Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [¹²⁵I]iodoestradiol, [³H]estradiol or [³H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at {difference between}90 mM and {difference between}155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4°C or addition of 10 mM GTP increased the proportion of the {difference between}90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 μM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [¹²⁵I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.