Estrogen-binding proteins of calf uterus. Purification to homogeneity of receptor from cytosol by affinity chromatography.

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.     

Properties of the estrogen receptors contained in the MtTF4 tumor whose growth is inhibited by estradiol: 17 beta-estradiol, 17 alpha-estradiol and DNA bindings

The steroid and the DNA bindings of the estrogen receptor of the MtTF4 tumor whose growth is inhibited by estradiol where characterized and compared to those of uterine estrogen receptors. In the tumor cytosol: E protects its binding sites against thermal denaturation, depending on the effects of sodium molybdate upon the dissociation rate of [3H]E at 20 degrees C and the ability of receptor to bind to DNA, the activation (or transformation) process, supposed to be necessary for the full action of estrogen ligand, occurs on estrogen receptor complexes and the calf thymus DNA interacts with estrogen receptor with an affinity similar to that of uterine estrogen receptor. Kinetic and equilibrium studies with 17 alpha-[3H]E both in uterus and tumor indicate that this ligand is fast-associating, fast-dissociating and that its affinity for ER is 2- to 4-fold lower than that of 17 beta-[3H]estradiol one. Competition experiments between 17 beta-[3H]estradiol and the unlabelled 17 alpha epimer reveal, in both uterus and tumor, a time-dependent decrease of the apparent potency of 17 alpha-E to inhibit the binding of [3H]E. It is concluded that the estrogen receptors are very similar in MtTF4 tumor and uterus and the diversity of the response of cell growth to E is due rather to differences at the post-receptor level.     

Structural characterization of the 9-10S estradiol receptor from calf uterus

An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.     

Isolation of untransformed bovine estrogen receptor without molybdate stabilization

A low concentration estrogen-derivatized affinity resin has been used in a rapid, single step purification of the untransformed estrogen receptor from calf uterine cytosols prepared without sodium molybdata. The procedure isolates the Mr 65,000 estrogen receptor in association with the bovine heat shock protein hsp90. Small amounts of proteolyzed receptor ranging in size from Mr 50,000 to 60,000 are also present in the purified extracts. Results from affinity chromatography of receptor cytosols either untreated or presaturated with estradiol suggest that two proteins of Mr 22,000 and 38,000 are co-purified with the untransformed receptor complex and may represent additional nonhormone-binding components of the native receptor form. Some indication of the stability of protein-protein interactions within the oligomeric complex has been derived from differential salt elution studies with heparin-sepharose and affinity gel-immobilized untransformed receptor. On size exclusion high performance liquid chromatography the untransformed complex eluted with a Stokes radius of 75 +/- 2 A (n = 18), but was shown to be sensitive to extended ultracentrifugal analysis dissociating to the receptor homodimer, sedimentation coefficient 5.3 +/- 0.3 s (n = 5). Preliminary data on urea- and heat-induced transformation of the isolated receptor to the DNA-binding state is presented.     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Mitotic apparatus and nucleoli compartmentalization of 50,000-dalton type II regulatory subunit of cAMP-dependent protein kinase in estrogen receptor negative MDA-MB-231 human breast cancer cells

Affinity purified RI and RII antibodies of regulatory subunits (R) of type I (RI) and type II (RII) cAMP-dependent protein kinase were utilized to determine the immunological characterization and specific compartmentalization of R in estrogen receptor negative MDA-MB-231 human breast cancer cells. The 8-azido-(32P)-cAMP binding analysis of MDA-MB-231 cell extracts exhibited 47,000- and 50,000-dalton cAMP receptor proteins. RI and RII antibodies, by immunoprecipitation, detected the 47,000- and 50,000-dalton proteins, respectively. The 47,000-dalton protein was identified as RI as it showed a similar molecular weight as of bovine RI on SDS-polyacrylamide gel electrophoresis. Although 50,000-dalton protein did not co-migrate with bovine heart 54,000-dalton RII, it was identified as RII of MDA-MB-231 cells since it was specifically precipitated with RII antibody but not with RI antibody. An indirect immunofluorescence revealed that during different phases of growth of MDA-MB-231 cells, 50,000-dalton RII was specifically compartmentalized in the mitotic spindle and nucleoli of the cells whereas RI did not exhibit a specific compartmentalization in the cells, but was distributed throughout the cell components. These results suggest specific role(s) of 50,000-dalton RII at the nuclei of MDA-MB-231 cells.     

