Binding characteristics of estrone,estradiol and estriol to the human myometrial estrogen receptor

The human myometrial estrogen receptor in cytosol from pre-menopausal uterine samples has been characterized. At 0° estradiol (K_D 0.38 × 10⁻¹⁰M) has the highest affinity to the receptor followed by estrone (K_D 0.76 × 10⁻¹⁰M) and estriol ((K_D 1.33 × 10⁻¹⁰M). The association rate constant is 2.8 × 10⁵M⁻¹s⁻¹ for estradiol, 2.1 × 10⁵M⁻¹s⁻¹ for estrone and 0.79 × 10⁵M⁻¹s⁻¹ for estriol. The dissociation constants and the association rate constants increase with temperature. The calculated thermodynamic parameters indicate a positive change in entropy for the formation of the estrogen receptor complex.The cytoplasmic estrogen receptor has a sedimentation coefficient of 4 s in low salt sucrose gradients. In buffer containing diisopropylfluorophosphate (DFP) to inhibit proteolytic activity the estrogen receptor complex sediments solely as an 8 s peak if [³H]-estradiol is added to the buffer prior to homogenization and the tissue sample is used immediately after hysterectomy. Estrogen receptor complexes that sediment at 4 s and 8 s are found if [³H]-estradiol is omitted from the homogenization buffer and instead added after the cytosol preparation. Most likely a protease is involved the activity of which is not completely inhibited by DFP.Addition of low concentrations of Cu²⁺ (10 μM) to the cytosol increases the dissociation constant and decreases the estrogen-binding capacity of the receptor. The rate of association is reduced in the presence of 20 μM Cu²⁺. The estrogen receptor complexes do not show any change in their sedimentation profiles in the presence of Cu²⁺.