Identification of high affinity estrogen binding sites in calf uterine microsomal membranes.

Abstract

Membrane-associated binding sites with high affinity and specificity for estrogens have been identified in calf uterine microsomes. The binding of 17 beta-[3H]estradiol is specific and saturable at low hormone concentration (2 nM) of high affinity (Kd = 0.5 nM) and sensitive to trypsin and other proteolytic enzymes. Binding of [3H]estradiol to membranes is inhibited by low concentrations of unlabeled 17 beta-estradiol and diethylstilbestrol (50 to 100 pM) while high concentrations of nonestrogenic steroids have little effect. The nondisplaceable binding is low and never exceeds 15% at the half-maximal point of specific binding. The maximum amount of ligand bound per mg of membrane protein is in the range of 0.4 to 1.0 pmol. Specific estradiol binding associated with microsomal fractions varies between 7 to 15% of the total binding sites. Estradiol appears not to be metabolized to any significant extent after binding to uterine membranes. Whereas the affinities of the estrogens tested are similar, the affinity of the antiestrogen, Tamoxifen, for the cytosolic receptor is at least 10 times higher than for the microsomal binding sites. In contrast to rat uterus and ovaries, the microsomal membranes from various nontarget rat tissues do not show any specific binding.