Low Salt Sucrose Gradients Research Articles

A role for estrogen receptor beta in the regulation of growth of the ventral prostate.

In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor beta (ERbeta). In rat ventral prostate, 530- and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ERbeta does not contain the same protein chaperones that are associated with ERalpha. Estradiol (E(2)) binding and ERbeta immunoreactivity coincide on the gradient, with no indication of ERalpha. In prostates from mice in which the ERbeta gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigen Ki-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells express Ki-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5alpha-androstane-3beta,17beta-diol (3betaAdiol). This compound, which competes with E(2) for binding to ERbeta and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ERbeta knockout (BERKO) mice. Thus ERbeta, probably as a complex with 3betaAdiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ERbeta may be useful in the prevention and/or clinical management of prostatic hyperplasia and neoplasia.

The two native estrogen receptor forms of 8S and 4S present in cytosol from human uterine tissues display opposite reactivities with the antiestrogen tamoxifen aziridine and the estrogen responsive element

We have investigated the capability of the different native ER forms, present in cytosols from human uterine tissues, of reacting with the antiestrogen [ 3H]Tamoxifen aziridine ([ 3H]TA) and with the Estrogen Responsive Element (ERE). Cytosols from uterine leiomyoma (myoma) prepared in buffer containing 40 mM molybdate and protease inhibitors, labelled with [ 3H]estradiol and analyzed in low-salt sucrose gradient showed 8S and 4S ER forms. The same cytosols labelled with [ 3H]TA only showed a 4S ER form, whereas the ERE only reacted with fractions from the 8S peak. The band of ERE reaction in the EMSA assay showed a lower relative mobility than the band labelled with [ 3H]TA, but both bands contained immunoreactive ER of 65 kDa. Electrophoretic mobility of the [ 3H]TA-labelled band in that system was not affected by cytosol treatment with cross-linkers or SDS, which suggests that it is a monomeric protein. The [ 3H]TA-binding 4S ER form was found in all studied myoma samples, as well as in human endometrium or myometrium, but not in rat tissues. These results suggest that the 8S and 4S ER form were already present before cytosol from human uterine tissues comes into contact with the molybdate buffer. They both contain the same ER molecule of 65 kDa, either in the free form or as an oligomer. Only the ER dimers, which have been described both in the cytosolic 8S form and in the nuclear 4-5S form, react with the ERE. [ 3H]TA only binds to the 4S ER monomer probably because its binding site is concealed in the 8S form under these experimental conditions. The opposite reactivity of the 8S and 4S ER forms with [ 3H]TA and the ERE support the hypothesis that they may constitute separate entities with a different physiological role.

Transformation of the estrogen receptor detected by two monoclonal antibodies

The estrogen receptor from fetal guinea-pig uterus is recognised by two monoclonal antibodies (H222 and H226) developed against the human estrogen receptor but it interacts differently with each of them. The H222 antibody, whose epitope is located in the hormone-binding domain of the receptor, shifts the sedimentation coefficient of the nonactivated oligomeric receptor in low salt sucrose gradients from 9S to 11S. When this oligomeric receptor-H222 complex is centrifuged in high salt gradients, it dissociates to an 8S monomer-H222 complex, indicating that all the estradiol-binding units present in the nonactivated receptor can bind the H222 antibody. In contrast, the H226 antibody, whose epitope is located close to the DNA-binding domain, shifts the sedimentation coefficient of the nonactivated receptor only to 9.4S and when this complex sediments in high salt gradients, it dissociates to a 7S monomer-H226 complex plus a 4.5S monomeric receptor not bound to the antibody. This observation suggests that not all the H226 epitopes are accessible in the nonactivated receptor. On the other hand, the temperature-activated receptor reacts with the H226 antibody to form two complexes sedimenting at 7S and 9S in high salt gradients. This 9S complex indicates the formation of a homodimer that binds two molecules of the H226 antibody. However, only one H222 epitope seems to be accessible in this dimeric form of the receptor, since only one 8S complex is observed when the activated receptor reacts with the H222 antibody. In addition, binding to the H222 antibody before activation prevents the dimerisation. This suggests that the H222 epitope is near or directly involved in the dimerisation domain. Interaction of the H222 and H226 antibodies with the estrogen receptor reveals modifications of its structure during activation, and consequently of the exposure of its functional domains.

