[show abstract][hide abstract] ABSTRACT: The retinoblastoma-interacting zinc-finger gene RIZ is expressed in two forms (RIZ1 and RIZ2) that differ for the presence near the N-terminus of RIZ1 of a conserved domain, defined PR (PRDI-BF1-RIZ homology), homologous to a similar domain present in other proteins recognized as tumor suppressor gene products. The RIZ1 form is usually absent or expressed at low levels in tumor cells, whereas RIZ2 is frequently expressed. We investigated a possible involvement of RIZ1 in differentiation control using a myeloid cell maturation model that is easily modulated by retinoids and other agents.
HL60 or NB4 cell lines or patients' leukemic promyelocytes were treated with all- trans -retinoic acid or other agents to induce differentiation. RIZ gene expression was determined with reverse transcriptase polymerase chain reaction (RT-PCR) and RNase protection assay. Immunocytochemistry was performed to assess variation of the intracellular distribution of RIZ protein on all- trans-retinoic acid treatment. Forced expression of RIZ1 protein was obtained with a recombinant adenovirus containing RIZ1 cDNA.
Treatment with retinoic acid induced a selective expression of RIZ1 in HL60 cell line. Retinoic acid effect was maximal at 7 days and correlated to the granulocytic differentiation of cells. A similar effect was obtained in retinoic acid-sensitive NB4 cell line or in patients' leukemic promyelocytes, but not in the retinoic acid-resistant cell line NB4.007/6 or in the U937 cell line. Selective expression of RIZ1 was also induced by 12-O-tetradecanoyl-phorbol-13-acetate in the U937 and HL60 cell lines and by 1,25-dihydroxyvitamin D(3) only in HL60 cells. In HL60 cells, RIZ1 was also induced by activation of a retinoid alpha receptor-independent maturation pathway based on retinoid X receptor agonist and protein kinase A synergism. In addition, retinoic acid produced a redistribution of the antigen within the nucleus in these cells. Forced expression of RIZ1 protein induced growth arrest and death of HL60 cells.
The correlation between the selective expression of RIZ1 induced by retinoic acid, 12-O-tetradecanoyl-phorbol-13-acetate, or 1,25-dihydroxyvitamin D(3) and differentiation suggested that RIZ protein was involved in myeloid cell differentiation induced by these agents.
Molecular Medicine 09/2001; 7(8):552-60. · 4.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: As reported earlier, p53 antibodies are detected in the sera of patients with different types of cancer, including lung cancer. In contrast, in the serum of healthy subjects the presence of anti-p53 antibodies is extremely rare. We collected the venous blood samples of 109 patients affected with lung cancer (LC): 57 patients (46 M, 11 F) with non-small-cell carcinoma (NSCLC), 52 others (40 M, 12 F) with small-cell carcinoma (SCLC). Serum p53 antibodies were assayed using ELISA method and all positive sera were confirmed by Western-blot method. In addition, using IRMA methods we assayed serum CEA, TPA, CYFRA21-1 and NSE. Serum p53Ab are detectable (p53Ab-positive) in 35/109 (32.1%) patients with lung cancer. About 17/57 (29.8%) patients affected with NSCLC and 18/52 (34.6%) with SCLC were p53Ab-positive. CEA, TPA, CYFRA21-1 and NSE sensitivity in LC patients (NSCLC+SCLC) is 50.5%, 58.7%, 42.2%, 35.8%, respectively. The lower sensitivity (32.1%) of serum p53Ab is connected with the higher specificity and diagnostic accuracy (100% and 69%, respectively). Out of 35 patients p53Ab-positive, five (14.3%) exhibit only serum p53Ab, while serum values of the established tumor markers were lower than cut-off. Serum p53Ab assessment is a simple and a low-cost assay with a good specificity and diagnostic accuracy that in LC patients can be used at least in association with established tumor markers.
Lung Cancer 01/2001; 33(2-3):163-9. · 3.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm.
Cancer Research 06/2000; 60(10):2594-7. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor.
The EMBO Journal 04/1996; 15(6):1292-300. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: A tyrosine kinase purified from calf uterus activates the hormone binding of endogenous estradiol receptor (ER) predephosphorylated and preinactivated by a nuclear phosphotyrosine phosphatase. The kinase also activates and phosphorylates the human estradiol receptor HEO synthesized in vitro, which differs from the wild type receptor HEGO because a glycine is replaced by a valine at position 400. Moreover, the kinase activates and phosphorylates a deletion mutant of HEO which consists almost exclusively of the hormone binding domain. Using HEGO and HEO in parallel and measuring both binding activation and phosphorylation of ER we now observe that the wild type receptor is a good kinase substrate, slightly better than HEO. Furthermore, HEGO like the calf uterus receptor in the presence of estradiol, stimulates the kinase. From present findings it appears that ER and uterus tyrosine kinase are functionally associated and that this association is abolished by glycine to valine substitution at position 400 of ER.
