Presence of two types of estrogen binding sites in mouse testis

The characteristics of cytosol estrogen binding sites in BALB/c mouse testis were investigated. The cytosol prepared from the whole testis contained two classes of the specific estrogen binding sites by Scatchard and Rosenthal plot analyses. The first binding site (first binder) had high affinity for 17 beta-estradiol (E2; Kd = 4.9 X 10(-9) M) and binding specificity as observed in the typical estrogen receptor. The second binding site (second binder) had lower affinity for E2 (Kd = 4.8 X 10(-8) M) and the binding was inhibited less vividly by diethylstilbestrol (DES) and antiestrogens in comparison with that for the first binder. Postlabeled sucrose density gradient analysis in a low salt medium revealed that the major radioactive peak of the first binder appeared at 7S region, while that of the second binder sedimented at 4S region. The 7S component showed an appreciable binding to the nuclei, while the 4S component did not show a significant binding ability to the nuclei. Much higher concentrations of the first and the second binders were found in Leydig cells preparations. These results demonstrate the presence of two types of the specific estrogen binding sites in the mouse testis especially in Leydig cells.     

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.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Estrogen receptor in chicken oviduct: receptor dissociation kinetics and transformation

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22 degrees C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate (k = 120 X 10(-3) min-1) decreases as a function of the pre-incubation at 22 degrees C; after prolonged pre-incubation only the slowly dissociating (k = 12.3 X 10(-3) min-1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30 degrees C is similar, showing a transition of a fast dissociating form (k = 120 X 10(-3) min-1) to a slowly dissociating form (k = 7.6 X 10(-3) min-1) as a result of pre-incubation at 30 degrees C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0-35%) shows monophasic dissociation kinetics of estradiol (k = 39.5 X 10(-3) min-1) and for moxestrol (k = 10.8 X 10(-3) min-1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0 degree C at 4.8 S. Only after subsequent incubation at 30 degrees C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22 degrees C (k = 70 X 10(-3) min-1; k = 14.0 X 10(-3) min-1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22 degrees C. Moxestrol dissociates from the nuclear receptor at 30 degrees C monophasically with a slow rate (k = 6.1 X 10(-3) min-1), suggesting that it is extracted as an activated hormone-receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for separate binding sites for progesterone and RU486 in the chick oviduct

Binding characteristics of synthetic steroid, mifepristone (RU38486 - also referred to as RU486), were examined in cytosol prepared from the chick oviduct and the calf uterus, and were compared with those of progesterone and synthetic progestin R5020. Unlike [³H]progesterone binding, the [³H]RU486 binding in the oviduct cytosol did not saturate at 50 nM ligand concentration. The [³H]progesterone binding could not be eliminated in the presence of excess RU486, and [³H]RU486 binding was seen to be indisplaceable upon pretreatment of the chick oviduct cytosol with a 1000-fold excess progesterone. It is apparent that the chick oviduct cytosol is endowed with two separate sets of sites which interact with progesterone and RU486 independently. Furthermore, [³H]RU486 binding in the chick oviduct cytosol remained intact when incubated for 60 min at 37°C; it exhibited a single ionic form upon elution from DEAE-Sephacel and the [³H]RU486-associated radioactivity sedimented in the 4 S region both in salt-free and 0.3 M KCl-containing 5–20% sucrose gradients. In the calf uterus cytosol, both steroids exhibited comparable binding profiles. Our results provide evidence that chick oviduct possesses distinct binding sites that accept either progesterone or RU486, but not both, as is the case in the calf uterus.     