Analysis of the disruptive action of an epididymal protease and the stabilizing influence of molybdate on nondenatured and denatured glucocorticoid receptor.

The nucleomyofibrillar fraction of epididymides from sexually mature rabbits contains a novel leupeptin-sensitive protease that disrupts the oligomeric conformation of cytosolic estrogen and progesterone receptors, and molybdate inhibits this process. In this report we used the AtT-20 cell glucocorticoid receptor as substrate and performed analyses under nondenaturing vs. denaturing conditions to further investigate the effects of the epididymal protease and molybdate on steroid receptor structure. Analysis on low salt sucrose gradients indicated that the protease partially converted the oligomeric (9-10S) glucocorticoid receptor to several more slowly sedimenting forms (3-7S), and this effect was not observed in the presence of molybdate. Paradoxically, gradient analysis under high salt conditions revealed that the protease induced a discrete, quantitative and molybdate-insensitive conversion of the 4-5S steroid-binding subunit to a 3S form. Further studies were done using denaturing polyacrylamide gel electrophoretic analysis of receptor that had been labeled covalently with [3H]dexamethasone 21-mesylate and partially purified by DNA/cellulose chromatography. At 0-4 C, the protease cleaved the steroid-binding subunit (mol wt, 96,900) of the receptor to a single steroid-labeled fragment (mol wt, 42,600). Under these conditions, digestion was complete within 30-60 min and was inhibited by leupeptin, but was unaffected by thiol-reactive reagents or molybdate. The epididymal protease and alpha-chymotrypsin produced steroid-labeled receptor fragments that were indistinguishable in size, shared an epitope recognized by our BuGR-2 monoclonal antibody, and retained DNA-binding activity. Despite the apparent similarity of these two enzymes, they are distinct, since the chymotrypsin-dependent cleavage event was not inhibited by leupeptin. These studies show that the epididymal protease attacks a site on the steroid-binding subunit of glucocorticoid receptors as well as estrogen and progestin receptors. It also appears that the cleavage site is situated close to that most readily attacked by alpha-chymotrypsin. Finally, our data provide independent confirmation of a recent report indicating that molybdate ions interact directly with the cytosolic steroid receptor to stabilize its oligomeric structure even after proteolysis within the steroid-binding subunit.

Characterization of molybdate-stabilized estrogen receptors by hydrophobic interaction HPLC: resolution of two 8S subunits.

This report describes the separation and characterization of estrogen receptors (ER) according to their degree of hydrophobicity and surface charges. Molybdate-stabilized [3H]ER from rabbit uterine cytosol was sequentially purified by passage through a size-exclusion pre-column, an anion-exchange column, and a hydrophobic interaction column. With fresh cytosol, a major radioactive peak was eluted from the DEAE columns; a major peak and a minor, less hydrophobic, peak were eluted from the hydrophobic column. In contrast, ER from frozen cytosol showed one peak in the DEAE-column and exhibited four radioactive peaks in the hydrophobic column. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, [3H] tamoxifen aziridine(TA)-labelled ER showed radioactive bands at 62 and 48 kd. The subunits which were characterized by these radioactive bands were successfully separated by the hydrophobic column; the more hydrophobic subunit corresponded to the 62 kd band. The HPLC-purified [3H]TA-labelled ER subunits sediment at a 7.4-8.5S region in a low-salt sucrose gradient. These results show that differential negative surface charge and hydrophobic areas exist in the holo-receptor and its subunits, and the hydrophobic interaction HPLC column separates the two major 8S steroid binding subunits of ER.

Biochemistry and pathogenicity of echovirus 9 II. Mutants of echovirus 9, strain Hill, with altered capsid surface properties

The two echovirus 9 strains Hill and Barty have been shown previously not only to differ in pathogenicity for newborn mice but also in a number of in vitro characteristics which depend on viral capsid structure. Three spontaneously occurring mutants of the mouse-apathogenic echovirus 9 prototype strain Hill being resistant to an inhibitor of plaque formation present in agar were isolated and compared biochemically and biophysically to their parent strain and to the mouse-pathogenic echovirus 9 strain Barty, which is resistant to this inhibitor. The mutants differ from their apathogenic parent strain Hill in most of the same in vitro characteristics as strain Barty, namely adsorption to cells in culture, sedimentation behavior in low salt sucrose gradients, distribution of mutant virus particles in isoelectric focusing, and antigenic determinants inducing neutralizing antibodies. For two of the three mutants, Ag2 and Ag3, evidence was obtained from fingerprinting that they differ from their parent strain Hill in VP1; thus, the observed in vitro properties may be caused by the change in this capsid protein. All mutants, however, were found to be apathogenic for newborn mice and do not replicate in the tissues of these animals. It is concluded that the observed changes in capsid structure do not covary with virulence.

