The Molecular Mechanism of the in Vitro 4 S to 5 S Transformation of the Uterine Estrogen Receptor

Abstract

Abstract The rat uterine cytosol contains two estrogen-binding proteins (EBP) referred to as the and EBP. The 5 S EBP was produced by incubating the uterine cytosol-[3H]estradiol mixture at 28° for 30 min to promote the S to 5 S transformation as described by Jensen et al. ((1971) Biochem. Soc. Symp. 32, 133). These two EBP species were characterized in high ionic strength buffers using sucrose gradient centrifugation and Sephadex G-200 gel chromatography. With these analytical methods the sedimentation coefficient, molecular Stokes radii, and molecular weights were determined. In the presence of 0.4 m KCl at pH 7.4 the 4 S EBP has a sedimentation coefficient of 4.2 ± 0.04 S, a molecular Stokes radius of 44.0 ± 0.4 A, and a molecular weight of 76,200. While in the identical buffers the 5 S EBP has a sedimentation coefficient of 5.5 ± 0.02 S, a molecular Stokes radius of 58.5 ± 0.5 A, and a molecular weight of 132,700. In the presence of 0.4 m KCl and 3 m urea at pH 7.4 the 4 S EBP shows a decrease in its sedimentation to 3.6 ± 0.04 S, but an increase in its molecular Stokes radius to 53.8 ± 0.9 A. The estimated molecular weight is 79,900. The 5 S EBP has a sedimentation coefficient of 4.6 ± 0.9 S and a molecular Stokes radius of 70.6 ± 1.0 A. The molecular weight is 133,900. In addition, sucrose gradient centrifugation analyses, in the presence of 0.4 m KCl, 3 m urea at pH 7.4 showed that a moderate fraction (25 to 50%) of the 5 S EBP is dissociated to the 4 S EBP. In the presence of 0.4 m KCl at pH 6.8 the 4 S and 5 S EBPs sediment at 4.7 ± 0.04 and 5.6 ± 0.05 S, respectively. The 5 S EBP also shows a tendency to revert to the 4 S EBP. Consequently, it was observed that a combination of 0.4 m KCl and 3 m urea at pH 6.8 is extremely effective, in contrast to using each of these reagents alone, in dissociating the 5 S EBP to the 4 S EBP without causing a loss of [3H]estradiol binding. The dissociation process is first order with a half-life of approximately 5 hours. These results indicate that the in vitro S to 5 S transformation is an association of the 4 S EBP, having a molecular weight of ∼80,000, with a second component or subunit of ∼50,000 to form the 5 S EBP (∼130,000). Concurrent with the association process, the 4 S and 5 S EBPs are also capable of marked conformational changes that were discerned when the EBPs were compared to the protein standards under identical conditions. The protein standards used were myoglobin, trypsin, ovalbumin, serum albumin, alkaline phosphatase, aldolase, γ-globulin, and ferritin. The conformational changes of the EBPs were shown by their sedimentation coefficients and molecular Stokes radii varying in a reciprocating manner while their molecular weights remained constant.