Characterization of R5020 and RU486 binding to progesterone receptor from calf uterus

We have examined and compared the binding characteristics of the progesterone agonist R5020 [promegestone, 17,21-dimethylpregna-4,9(10)-diene-3,20-dione] and the progesterone antagonist RU486 [mifepristone, 17 beta-hydroxy-11 beta-[4-(dimethylamino) phenyl]-17 alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] in calf uterine cytosol. Both steroids bound cytosol macromolecule(s) with high affinity, exhibiting Kd values of 5.6 and 3.6 nM for R5020 and RU486 binding, respectively. The binding of the steroids to the macromolecule(s) was rapid at 4 degrees C, showing saturation of binding sites at 1-2 h for [3H]progesterone and 2-4 h for both [3H]R5020 and [3H]RU486. Addition of molybdate and glycerol to cytosol increased the extent of [3H]R5020 binding. The extent of [3H]RU486 binding remained unchanged in the presence of molybdate, whereas glycerol had an inhibitory effect. Molybdate alone or in combination with glycerol stabilized the [3H]R5020- and [3H]RU486-receptor complexes at 37 degrees C. Although the rate of association of [3H]RU486 with the cytosolic macromolecule was slower than that of [3H]R5020, its dissociation from the ligand-macromolecule complex was significantly slower than [3H]R5020. Competitive steroid binding analysis revealed that [3H]progesterone, [3H]R5020, and [3H]RU486 compete for the same site(s) in the uterine cytosol, suggesting that all three bind to the progesterone receptor (PR). Sedimentation rate analysis showed that both steroids were bound to a molecule that sediments in the 8S region. The 8S [3H]R5020 and [3H]RU486 peaks were abolished by excess radioinert progesterone, RU486, or R5020.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of estradiol-17 beta and estriol on their binding sites in the rabbit uterus

1. Receptors for estradiol-17 beta (E2) and estriol (E3) were detected in the rabbit uterus. 2. Saturation analysis of estrogen binding sites in the cytosol showed that the dissociation constants of E2 and E3 for the high affinity binding sites were 1.8 +/- 0.5 nM and 2.3 +/- 0.3 nM, respectively, when dextran-coated charcoal was used to isolate free and bound ligands. 3. To eliminate non-specific (cross) bindings to their receptors, effects of unlabeled E2 and E3 on [3H]E3 and [3H]E2 bindings was examined. 4. [3H]E2 cytosol binding was observed to be specific for E2 and [3H]E3 cytosol binding was more specific for E3. 5. E2 priming to rabbits increased the binding sites for both E2 and E3, which was also more potent than E3 priming. 6. Moreover, the increase in E2 binding sites was greater than that in E3 binding sites. 7. These findings may suggest that there are separate binding sites for E2 and E3 in rabbit uterus and that synthesis of their binding sites is regulated by E2 but not E3.     

Evidence for an androgen binding protein in the testis of a teleost fish (Salmo gairdneri R.): a potential marker of Sertoli cell function

A factor binding tritiated testosterone was detected using "steady-state" polyacrylamide-gel electrophoresis, in rainbow trout genital tract. It migrated with a Rf identical to that of rat ABP. This binding was thermolabile, and was competitively inhibited by unlabelled testosterone. The steroid binding protein was found in cytosols from trout testes which had been previously perfused to avoid blood contamination, trout seminal plasma and in testicular explants incubation media. Using a quantitative assay and a Scatchard analysis, 25-50 pmol binding sites per gram gonad were found in testis cytosol. Binding affinity constant for testosterone in the various samples was close to 4 x 10(8) M(-1). The dissociation of steroid-protein complex was rapid (t 1/2 approximately 1.5 min). Hormonal specificity was studied by the competition of 3H-T binding with several concentrations of unlabelled competitors and the following order for affinities was obtained: dihydrotestosterone approximately androstenedione greater than testosterone greater than oestradiol greater than 17 alpha, 20 beta DHP greater than 11KT greater than cyproterone acetate greater than cortisol. High testicular cytosol and seminal plasma concentrations and apparent in vitro production indicate that the testis may synthesize an ABP-like protein in the trout. Such a factor would provide a unique marker of Sertoli cell activity and regulation in various physiological or experimental situations.     

