Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei.
Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.
W
W
de Boer
L
L
Snippe
G
G
Ab
M
M
Gruber
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002642:Chick Embryo; D002645:Chickens; D004958:Estradiol / Q000378:metabolism; D045166:Estradiol Congeners / Q000378:metabolism; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D009994:Osmolar Concentration; D011960:Receptors, Estrogen / Q000378:metabolism*; D014599:Uterus / Q000378:metabolism*
D045166:Estradiol Congeners; D011960:Receptors, Estrogen; D004997:Ethinyl Estradiol; D004958:Estradiol; C011310:moxestrol