A spin-column procedure for estrogen receptor equilibrium and competition binding analysis.
Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17beta-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERalpha was used to validate the protocol. We expressed the full-length ERalpha protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERalpha. At physiological conditions (150 mM salt, 37 degrees C) we determined the 17beta-estradiol Kd for ERalpha to be 281 +/- 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37 degrees C. In homologous competition binding experiments using 17beta-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.
Shantz
J
J
Roche Vitamins Ltd., Human Nutrition & Health, Research and Development, CH-4070 Basel, Switzerland.
Heim
M
M
Kampmann
G
G
Sochocky
N
N
Fuchs
P
P
Schweitzer
C h
Ch
Weber
P
P
Lundstrom
K
K
Hunziker
W
W
Bendik
I
I
D018354:Alphavirus Infections; D001667:Binding, Competitive; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004958:Estradiol / Q000378:metabolism*; D047628:Estrogen Receptor alpha; D006801:Humans; D020128:Inhibitory Concentration 50; D011960:Receptors, Estrogen / Q000032:analysis*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics; D014158:Transcription, Genetic
D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D004958:Estradiol