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. 1981 Nov 1;199(2):371–381. doi: 10.1042/bj1990371

Kinetic and physicochemical characteristics of an endogenous inhibitor to progesterone--receptor binding in rat placental cytosol.

T F Ogle
PMCID: PMC1163380  PMID: 7340809

Abstract

This study describes the kinetic behaviour and physicochemical aspects of an endogenous inhibitor of progesterone--receptor binding in trophoblast cytosol from day-12 embryos. The progesterone cytosol receptor was partially purified and isolated from the inhibitor as the 0--50%-satd. (NH4)2SO4 fraction. The inhibitory substance was shown to reside in the 50--70%-satd. (NH4)2SO4 fraction. Equilibration of the inhibitor preparation with the receptor fraction increased the Kapp.D of the ligand--receptor binding reaction in a concentration-dependent manner (26 +/- 3-fold increase in Kapp.D per mg of protein of the (NH4)2SO4 fraction, n = 16). However, the inhibitor did not alter the concentration of binding sites. Studies of other physicochemical aspects of the inhibitor showed it to be non-diffusible, excluded from Sephadex G-25, stable at 35 degrees C for 30 min, but irreversibly denatured at 70 degrees C for 30 min. The Stokes' radius was estimated by gel chromatography to be 2.8 +/- 0.11 nm (n = 5). Inhibitory activity was destroyed by HgCl2, suggesting that disulphide bridges play an essential role in the biological activity of this molecule. The inhibitor is a macromolecule which does not bind progesterone and differs from albumin. The kinetic mechanism by which the inhibitor enhanced Kapp.D was investigated by measuring association and dissociation rate constants and the energy of activation (Ea) for each reaction. The association rate (k+1) for progesterone and receptor was (1.3 +/- 0.2) x 10(4) M-1 . s-1 but declined to (0.4 +/- 0.1) x 10(4) M-1 . s-1 (n = 5) when exposed to the inhibitor (P less than 0.01). The dissociation rate (k-1) was (3.2 +/- 0.6) x 10(-5) s-1 for progesterone--receptor complex and was unchanged by the inhibitor. The Ea for the association of complex was 33.6 +/- 4.2 kJ/mol and was increased to 63.0 +/- 8.4 kJ/mol by the inhibitor (P less than 0.05). The Ea of dissociation was unaltered. Thus, an inhibitor is present in trophoblast cytosol which specifically enhances Kapp.D without altering availability of binding sites. The mode of action of inhibitor is to increase the energy of activation for association of complex without influencing the dissociation reaction.

