Purification and characterization of estrogen-binding proteins from calf uterus. 4. Estrogen-binding proteins of calf uterus. Purification to homogeneity of receptor from cytosol by affinity chromatography

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Abstract
The estrogen receptor was purified to homogeneity from calf uterus cytosol by sequential affinity chromatography by using heaprin-Sepharose 4B and 17-hemisuccinyl-17.beta.-estradiol ovalbumin-Sepharose 4B. The procedure yielded about 1.2 mg of receptor protein from 1 kg of calf uteri, with a recovery of 53%. The receptor protein, as a complex with 17.beta.-[3H]estradiol [E2], was purified more than 99%. A single band was seen on polyacrylamide gel electrophoresis under nondenaturing conditions. 17.beta.-[3H]E2 comigrated with the protein band. As computed from the specific activity of radioactive hormone, 64,450 g of purified receptor protein bound 1 mol of 17.beta.-E2. 17.beta.-[3H]E2 bound to the protein was displaced by estrogenic steroids but not by progesterone, testosterone or cortisone. As judged by chromatography on calibrated Sephadex G-200 columns, the purified receptor was identical with native receptor in crude cytosol: both show a Stokes radius of 6.4 nm. On sucrose gradient in low-salt buffer, the purified receptor sedimented at 8 S. On electrophoresis in NaDodSO4 gels, the purified receptor migrated as a single protein band with an apparent MW of 70,000. The sedimentation coefficient measured on sucrose gradients in the presence of chaotropic salts [1 M NaBr or NaSCN (0.1 M)] was 4.2 S. The estrogen receptor of cytosol consists of a single subunit weighing about 70,000 daltons and endowed with 1 estrogen binding site. Under native conditions in cytosol, several subunits associate to form a quaternary structure with a Stokes radius of 6.4 nm.