Isoelectric heterogeneity of human uterine estrogen binding proteins

Upon Isoelectric Focusing (IEF) of premenopausal uterine myometrial cytosol, specific binding of estradiol (E2) can be shown at elution pH's (EpH) of 4.0-4.4, 5.0-5.2, 5.8-6.2 and 7.5-8.0. Pre-adsorption of premenopausal uterine cytosol by Concanavalin A Sepharose (Con-A) or precipitation with 30% ammonium sulfate results in loss of estradiol binding at EpH's 4.4 and 5.0. The estradiol binding sites that bind to Con-A are present in plasma and have been shown to be Sex Hormone Binding Globulin (EpH = 5.0) and Estrogen Binding Protein (EpH = 4.4). After Con-A adsorption premenopausal cytosol preincubated with 2 nM 3HE2 reveals a single peak on IEF at EpH's congruent to 6.0, while preincubation with 40 nM 3HE2 reveals specific binding peaks at EpH's of congruent to 6.0 and 7.5-8.0. Postmenopausal uterine cytosol preincubated with either 2 or 40 nM 3H-E2 on IEF reveals EpH = 5.8-6.0 binding only. Post-labeling of IEF fractions with 20 nM 3HE2 demonstrates one peak at EpH 5.8-6.0 in postmenopausal tissue and two peaks (5.8-6.2 and 7.5-8.0) in premenopausal tissue. Scatchard analysis of postmenopausal cytosol demonstrates a single population of binding sites with a dissociation constant (Kd) of 10(-10) M. Premenopausal cytosol on Scatchard analysis contains two estradiol binding populations with Kd's of 10(-10) and 10(-9) M. The data suggest that the 10(-10) M E2 binding population has a EpH of 5.8-6.2, while the 10(-9) M component has an EpH of 7.5-8.0.