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex.

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.

Physicochemical and Functional Properties of Chicken Breast Muscle Proteins

Dexamethasone-receptor complexes can be extracted from HeLa cell nuclei by mild sonication. These complexes can account for 800–1000 binding sites/cell, and are indistinguishable from salt-extractable ones as judged by sucrose gradient centrifugation in the presence of 0.3 M KCl, showing a sedimentation coefficient of 3.6 S. These complexes, however, broadly sediment in the 7 to 3.6 S region of low salt sucrose gradients. Enzymatic treatment of soluble extracts from nuclear sonicates shows that RNA is associated to dexamethasone-receptor complexes.

RNase a effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25°C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment of HeLa cell cytosol at 2°C results in receptor complexes which sediment at 6.9S in low salt gradients. RNase A treatment at 25°C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2°C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25°C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25°C, but not at 2°C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating.Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25°C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2°C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25°C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A.These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.

Steroid receptor status of human testicular tumors.

Biopsies of 9 testicular tumors were examined for the presence of cytoplasmic receptor proteins for estrogens, progestins, androgens, and glucocorticoids. Using an arbitrary threshold value of 10 fmol/mg cytosol protein for a postive assay, only 1 of 9 tumors was estrogen-receptor positive. However, no receptor-specific 8S binding could be detected by low salt sucrose gradient centrifugation. 1 tumor was progesterone-receptor positive. No androgen receptors could be demonstrated. In contrast, 8 of 9 neoplasms contained significant quantities of glucocorticoid receptors exclusively sedimenting at 8S. Our findings suggest that androgens, estrogens, and progestins are unlikely to play a major role in the natural history of testicular tumors. The presence of glucocorticoid receptors might offer a chance for endocrine manipulation of the development and growth of these neoplasms by glucocorticoids.

Steroid receptor status of focal-nodular hyperplasia of the human liver.

As with normal human liver, both focal nodular hyperplasia (FNH) of the liver and a hepatocellular carcinoma were found to contain low levels of high affinity estrogen-binding components. In low-salt sucrose gradients the estrogen binders sedimented at 4S and 8S. Specificity studies indicated a requirement for estrogenic hormones. Progestin-binding studies using labeled and unlabeled ORG 2058 revealed high levels of specific binding in FNH and normal liver cytosol, whereas only insignificant amounts of ORG 2058 binding was found in the hepatocellular carcinoma. As judged by the high apparent dissociation constant (4-14 X 10(-8) mol/l) of the ORG 2058 - binder complex, the apparent lack of 8S binding in low-salt sucrose gradients and the atypical ligand specificity, the progestin receptor nature of these binding entities is unlikely. In contrast, normal liver, FNH, and the hepatocellular carcinoma were found to contain significant quantities of glucocorticoid receptors.

Estrogen and progesterone receptors in human uterus and oviduct.

The binding of 3H-estradiol and 3H-R5020 (dimethyl-19-norpregna-4,9-diene-3,20-dione) to estrogen (ER) and progesterone (PR) receptors in human uterus and oviduct were studied. The binding of the ligands to their respective receptors were of high specificity and affinity. The equilibrium dissociation constants for ER and PR were 0.07 +/- 0.025 nM respective 0.76 +/- 0.22 nM in the oviduct and 0.065 +/- 0.015 nM respective 0.82 +/- 0.25 nM in the uterus (mean +/- SE) (n = 12). Sucrose density gradient centrifugation revealed in both tissues specific binding in the area sedimenting at 8 S whereas the slower sedimenting 4 S and 5 S peaks contained both specific and non-specific binding. The oviductal cytosol contained a similar proteolytical activity that has been described in human uterine and breast cancer tissue. The protease, which can be inhibited by diisopropylfluorophosphate, converts the 8 S receptor from into a 4.2 S proteolytic fragment. The KCl-extracted uterine and oviductal estrogen and progesterone receptors sedimented at 5.2 S in both low salt (0.01 M KCl) and high salt (0.4 M KCl) sucrose gradients. It is well known that the suppression of the estrogen receptor system by progesterone correlates with suppressed function in the oviduct but with increased growth and secretory activity in the uterus. In the light of the present data it is concluded, that the difference in the hormonal response in the two tissues is most likely not due to differences in the binding characteristics of the estrogen or progesterone receptors.

