Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen ( sup 3 H)desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex

Journal Article · Wed Jan 31 23:00:00 EST 1990 · Molecular Endocrinology; (USA)

DOI:https://doi.org/10.1210/mend-4-2-255· OSTI ID:6774854

Hutchens, T W; McNaught, R W; Yip, T T; Suzuki, T; Li, C M; Besch, P K ^[1]

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1. Baylor College of Medicine, TX (USA)

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 (greater than 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 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with (3H)desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl 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 steroid-binding 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.

• OSTI ID: 6774854
• Journal Information: Molecular Endocrinology; (USA), Journal Name: Molecular Endocrinology; (USA) Vol. 4:2; ISSN 0888-8809; ISSN MOENE
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMIDES
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBONIC ACID DERIVATIVES
CATTLE
CENTRIFUGATION
CHROMATOGRAPHY
COMPARATIVE EVALUATIONS
DNA
DOMESTIC ANIMALS
ELECTROPHORESIS
ESTRADIOL
ESTRANES
ESTROGENS
FEMALE GENITALS
HORMONES
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LIGANDS
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PROTEINS
REACTION KINETICS
RECEPTORS
RUMINANTS
SALTS
SEPARATION PROCESSES
STEROID HORMONES
STEROIDS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRACENTRIFUGATION
UREA
UTERUS
VERTEBRATES