Synthesis and characterization of liquid chromatographic columns containing the immobilized ligand binding domain of the estrogen related receptor alpha and estrogen related receptor gamma.
The ligand binding domains of the estrogen related receptors, ERRalpha and ERRgamma were covalently immobilized onto the surface of an aminopropyl silica liquid chromatography stationary phase to create the ERRalpha-silica and ERRgamma-silica columns and onto the surface of open tubular capillaries to create the ERRalpha-OT and ERRgamma-OT columns. The ERR-silica and ERR-OT columns were characterized using frontal chromatographic techniques with diethylstibesterol and the binding affinities, K(d) values, to the immobilized receptors were consistent with the values obtained by a radioligand binding assay. The ERRgamma-silica column was also characterized using non-linear chromatographic techniques using a series of tamoxifen derivatives. The relative K(d) values obtained for the derivatives were consistent with the relative ability of the compounds to inhibit the cellular proliferation of the human-derived T98G glioma cell line, expressed as IC(50) values. The results indicate that the columns containing immobilized ERRalpha and ERRgamma can be created and used to characterize the binding of compounds to the immobilized receptors and that the relative retention of compounds on these columns reflects the magnitude of their inhibitory activity.
Sanghvi
M
M
Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
Moaddel
R
R
Frazier
C
C
Wainer
I W
IW
D001665:Binding Sites; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008024:Ligands; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry*; D000097983:ERRalpha Estrogen-Related Receptor
D008024:Ligands; D011960:Receptors, Estrogen; D012822:Silicon Dioxide; D000097983:ERRalpha Estrogen-Related Receptor; C490917:ESRRG protein, human; C021611:aminopropyl silica gel