Properties of biospecific adsorbents, obtained by immobilization of oestradiol 7 alpha-derivatives, for purification of calf-uterine cytosol oestradiol receptor.
The properties of three types of adsorbents obtained by coupling oestradiol 7 alpha-derivatives to agarose were compared. The adsorbents examined were: oestradiol 7 alpha-decamethylene-agarose, oestradiol 7 alpha-decamethylene-poly(anayl-lysine)-agarose and oestradiol 7 alpha-trimethylene-poly(alanyl-lysine)-agarose. The following results were obtained. (1) All these adsorbents are stable at 0 degrees C for a least a year when stored in water. In the presence of cytosol they are stable for several hours and are reusable after a simple wash. (2) A new method allowing the calculation of the maximala receptor binding capacity of an absorbent was developed. (3) The geometry of the column and the dynamics of the loading have no influence on the binding capacity of the adsorbents. (4) Binding of the cytosol receptor to the adsorbent depends on whether the receptor had previously been partially purified by heparin-Ultrogel chromatography or treated with low or high salt concentration or trypsin. It was demonstrated that aggregation decreases the binding of the receptor to the adsorbents. (5) A satisfactory recovery of receptor upon elution is possible only with biospecific adsorbents containing low concentrations of coupled steroid (less than or equal to 0.2 mg/ml). The use of these adsorbents for the purification of the trypsin-treated receptor directly from cytosol allowed a 2500-fold purification corresponding to 5% pure protein (assuming one oestradiol binding site per molecule of Mr 60000). When starting from a low salt preparation containing the native 8-S receptor, partially purified by heparin-Ultrogel chromatography, preliminary experiments using affinity chromatography gave a further purification of 250--500-fold and led to a 50--90% pure protein (assuming one oestradiol binding site per molecule of Mr 70000).
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D011134:Polysaccharides / Q000494:pharmacology*; D011960:Receptors, Estrogen / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives / Q000494:pharmacology*; D014599:Uterus
D011134:Polysaccharides; D011960:Receptors, Estrogen; C026319:estradiol 7 alpha-decamethylene-poly(alanyllysine)-sepharose; C026318:estradiol 7 alpha-decamethylene-sepharose; C026320:estradiol 7 alpha-trimethylene-poly(alanyllysine)-sepharose; D004958:Estradiol; D012685:Sepharose