RNase-sensitive glucocorticoid-receptor complexes from HeLa cell nuclei

Dexamethasone-receptor complexes can be extracted from HeLa cell nuclei by mild sonication. These complexes can account for 800–1000 binding sites/cell, and are indistinguishable from salt-extractable ones as judged by sucrose gradient centrifugation in the presence of 0.3 M KCl, showing a sedimentation coefficient of 3.6 S. These complexes, however, broadly sediment in the 7 to 3.6 S region of low salt sucrose gradients. Enzymatic treatment of soluble extracts from nuclear sonicates shows that RNA is associated to dexamethasone-receptor complexes.

The effects of ribonuclease on rat liver dexamethasone receptor: increased affinity for deoxyribonucleic acid and altered sedimentation profile.

The ability of the dexamethasone (9 alpha-fluoro-11 beta, 17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3,20-dione)-receptor complex to bind to DNA-cellulose is stimulated by RNase treatment of the activated receptor. Both RNase A and RNase T1 can induce the stimulation. The enhancement of the DNA binding ability occurs concomitantly with an alteration of the sedimentation profile of the dexamethasone-receptor complex from the 7-8S form to the 3-4S form in low salt sucrose gradients. If RNase treatment occurs in the presence of sodium molybdate, both the increase in DNA binding ability and the alteration in sedimentation profile fail to occur. Treatment of the receptor with high salt suggests that the 3-4S form can reversibly combine with a factor in a salt-sensitive association. These experiments indicate that the 7-8S form of the dexamethasone-receptor complex is associated with a RNA molecule(s) that can be removed by RNase treatment or salt dissociation, and that this RNA inhibits the binding of the receptor to DNA.

Sulfhydryl sensitivity and [ 125I]-16α-iodo-17β-estradiol binding of estrogen receptor in ovarian epithelial carcinomas

We find that estrophilin from either the cytosolic or nuclear fractions of ovarian epithelial carcinomas (OVCA) binds irreversibly to controlled-pore glass beads (CPG). The CPG-adsorbed estrophilin releases [ 3H]-estradiol at 4°C in the presence of 1.25 mM AgNO 3; estradiol binding capacity from the nuclear fraction is restored by 10 mM dithiothreitol. The number of available estradiol binding sites in cytosol that are sensitive to AgNO 3 correlate with (a) the number of estradiol-inhibitable binding sites found in the 8S region in low-salt sucrose gradients ( r = 0.9) but not with (b) estradiol-inhibitable binding in the 4S region ( r = 0.3) and thus, as expected, only poorly with (c) estimates of available cytoplasmic estrogen binding sites using a standard dextran-coated charcoal analysis ( r = 0.7). Total estrogen binding extracted from the nuclear fraction with 0.5 M KCl and adsorbed to hydroxylapatite agreed with the sulfhydryl blocking reagent sensitive moiety ( r = 0.9). Estrophilin from OVCA cytosol or nuclear fractions bound [ 125I]-16α-iodo-estradiol indistinguishably from [ 3H]-estradiol. The two forms of radiolabelled-estradiol yielded equivalent data that demonstrated a shift in the estrogen binding moiety from the 8S region to the 4–5S region when 0.5 M KCl was added to gradient analyses of OVCA cytosols. From these observations we conclude that the estrophilin found in OVCA is similar to that found in normal and cancerous tissues of other female reproductive organs and that this estrophilin can bind a biologically active radiohalogenated estrogen potentially useful for imaging or treating these tumors.

