Dynamic pattern of estradiol binding to uterine receptors of the rat. Inhibition and stimulation by unsaturated fatty acids

The binding of estradiol to uterine cytosoluble receptors from 24-day-old rats was reduced or potentiated by unsaturated fatty acids (NEFAs), depending on the concentrations of estradiol and unsaturated NEFAs. At estradiol concentrations of up to 1.5 x 10(-8) M, unsaturated NEFAs inhibited estradiol binding to the 8 S cytosol receptor. This inhibition was dose-dependent (10-70%, p less than 0.001) and a function of NEFA unsaturation. Scatchard analysis indicated that unsaturated NEFAs caused a large decrease in receptor affinity for estradiol. Polyunsaturated NEFAs had no apparent effect on estradiol binding at estradiol concentrations of 2-4 x 10(-8) M. At high estradiol concentrations (above 4 x 10(-8) M), estradiol binding was increased 130-250% (p less than 0.01) by polyunsaturated NEFAs. This increased binding was particularly associated with proteins sedimenting at 12.5 S and the 8 S binding was, in fact, reduced. Metabolic studies showed that the reduced binding in the presence of unsaturated fatty acids was correlated with a decrease in reversibly bound estradiol at low estradiol concentrations. The increase in estradiol binding at high estradiol concentrations is the result of a reduction in reversibly bound estradiol and an increase in nonorganic solvent-extractable (water-soluble) estradiol. The amounts of these water-soluble estradiol derivatives depended on both estradiol and unsaturated NEFA concentrations. 70% of the water-soluble estradiol derivatives were trichloroacetic acid-precipitable, suggesting a covalent protein-steroid link. Thus, changes in the hydrophobic fatty acid environment of the uterine cytosol estrogen receptor could modify estrogen-receptor function by altering binding site conformation and/or by inducing changes in estradiol metabolism.     

Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at congruent to 90 mM and congruent to 155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4 degrees C or addition of 10 mM GTP increased the proportion of the congruent to 90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 microM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.     

Inhibition of rat uterine estradiol receptor by aurintricarboxylic acid

Estradiol-receptor complex from rat uterus has been shown to have an affinity for DNA-cellulose and ATP-Sepharose. This DNA and ATP binding of estradiol receptor was observed to be sensitive to low concentrations (0.01–0.2mM) of aurintricarboxylic acid. The inhibitor was more effective when added to preparations that contained activated estradiol-receptor complex. Steroid binding properties of the receptor remained intact under the above conditions as judged by charcoal adsorption assays and sucrose gradient analysis. In addition, a 40% inhibition in the nuclear translocation of cytosol estradiol receptor was observed when rat uteri were incubated with 10nM [³H] estradiol under an atmosphere of 95% O₂ and 5% CO₂ in the presence of aurintric-carboxylic acid. Our results suggest that aurintricarboxylic acid is an effective inhibitor of rat uterine estradiol receptor and that it may be acting by interfering with site(s) on the estradiol receptor which may be exposed upon activation and are subsequently involved in processes such as ATP binding, nuclear uptake and DNA binding.     

Estrogenic effect of phenol red in MCF-7 cells is achieved through activation of estrogen receptor by interacting with a site distinct from the steroid binding site

MCF-7 cells serially subcultured in media containing phenol red show poor stimulation of progesterone receptor (PR) synthesis in response to estradiol compared to cells grown in phenol red-free media. Phenol red, when added to cytosol, did not compete with [3H]estradiol for estrogen binding sites in concentrations ranging from 2 microM-1 mM. However 25 microM of the dye was sufficient to increase nuclear translocation of estrogen receptor (ER) in the intact cell. Phenol red activates cytoplasmic ER as indicated by DNA-cellulose binding studies. When cells grown in phenol red-free medium were exposed to phenol red for 48 h, PR levels increased in a dose dependent manner. From these data, it may be concluded that phenol red causes estrogenic effect in MCF-7 cells through activation of cytoplasmic receptor by interacting at a site distinct from the steroid binding site.     

Investigation by fluorescence spectroscopy, resonance rayleigh scattering and zeta potential approaches of the separate and simultaneous binding effect of Paclitaxel and estradiol with human serum albumin

Separate and simultaneous binding effects of paclitaxel (a drug with anti-tumor activity) and estradiol (used for treating multiple maladies) with human serum albumin (HSA) were investigated by fluorescence quenching, UV absorption, circular dichroism, zeta potential and molecular dynamic techniques. An extensive fluorescence quenching was observed during the reaction of drugs and HSA and was rationalized in terms of a static quenching mechanism. The molecular distances between the donor (HSA) and acceptors (paclitaxel or estradiol) in binary and ternary systems were estimated according to F?rster's theory of dipole-dipole non-radiation energy transfer. The features of drug-induced structural disturbances of HSA have been studied in detail by synchronous fluorescence and circular dichroism (CD) analysis. The resonance Rayleigh scattering (RRS) intensities were proportional to the paclitaxel and estradiol concentrations in the range of respectively (0-8)×10(-6) and (0-1)×10(-4) mM in binary systems. The critical induced aggregation concentrations (C(CIAC)) of paclitaxel and estradiol for binary and ternary systems were determined by nonlinear relationships between the enhancement of the RRS intensities and the drug concentrations. A comparison between binary and ternary systems for two drugs allowed us to estimate the effect of a drug on the initial formation aggregation of the second drug. The zeta potential results were used to verify the existence of complexation and confirmed the C(CIAC) values obtained by the RRS technique. This phenomenon was supported by a progressive rise of the protein charge to a reversal point as a consequence of drug binding. The quantitative analysis data of circular dichroism (CD) spectra demonstrated that the binding of paclitaxel and/or estradiol to HSA induced conformational changes in HSA. Moreover, the α-helix content in HSA greatly decreased in the presence of paclitaxel as opposed when estradiol was present. Protein-ligand docking suggested that estradiol bound to residues situated in subdomain IIA of HSA. On the other hand, in the ternary system, the presence of the first drug decreased the binding affinity of the second drug to HSA. Therefore binding effects of paclitaxel and estradiol with HSA alone have different behavior than simultaneous interaction.     

Calcium-induced membrane metabolic alterations modify the sex steroids binding into dog brain synaptosomal plasma membranes

The binding of 45Ca2+ into synaptosomal plasma membranes (SPM) of dog brain follows a sigmoid path. In graphical analysis of this binding the mean Hill coefficient (h) was 1.64 +/- 0.09 (r2 = 0.96 +/- 0.02). Binding of Ca2+ into SPM was saturable, with an apparent binding constant of 1.2 +/- 0.1 microM. At saturation, such calcium specific binding sites corresponded to 11.2 +/- 0.9 nmol/mg SPM protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of calcium into SPM of at least one class of high affinity specific binding sites. [14C]estradiol, [14C]estrone and [14C]progesterone, when incubated with SPM up to a concentration of 10 microM for 2 hr at 37 degrees C, bind into SPM at nmolar concentrations. Ca2+ ions up to 5 mM considerably increase steroids binding into SPM. This effect of calcium was concentration-dependent, reached saturation at approx 4-5 mM. Once calcium has promoted steroids binding, the subsequent addition of 25 mM EGTA failed to displace bound steroids. Molecular interactions between calcium and SPM was assessed by measuring the steady-state fluorescence polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), and by estimating the production of malondialdehyde (MDA) during 2 hr incubation of Ca2+ (5 mM) with SPM at 37 degrees C. The effect of Ca2+ on the SPM structure was to increase both the rigidity of the membrane and the MDA production. Chelation of Ca2+ (5 mM) with EGTA (25 mM) did not reverse the increase in the rigidity owing to metabolic alterations of SPM lipids (e.g. production of MDA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear estradiol binding in rat adipocytes. Regional variations and regulatory influences of hormones.

The nuclear estrogen receptor was characterised in isolated rat adipocytes. The binding reaction with [3H]estradiol was performed with intact isolated rat adipocytes and the radioactivity associated with the nucleus was subsequently determined after cell lysis. The nuclear uptake of [3H]estrogen in rat adipocytes was temperature dependent and steroid specific. The steady-state binding was achieved after 30 min at 37 degrees C and was constant for several hours. Estradiol was found to bind to a homogeneous class of nuclear receptors in epididymal adipocytes with an apparent Kd of 3.1 +/- 0.76 nM and a Bmax of 7.98 +/- 1.11 fmol/10(6) cells corresponding to about 4800 receptors per nucleus. The estradiol binding exhibited regional variations in isolated adipocytes. In lean rats the highest receptor number was found in epididymal adipocytes, whereas there was a significantly lower number of nuclear binding sites in perirenal and subcutaneous adipocytes (P less than 0.05), unlike in older and more obese rats where the nuclear estradiol binding was greatest in adipocytes from the perirenal fat depot. Incubations with isoproterenol (10 microM) and dibutyryl-cAMP (2.5 mM) both reduced estradiol binding by 56% (P less than 0.005), while insulin (1 nM) enhanced the estradiol binding by 37% (P less than 0.01). In conclusion, a specific and high affinity nuclear estradiol receptor was demonstrated in rat adipocytes and regional differences in nuclear estradiol binding were detected. Furthermore, it was demonstrated that nuclear estradiol binding could be modulated by other agents known to affect adipocyte metabolism.     