Purification of Choline Acetyltransferase from the Locust Schistocerca gregaria and Production of Serum Antibodies to This Enzyme

Choline acetyltransferase (ChAT; EC 2.3.1.6) was purified from the heads of Schistocerca gregaria to a final specific activity of 1.61 mumol acetylcholine (ACh) formed min-1 mg-1 protein. The molecular mass of the enzyme as determined by gel filtration is 66,800 daltons. The final enzyme preparation showed one major band at 65,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which corresponds with the native molecular mass of the enzyme, a band at 56,000 daltons, and two bands at 40,500 and 38,000 daltons. Antibodies raised against ChAT in rabbit react only with the active band on native gel after Western blotting. They strongly react with the 65,000-dalton polypeptide band on Western blots of SDS gel separation of pure preparation of enzyme and with both the 65,000- and 56,000-dalton bands after SDS gel separation of crude extract.     

The relative binding affinity of diethylstilbestrol to uterine nuclear estrogen receptor: effect of serum and serum albumin

The relative binding affinity (RBA) of diethylstilbestrol (DES) was determined in nuclear fractions of the rat uterus. DES displayed a two- to threefold greater affinity (RBA = 245 +/- 36) than estradiol (RBA = 100) for nuclear E receptor. The RBA of DES to nuclear E receptor was lowered significantly in the presence of rat serum (43 +/- 1) or human serum (52 +/- 7). Dilution of human serum resulted in a progressive increase in the RBA of DES which approached that observed in the absence of serum. Addition of purified human serum albumin mimicked the decrease in RBA of DES that was observed with serum. The IC50 of estradiol was not changed in the presence of either rat serum or albumin. These data show that DES possesses a greater affinity for nuclear E receptor than estradiol and that serum albumin can modulate DES binding to uterine E receptor.     

Affinity chromatography of estrogen receptors on diethylstilbestrol-agarose

Diethylstilbestrol was coupled to epoxy-activated agarose yielding an affinity resin which is highly efficient for the isolation of estrogen receptors. This resin, diethylstilbestrol-agarose (DES-agarose), bound two proteins (Mr = 50,000 and 65,000) from rabbit uterine cytosol that show a specific interaction with estradiol. A two step procedure--adsorption on DES-agarose followed by a selective elution with p-sec-amylphenol and NaSCN, yielded highly purified estrogen receptors which can be used in the studies of estradiol-receptor interactions with other cell constituents.     

Characterization of monoclonal nonspecific suppressor factor (MNSF) with the use of a monoclonal antibody

The secretion of immunoglobulin (Ig) from cultured mononuclear cells by lipopolysaccharide (LPS) stimulation is inhibited by monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma. In an attempt to develop a murine monoclonal antibody (MAb) with specific reactivity against MNSF, a cell fusion technique that incorporated immune murine splenocytes and HAT-sensitive murine myeloma cells was used. Cross-reactivity experiments confirmed that the MAb (MO6) does not bind to unrelated proteins such as bovine serum albumin, mouse IgG, and murine interferon-gamma (IFN-gamma). There are no effects when anti-IFN-gamma antibodies are used with MNSF. As far as biological activity is concerned, MO6 inhibits in vitro the activity of MNSF in terms of the Ig secretion from cultured lymphocytes. By using MO6, affinity chromatography and immunoblotting were performed. The MNSF on the SDS-PAGE showed a band with m.w. of approximately 70,000, indicating the formation of an aggregate in saline; but after treatment with 0.4 M pyridine-acetic acid buffer, separate bands of 24,000 and 16,000 daltons were evident. Therefore MO6 recognizes 70,000 and both 24,000 and 16,000 daltons. Thus we confirmed by using this MAb and affinity chromatography, the existence of human counterpart, human nonspecific suppressor factor (hNSF), in supernatant from concanavalin A-stimulated T cells. When hNSF was fractionated by high pressure liquid chromatography (HPLC), the activity was found in a region corresponding to 70,000 daltons. However, when fractionated in pyridine-acetic acid buffer, hNSF activity was distributed in a slightly wider range of 15,000 to 30,000 daltons. Physicochemical analysis showed that the purified hNSF is resistant to either heating at 56 degrees C or to 2-mercaptoethanol treatment; however, it is labile to acidification at pH 2.0 and is also sensitive to protease treatment, the characteristics of which were similar to those of murine MNSF. Thus MO6 was confirmed to be a pertinent tool for isolation of hNSF, as well as for murine MNSF.     

