A High-Affinity Estrogen-Binding Protein in Rat Placental Trophoblast

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Abstract
A high-affinity, low-capacity estradiol-binding molecule (RE) was demonstrated in the basal zone trophoblast (BZT) of the pregnant rat. On day 11 of pregnancy (day 0 = 1st sperm-positive day) RE is present in BZT cytosol, where it has a ka [association constant] of 1.2 .times. 106 M-1 s-1, t1/2 [half-life] = 12.7 min, at 20.degree. C. The kd [dissociation constant], under similar conditions, consists of 2 components, 1.3 .times. 10-4 s-1, t1/2 = 90 min, and 5.9 .times. 10-5 s-1, t1/2 = 196 min. When one uses the faster component, the equilibrium constant, Kd, obtained from kd/ka is 1.1 .times. 10-10 M, in close agreement with that obtained from Scatchard analysis of specific estradiol (E2) binding at 20.degree. C. On day 11 there were .apprx. 12,000 sites/cell in BZT cytosol. Scatchard analysis of nuclear RE on day 11 indicated a Kd of 1.85 .times. 10-10 M and .apprx. 21,000 sites/nucleus, but, in day 15 BZT, nuclear RE was undetectable. Neither cytosol nor nuclei prepared from placental labyrinthine zone (LZT) tissue (fetal placenta) showed evidence of high-affinity, low-capacity E2 binding. Sucrose density gradient analysis on 5-20% linear gradients showed the cytosol RE to be .apprx. 4S whether in high or low-salt conditions. When measured against binding by 3H-labeled E2, the cytosol BTZ RE was competed for strongly (80-90%) by estrone, estriol, diethylstilbestrol and estradiol-17.alpha. at 200 times excess. Nafoxidine-HCl, also at 200.times. excess, competed to .apprx. 50%. Corticosterone, progesterone, testosterone, dehydroepiandrosterone and pregnenolone did not compete. The hormone specificity of nuclear BZT RE was similar to that of the comparable cytosol RE with the exception that nafoxidine did not compete. This was probably due to differences in kinetics, nafoxidine requiring a longer time to reach equilibrium than the other estrogens. The size of the nuclear RE by sucrose density gradient analysis was .apprx. 2S by KCl extraction (which was inefficient) or 4S by trypsin extraction. The BZT of the day-11 rat placenta contains an estrogen-binding molecule with many of the attributes of a true receptor.