J. Biol. Chem., Vol. 260, Issue 10, 5936-5941, May, 1985
Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus
V Atrache, T Ratajczak, S Senafi and R Hahnel
The molybdate-stabilized calf uterine estradiol receptor has been purified
to near-homogeneity by a three-step procedure. Initial purification by
heparin-Sepharose chromatography provides a concentrated receptor extract
in 40% yield with a 5-10-fold increase in purity. The inclusion of
molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability
in high salt and allows elution of the oligomeric receptor complex from
heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone
carboxymethyloxime coupled to diaminoethyl
bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a
further 1600-fold. High performance liquid chromatography gives homogeneous
receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis as a polypeptide of Mr approximately 89,000. The purified
molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in
glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a
calculated Mr approximately 290,000. These properties and the
steroid-binding specificity of the purified receptor bear a close
similarity to those found for the 9-10 S receptor in crude cytosol.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