Somatostatin enhances binding of [3H]estradiol to a cytosolic protein in rat pancreas. Possible role of oligopeptide coligands in secretion

The cytosol fraction of rat pancrease can bind [3H] estradiol specifically and extensively. In contrast to the rat uterus, the binding protein in pancreas requires an accessory factor as a coligand in the steroid-binding reaction. Removal of this accessory factor by passage of the cytosol through CM Affi-Gel blue columns renders eluate fractions virtually incompetent with respect to binding of [3H]estradiol (10 nM). Certain synthetic oligopeptides such as N-benzoyl-L-argininyl-p-nitroanilide, as well as an endogenous accessory factor, can reactivate binding of [3H]estradiol. Thus, localization of the protein that binds [3H]estradiol following chromatography with CM Affi-Gel blue columns can be determined readily by assaying eluate fractions in the absence and presence of either accessory factor or N-benzoyl-L-argininyl-p-nitroanilide. Addition of somatostatin (tetradecapeptide referred to as SRIF14; somatotropin release inhibiting factor) to the activatable, but incompetent, eluate fractions, also enhanced binding of [3H]estradiol. The effect of SRIF14 was biphasic. The threshold concentration required for activation of [3H]estradiol binding was about 1 microM, and maximal stimulation occurred at 25 microM. At higher concentrations of SRIF14, binding declined and reached basal levels at about 75 microM. The concentrations of somatostatin required for activation of binding of [3H]estradiol in vivo may be lower than those indicated above since 1) preparations containing [3H]estradiol-binding protein also contained an SRIF14 peptidase. Following incubation of [125I-Tyr1]SRIF14 with these preparations there was loss of binding of radiolabeled peptide with SRIF14 antiserum. 2) The biphasic nature of SRIF14 activation may reflect feedback inhibition of [3H]estradiol binding by a degradation product of SRIF14. Since SRIF14 has been identified in the delta- (or D-) islet cells of the pancreas, and in concentrations that may be in the microM range, the possibility is raised that these cells serve a paracrine function with respect to acinar cell secretion.     

The Use of Ascorbate as a Probe of Opioid Receptor Structure: Evidence for two Independent Mechanisms of Receptor Destruction by Ascorbate

Abstract

Ascorbate (20 mM) pretreatment of brain membrane suspensions at 37° produced a rapid irreversible loss of specific opioid binding. There was no reduction in specific ³H-haloperidol binding. Ascorbate induced loss of opioid binding under these experimental conditions was not blocked by low concentrations of EDTA or Mn⁺⁺. In contrast, the slowly developing loss of opioid binding during exposure to 1 mM ascorbate at 23° was completely inhibited by 10^?5M EDTA or Mn⁺⁺. At 37°, D-isoasoorbate, and several other reducing agents (glutathione, dithiothreitol, oysteine) produced a loss of opioid binding similar to that seen with ascorbate. It is concluded that 1 mM ascorbate at 23°, and 20 mM ascorbate at 37°, destroy opioid binding sites by two independent mechanisms. Lipid peroxidation is implicated at low ascorbate concentrations; a reductive process appears to be responsible for the ascorbate induced loss of binding at higher concentrations.     

Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain

Biologically active, mouse estrogen receptor hormone-binding domain (residues 313–599) overexpressed in Escherichia coli was purified to apparent homogeneity as a single component with a molecular mass of 32.831 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The intact domain was isolated using a novel, rapid purification scheme without recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity for estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [³H]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroid unoccupied ERhbd (from dynamic light scattering) was 72 kDa, suggesting that the pure, unlabelled ERhbd formed homodimers. Steroid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [³H]estradiol was unaffected by Ca²⁺ and Mg²⁺ ions up to 1 mM but was significantly inhibited by Zn²⁺ ions at concentrations above 10 μM, an effect reversed by EDTA.     

Effects of calcium and magnesium ions on the interaction of corticosterone with rat brain cytosol receptor(s).

