Estrogen binding proteins of calf uterus. Inhibition of aggregation and dissociation of receptor by chemical perturbation with NaSCN

Sodium thiocyanate up to 0.5 M is compatible with a stable estradiol-t-receptor complex during sucrose gradient centrifugation; however, the maximum permissible concentration in 0.1 M during Sephadex G-100 and G-200 chromatography. When NaSCN 0.1 M is added to low-salt cytosol (approximately 7 mg of protein/ml); (1) age-dependent aggregation of receptor is inhibited; (2) peaks of estrogen-binding activity in sucrose gradients and on Sephadex chromatography are sharp; (3) instead of the usual larger molecular states ("8S") found in low salt, most of estrogen receptor is under the following form: 4.1S; Stokes radius, 36 A; mol wt 61 000; flfo, 1.25; homogeneous at electrofocusing, with isoelectric point at 6.0. When cytosol containing NaSCN 0.1 M is diluted down to 2-3 mg of protein/ml or, only for sucrose gradients, NaSCN concentration is increased to 0.4-0.5 M, the 61000 dalton species decreases, being substituted, without loss of bound estradiol-t, by the following estrogen-binding entity: 28S; Stokes radius, 28 A; mol wt 32 000; flfo, 1.44. In the presence of NaSCN, KCl up to 0.4 M does not affect in a significant manner the molecular properties of the above forms. When NaSCN is dialyzed out, most receptor reverts to a 8-9S state. When cytosol is preincubated with Ca2+ (4 mM) and KCl (0.4 M) before addition of NaSCN, the above picture is modified only in the following aspects: (1) Sephadex chromatography peaks are broader and slightly but reproducibly shifted toward higher elution volumes; (2) the electrofocusing pattern consists of a two-peak heterogeneous band shifted toward higher pH (isoelectric points 6.4 and 6.6); (3) upon dialysis of NaSCN there is little or no reversion to faster sedimenting states. These modifications appear to depend on limited proteolytic attack of receptor by Ca2+ -activated receptor transforming factor (RTF), not on binding of Ca2+ to receptor. Present data suggest that the 4.1S entity is a dimer resulting from side-by-side pairing of 2.8S subunits. Molecular dimension of larger receptor forms purified from cytosol are consistent with the hypothesis that under native conditions in vivo dimers are coupled end-by-end into tetrameric structures with two stronger (between subunits) and two weaker (between dimers) bonding regions, and that tetramers may further self-associate. While NaSCN reversibly releases native dimers and subunits by direct impairment of intersubunit bonds, Ca2+ activated RTF irreversibly and specifically rel