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Unique Molecular Properties of a Urea- and Salt-Stable DNA-Binding Estrogen Receptor Dimer Covalently Labeled with the Antiestrogen [³H] Desmethylnafoxidine Aziridine. A Comparison with the Estrogen- Receptor Complex.

The Reproductive Research Laboratory, Department of Obstetrics and Gynecology and The USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital Houston, Texas 77030

Address requests for reprints to: T. William Hutchens, Department of Pediatrics (CNRC), Baylor College of Medicine, 1100 Bates, Houston, Texas 77030.

Abstract

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [³H]desmethylnafoxidine 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 DNAagarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (K_d = 0.34 nM) unchanged by ureainduced transformation and purification. The DNAbinding receptor form labeled with [³H]desmethylnafoxidine 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; M_r = 136,800) heterogeneity. Sodium dodecyl sulfatepolyacrylamide 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 DNAbinding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.

FOOTNOTES

Supported in part by the U.S. Department of Agriculture, Agricultural Research Service Agreement No. 58-7MNI-6-100. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Received for publication September 8, 1989. Revision received November 10, 1989. Accepted for publication November 28, 1989.