Search

It was previously shown that calf uterus cytosol contains a Ca2+-activated receptor transforming factor (RTF) which irreversibly converts the larger molecular states of estrogen receptor (5.3 to 8.6 S, depending on ionic strength) into a smaller, salt-stable form (4.5 S, independent of ionic strength). We now describe a method for rapid and reliable separation of precursor and RTF-transformed receptor forms, which takes advantage of a difference in isoelectric point between the two: the more acidic precursor (isoelectric point, 6.2) is still retained by DEAE-cellulose under conditions (0.12 M KCl, pH 8.3) which produce release from cellulose of the less acidic transformed form (isoelectric point, 6.6 to 6.8). Based on this method of separation, RTF activity can be assayed easily and we could thus progress in the purification and physical and functional characterization of this factor, RTF has been purified about 100-fold. Molecular properties, as assayed by methods suited to partially purified preparations, are as follows: sedimentation coefficient, 6.4 S; Stokes radius, 45 A; molecular weight, 115,000; isoelectric point, 4.9. The Michaelis constant, expressed as moles/liter of estradiol binding sites, is 1.25 X 10(8), at pH 7.5 and 4 degrees, pH 8.5 is optimum for activity. RTF attacks native casein (Km, 1.25 X 10(-5) mol/liter at pH 7.5 and 22 degrees) but not hemoglobin, ovalbumin, or albumin. N-Benzoylarginine methyl ester is a competitive inhibitor of RTF-induced receptor transformation, while L-leucylglycylglycine and N-benzoyltyrosinamide are not. RTF activity is protected by -SH compounds. RTF activity is Ca2+-dependent. Ca2+ starts an activation-inactivation cycle of RTF, with permanent loss of transforming activity which proceeds at a particularly fast rate in the absence of substrate. Mg2+ is inactive, while Sr2+ and Mn2+ may in part substitute for Ca2+. RTF is present in both endometrium and myometrium. RTF is not a lysosomal hydrolase, as shown by its alkaline pH optimum (8.5) and exclusive location in cytosol, nor is it trypsin or a protease of the trypsin group. Also, it is distinct from known proteases of human uterus. The functional significance of this Ca2+-activated protease of cytosol with alkaline pH optimum and high affinity for the larger native form of receptor is still unknown.

The estrogen receptor has been purified to homogeneity from calf uterus cytosol by sequential affinity chromatography by using heparin--Sepharose 4B and 17-hemisuccinyl-17beta-estradiol-ovalbumin--Sepharose 4B. The procedure yields about 1.2 mg of receptor protein from 1 kg of calf uteri, with a recovery of 53%. The receptor protein, as a complex with 17beta-[3H]estradiol, is purified more than 99%. A single band is seen on polyacrylamide gel ectrophoresis under nondenaturing conditions. 17beta-[3H]Estradiol comigrates with the protein band. As computed from the specific activity of radioactive hormone, 64,450 g of purified receptor protein binds 1 mol of 17beta-estradiol. 17beta-[3H]Estradiol bound to the protein is displaced by estrogenic steriods but not by progesterone, testosterone, or cortisone. As judged by chromatography on calibrated Sephadex G-200 columns, the purified receptor is identical with native receptor in crude cytosol: both show a Stokes radius of 6.4 nm. On sucrose gradient in low-salt buffer, the purified receptor sediments at 8 S. On electrophoresis in NaDodSO4 gels, the purified receptor migrates as a single protein band with an apparent molecular weight of 70,000. The sedimentation coefficient measured on sucrose gradients in the presence of chaotropic salts [1 M NaBr or NaSCN (0.1 M)] is 4.2 S. We conclude that the estrogen receptor of cytosol consists of a single subunit weighing about 70,000 daltons and endowed with one estrogen binding site. Under native conditions in cytosol, several subunits associate to form a quaternary structure with a Stokes radius of 6.4 nm.

