[Inhibition of the uterine binding of estrogens by unsaturated fatty acids in immature female rats]. / Inhibition de la fixation utérine des aestrogènes par les acides gras insaturés chez la ratte immature.

We demonstrate that the interactions between the uterine cytosol proteins of 24 day old Rats and estradiol-17 beta (E2) are significantly influenced by physiological concentrations of non esterified fatty acids (2. 10(-4)M). We show that the specific binding of the hormone to the 8S uterine receptor, is markedly inhibited (50-80%) by the unsaturated fatty acids. The observed inhibition is a function both of acid dose and acid unsaturation degree. The polyunsaturated arachidonic and docosahexaenoic acids are the strongest inhibitors, whereas the saturated palmitic and stearic acids do not displace the E2 from the 8S receptor.

alpha-Fetoprotein is not a component of the estradiol receptor of the rat uterus.

In high-salt medium, cytosol from immature rat uteri displays two main high-affinity estradiol-binding peaks after ultracentrifugation in a sucrose gradient. The two components are the estradiol receptor which has a sedimentation coefficient of 5.5 S, and the alpha-fetoprotein which sediments at 4.5 S. The dissociation rate constants (k-1) of plasma alpha-fetoprotein-estradiol complexes measured at 0 degrees in the absence or presence of 0.4 M KCl were found to be 7 X 10(-5) and 8 X 10(-5) sec-1, respectively. The half-time of dissociation of these hormone-plasma protein complexes is 100-200 times more rapid than that of the estradiol-receptor complexes. These data led to the use of two "differential dissociation" methods for the measurement of the hormone-binding protein complexes. In a high-salt cytosol, the charcoal technique measured selectively the receptor binding sites; the hydroxylapatite technique measured the sum of the alpha-fetoprotein plus receptor binding sites. Under these conditions, binding specificity studies provided evidence that alpha-fetoprotein is not a subunit of the receptor. This was confirmed by binding specificity studies in high-salt medium of the receptor separated from alpha-fetoprotein by ultracentrifugation.

[Characterization of the "4 S-trypsin" form of the cytosoluble estradiol receptor of calf uterus in a nondenaturing system]. / Caractérisation par électrophorèse en système non dénaturant de la forme "4 S-trypsine" du récepteur cytosoluble de l'oestradiol d'utérus de Veau

The characterisation of the "4 S-trypsin" form of the estradiol-receptor from calf uterus cytosol was carried out by polyacrylamide gel electrophoresis under non-denaturing conditions. In a multiphasic buffer system (upper buffer Tris-glycine pH25degrees C = 8,6, lower buffer Tris HCl pH25degrees C = 7,4), three radioactive estradiol peaks were observed. The first was free estradiol, the second estradiol initially complexed with a macromolecule and dissociated during electrophoresis, and the third the hormone-receptor complex. The sedimentation coefficient (4 S) of this complex was the same before and after electrophoresis. Its mean geometric radius is R = 2.53 + 0.08 nm and its molecular weight was estimated to be 55,000.

Affinity chromatography of the uterine estradiol receptor on estradiol-PAB-cellulose: an artefact.

Estradiol-PAB-cellulose, an easily prepared adsorbent, has been proposed to purify the uterine estradiol receptor according to the principle of biospecific affinity chromatography. It apparently removes all hormone binding sites when cytosol preparations are incubated with it. A systematic study of this adsorbent was undertaken, including the synthesis and testing of the radioactive material. Two main results were obtained: 1) Estradiol-PAB-cellulose is heavily contaminated with free ligand and releases it during the normal chromatographic conditions. 2) Estradiol spacer derivatives (hydroxyethylphenyl-diazo (2 or 4)-estradiol) have a very low affinity for the receptor (Ki = 10 muM). The conclusion is that estradiol-PAB-cellulose is unsuitable for affinity chromatography of estradiol receptor.