Smith J S, Thomas P
Marine Science Institute, University of Texas at Austin, Port Aransas 78373.
Gen Comp Endocrinol. 1990 Jan;77(1):29-42. doi: 10.1016/0016-6480(90)90203-x.
Estrogen receptors were identified in cytosolic and nuclear extracts of livers of adult female spotted seatrout, Cynoscion nebulosus. A single class of high affinity binding sites was found, Kd = 1.26 +/- 0.55 nM (N = 55) for the cytosolic extract and Kd = 1.96 +/- 0.42 nM (N = 8) for the nuclear extract. The Kd did not differ between males and females or between vitellogenic and nonvitellogenic females. The binding in both the cytosolic and nuclear extracts was specific for estrogens (DES greater than E2 much greater than E1 = E3). Receptor concentrations in cytosolic extracts from late vitellogenic females (14.61 +/- 1.07 pmol/g liver, N = 40) were significantly higher than those from nonvitellogenic females (3.91 +/- 0.73 pmol/g liver, N = 7). The nuclear binding capacity of livers from midvitellogenic females (1.12 +/- 0.45 pmol/g liver, N = 10) was significantly higher than the binding capacity in livers from nonvitellogenic females (0.16 +/- 0.07 pmol/g liver, N = 26), but not that of late vitellogenic females (0.80 +/- 0.09 pmol/g liver, N = 77). The concentration of estradiol-binding sites was greatest in the liver (liver much greater than ovary greater than heart greater than spleen greater than muscle greater than brain). No interference from other steroid-binding proteins was detected using a simple dextran-coated charcoal method to separate bound from free hormone. Approximately 14% of the binding in the cytosolic extract had DNA-binding affinity. Estrogen receptor binding activity was maximally extracted from nuclei with buffer containing 0.6 M KCl. Nuclear receptors eluted from gel filtration columns with an apparent molecular weight of 95 kDa.
