Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1970 Aug;118(5):695–701. doi: 10.1042/bj1180695

The use of protamine to study [6,7-3H]-oestradiol-17β binding in rat uterus

A W Steggles 1, R J B King 1
PMCID: PMC1179277  PMID: 4248960

Abstract

1. The binding of [6,7-3H]oestradiol-17β to uteri has been studied by using sucrose-gradient analysis and also the property of oestradiol receptors to form insoluble complexes with protamine. 2. Protamine precipitates the 8S and part of the 4S oestradiol-binding proteins in uterine cytoplasm from mature rats. It does not precipitate the oestradiol-17β-binding proteins present in cytoplasm from non-target tissues or serum. No tritium-labelled material was precipitated by protamine after equilibration of [6,7-3H]oestradiol-17β with either serum albumin or phosvitin. 3. Protamine precipitated a small amount of progesterone but not testosterone or cortisol that had been equilibrated with uterine cytoplasm. It did not precipitate any tritium radioactivity from muscle cytoplasm that had been equilibrated with either [1,2-3H]testosterone sulphate or [1,2-3H]dehydroepiandrosterone. 4. A simple method has been devised for measuring binding constants of tissue extracts for [6,7-3H]oestradiol-17β, based on precipitation with protamine. Reasonable agreement was obtained between the values obtained by this method and those obtained by sucrose-gradient analysis. 5. This method has been used to study the effect of maturity, ovariectomy, adrenalectomy and hypophysectomy on the cytoplasmic binding of [6,7-3H]oestradiol-17β. None of these procedures affected the dissociation constant Kd or the number of binding sites/mg of cytoplasmic protein. When measured per uterus or per mg of DNA, ovariectomy and hypophysectomy decreased the number of binding sites. Adrenalectomy had no effect. 6. The properties of the 4S oestradiol-binding protein present in cytoplasm from mature uteri have been studied. It is not present in uteri from immature, ovariectomized, or hypophysectomized rats and it does not bind testosterone or cortisol. Unlabelled oestradiol-17β, U-11,100A, N-ethylmaleimide and N-bromosuccinimide all decrease the binding of [6,7-3H]oestradiol-17β to both 8S and 4S receptors. Binding to both 8S and 4S receptors decreases when oestradiol is transported to the nucleus. The 4S receptor is not the same as the 4S binding component formed by salt dissociation of the 8S receptor.

Full text

PDF
695

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cathro D. M. The effect of clomiphene on the androgen production of pubertal and prepubertal boys. J Endocrinol. 1969 Sep;45(1 Suppl):xxii–xxii. doi: 10.1530/acta.0.061s204. [DOI] [PubMed] [Google Scholar]
  3. King J. B., Gordon J. The localization of [6,7-3H]oestradiol-17-beta in rat uterus. J Endocrinol. 1966 Apr;34(4):431–437. doi: 10.1677/joe.0.0340431. [DOI] [PubMed] [Google Scholar]
  4. King R. J., Gordon J., Steggles A. W. The properties of a nuclear acidic protein fraction that binds [6,7-3H]oestradiol-17beta. Biochem J. 1969 Sep;114(3):649–657. doi: 10.1042/bj1140649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Korenman S. G., Perrin L. E., McCallum T. P. A radio-ligand binding assay system for estradiol measurement in human plasma. J Clin Endocrinol Metab. 1969 Jul;29(7):879–883. doi: 10.1210/jcem-29-7-879. [DOI] [PubMed] [Google Scholar]
  6. Korner A. Anabolic action of growth hormone. Ann N Y Acad Sci. 1968 Feb 5;148(2):408–418. doi: 10.1111/j.1749-6632.1968.tb20366.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Peterson R. P., Spaziani E. Cycloheximide and cortisol inhibiton of estradiol-stimulated uterine uptake and distribution of homologous serum albumin and alpha globulin in the rat. Endocrinology. 1969 Nov;85(5):932–940. doi: 10.1210/endo-85-5-932. [DOI] [PubMed] [Google Scholar]
  9. Rosner W., Deakins S. M. Testosterone-binding globulins in human plasma: studies on sex distribution and specificity. J Clin Invest. 1968 Sep;47(9):2109–2116. doi: 10.1172/JCI105896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sombre E. R., Puca G. A., Jensen E. V. Purification of an oestrophilic protein from calf uterus. Biochem J. 1969 Dec;115(5):47P–47P. doi: 10.1042/bj1150047pb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Toft D., Gorski J. A receptor molecule for estrogens: isolation from the rat uterus and preliminary characterization. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1574–1581. doi: 10.1073/pnas.55.6.1574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Toft D., Shyamala G., Gorski J. A receptor molecule for estrogens: studies using a cell-free system. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1740–1743. doi: 10.1073/pnas.57.6.1740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wool I. G., Stirewalt W. S., Kurihara K., Low R. B., Bailey P., Oyer D. Mode of action of insulin in the regulation of protein biosynthesis in muscle. Recent Prog Horm Res. 1968;24:139–213. doi: 10.1016/b978-1-4831-9827-9.50010-1. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES