Estradiol receptor of calf uterus: interactions with heparin-agarose and purification.

Abstract

Heparin attached covalently to agarose beads binds the "native" form of the estradiol receptor with very high affinity. Chondroitin sulfate does not bind to the receptor. When the receptor is corplexed with hormone, the affinity is at least 10 times higher. Only the "native" and not the "nuclear" or the "derived" (i.e., after activation by a calcium-dependent enzyme) forms of the estradiol receptor interact with heparin. The "native" estradiol-receptor complex is purified to homogeneity after chromatography on columns of heparin-agarose, Sephadex G-200, and DEAE-cellulose, followed by two more Sephadex G-200 columns. The purified molecule is a single polypeptide of molecular weight 69,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The sedimentation coefficient on sucrose gradients is 4.3 S, the Stokes radius from gel filtration is 36.5 A, and the isoelectric point is 6.4. The purified [3Hles-tradiol-receptor complex exchanges the radioactive hormone with estradiol or other estrogenic steroids, but not with testosterone, 5a-dihydrotestosterone, or progesterone. Various forms of the estrogen receptor have been identified in uterine tissue: (i) the "native" receptor, localized in the extranuclear space of the cell, which is characterized by the reversible change of its sedimentation coefficient on sucrose gradients from 8 to 4 S when the salt concentration is increased from 0.1 to 0.4 M (1); (ii) the "derived" cytosol receptor, sedimenting at 4 S in low or high salt, which is obtained from the "native" receptor on activation by a Ca2+-dependent enzyme (2); (iii) the "nuclear" receptor, the form that migrates from the cytosol into the nucleus after formation of the hormonereceptor complex (3).Although the "derived" form of the receptor of calf uterus can be purified by affinity chromatography on estradiol adsorbents (4), the propensity of the "native" receptor to form large and irreversible aggregates has until now blunted all attempts at its purification. This paper describes a property which is peculiar to the "native" form of the receptor, its ability to interact specifically with heparin bound covalently to Sepharose. This property permits the facile purification of this form of the receptor, and it may also reflect important, previously unrecognized physiological functions. METHODS 173-[3H]Estradiol at 41 Ci/mmol was from Amersham, and nonradioactive steroids were from Steraloids. Values of molecular weight (Mr) and Stokes radius of standard proteins were from the literature (5, 6). The following buffers were used: Buffer A: Tris-HCI, 20 mM, pH 7.5/dithiothreitol, 1 mM/ EDTA, 0.5 mM; Buffer B: same as A plus MgCl2, 5 mM, and additional salts as specified.Calf uteri were frozen at -20°and processed within 2 weeks from collection. Preparation of cytosol, determination of bound hormone, sucrose gradient centrifugation, electrofocusing, and gel filtration were carried out as described earlier (7). Proteins were assayed with the method of Lowry as modified for samples in which sulfhydry...

Purification of chicken intestinal receptor for 1,25-dihydroxyvitamin D.

“…DNA-cellulose has been used to study and purify an extensive number of nuclear proteins and steroid hormone receptors such as the progesterone A subunit (31), the estrogen receptor (32,33), the glucocorticoid receptor (34), and the androgen receptor (35). In addition, heparin-Sepharose has been used to purify the "native" form of the estrogen receptor (21). Thus, the observation that the 1,25-(OH)2D receptor can be extensively purified by these techniques suggests that this receptor belongs to a class of proteins which bind to, and possibly modify, DNA.…”

The binding of [3H]oestradiol-receptor complexes to calf uterine chromatin

“…The heparinSepharose pellet was resuspended in 5vol. of TED buffer/0.9M-KCl for 60min to extract the oestrogen receptor (Molinari et al, 1977). The extract obtained after centrifugation was treated with (NH4)2SO4 as described above; pellets were stored at -70°C until the day of the experiment.…”

A comparison of heparin agarose and DNA cellulose for the characterization and partial purification of androgen receptors from rat prostate

“…Also the nuclear form of the receptor was bound. A specific interaction of calf uterine oestradiol receptor was observed [3] only for the native cytoplasmic form. In this respect differences might exist between the oestradiol and androgen receptor.…”

“…From recent studies on the interaction of the uterine oestradiol receptor with heparin agarose [3], it appeared that the cytoplasmic, but not the nuclear receptor was strongly adsorbed by heparin agarose and could be purified several fold by selective elution from heparin agarose.…”