The Journal of Steroid Biochemistry and Molecular Biology 05/1991; 38(4):407-13. · 3.98 Impact Factor
[show abstract][hide abstract] ABSTRACT: Estradiol receptor (ER) activity requires interaction with hormone and specific DNA sequence. We now report that this receptor also interacts with calmodulin (CaM), the major intracellular mediator of Ca2+ action in eucaryotic cells. This interaction has been observed using both CaM-Sepharose and [125I]CaM. Crude and purified [3H]ER complex show high affinity interaction with CaM-Sepharose [dissociation constant (Kd) 0.12 and 0.16 nM, respectively]. Unoccupied receptor shows a similar high affinity interaction. Tamoxifen-ER complex also binds to CaM-Sepharose. Several findings show that this CaM-ER interaction is very specific: lack of this interaction has been observed in the presence of trifluoperazine, an inhibitor of protein binding to CaM; the receptor binds neither Sepharose, nor parvalbumin-Sepharose; competition of interaction of [3H]ER complex with CaM-Sepharose is observed by cold ER complex; rat liver glucocorticoid receptor does not bind to CaM-Sepharose. The interaction of purified receptor with 125I-labeled CaM has been detected by various techniques: centrifugation through sucrose gradient of CaM incubated with receptor shows that CaM binds to a protein forming a complex sedimenting at 5 S. This complex is shifted to the 7.5 S region by a monoclonal antireceptor antibody. Incubation of CaM with receptor followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography of the immunoprecipitated receptor shows that [125I]CaM coprecipitates with the receptor. Competition of this interaction by an excess of cold CaM is observed. Interaction of the receptor with CaM is also observed by the overlay technique.(ABSTRACT TRUNCATED AT 250 WORDS)
[show abstract][hide abstract] ABSTRACT: Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new.
The EMBO Journal 11/1987; 6(10):2923-9. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: In whole rat uterus incubated in the presence of [32P]orthophosphate the oestradiol receptor is [32P]phosphorylated on tyrosine. This finding follows our previous observation that in vitro this receptor can be phosphorylated on tyrosine by a uterus kinase that endows the receptor with oestradiol-binding activity. The calf uterus oestradiol receptor interacts with high affinity with 2G8 and 1G2 antiphosphotyrosine antibodies coupled to Sepharose (Kd values of 0.28 and 1.1 nM, respectively). The interaction with 2G8 antibody has been exploited to purify the oestradiol receptor. This interaction disappears after inactivation of the oestradiol receptor by the nuclear phosphatase that hydrolyses phosphotyrosine of the receptor. This fact substantiates the evidence that the oestradiol receptor in uterus is phosphorylated on tyrosine and that this phosphorylation is required for hormone binding to the receptor. The rat liver glucocorticoid receptor also interacts with high affinity with 2G8 antiphosphotyrosine antibody coupled to Sepharose (Kd value of 0.21 nM). This receptor has been purified by using in sequence heparin-Sepharose and antiphosphotyrosine antibody-Sepharose.
Journal of Steroid Biochemistry 02/1987; 27(1-3):245-53.
[show abstract][hide abstract] ABSTRACT: In whole rat uterus incubated in the presence of [32P]orthophosphate the oestradiol receptor is [32P]phosphorylated on tyrosine. This finding follows our previous observation that in vitro this receptor can be phosphorylated on tyrosine by a uterus kinase that endows the receptor with oestradiol-binding activity.The calf uterus oestradiol receptor interacts with high affinity with 2G8 and 1G2 antiphosphotyrosine antibodies coupled to Sepharose (Kd values of 0.28 and 1.1 nM, respectively). The interaction with 2G8 antibody has been exploited to purify the oestradiol receptor. This interaction disappears after inactivation of the oestradiol receptor by the nuclear phosphatase that hydrolyses phosphotyrosine of the receptor. This fact substantiates the evidence that the oestradiol receptor in uterus is phosphorylated on tyrosine and that this phosphorylation is required for hormone binding to the receptor.The rat liver glucocorticoid receptor also interacts with high affinity with 2G8 antiphosphotyrosine antibody coupled to Sepharose (Kd value of 0.21 nM). This receptor has been purified by using in sequence heparin-Sepharose and antiphosphotyrosine antibody-Sepharose.