The apparent maximum corticosterone binding (B max) with rat brain cytosol and the apparent dissociation constant of this steroid-receptor binding (Kd) estimated with a Scatchard plot was 2.9 X 10(-13) moles/mg cytosol protein and 4.0 X 10(-9) M, respectively. When increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, a specific [3H] corticosterone binding increased 4-fold by CaCl2 at concentrations of 1.0-2.0 mM and 1.5-fold by MgCl2 at concentrations of 0.5-5.0 mM. The addition of MnCl2 and KCl did not affect this binding. Binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EGTA and complete inhibition was observed at concentrations equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.     

Phosphorylation of the isolated high-affinity (Ca2+ + Mg2+) ATPase of the human erythrocyte membrane

Solubilized and purified high-affinity (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the human erythrocyte membrane (Wolf, H.U., Dieckvoss, G. and Lichtner, R. (1977) Acta Biol. Ger. 36, 847) has been phosphorylated and dephosphorylated under various conditions with respect to Ca2+ and Mg2+ concentrations. In the range, 0.001--100 mM, the rate of phosphorylation was dependent on Ca2+ concentration, showing a maximum at 10 mM. The phosphorylation rate was nearly independent of the Mg2+ concentration within the range 0.01-1 mM. This enzyme has at least three Ca2+ binding sites with different affinities and regulatory functions: (1) binding to the high-affinity site yields phosphorylation of the enzyme; (2) binding to a low-affinity site (Ca2+ concentrations higher than 40 microM) inhibits dephosphorylation or the conformational change which is necessary for dephosphorylation; (3) by binding to an additional low-affinity site, Ca2+ at concentrations higher than 1 mM abolishes negative cooperative behaviour (shown below 1 mM Ca2+) and causes weak positive cooperativity between at least two catalytic subunits in the phosphorylation reaction. The phosphoprotein obtained at Ca2+ concentrations above 1 mM dephosphorylates spontaneously after removal of the divalent metal ions. Addition of Mg2+ accelerates the dephosphorylation rate. Affinities of the inhibitory Ca2+ binding sites are reduced by the binding of substrate or K+.     

Melatonin blocks the activation of estrogen receptor for DNA binding.

The present study shows that melatonin prevents, within the first cell cycle, the estradiol-induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol-estrogen receptor complexes to estrogen-responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol-estrogen receptor complex to the estrogen responsive element.     

Estradiol- and estriol-binding in human benign prostatic hyperplasia.

Binding of the natural estrogens, estradiol and estriol, was investigated, in 34 samples of human benign prostatic hypertrophy (BPH) tissue, using Scatchard analysis and agar gel electrophoresis. Saturation binding analysis using a wide range of concentrations of both ligands resulted in curvilinear Scatchard plots. This confirmed the presence of two binding forms for estradiol: a true estrogen receptor, and a protein with lower affinity and higher capacity. Both binding species were also demonstrated and quantified with estriol. The electrophoretic process, after incubation at low and high ligand concentrations also resulted in separation, for both estrogens, of two binding peaks. They are probably two distinct forms of the low affinity, high capacity binding measured by Scatchard. The procedure used in our laboratory was not able to provide accurate determination of the concentrations of these binding forms. Possible modifications to alleviate these drawbacks are discussed.     

The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.     

Inhibitory effect of Ca2+ on formation of Mg2(+)-mediated two-dimensional hexagonal lattice structure by an R-form lipopolysaccharide from Klebsiella pneumoniae

When the R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was suspended in 50 mM Tris buffer at pH 8.5 containing 0.1 mM or higher concentrations of MgCl2, it formed an ordered two-dimensional hexagonal lattice structure and its center-to-center distance (lattice constant) depended upon the concentration of MgCl2 and reached the shortest value (14 nm) at 10 mM. In contrast, in the presence of 0.1 to 10 mM CaCl2 in place of MgCl2, the electrodialyzed LPS did not form such an ordered hexagonal lattice structure but formed an irregular network structure with a center-to-center distance of 19 to 20 nm. We investigated interaction of Mg2+ and Ca2+ in formation of the hexagonal lattice structure by the electrodialyzed LPS suspended in 50 mM Tris buffer at pH 8.5. When 0.1 mM or higher concentrations of CaCl2 were mixed with 1 mM MgCl2 or when 1 mM or higher concentrations of CaCl2 was mixed with 10 mM MgCl2, the electrodialyzed LPS did not form the hexagonal lattice structure of the magnesium salt type but formed the irregular network structure of the calcium salt type. In the coexistence of equimolar or higher concentrations of CaCl2 together with 1 or 10 mM MgCl2, the binding of Mg to the electrodialyzed LPS was significantly inhibited and, conversely, the binding of Ca was enhanced as compared with when MgCl2 or CaCl2 was present alone. However, the coexistence of 10 times less molar concentrations of CaCl2 did not significantly inhibit the binding of Mg to the electrodialyzed LPS. Therefore, the inhibition of formation of the Mg2(+)-mediated hexagonal lattice structure of the electrodialyzed LPS by equimolar or higher concentrations of CaCl2 accompanied the inhibition of binding of Mg but that by 10 times less molar concentrations of CaCl2 did not accompany it.     

