Human Breast Tumor Estrogen Receptor: Effects of Molybdate and Electrophoretic Analyses*

Abstract

The largest and smallest discrete forms of the estrogen receptor in human breast tumor cytosol were characterized by competitive steroid binding, ultracentrifugation, gel filtration, and electrophoresis in polyacrylamide gels of several concentrations. Incubation of cytosol with [3H]estradiol and centrifugation in glycerol gradients containing 20 mM Na2MoO4 and 0 or 150 mM KCl revealed a 9-10S form of the receptor. It resembles the molybdate-stabilized complexes in cytosols of other human and rodent, malignant and healthy tissues, and the complex detected in breast tumor cytosol containing leupeptin, a bacterial protease inhibitor. Preservation of receptor integrity during purification and discrimination from serum steroid-binding components are facilitated by inclusion of molybdate in all buffers. Possible mechanisms of action of molybdate include the inhibition of ribonuclease action on RNA-associated receptor forms and protection against specific proteolytic cleavage by stabilization of a phosphate group on the vulnerable residue or a neighboring one. During fractionation of tumor cytosol in the absence of molybdate, the receptor is converted to a mixture of fragments. The smallest that retains the bound steroid, the mero-receptor, resembles the products of endogenous and exogenous protease action on receptors for all classes of steroids in a wide range of tissues. The similarities between both the largest and the smallest known forms of the breast tumor estrogen receptor and corresponding forms of other receptors support the notion of the common architecture of steroid receptors in normal and malignant tissues of diverse origins.

Effects of Temperature, Storage and Sodium Molybdate on the Analysis of Estrogen and Progesterone Receptors in Rabbit Uterine Tissue and Gynecologic Tumor

“…Consistent with other reports [3][4][5]12. 13], we have presented data showing that at least two forms of ERC and PRC exist in patient tumors; the smaller moiety (the 45 form) apparently subsists as a subunit of the holo-receptor (the 85 form).…”

Monoclonal antibodies against the rat liver glucocorticoid receptor.

“…Seven of seven of our polyclonal rabbit antisera tested bound to the latter domain but not to the other two GR domains (12 [3H]TA-GR complex (0.1 pmol) from crude rat liver cytosol was incubated with 250 ,ul of control ascites, Sp 2/0 (x), 30 (wt/vol) glycerol density gradients and centrifuged as described in Fig. 3. bind to the 3.3-nm steroid-and DNA-binding entity (data not shown).…”

Characterization of Estrogen Receptor in Estrogen-Dependent Transplantable Rat Pituitary Tumor MtT/F84.

“…Several investigators have shown that sodium molybdate is a significantly effective stabilizing agent for glucocorticoid Weatherill and Bell, 1982) , progesterone (Bevins and Bashirelahi, 1980;Grody et al, 1980;Nishigori and Toft , 1980;Wolfson et al, 1980;Chen et al ., 1981;Niu et al, 1981;Ogle, 1983), androgen (Gaubert et al, 1980) and estrogen (Shyamala and Leonard, 1980;Miller et al, 1981;Ratajczak et. al., 1981) (Puri et al, 1982;Renoir et al, 1982;Grandics et al, 1984;Atrache et al, 1985;Redeuilh et al, 1987).…”