[show abstract][hide abstract] ABSTRACT: Estradiol and progesterone receptors were assayed in tumors from 79 patients with primary colorectal and 56 patients with stomach adenocarcinomas. Eighteen of 79 colorectal cancers contained estradiol receptor, while 34 specimens were positive for progesterone receptor. In stomach cancer, the positive samples were 8 for estradiol and 14 for progesterone receptors. In both types of tumors, the Kd was in the range of 10(-10) M for estradiol and 10(-9) M for progesterone receptor, respectively. In colorectal adenocarcinomas, the presence of progesterone receptor seems to be partially correlated to the presence of estradiol receptor while, in stomach tumors, this correlation is lost. The positivity of at least one receptor in colorectal cancers is higher in the female sex. The contrary occurs for stomach cancer. Sucrose gradient centrifugation showed that cytoplasmic estradiol receptor of stomach cancer sedimented at 8S or 4 to 5S at low ionic strength. The isoelectric point of stomach cancer estradiol receptor is 6.5.
Cancer Research 11/1984; 44(10):4670-4. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The relationship of the cytosolic estradiol receptor with membranous and cytoskeletal structures of the cell matrix has been studied using a model system formed by the erythrocyte membrane. Extraction of erythrocyte ghosts with various procedures and also with the nonionic detergent Triton X-100 under conditions that yield a cytoskeletal matrix reveals the presence of binding sites for the soluble estradiol receptor of calf uterus.The interaction between the estradiol receptor and the cytoskeleton is critically dependent on temperature which is required for the interaction itself and not for a modification of the receptor molecule. Ionic strength and cations, with selectivity for Mg2+, also influence the interaction which has an optimum at pH 7.5. Saturation experiments reveal the presence of a limited number (less than 100) of sites per cell having high affinity () for the estradiol-receptor complex. The affinity of the receptor for the steroid does not change after it has bound to the cytoskeleton. Limited proteolysis of the receptor leads to a loss of its binding capacity for the cytoskeleton without altering its estradiol binding properties, indicating that the cytoskeletal binding domain is different from the steroid binding domain. We suggest that the estradiol receptor, generally considered ‘soluble’ in the cytoplasm, might be physiologically associated with cytoskeletal components of the cell cytoplasm.
Journal of Steroid Biochemistry 01/1982; 15:307-12.
[show abstract][hide abstract] ABSTRACT: Estradiol receptor (ER) and progesterone receptor (PgR) were assayed in tumors from 20 patients with primary colorectal cancer. Ten of 20 tumors contained high affinity sites for 17 beta-estradiol and progesterone. The highest concentration of ER was 56 fmol/mg of protein. The ER dissociation constant ranged from 1.6 X 10(-10) M (mean 4.6 +/- 2.6). The highest concentration of PgR was 42 fmol/mg of protein. The PgR dissociation constant ranged from 3 X 10(-9) to 9 X 10(-9) M (mean 5.65 +/- 2.1). Four out of 20 specimens analyzed were from male patients and all resulted negative for both receptors. Sixty per cent of ER positive tumors were also PgR positive, whereas only 20% of ER negative were PgR positive. Sucrose gradient centrifugation showed that cytoplasmic ER of colorectal cancer sedimented at 3 S in the absence of protease inhibitors and at 4.5 S in the presence of 1 mM phenylmethylsulphonyl fluoride (PMSF) both in low and in high ionic strength. When chromatographed on Sephadex G-200 almost all ER was quantitatively recovered in the included fractions. Molecular weights of ER eluted from Sephadex G-200 ranged from 90,000 to 50,000 daltons. Elution profile and molecular weight heterogeneity suggest that, in spite of the presence of PMSF, there is a limited proteolysis of ER. Partially purified colorectal cancer ER did not bind to sepharose-heparin. The isoelectric point of ER was 6.4-6.5.
