Estrogen binding is a noncooperative process in primary rat uterine cells.
Estrogen binding in a primary rat uterine cell culture system did not exhibit positive cooperativity as judged by Hill coefficients. Under defined culture conditions, total specific [3H]estradiol (E2) binding in intact cells attained apparent equilibrium within 3 h after E2 had been varied from 5 to 250 pM. Thereafter, at each concentration of E2 studied, estrogen receptor (ER) levels were constant for up to 6 additional h. We have characterized a single high affinity E2 binding site with a dissociation constant (Kd alpha) of 0.01-0.05 nM E2. The binding capacity of the alpha-site is approximately 5-8 fmol/micrograms DNA, corresponding to approximately 20,000 receptors per cell. In extensive studies, we have seen no evidence for positive cooperative E2 binding. The Hill coefficient was never significantly (95% confidence level) greater than 1. Data are also presented which indicate that [3H]E2 exogenously added to the extracellular medium is in simple equilibrium with the intracellular compartment and the ER. When saturation analyses were conducted using medium that contained BSA, increased concentration of serum protein increased the apparent Kd of E2 for the receptor. No effect was observed on either the shapes of the binding curves or on the calculated Hill coefficients. Furthermore, dose correlations between E2 binding and stimulated response(s) indicated that the ER in these cells was biologically active, which was a major advantage of this culture system over freshly dispersed cells or cytosolic extracts.
Walent
J H
JH
Department of Biochemistry, University of Wisconsin, Madison 53706.
Gorski
J
J
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003198:Computer Simulation; D003513:Cycloheximide / Q000494:pharmacology; D004958:Estradiol / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D011397:Promegestone / Q000378:metabolism; D051381:Rats; D011960:Receptors, Estrogen / Q000378:metabolism; D011980:Receptors, Progesterone / Q000187:drug effects / Q000378:metabolism; D014599:Uterus / Q000378:metabolism*
D046911:Macromolecular Substances; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D004958:Estradiol; D003513:Cycloheximide; D011397:Promegestone