By means of the DNA-cellulose competitive binding assay, the interaction of estrogen receptor complexed to 17 beta-estradiol with fragments of a cloned DNA region of the estrogen responsive chicken ovalbumin gene spanning from 1343 bps upstream to 373 bps within the transcribed region of the gene (p0V 1.7) was investigated. Only DNA fragments including either the 5'-flanking region from -21 to -140 bps or the region within the gene from +41 to +143 bps showed binding affinity for the estrogen receptor higher than calf thymus DNA. DNA fragments from human alpha 1-globin gene and glucocorticoid responsive murine mammary tumor provirus corresponding to the same DNA region investigated for ovalbumin showed affinity for the estrogen receptor no higher than that of calf thymus DNA. These results suggest that two specific binding sites for estrogen receptor are located upstream and within the ovalbumin gene, near the start-site of transcription. These receptor binding sites overlap with the 'estrogen response element' identified by Dean et al. (1) and the DNase I Hypersensitive region I found by Kaye et al. (2).

A macromolecular component binding 3H-labelled 17 beta-oestradiol in a specific manner and sedimenting in the 8-10-S region on sucrose gradient has been detected in the mammary gland cytosol of ovariectomized adult virgin mice. The dissociation constant of the macromolecule-oestradiol complex is 4.2 times 10(-10)M at 4 degrees C. The binding sites for 17beta-oestradiol of cytosol are 3.7 times 10(-14) mole/mg of protein. Incubation of cytosol with different enzymes suggests that the oestrogen-binding cytosol component is proteinaceous. The binding activity is destroyed by incubation at high temperatures and by some but not all SH-reagents tested. Competition studies show a specificity for oestrogens relative to other steroid hormones. The conclusion is that mammary gland cytosol of virgin mice contains oestradiol receptor. The receptor content does not increase in a specific manner during pregnancy and lactation but rather proportionally to total mammary gland protein.

1. When NaBr, a chaotropic salt, is added, in concentrations ranging from 0.5m to 2m, to low-salt mammary cytosol, (i) age-dependent aggregation of oestrogen receptor is inhibited, (ii) the receptor sediments as a sharp peak at 4.2S on sucrose-gradient centrifugation, with complete disappearance of heavier forms, and (iii) on gel filtration with Sephadex G-200, the receptor is included in the gel matrix. On a calibrated column, the receptor has a Stokes radius of 3.7nm (+/-6%). 2. Because NaBr inhibits interaction of receptor with other components of cytosol, the values of the sedimentation coefficient, measured by sucrose-gradient sedimentation, and of the Stokes radius, measured by gel filtration, can be accepted with confidence. From these values, it can be computed that the oestrogen-receptor form in NaBr has a mol.wt. of 64000, with a frictional ratio of 1.4. 3. Also, inhibition of aggregation by NaBr allows a 30-90-fold purification of oestrogen receptor. Analysis of this partially purified receptor by sucrose-gradient sedimentation and gel filtration in NaBr gives the same results as for receptor in crude cytosol. On electrofocusing on a pH5-8 gradient, the partially purified oestrogen receptor focuses at pH6.2. On removal of NaBr, receptor aggregates even in this partially purified state. It seems likely that at the protein and ionic concentrations of cytoplasm in vivo, the 64000-mol.wt. receptor form is part of higher states of self- and/or hetero-association with other cytoplasmic components. 4. NaBr up to a concentration of 2m does not inhibit binding of oestrogen by receptor, nor does it decrease the affinity of the interaction (K(D) approximately 8.9x10(-10)m). The total number of binding sites in cytosol, however, decreases by approx. 10%, but this decrease may actually be the result of elimination of lower-affinity binding by non-receptor components of cytosol. 5. NaSCN, another chaotropic salt, was also tested but gave less satisfactory results with the mammary cytosol than with uterine cytosol. EDTA was omitted from the buffers because it favours aggregation of mammary oestrogen receptor. KCl (0.4m), sucrose (15%) and ZnSO(4) (3mm) did not prevent aggregation of receptor.

In normal alveolar cells of the mammary gland of C3H/HeJ female mice, DNA synthesis lasts an average of 20.7 hours with a coefficient of variation of 26.6 percent. Treatment of the mice with I microgram of 17-beta-estradiol and 1 milligram of progesterone per day for 3 to 4 days decreases the average duration to 10.7 hours and the coefficient of variation to 13.8 percent.