Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment
The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.
- Research Organization:
- Baylor College of Medicine, Houston, TX
- OSTI ID:
- 6524645
- Journal Information:
- Biochemistry; (United States), Vol. 26:3
- Country of Publication:
- United States
- Language:
- English
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RADIORECEPTOR ASSAY
CALVES
CHROMATOGRAPHY
CONFIGURATION INTERACTION
ION EXCHANGE
LIGANDS
RECEPTORS
TRITIUM COMPOUNDS
UTERUS
ANIMALS
BODY
CATTLE
DOMESTIC ANIMALS
ESTRANES
ESTROGENS
FEMALE GENITALS
HORMONES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
ORGANIC COMPOUNDS
ORGANS
PROTEINS
RUMINANTS
SEPARATION PROCESSES
STEROID HORMONES
STEROIDS
TRACER TECHNIQUES
VERTEBRATES
550601* - Medicine- Unsealed Radionuclides in Diagnostics