Soluble biospecific macromolecule for purification of estrogen receptor.

Abstract

An alternative to affinity chromotography purification of proteins based on the use of a water-soluble biospecific polymer has been applied to the estrogen receptor from calf uterus. The receptor (4S-sin form) was bound to a dextran-estradiol conjugate (molecuLar weight 500,000) and the complex was isolated by gel filtration. Highly purified receptor was subsequently released from the dextran-estradiol conjugate by exchange with [3Hkestradiol. Biospecific adsorbents, i.e., insoluble materials containing covalently linked radicals capable of binding specific macromolecules (e.g., enzymes, antibodies), have been used extensively for purification of these macromolecules (reviews in refs. 1-3). In comparison with classical methods, affinity chromatography allows for substantially higher purification in a single step and is therefore particularly suitable for biological samples containing very low concentrations of the macromolecule of interest. This is the case with intracellular steroid hormone receptors, which are present in crude target tissue extracts at a concentration about 1/105 that of most other proteins. A biospecific step, therefore, is necessary for their purification (4-6). However, certain difficulties have been encountered due to properties of the insoluble support of the ligand. In particular, such material has a relatively high ion-exchange capacity, which results in considerable retention of nonspecific proteins along with receptor. These proteins subsequently contaminate the receptor preparation at elution. Moreover, the local concentration of ligand residues in the solid support used for affinity chromatography does not depend only on their overall concentration in the column or suspension, but also on the manner in which synthesis was performed. Due to the geometry of distribution of residues within the adsorbent, only a fraction may be accessible for binding to receptor molecules. Because of this, conditions for binding, as well as for release of receptor retained by the affinity adsorbent, must be established empirically.In the present work we describe an alternative to affinity chromatography in which the usual solid support was replaced by a water-soluble macromolecule of dextran (molecular weight -500,000). Estradiol was attached to the dextran molecule at the 7a position via an eight-atom spacer chain. 7a-Estradiol derivatives have proved to be most appropriate for purification of estrogen receptor by solid-support affinity chromatography in previous work (7,8). Binding of receptor to the estrogen residue covalently linked to the dextran macromolecule yielded a complex (molecular weight >500,000) that was easily separated by gel filtration chromatography from other proteins present in the preparation. Receptor was subsequently releasedThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3143by exchange with free [3...

Immunohistologic and histochemical methods for detection of steroid binding in breast cancer: A reappraisal

“…Solid support affinity chromatography using 17-hemisuccinyl-17B-estradiol-ovalbuminsepharose 4B was utilized for the purification of ER (35). Soluble estradiol-dextran polymer derivatives have also been utilized for receptor purification assay (36). Chick oviduct PgR has been shown to bind specifically with 21-hemisuccinyloxyprogesterone-BSA-agarose (37).…”

Ανοσοιστοχημικη Ανιχνευση Υποδοχεων Οιστρογονων Σε Καρκινο Του Μαστου. Συγκριτικη Μελετη

“…Πειράματα κάθαρσης του υποδοχέα (Kuhn, et al, 1975, Hubert, et al, 1978, Sica and Bresciani, 1979) έθεσαν τη βάση για τη σύνθεση ουσιών σημασμένων με φθορισμό και υπεροξειδάση, οι σκοίες χρησιμοποιήθηκαν για τις κυτταροχημικές δεσμευτικές μεθόδους.…”