Properties of Biospecific Adsorbents, Obtained by Immobilization of Oestradiol 7α‐Derivatives, for Purification of Calf‐Uterine Cytosol Oestradiol Receptor

Abstract

The properties of three types of adsorbents obtained by coupling oestradiol 7a-derivatives to agarose were compared. The adsorbents examined were : oestradiol 7x-decamethylene-agarose, oestradiol 7r.-decamethylene-poly(alanyl-lysine)-a~arose and oestradiol 7a-trimethylene-poly(alanyl1ysine)-agarose. The following results were obtained.(1) All these adsorbents are stable at 0 -C for at 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 maximal receptor binding capacity of an absorbent was developed. The use of these adsorbents for the purification of the trypsin-treated receptor directly from cytosol allowed a 2500-fold purification corresponding to 5 Y! pure protein (assuming one oestradiol binding site per molecule of M , 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 M , 70000).The use of affinity chromatography has been proposed as a necessary step for purification of hormone receptor. We have attempted to find an adsorbent convenient for the purification of oestradiol receptor from calf uterine cytosol. Two different forms of receptor may be characterized by their sedimentation coefficients: 8-S receptor [l] and 4-5-S receptor [2] in low and high salt concentration, respectively. This oestradiol receptor is difficult to purify because of its non-specific and irreversible aggregation with cytosol proteins. Mild trypsin treatment leads to a nonaggregatable form of the receptor [3] with a sedimentation coefficient of 4 S, whatever the ionic conditions [4]. For this reason the 4-S receptor was used for a systematic investigation of the parameters influencing purification by affinity chromatography. This 4-S Abbreviution. Poly(Ala-Lys), poly(oL-alanyl-L-lysine) copolymer. molecular form of the receptor is probably biologically inactive (unable to translocate to nucleus or to bind DNA), but its binding properties to the hormone remain unchanged.New biospecific adsorbents have been previously synthesized and studied in this laboratory [5]. The affinity of the receptor for the adsorbent was found to depend on the position at which the spacer is fixed onto the steroid, and therefore an oestradiol derivative carrying a long spacer chain i.e. decamethylene, ( C H Z )~~, attached at the 7a position was coupled to an insoluble matrix [5]. Such adsorbents offer many advantages in comparison with some previously described [6,7], in particular: (a) great stability due to the absence of ester bonds in the spacer chain, and (b) high degree of selectivity for the receptor since the 7a position of fixation of the spacer chain does not interfere with the high affinity of the receptor for the immobilized ligand.

Immunorecognition of the active form of the oestrogen receptor by using a monoclonal antibody

“…On the other hand, we found only a weak inhibition of the KCl-induced activation when Na2MoO4 was present during preparation of the oestradiol-receptor complex, and only a partial diminution of the a form. This partial inhibitory effect of Na2MoO4 on the high-salt-induced activation was also observed by other authors (Nishigori & Toft, 1980;Redevilh et al, 1981;Mauck et al, 1982;Muller et al, 1983). The different effect of Na2MoO4 on activation induced by temperature or high ionic strength suggests that these processes of activation are not identical.…”

Progesterone Receptor from Chick Oviduct:. Purification of Molybdate-Stabilized Form and Preliminary Characterization

“…The efficiency and the capacity of the gels to bind progesterone receptor were determined according to Redeuilh et al [34]. Cytosol was loaded onto affinity resins packed in columns and fractions were collected as described in Materials and Methods.…”

“…Depending on the preparation, the value of the product p N (i.e. the tangent at the origin of the exponential curve obtained by plotting bound vs loaded receptor [34], varied between 0.92 and 1.00 (five measurements over 6 months). The capacity was calculated to be 0.25 mmol/ml of resin under our experimental conditions.…”