Differences in aromatase activity between Leydig cells from the scrotal and abdominal testis in the naturally unilateral-cryptorchid boar

In an earlier study, estrogen production was much lower in Leydig cells from the abdominal than from the scrotal testis in naturally occurring unilateral cryptorchidism in the boar. A more direct assessment of aromatase activity was made in thirty-two mature male pigs to examine this observation further, using nonradioactive androstenedione (delta 4A 1.0 x 10(-6) M - 1.5 x 10(-5) M) and [1 beta, 2 beta-3H] delta 4A as substrates. Purified Leydig cells were prepared from normal boars and from unilaterally and bilaterally cryptorchid animals. Combined estrone sulfate (E1S) and estrone (E1) formation from delta 4A were measured by radioimmunoassay. Little or no estrogen secretion was seen with cells from the abdominal testis in unilaterally cryptorchid boars (n = 7), and E1S formation from delta 4A was 6- to 14-fold higher for scrotal cells (n = 6). Aromatase activity as reflected in percent conversion of substrate to [3H]-labeled water was clearly lower in cells from the abdominal testis (1.10 +/- 0.08 and 11.22 +/- 0.7%, respectively, p less than 0.01, n = 6). No marked reduction was noted for unilaterally cryptorchid boars with an inguinally located testis (10.18 +/- 0.27 and 13.09 +/- 0.58% for inguinal and scrotal testes, respectively, n = 3). Concentrations of E1S in testicular arterial and venous blood (n = 9) gave additional evidence of lower estrogen production by the undescended testis of the cryptorchid boar. It was concluded that lower aromatase activity is present in Leydig cells of the abdominal testis.     

Evidence for an androgen receptor in the seminiferous tubules of the human testis

Androgen receptors (AR) were studied in seminiferous tubule cytosol and testicular nuclear extracts prepared from testes of previously untreated elderly men undergoing orchiectomy as therapy for prostatic carcinoma. Cytosol exhibited high affinity (Kd = 0.8 nM), saturable binding of [3H]methyltrienolone; however, the synthetic progestin, promegestone was a stronger competitor for MT binding sites than were 5 alpha-dihydrotestosterone (DHT) or testosterone (T), suggesting the presence of progesterone-like binding sites. Addition of triamcinolone acetonide (TA) produced the usual relative steroid specificity for AR binding and reduced the measured AR binding capacity by 19 +/- 8% (Mean +/- SD, n = 3). The umber of MT binding sites was 30-40 fmol/mg protein, or an average of 65 fmol/g testis, and the equilibrium dissociation constant at 0 degrees C was 0.6-1.4 nM. In the presence of sodium molybdate, binding was stable for 40 h at 0 degrees C and the half-time of dissociation of the MT-AR complex was 12-16 h. The binding of salt extractable (600 mM KCl) nuclear sites to MT was saturable and was specific for androgens. The number of binding sites in nuclear extracts was 170 fmol/g testis and the apparent equilibrium dissociation constant was 4.2 nM. Thus, the binding of MT to human seminiferous tubule cytosol and testicular nuclear extract exhibits properties which are nearly identical to those of the prostate AR. Further study of this androphilic protein may provide insight into the role of androgen in normal and abnormal spermatogenesis in man.     

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.     

Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

RU486 inhibits induction of aromatase by dexamethasone via glucocorticoid receptor in cultured human skin fibroblasts

Effects of RU486 on the induction of aromatase by dexamethasone via glucocorticoid receptor were determined using cultured human skin fibroblasts. Competition of [3H]dexamethasone binding to the cytosol receptor was 7 times stronger with RU486 than with dexamethasone. The order of the strength of competition was RU486 greater than dexamethasone greater than betamethasone greater than prednisolone greater than hydrocortisone. RU486 abolished a specific 8.6 S [3H]dexamethasone binding peak in the cytosol, determined using a sucrose density gradient analysis. Dexamethasone markedly induced aromatase and this event was strongly suppressed by RU486, in a dose-dependent manner, in the cultured skin fibroblasts. A linear correlation between the strength of competition and the induction of aromatase of various glucocorticoids was observed. RU486 non-competitively inhibited aromatase induction by dexamethasone determined from a double reciprocal plot of aromatase activity, with respect to [3H]androstenedione concentration in the presence of RU486. These results show that RU486 is a peripheral noncompetitive antiglucocorticoid on aromatase induction by glucocorticoid in human skin fibroblasts and that aromatase induction is a good marker for the biological function of glucocorticoid receptor in human skin fibroblasts.     

