Endocrinology, Vol 118, 2276-2283, Copyright © 1986 by Endocrine Society

ARTICLES

Dissimilarities between the uterine estrogen receptor in cytosol of castrated and estradiol-treated rats

JC Faye, A Fargin and F Bayard

The estrogen receptor (ER) in its native state appears oligomeric and can be dissociated by salt into a monomer with a mol wt of 50,000- 80,000. Lower molecular weight fragments have also been observed but are considered to result from ER proteolysis by tissue proteases. Three- month-old Sprague-Dawley rats have been studied after castration, estradiol treatment (3 days at 25 micrograms/day), or during the normal estrus cycle. The uterine cytosol was labeled with [3H]estradiol, and the [3H]estradiol-receptor complexes were studied by sucrose gradient centrifugation, gel filtration, diethylaminoethyl (DEAE)-Trisacryl and phosphocellulose chromatography, and kinetic experiments. In low salt buffer in presence of molybdate (20 mM), uterine ER of castrated and treated rats could not be differentiated by sucrose gradient centrifugation (9.9S), gel filtration (7.7 nm), or DEAE-Trisacryl and phosphocellulose chromatography, but the dissociation rate at 23 C was lower for castrated than for treated rats (t1/2 = 45 and 27 min, respectively). In high salt buffer (0.4 M KCl) in presence of molybdate (20 mM), no difference was apparent by sucrose gradient centrifugation (3.7S) or gel filtration (3.4 nm), but again the dissociation rate at 23 C was lower for castrated than for treated rats (t1/2 = 102 and 45 min, respectively). In high salt buffer in presence of molybdate and ammonium thiocyanate (0.5 M), differences were observed by sucrose gradient centrifugation (3.6 and 3.2S), gel filtration (3.2 and 2.6 nm), and dissociation rate assays at 10 C (t1/2 = 24 and 12 min) for castrated and treated rats. The calculated wts were 50,000 and 35,000, respectively. Protection from limited proteolysis by molybdate, contained in all buffers used, plus various protease inhibitors, particularly leupeptin, as well as mixing tissues before homogenization, suggested that the 35,000-dalton monomer was not a product of protease action formed after cell breakage. ERs covalently labeled with [3H]tamoxifen aziridine also showed different entities of 62,000 and 47,000 mol wts, respectively, on denaturing polyacrylamide gels. In vitro activation and transformation were demonstrated for the two types of [3H]estradiol-receptor complexes by sucrose gradient centrifugation analysis and affinity for phosphocellulose and purified nuclei. Finally, it has been shown that both entities coexisted in different proportions during the diestrous (1:2) and proestrous (1:1) phases of the estrus cycle; only the low mol wt component was present during the estrous phase.

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