Interaction of estrogen receptor of calf uterus with a monoclonal antibody: probing of various molecular forms

A monoclonal antibody to estrogen receptor (JS34/32) is able to recognize, in the calf uterine cytosol, a protein (approximately 65 000 daltons) giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two molecules of this antibody are able to simultaneously interact with the native 8S form of the receptor present in the calf uterine cytosol ("twin antibody" assay). This indicates the presence of two antigenic determinants on the "low-salt" 8S form of the receptor. This form of the receptor shows an increase in Mr from 345 000 to 665 000 after interaction with the soluble antibody. Dissociating agents that induce the dissociation of the 8S form to smaller forms also induce the dissociation of the two antigenic determinants. The 4S "high-salt" form of the estrogen receptor has one determinant per molecule, appearing to be the smallest form of the receptor not containing repetitive structures associated with the steroid binding site. The nuclear receptor also shows the presence of more than one antigenic determinant on its molecule.     

The steroid binding domain of porcine estrogen receptor

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.     

Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.     

Estrogen receptor purification by affinity chromatography using an orange triazine dye

A rapid two-step procedure was devised for the purification of the estrogen receptor from the calf uterus. A 900- to 1700-fold purification of the estrogen receptor was obtained using ammonium sulfate precipitation followed by dye affinity chromatography with Reactive Orange 14 immobilized to Sepharose. The Reactive Orange 14-Sepharose was used to purify the estrogen receptor in the presence or absence of estradiol as well as to purify the progesterone receptor. The purified estrogen receptor retained its estradiol- and DNA-binding properties and sedimented into sucrose gradients as the 5 S receptor dimer. The Reactive Orange 14-Sepharose is easily prepared and offers a higher yield and purity of the estrogen receptor than that afforded by estrogen- or heparin-Sepharose chromatography.     

Localization of ionophore activity in a 20,000-dalton fragment of the adenosine triphosphatase of Sarcoplasmic reticulum.

The (Ca2+ + Mg2+)-dependent ATPase of sarcoplasmic reticulum has been shown to ast as a Ca2+-dependent and selective ionophore in artificial lipid bilayers. Four fragments of 55,000, 45,000, 30,000, and 20,000 daltons have been purified from tryptic digests of the enzyme and it has been shown that the 55,000- and 45,000-dalton fragments are obtained from a single cleavage of the 100,000-dalton ATPase, while the 30,000- and 20,000-dalton fragments are obtained subsequently by a cleavage of the 55,000-dalton fragment. The 55,000- and 20,000-dalton fragments have ionophore activity inhibited by ruthenium red and by mercuric chloride but not by methylmercuric chloride, an inhibitor of the hydrolytic site of the enzyme. Under standard conditions the 45,000-dalton fragment was not active as an ionophore, while the 30,000-dalton fragment acted as a nonselective ionophore. The 55,000- and 30,000-dalton fragments have been shown to contain the site of phosphorylation and of N-ethyl [2-3H]-maleimide binding indicative of the hydrolytic site in the enzyme, and this site is absent from the 20,000-dalton fragment. Therefore, the ionophoric and hydrolytic sites are localized in separate regions of the ATPase molecule and they have now been physically separated. The 20,000-dalton fragment was degraded with cyanogen bromide and fragments were separated by molecular sieving. Ionophore activity was found in fragments of molecular mass less than 2,000 daltons.     

Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.     

Production and characterization of single-chain tissue-type plasminogen activator produced by an established cell line from human uterine muscle

This report describes the purification and characterization of single-chain tissue-type plasminogen activator (sct-PA) present in tissue culture medium of a cell line established from human uterine muscle. The cell line used for the experiment, KW, had estrogen receptor. The PA fraction (KW-PA) was purified from the tissue culture medium of KW employing several steps of affinity chromatography and gel filtration in the presence of aprotinin. The final product (KW-PA) of purification, which predominantly contained the inactive form of sct-PA as well as active sct-PA to a lesser extent, revealed a single band with a molecular weight of 70,000 on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis both in the absence and presence of reducing agent. Electrophoretic enzymography demonstrated a single lytic zone at Mr 70,000. When KW-sct-PA was treated with plasmin, SDS-polyacrylamide gel electrophoresis revealed two bands of Mr 37,000 and 33,000 under reduced conditions. Such plasmin treatment of KW-sct-PA enhanced the enzymatic activity as well as the [3H]DFP incorporation significantly. The KW-sct-PA demonstrated a higher affinity for lysine than did melanoma-t-PA, but the fibrin affinity of KW-sct-PA was identical with that of melanoma-t-PA. Circular dichroism (CD) analysis showed that the CD spectra of KW-sct-PA were different from those of melanoma-t-PA. These results suggest that the single-chain inactive form of t-PA which was obtained from the tissue culture medium of the cell line from human uterine muscle is activated to a two-chain form on plasmin treatment, with an accompanying significant increase in enzymatic activity.     

Further characterization of estrogen binding to rat testis cytosol

Binding of estradiol (E2), estriol (E3), RU16117, and moxestrol to testis cytosol from adult male rats was investigated. High-affinity binding sites were identified in the 8-9S region of sucrose density gradients; a second, high-capacity binding component in the 4S region was probably due to contamination with serum. Thermodynamic properties of the testicular estrogen binding site were quite similar to those of the uterine receptor. E2 had the highest affinity for testicular cytosol binding sites (Ka: E2 much greater than moxestrol greater than E3 greater than RU16117). Comparison of association rate (E2 greater than E3 greater than moxestrol = RU16117) and dissociation rate constants (E3 = RU16117 greater than E2 much greater than moxestrol) as well as studies in vivo revealed moxestrol as a long-acting and RU16117 as a short-acting compound. This difference may be useful for evaluation of the mediation of estrogen effects in the rat testis.     

Cleavage of nascent UDP glucuronosyltransferase from rat liver by dog pancreatic microsomes

Antibody to mouse UDP glucuronosyltransferase, previously shown to cross-react with rat transferase [1], immunoadsorbed 3 electrophoretically distinct transferase forms from the microsomes of untreated and phenobarbital-treated rats and 4 forms from 3-methylcholanthrene treated animals. The forms from phenobarbital-treated or control animals ranged in molecular weights from 49,000 to 52,000 daltons, and those from 3-methylcholanthrene-treated rats ranged from 51,000 to 57,000 daltons. The intensity of the electrophoretic bands indicated that the levels of at least two forms were increased by the administration of either compound.In contrast, only a 52,000-dalton electrophoretic band was observed after immunoadsorption of in vitro translated products using poly(A) RNA isolated from either control, phenobarbital-, or 3-methylcholanthrene-treated rats. When dog pancreatic microsomes were included in the in vitro translation assay for either of the poly(A) RNA preparations, part of the 52,000-dalton band remained and a new band of about 50,000 daltons was generated. This processed transferase form(s) appeared to be inserted into or sequestered by the microsomes. These results indicate that some of the electrophoretic variants of rat liver transferase arise by posttranslational modifications and that at least one rat transferase form undergoes proteolytic cleavage of an approximate 2,000-dalton peptide fragment during insertion into the membrane.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.