Heterogeneity of [3H]estradiol binding sites in the rat prostate: properties and distribution of type I and type II sites

In order to assess the rat prostate as a target tissue for receptor-mediated estrogen action, we have studied the properties and distributions of estrogen binding sites in the dorsolateral (DLP) and ventral (VP) prostate. Saturation analyses over a wide range of [3H]estradiol ([3H]E2) concentrations (0.5-100 nM) revealed two distinct types of binding sites in the cytosol and nuclear fractions of DLP of intact rats. The high affinity (type I) estrogen binding sites saturated at 2-4 nM of [3H]E2 and had a capacity of 170 fmol/mg DNA in the cytosol and 400 fmol/mg DNA in the nuclei. DLP type I sites had ligand specificity similar to that described for the classical estrogen receptors (ERs) found in female target tissues. The moderate affinity (type II) estrogen binding sites saturated at 15-30 nM of [3H]E2 and had a capacity of 850 fmol/mg DNA in the cytosol and 1600 fmol/mg DNA in the nuclei. DLP type II sites shared some characteristics of the type II ERs described for the rat uterus; they were estrogen specific, heat labile, and sensitive to reducing agents such as dithiothreitol. Saturation analyses on VP cytosols and nuclear fractions revealed only high affinity sites but no moderate affinity sites in the tissue preparations. Our finding that prostatic type II estrogen binding sites are present exclusively in the DLP supports the concept that basic biological differences exist between the two major prostatic lobes of the rat. Furthermore, our findings may help elucidate the observed differences in susceptibility between these two lobes to the hormonal induction of proliferative prostatic lesions.     

A male specific hepatic estrogen binding protein: characteristics and binding properties

Mammalian liver is a sex-steroid responsive tissue in that androgen and estrogen receptors are present and mediate differential hepatic hormonal effects. Further, we and others have found a sexual dimorphism in the hepatic cytosolic content of estrogen binding proteins. In addition to the estrogen receptor, the male has a high-capacity (12.0-15.0 pmol/mg protein) estrogen binding protein (MEB) which demonstrates a moderate affinity for estradiol (Kd = 31.0-43.2 nM) if estradiol metabolizing enzymes are first precipitated with protamine sulfate. This protein exhibits a unique specificity for steroidal estrogens: 2-methoxyestriol greater than estradiol greater than estriol = 2-methoxyestradiol greater than 2-hydroxyestradiol greater than estrone greater than 2-methoxyestrone greater than estriol 3-glucuronide greater than 2-hydroxyestrone = 3-methoxyestriol greater than androstanediol greater than dihydrotestosterone greater than testosterone. Other androgens such as androstenedione and methyltrienolone, nonsteroidal estrogens such as diethylstilbestrol, and the antiestrogens tamoxifen and 4-hydroxytamoxifen do not compete for [3H]estradiol ([3H]E2) binding. MEB is a relatively small-molecular-weight protein with a Sr of 20.4 A as determined by gel filtration on Sephadex G-100. The kinetics of [3H]E2 association and dissociation at 4 degrees C are very rapid, with t1/2 values of less than 5 s. Sodium molybdate, generally used to stabilize steroid receptors, inhibits MEB-[3H]estradiol binding activity in cytosol in a time- and dose-dependent manner, an effect not observed with partially purified MEB. Magnesium chloride inhibits binding activity of the Sephadex G-100 MEB pool, an effect reversed by EDTA. Other divalent cations also inhibit binding: Mn2+ greater than Mg2+ greater than Ca2+. Furthermore, EDTA complexes of these cations slightly enhance binding relative to EDTA alone: Ca2+ EDTA greater than Mg2+ EDTA greater than Mn2+ EDTA. These results demonstrate that MEB is a unique sex-steroid binding protein, albeit of unknown function, which is distinct from hepatic steroid receptors.     

Ultraviolet photosensitivity of the estrogen binding protein from rat uterus. Wavelength and ligand dependence. Photocovalent attachment of estrogens to proteins.