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Selected References

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  1. Batra S. Uterine progesterone after progesterone treatment: on the disappearance of cytosolic progesterone receptors. J Steroid Biochem. 1979 Jul;11(1B):407–411. doi: 10.1016/0022-4731(79)90059-1. [DOI] [PubMed] [Google Scholar]
  2. Brown P. R., Edwards J. O. Reaction of disulfides with mercuric ions. Biochemistry. 1969 Mar;8(3):1200–1202. doi: 10.1021/bi00831a054. [DOI] [PubMed] [Google Scholar]
  3. Cornish-Bowden A., Eisenthal R. Estimation of Michaelis constant and maximum velocity from the direct linear plot. Biochim Biophys Acta. 1978 Mar 14;523(1):268–272. doi: 10.1016/0005-2744(78)90030-x. [DOI] [PubMed] [Google Scholar]
  4. Cornish-Bowden A., Eisenthal R. Statistical considerations in the estimation of enzyme kinetic parameters by the direct linear plot andother methods. Biochem J. 1974 Jun;139(3):721–730. doi: 10.1042/bj1390721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Evans R. W., Sholiton L. J., Leavitt W. W. Progesterone receptor in the rat anterior pituitary: effect of estrogen priming and adrenalectomy. Steroids. 1978 Jan;31(1):69–81. doi: 10.1016/0039-128x(78)90020-x. [DOI] [PubMed] [Google Scholar]
  6. Feil P. D., Glasser S. R., Toft D. O., O'Malley B. W. Progesterone binding in the mouse and rat uterus. Endocrinology. 1972 Sep;91(3):738–746. doi: 10.1210/endo-91-3-738. [DOI] [PubMed] [Google Scholar]
  7. Hansen P. E., Johnson A., Schrader W. T., O'Malley B. W. Kinetics of progesterone binding to the chick oviduct receptor protein. J Steroid Biochem. 1976 Oct;7(10):723–732. doi: 10.1016/0022-4731(76)90171-0. [DOI] [PubMed] [Google Scholar]
  8. Hsueh A. J., Peck E. J., Clark J. H. Receptor progesterone complex in the nuclear fraction of the rat uterus: demonstration by 3H-progesterone exchange. Steroids. 1974 Nov;24(5):599–611. doi: 10.1016/0039-128x(74)90014-2. [DOI] [PubMed] [Google Scholar]
  9. Korach K. S., Muldoon T. G. Characterization of the interaction between 17 beta-estradiol and its cytoplasmic receptor in the rat anterior pituitary gland. Biochemistry. 1974 Apr 23;13(9):1932–1938. doi: 10.1021/bi00706a024. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Li S. A., Li J. J. Estrogen-induced progesterone receptor in the Syrian hamster kidney. I. Modulation by antiestrogens and androgens. Endocrinology. 1978 Dec;103(6):2119–2128. doi: 10.1210/endo-103-6-2119. [DOI] [PubMed] [Google Scholar]
  12. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  13. McGuire J. L., Bariso C. D. Isolation and preliminary characterization of a progestogen specific binding macromolecule from the 273,000 g supernatant of rat and rabbit uteri. Endocrinology. 1972 Feb;90(2):496–506. doi: 10.1210/endo-90-2-496. [DOI] [PubMed] [Google Scholar]
  14. Mester J., Seeley D., Catelli M. G., Binart N., Geynet C., Sutherland R., Baulieu E. E. Chicken oviduct nuclear oestrogen receptors: aspects of steroid hormone action. J Steroid Biochem. 1979 Jul;11(1A):307–313. doi: 10.1016/0022-4731(79)90313-3. [DOI] [PubMed] [Google Scholar]
  15. Ogle T. F. Characteristics of high affinity progesterone binding to rat placental cytosol. Endocrinology. 1980 Jun;106(6):1861–1868. doi: 10.1210/endo-106-6-1861. [DOI] [PubMed] [Google Scholar]
  16. Porter W. R., Trager W. F. Improved non-parametric statistical methods for the estimation of Michaelis-Menten kinetic parameters by the direct linear plot. Biochem J. 1977 Feb 1;161(2):293–302. doi: 10.1042/bj1610293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sanborn B. M., Rao B. R., Korenman S. G. Interaction of 17 -estradiol and its specific uterine receptor. Evidence for complex kinetic and equilibrium behavior. Biochemistry. 1971 Dec 21;10(26):4955–4962. doi: 10.1021/bi00802a019. [DOI] [PubMed] [Google Scholar]
  18. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  19. Sprague E. D., Larrabee C. E., Jr, Halsall H. B. Statistical evaluation of alternative models: application to ligand--protein binding. Anal Biochem. 1980 Jan 1;101(1):175–181. doi: 10.1016/0003-2697(80)90057-3. [DOI] [PubMed] [Google Scholar]
  20. Walters M. R., Clark J. H. Cytosol and nuclear compartmentalization of progesterone receptors of the rat uterus. Endocrinology. 1978 Aug;103(2):601–609. doi: 10.1210/endo-103-2-601. [DOI] [PubMed] [Google Scholar]
  21. Walters M. R., Clark J. H. Cytosol progesterone receptors of the rat uterus: assay and receptor characteristics. J Steroid Biochem. 1977 Nov;8(11):1137–1144. doi: 10.1016/0022-4731(77)90064-4. [DOI] [PubMed] [Google Scholar]

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