Interaction of chick oviduct progesterone receptor with sodium tungstate: Inhibition of activation and DNA binding

The effects of sodium tungstate on the chick oviduct progesterone receptor were examined and found to be pH-dependent. When freshly prepared cytosol receptor preparations were activated by incubation at 23°C in presence of different concentrations of sodium tungstate, receptor activation was completely blocked by 0.2 and 2 mM inhibitor at pH 7 and pH 8, respectively. The presence of tungstate inhibited the maximum extent and not the rate of activation. DNA-cellulose binding of the activated cytosol receptor was also inhibited completely by 2.5 mM tungstate at pH 7, whereas at pH 8, no significant inhibition was observed with up to 10 mM tungstate. Tungstate was more effective in blocking the DNA binding when (NH 4) 2SO 4-fractionated receptor was used, causing a complete inhibition by 1 and 10 mM at pH 7 and pH 8, respectively. The effect of tungstate on the DNA binding of activated receptor appeared specific while several other phosphatase inhibitors were less or completely ineffective. Upon its adsorption onto DNA-cellulose, the receptor could be extracted in a specific manner by 0.2 and 2 mM tungstate at pH 7 and pH 8, respectively. The tungstate-extracted receptor sedimented in ihe 4.3–6S region in high- (0.3 M KCl) and low-salt (10mM KCl) sucrose gradients. Following the incubation with/or extraction from DNA-cellulose with tungstate, the DNA binding capacity of the receptor was completely lost and could not be recovered upon dialysis. In contrast to effects of previously more thoroughly studied molybdate ions, tungstate affects both the non-activated and activated progesterone receptor. The effects of tungstate appear direct and due to its interaction with the progesterone receptor and may not involve a mere inhibition of phosphatase activities in the receptor preparations.

Influence of tetracaine on the structure and function of estrogen receptors.

The local anesthetic agent tetracaine has been shown to relax the affinity of estrogen receptors for estradiol and unidentified ligands in uterine cytosols. This effect is dependent on both the temperature of the exchange reaction and the concentration of tetracaine. Increasing the concentration of tetracaine in the system from 0 to 6 mM while keeping the temperature at 10 C results in a stepwise increase in the fractions of receptors that engage in a rapid exchange. Increasing the temperature of the exchange reaction from 0 to 17 C in the presence of 4 mM tetracaine similarly results in a stepwise increase in the fraction of receptors exhibiting the rapid exchange. The combination of 4 mM tetracaine and 10 C achieves the displacement of [3H]estradiol by unlabeled estradiol from the majority of the receptors in 30 min. In addition, tetracaine appears to relax the specificity of the receptors so as to facilitate the displacement of [3H]estradiol from the receptor by otherwise low affinity ligands, such as tamoxifen, nafoxidine, and 17 alpha-(2',3'-dihydroxypropyl)17 beta-estradiol. Tetracaine also dissociates estrogen receptors from other macromolecules in the cytosol, so that the receptors sediment in low salt sucrose gradients an 4S complexes. The data support the view that estrogen receptors of uterine cytosols are functionally microheterogeneous, possibly by virtue of their interactions with other components of the cytosol, and that set fractions of receptors are transformed to a state with a lower affinity for estradiol by specific concentrations of tetracaine at a given temperature. A concept of receptor action is discussed in which the ability of receptors to mediate the interaction of macromolecules may regulate the activity of multienzyme systems or the function of specific chromatin complexes.

Activation of progesterone receptor by ATP.

Progesterone-receptor complex from freshly prepared hen oviduct cytosol acquired the ability to bind to isolated nuclei, DNA-cellulose and ATP-Sepharose when incubated with 5-10 mM ATP at 4 degrees C. The extent of this ATP-dependent activation was higher when compared with heat-activation achieved by warming the progesterone-receptor complex at 23 degrees C. The transformation of progesterone-receptor complex which occurred in a time-dependent manner was only partially dependent on hormone presence. The ATP effect was selective in causing this transformation whereas ADP, AMP and cAMP failed to show any such effect. The non-hydrolyzable analogs of ATP, adenosine 5'-[alpha, beta-methylene]triphosphate and adenosine 5-[beta, gamma-imido]triphosphate were also found to be ineffective. Presence of 10 mM sodium molybdate blocked both the ATP and the heat-activation of progesterone-receptor complex. Mn2+ and Mg2+ had no detectable effect on the receptor activation but the presence of Ca2+ increased the extent of ATP-activation slightly. EDTA presence (greater than 5 mM) decreased the extent of receptor activation by about 40% and was, therefore, not included in the buffers used for activation studies. Divalent cations were also ineffective when tested in the presence of 1-5 mM EDTA. The steroid-binding properties of progesterone-receptor complex remained intact under the above conditions when analyzed for steroid-binding specificity and Scatchard analysis. However, the ATP-activated progesterone-receptor complex lost the ability to aggregate when tested on low-salt sucrose gradients. ATP was equally effective in activating the rat-uterine-estradiol-receptor complex at 4 degrees C and influenced the transformation of 4-S receptor form into a 5-S form when analyzed on sucrose gradients containing 0.3 M KCl. The presence of ATP also increased the rate of activation of progesterone-receptor complex at 23 degrees C. These findings suggest a role for ATP in receptor function and offer a convenient method of studying the process of receptor activation at low temperature and mild assay conditions.