Ultraviolet irradiation of the estrogen binding protein in rat uterine cytosol results in a progressive photoinactivation which is rapid at 254 nm and slower at greater than 315 nm. Both unfilled and estradiol-filled sites are inactivated at approimately the same rates at 254 nm (t 1/2 equals 8 min and 11 min, respectively), but at 315 nm, empty sites are consumed much more rapidly (t 1/2 equals 3.4 hr) than filled ones (t 1/2 equals 24 hr). The protective effect of the estrogen ligand at this wavelength appears to depend on its binding to the estrogen-specific binding site, as inactivation rate studies at different concentrations of estrone, estradiol, and estriol show a good correlation between the extent of protection and the fractional saturation of the high affinity estrogen binding sites. Scatchard analysis indicates that inactivation is the result of a loss of binding sites and not a decrease in their affinity, and sedimentation analysis shows that increased heterogeneity and aggregation of the estrogen binding species accompanies the photoinactivation process. Photoinactivation appears to be the result of direct irradiative damage of the animo acid residues, as the inactivation rate is the same under air and nitrogen atmospheres, and is unaffected by nucleophiles, reductants, and radical scavengers. When photoinactivation is measureed by irradiation of cytosol containing [3-H]estradiol, a concurrent photocovalent attachment process is noted; the steroid becomes linked to protein in a solvent-extractable manner (boiling ethanol inextractable). This attachment, however, does not appear to be related to the steroid binding at the estrogen binding site. Its rate is affected by reductants and scavengers. A similar photocovalent attachment reaction occurs when bovine serum albumin or ovalbumin is irradiated in the presence of [3-H]estradiol or [3-H]diethylstilbestrol. The detailed reactions involved in this photocovalent attachment process have not been defined at present.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15 beta, 16 beta-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of [3H] ZK91587 to the total hippocampal corticosteroid receptor sites with high affinity (Kd 1.9 nM), and low capacity (Bmax 17.3 fmol/mg protein). When 100-fold excess RU28362 was included simultaneously with [3H] ZK91587, the labelled steroid binds with the same affinity (Kd 1.8 nM) and capacity (Bmax 15.5 fmol/mg protein). Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) greater than cortisol (F); Type II: B greater than F much greater than ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat (20.0 x 10(7) M-1 min-1). The steroid dissociates following a one slope pattern (t 1/2 30 h), indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like) receptor.     

Mammalian progesterone receptor shows differential sensitivity to sulfhydryl group modifying agents when bound to agonist and antagonist ligands

Modulation of calf uterine progesterone receptor (PR), in relation to its binding to synthetic steroids with known agonist (R5020) and antagonist (RU486) properties, was studied in the presence of iodoacetamide (IA), N-ethylmaleimide (NEM), beta-mercaptoethanol (MER), and dithiothreitol (DTT). Pretreatment of uterine cytosol at 4 degrees C with NEM (4-10 mM) reduced the binding of [3H]RU486 to PR by 40%, but [3H] R5020 binding was completely abolished. Whereas IA (2-10 mM) treatment did not affect [3H]RU486 binding, [3H]R5020 binding was totally eliminated. DTT or MER increased the binding of both steroids slightly (15%). [3H]R5020- or [3H]RU486-receptor complexes (Rc) migrated in the 8 S region and were eliminated upon pretreatment with NEM. At 23 degrees C, DTT increased the amount of 4 S [3H]R5020-Rc, but had no effect on the [3H]RU486-Rc. In the control, [3H]RU486 binding to the 8 S PR could be competed with radioinert R5020 or RU486, but R5020 failed to compete in the presence of IA. The heat-treated [3H]R5020- and [3H]RU486-Rc showed reduced binding to DNA-cellulose in the presence of NEM and IA. The results of our study suggest that SH group modifications differentially influence the properties of mammalian PR complexed with either R5020 or RU486. In the presence of IA, the [3H]RU486-Rc remained in the 8 S form when incubated at 23 degrees C, indicating that RU486 binding causes conformational changes in PR which are distinct from those that result upon R5020 binding.