| Abstract: | 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. |