Uterine progesterone receptor: stabilization and physicochemical alterations produced by sodium molybdate.

Incubation of hamster uterine cytosol with millimolar concentrations of sodium molybdate prior to addition of labeled steroid increased recovery of progesterone receptor 2-fold. This stabilizing effect of molybdate was also manifest on gel electrophoresis of the receptor. In the absence of molybdate, no specific [3H]progesterone binding was detectable on polyacrylamide gel electrophoresis. But, in the presence of 5 mM sodium molybdate, a [3H]progesterone-binding species was clearly evident on the gels. The radioactivity associated with this binding was displaceable by unlabeled progesterone but not by cortisol and depended on the concentration of [3H]progesterone employed, suggesting that this binding species is a progesterone receptor. Molybdate treatment produced a small increase in receptor size on low-salt sucrose gradients (from 6-7 to S to 7.5 S). There was no effect of molybdate of receptor sedimentation in the presence of high salt (0.3 M KCl). Further analysis of this phenomenon by gel filtration suggested that this molybdate-mediated increase in receptor size was due to receptor aggregation. In low-salt buffers, molybdate treatment markedly increased the proportion of receptors contained in large aggregates (Stokes radius greater than 8.0 nm). Again, this effect was abolished in the presence of high salt. In conjunction with receptor stabilization, molybdate prevented binding of uterine progesterone receptor to DNA--cellulose. These findings suggest that sodium molybdate stabilizes the unliganded, unactivated form of the receptor. Moreover, these effects seem to be mediated through a direct interaction of molybdate with the receptor, one which results in receptor aggregation.

Interaction of a high affinity anti-estrogen (alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbene, CI628M) with uterine estrogen receptors.

The polar metabolite of the anti-estrogen alpha-[4-pyrrolidinoethoxy]phenyl-4-methoxy-alpha'-nitrostilbene (CI628), that is selectively accumulated by the nuclear estrogen receptor in vivo, is identified as the corresponding free phenol (alpha-[4-pyrrolidinoethoxyl]phenyl-4-hydroxy-alpha'-nitrostilbene (CI628M) and has been prepared in high specific activity tritium-labeled form to study its interaction with the rat uterine estrogen receptor. CI628M has an affinity for the cytosol receptor that is comparable to that of estradiol (Kd (CI628M) = 0.16 nM; Kd (estradiol) = 0.28 nM), and the binding of these two compounds is mutually competitive. Dissociation of the CI628M complex with the estrogen receptor is somewhat slower than that of the estradiol complex, and prewarming the complexes at 28 degrees C causes a characteristic decrease in the rate of dissociation. On low salt sucrose gradients, the CI628M and estradiol cytoplasmic receptor complexes both sediment as 8S species; their sedimentation profiles on high salt gradients are somewhat different, however. While the estradiol receptor complex shows mainly a 4.6S species that shifts to a 5.8S species upon warming, the unwarmed CI628M complex is predominantly 5.8S and becomes somewhat more disperse upon warming. Under high salt conditions, there is a progressive shift of the 4.6S to the 5.8S species as cytosol concentration increases; the CI628M complex shows a greater tendency to form the more rapidly sedimenting form at all cytosol concentrations. After in vivo injection of [3H]CI628M, the uterine nuclear receptor sediments as a characteristic 5S species. Cytosol receptor complexes with estradiol and CI628M, maintained at 0 degrees C, show very little binding to ATP-Sepharose, while after warming, 50-60% of both complexes are bound by the resin. Both complexes also show similar elution profiles from DNA-cellulose columns; however, the extent of binding of the [3H]CI628M receptor complex to the column is consistently only 40-50% that of the estradiol receptor complex. These results indicate that the properties of the uterine estrogen receptor complexed with estradiol and with the high affinity anti-estrogen CI628M are very similar. The differences observed in the behavior of the CI628M cytosol receptor complex on high salt sucrose gradients and on DNA-cellulose columns, though quantitative rather than qualitative, may be indicative of differences in receptor properties that are important to the antagonist activity of this compound.