A low-affinity estrogen-binding site in pregnant rat uteri: analysis and partial purification

We have identified a low-affinity (type II) estrogen-binding site (EBS) that is expressed at high levels during pregnancy in rat uteri. Although this activity was detectable in nonpregnant rat uteri, it was present in amounts (0.094 pmol/g of uteri) that were severalfold lower than the high-affinity type I estrogen receptor (0.57 pmol/g of uteri). During pregnancy, at 19-20 days of gestation, the low-affinity type II EBS became the major (> or = 88%) estrogen-binding site in rat uteri. The increase in the level of low-affinity EBS (7.9 pmol/g) in uteri was approximately 85-fold with an approximately 20-fold increase in the specific activity (0.39 pmol/mg) of this form, whereas the high-affinity form remained relatively unchanged. We report here a method of purification of type II EBS from pregnant rat uteri and present an analysis of its DNA and steroid-binding properties. Estradiol-binding studies and Scatchard analysis showed that the type II EBS had an apparent estradiol-binding affinity of > or = 24 nM. Gel filtration and SDS/PAGE analysis indicated that the type II EBS was a monomeric 73-kDa protein. The estradiol binding remained apparently uninhibited in the presence of a large excess of tamoxifen, nafoxidine, or dihydrotestosterone. Estradiol, diethylstilbestrol, and quercitin (a type II EBS-specific inhibitor) competed efficiently. The purified low-affinity EBS did not have sequence-specific DNA-binding activity with the estrogen-responsive element, which indicated that it differs in function from the type I estrogen receptor.