradients and it had a rapid rate of dissociation with a half-life (T)) of 62 min. When the ionic strength of the CR was raised by addition of KC1 (CR + KCl), a sedimentation value of 4.6 If: 0.4 S and a T) of 112 min were found. These values were identical with those of a KC1 extract of NR. Rapid dialysis to remove the salt from CR+KCl and NR+KCl produced complexes which sedimented as 6.6 + 0.5 S and 5.4 f 0.4 S molecules, respectively. In addition, a significant difference in the T+ values was again noted following dialysis of the two receptors. Interestingly, dialysis of CR did not change the S value but increased ‘It. Conclusion: In the presence of high ionic strength, the CR and NR appear to be identical. However, removal of the salt by rapid dialysis has revealed a clear difference in both their S and Tf values by which they may be distinguished.
Abstracts microsomal progesterone binders are distinctly different from the known cytosolic progesterone receptors of rat myometrium in regard to binding affinity and steroid specificity.
5B 2. Progestogen receptors-11 147. Characterization and purification of the progesterone receptor from human uterus SMITH, R. G., IRAMAIN, C. A. and BUTTRAM,V. C., Departments of Cell Biology and Obstetrics and Gynaecology, Baylor College of Medicine, Houston, Texas, U.S.A. For the first time a specific high affinity progesterone receptor has been substantially purified (> 10,000 fold) from human uterus. The progesterone receptor was initially isolated by ammonium sulphate fractionation of human uterine cytosoi. The receptor was characterized as a macromolecule using sucrose density gradient analysis ( _ 3.7 S) and Sephadex gel filtration. Its high specificity and the absence of CBG contamination was confirmed using a charcoal adsorption assay in the presence of estradiol, dihydrotestosterone, corticosterone and cortisol. The receptor has an association constant for progesterone of 1.78 x lo9 M and a binding site concentration of 4.34 x 10 -9 M/mg protein. The receptor was further purified by affinity chromatography using deoxycorticosterone hemisuccinatecoupled to Sepharose-albumin. Scatchard analyses of the material which was not retained by the affinity column showed that all the receptor was tightly bound by the column; elution was accomplished by displacement with excess 3H-progesterone. Sephadex chromatography and sucrose density gradient analysis in the presence of competing steroids showed that the purified human receptor had retained its native physical-chemical characteristics. 148. Progesterone receptor in calf uterus cytosol BEST-BELPOMME, M., Laboratoire d’Enzymologie, 91190, Gif sur Yvette, France; Present address: Unite 33 INSERM, Lab Hormones, 94270, Bicetre, France The presence of a proteic receptor which binds specifically progesterone is demonstrated in calf uterus cytosol by the method of adsorption on small hydroxylapatite columns. The progesterone receptor does not,bind corticosteroids. It is equally distributed between the endometrium and the myometrium and exhibits an identicai affinity constant in both cases. In crude extracts (at 4”C), the progesterone receptor is unstable (half degradation in 48 h). Stabilization is obtained by precipitating with 35% ammonium sulfate. Its activity decreases with increasing KC1 concentration (half inactivation obtained at -0.25 M KCI). After sucrose gradient ultracentrifugation (low ionic strength), analysis by the hydroxylapatite method permits the distinction between the CBG-like-protein (~“4s”) and the non-specific sites (majority in the “45 region”) and the progesterone receptor which is clearly present in two active conformations: “8 s” and “aggregates”. Several parameters of the interaction of the receptoiand itsligand have been measured at 0°C: (a) Intrinsic affinity constant Ka = lOaM-‘. (b) Second order association rate constant k, = I.8 x lo* M-’ see- i. (c) Respective dissociation rate constants
(dissociation of the complex progesterone-receptor proceeds by two steps) kdl = 1.6 x 10e4 set-’ and kd2 = 8 x low6 see-‘. These kinetic and equiiib~um data and others obtainedwith calfuterus oestradiol receptor can be explained by a model which implicates a change in the information of the receptor induced by binding of steroids. 149. Synthesis and decay of estrogen-induced progesterone receptors in the hamster uterus REEL, J. R. and SHIH, Y., Department of Pharmacology, Parke-Davis Research Laboratories, Ann Arbor, Michigan 48106, U.S.A. Using sucrose-glycerol density gradient analysis, we present here data concerned with the kinetics of synthesis and decay of estrogen-induced uterine progesterone (P) receptors in the ovariectomized (ovx) immature hamster. Hamsters ovx 2 weeks prior were injected S.C.with 100 pg of 17~~-estradiol (E-1 oer ke bw at 6. 12. 16. 20. 24. 24. 72. 96 and 12Oh . before autopsy. A 7 S P receptor in the uterine cytosol was elevated 6h and continued to rise until a maximum was attained at 20 h. This new steady state level persisted for at least another 52 h; thereafter, the 7 S binder steadily declined whereas a 4.5 S binder. which was a minor component during the induction phase, gradually increased becoming the predominant species during the decay phase. Actinomycin D and cycloheximide blocked Ez induction of the 7 S receptor, suggesting that RNA and protein synthesis is involved in this response. Ancillary results suggest that the 4.5 S component is derived from the 7S species. Incubation of the cytosol at 25 “C or exposure to O-l 5-0.4M KC1 dissociated the 7 S species into the 4.5 S form. In summary, induction of P receptors is one of the early uterine responses to estrogen. During the induction and decay phases there are quantitative and qualitative changes in the molecular forms of the receptors, raising further questions as to their interrelationship and physiological significance. *a
.
150. Multiple forms of the progesterone receptor in the guinea pig uterus CEIL.P. D.. ACKER. G. and BARDIN. C. W.. The Milton S. Hershey Medical Center. The Pennsylvania State University, Hershey, Pennsylvania, U.S.A. Studies of the progesterone receptor in the guinea pig uterus indicated that the cytoplasmic (CR) and the nuclear (NR) forms of this intracellular binder were similar when prepared in high-salt buffers. It was, therefore, pertinent to distinguish CR from NR as a function of ionic strength. The CR complex sedimented as a 6.9 f 0.2 S (f SD) molecule in low-salt sucrose-glycerol gradients and it had a rapid rate of dissociation with a half-life (T)) of 62 min. When the ionic strength of the CR was raised by addition of KC1 (CR + KCl), a sedimentation value of 4.6 If: 0.4 S and a T) of 112 min were found. These values were identical with those of a KC1 extract of NR. Rapid dialysis to remove the salt from CR+KCl and NR+KCl produced complexes which sedimented as 6.6 + 0.5 S and 5.4 f 0.4 S molecules, respectively. In addition, a significant difference in the T+ values was again noted following dialysis of the two receptors. Interestingly, dialysis of CR did not change the S value but increased ‘It. Conclusion: In the presence of high ionic strength, the CR and NR appear to be identical. However, removal of the salt by rapid dialysis has revealed a clear difference in both their S and Tf values by which they may be distinguished.