UNIQUE MOLECULAR-PROPERTIES OF A UREA-STABLE AND SALT-STABLE DNA-BINDING ESTROGEN-RECEPTOR DIMER COVALENTLY LABELED WITH THE ANTIESTROGEN [H-3] DESMETHYLNAFOXIDINE AZIRIDINE - A COMPARISON WITH THE ESTROGEN-RECEPTOR COMPLEX

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafox-idine aziridine, has been used to investigate the;salt- and temperature-independent formation of;DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or;[3H]desmethylnafoxidine aziridine were quantitatively transformed (>90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 m urea at 0 C. The urea effect was;hormone-dependent and partially reversible. The;transformed receptors were purified (ca 250-fold);by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3;m;urea to;prevent transformation reversal. Scatchard analyses;revealed a single class of high affinity radioligand;binding sites (Kd;= 0.34 mu) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethyl-nafoxidine aziridine was stable as a probable dimer;in 3;m;urea with 0.4;m;KCI and displayed no evidence;of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr;= 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroidbinding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4;and phenyl stationary phases revealed no evidence;of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 m urea. Thus, exposure to;urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer;configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both;electrostatic and hydrophobic or hydrogen bonding;receptor domains are influenced by ligand binding. © 1990 by The Endocrine Society.