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Barbara Attardi 《Molecular and cellular endocrinology》1983,29(2):147-157
We have examined the sedimentation properties of hypothalamic and pituitary estrogen receptors (ER) translocated to the nucleus by in vivo estradiol (E2) or nafoxidine treatments in the immature female rat. Nuclear ER were extracted with 0.4 M KC1 and incubated in vitro with saturating concentrations of [3H]E2under conditions which allowed exchange with endogenous unlabeled ligands (23°C for 1–16 h). Sucrose gradient centrifugation of pituitary extracts from rats treated for 1, 24 or 48 h with E2 yielded two components, sedimenting at 3 and 4–5S; in hypothalamic extracts the 3S form and variable amounts of a heavier shoulder were observed. After nafoxidine treatment the 4–5S species was present in greater proportions than after E2. Both species interacted with monoclonal antibodies prepared against cytoplasmic ER from MCF-7 cells to yield more rapidly sedimenting complexes (7–8S). The 4–5S receptor was preferentially labeled in nuclear extracts subjected to exchange with [3H]E2 for l h in vitro. In addition, nuclear ER from rats treated with [3H]E2for l h in vivo sedimented at about 5S. We also investigated the direct binding of [3H]4-hydroxytamoxifen to nuclear ER. A single 4.2–4.3S component was present in both the hypothalamus and pituitary; this peak was displaced to about 7.5S after incubation with antibodies to MCF-7 ER. In the same nuclear preparations the 3S receptor predominated after exchange with [3H]E2;however, a 1000-fold excess of unlabeled nafoxidine or hydroxytamoxifen abolished the 3S peak. These results suggest that 4–5S nuclear ER may give rise to the 3S form through degradation by endogenous proteases and that binding of antiestrogens to the larger species may interfere with this conversion. 相似文献