Inhibition of protein synthesis by Cl-

Optimum K+ concentration for protein synthesis in four eukaryotic cell-free systems is obtained with 70 to 80 mM added KCl or with 110 to 150 mM added K(OAc). The different K+ optima are due to inhibition of protein synthesis by Cl- at concentrations higher than those present in the cytoplasm of eukaryotic cells. Initiation of protein synthesis is severely inhibited with 150 mM added KCl. This inhibition results from an impairment of mRNA binding to ribosomes. The binding of initiator Met-tRNAt, however, is only slightly inhibited by 150 mM KCl.     

Mechanism of biphasic action of mono- and bivalent salts on the binding of prolactin to the receptor in the rabbits mammary gland

The influence of NaCl, KCl, CaCl2, and MgCl2 on the binding of prolactin (PRL) to its receptor was investigated. The salts were dissolved in a metallic ion-free binding buffer and had biphasic effects on changes in the association rate constant (k+1) of PRL binding, depending on their concentrations: there was an increase in the k+1 at lower concentrations and a decrease at higher concentrations. The dissociation rate of bound PRL was unaffected. NaCl at any concentration did not change the binding capacity. Bivalent salts, at higher than 25 mM, increased the capacity about 1.6-fold as compared to the 0 mM control. By cross-linking the PRL-receptor complex, the band of a molecular weight (Mr) 34,500 receptor could always be detected on the autoradiogram. An Mr 78,000 receptor appeared only after incubation with bivalent salts. Data indicate that the binding of PRL to an Mr 78,000 receptor is directly regulated by bivalent cation.     

A recognition system for sex-hormone-binding protein-estradiol complex in human decidual endometrium plasma membranes

We have studied the specific binding of human serum sex-hormone-binding protein and its complexes with estradiol and testosterone to an enriched membrane fraction isolated from human decidual endometrium. The specific binding of sex-hormone-binding protein to these membranes has been found to be dependent on it being bound with estradiol. When estradiol concentration in the medium is high enough to provide for the saturation of sex-hormone-binding protein with the steroid, the protein exhibits specific binding to the membranes with an apparent equilibrium constant, Kd = (3.5 +/- 2.0) X 10(-12) M. In the absence of estradiol, the binding protein devoid of steroid or complexed with testosterone does not interact with the membranes. At low concentrations of sex-hormone-binding protein and estradiol, when their complex is almost completely dissociated in solution, it can be reconstituted on the membranes with the optimum estradiol to binding protein ratio being 1:1 (mol/mol). It is proposed that, in plasma membranes of the estradiol target cells, there is a recognition system for the sex-hormone-binding protein-estradiol complex which may allow these cells to take up from blood not only free estradiol, but also estradiol complexed with the binding protein.     

Partial purification of estradiol receptor from Xenopus laevis liver and levels of receptor in relation to estradiol concentration

We have used ammonium sulphate precipitation followed by affinity chromatography to partially purify the estrogen receptor from Xenopus laevis liver which may control the genes for vitellogenin, the precursor of the egg yolk proteins. The rate at which receptor binds estradiol explains the kinetics of the induction of vitellogenin synthesis by estradiol, and the dissociation constant (0.5 X 10(-9) M) explains the concentration dependence of the response, which has a threshold of 10(-9) M estradiol, when 67% of the receptor is bound to estradiol. The estradiol concentration in male liver, which does not make vitellogenin, is 0.18 X 10(-9) M, sufficient to saturate 26% of the receptor, while in female liver, which makes vitellogenin continuously, the estradiol concentration is 3.5 X 10(-9) M, giving 88% saturation of receptor, suggesting that the proportion of occupied receptor decides whether or not the vitellogenin genes are active. In the physiological concentration range, estradiol modulates the level of receptor, which varies between 100 binding sites per nucleus in males and 440 in females, but artificially high concentrations of estradiol raise the level to approximately 1000 sites per nucleus. This suggests that the small increase in vitellogenin mRNA induced by physiological concentrations of estradiol is due to pre-existing receptor and that the much larger increases induced by very high concentrations depends on newly-synthesized receptor.