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

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.