[show abstract][hide abstract] ABSTRACT: We describe our method of purification of the “native” form of estradiol receptor of calf uterus. The high speed supernatant, after addition of 5mM MgCl2. is incubated batchwise with agarose to which heparin has been covalently bound. Elution of the estradiol binding activity is obtained by heparin. The volume of the eluate is reduced to one-tenth the volume of the original cytosol but presence of heparin avoids the aggregation of the receptor. Immobilization of receptor on 17β-estradiol-17-hemisuccinylhexane-agarosc (a very simple derivative to prepare) is best carried out on column. The washing of the specific adsorbent is very critical and must be extensive. Elution is performed with the chaotropic salt, NaSCN, in presence of low concentration of estradiol. A third step is finally required to eliminate some residual contaminating proteins. Sephadex G-200 chromatography in low salt buffer gives good results.“Native” receptor maintains, even after complete purification, the tendency to aggregate in very large forms which do not penetrate the polyacrylamide gel in the absence of sodium dodecyl sulphate, and are eluted in the void volume of Sephadex G-200 columns and very often sediment at the bottom of sucrose gradients. Presence of cations, in our case MgCl2, during the purification procedure decreases the aggregation phenomenon.
Journal of Steroid Biochemistry 02/1980; 12:105-13.
[show abstract][hide abstract] ABSTRACT: A method is described for the purification to homogeneity of oestrogen receptor from calf uterus cytosol. This method consists of affinity chromatography on heparin-agarose followed by affinity chromatography on 17β-oestradiol-17-hemisuccinyl-ovalbumin-agarose, by a second adsorption on heparin-agarose and finally by gel filtration on Sephadex G-50. Elution from heparin-agarose was obtained using buffer containing heparin (4 mg/ml), and from the oestradiol-adsorbent using buffer containing NaSCN 0.5 M and oestradiol. The yield from 1 kg of uteri was about 1.2 mg of receptor protein with more than 50% recovery. A single protein band, co-migrating with the [2H]oestradiol, is seen in polyacrylamide gel electrophoresis. A single protein band, with an apparent mol. wt. of 70,000, is also seen on electrophoresis in sodium dodecyl sulfate gels. As computed from specific activity, there is one oestradiol binding site per 70,000 receptor subunit. The pure receptor on low salt sucrose gradient sediments at 8 S and on high salt or in chaotropic salt containing gradient sediments at about 4 S. The high tendency to form aggregates is still maintained by the pure receptor as judged by the elution pattern from Sephadex G-200 columns. Aggregation is avoided by small concentrations of heparin in the buffers.
Journal of Steroid Biochemistry 08/1979; 11(1A):301-6.
[show abstract][hide abstract] ABSTRACT: It was previously shown that calf uterus cytosol contains a Ca2+-activated receptor transforming factor (RTF) which irreversibly converts the larger molecular states of estrogen receptor (5.3 to 8.6 S, depending on ionic strength) into a smaller, salt-stable form (4.5 S, independent of ionic strength). We now describe a method for rapid and reliable separation of precursor and RTF-transformed receptor forms, which takes advantage of a difference in isoelectric point between the two: the more acidic precursor (isoelectric point, 6.2) is still retained by DEAE-cellulose under conditions (0.12 M KCl, pH 8.3) which produce release from cellulose of the less acidic transformed form (isoelectric point, 6.6 to 6.8). Based on this method of separation, RTF activity can be assayed easily and we could thus progress in the purification and physical and functional characterization of this factor, RTF has been purified about 100-fold. Molecular properties, as assayed by methods suited to partially purified preparations, are as follows: sedimentation coefficient, 6.4 S; Stokes radius, 45 A; molecular weight, 115,000; isoelectric point, 4.9. The Michaelis constant, expressed as moles/liter of estradiol binding sites, is 1.25 X 10(8), at pH 7.5 and 4 degrees, pH 8.5 is optimum for activity. RTF attacks native casein (Km, 1.25 X 10(-5) mol/liter at pH 7.5 and 22 degrees) but not hemoglobin, ovalbumin, or albumin. N-Benzoylarginine methyl ester is a competitive inhibitor of RTF-induced receptor transformation, while L-leucylglycylglycine and N-benzoyltyrosinamide are not. RTF activity is protected by -SH compounds. RTF activity is Ca2+-dependent. Ca2+ starts an activation-inactivation cycle of RTF, with permanent loss of transforming activity which proceeds at a particularly fast rate in the absence of substrate. Mg2+ is inactive, while Sr2+ and Mn2+ may in part substitute for Ca2+. RTF is present in both endometrium and myometrium. RTF is not a lysosomal hydrolase, as shown by its alkaline pH optimum (8.5) and exclusive location in cytosol, nor is it trypsin or a protease of the trypsin group. Also, it is distinct from known proteases of human uterus. The functional significance of this Ca2+-activated protease of cytosol with alkaline pH optimum and high affinity for the larger native form of receptor is still unknown.
Journal of Biological Chemistry 03/1977; 252(4):1358-66. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sodium thiocyanate up to 0.5 M is compatible with a stable estradiol-t-receptor complex during sucrose gradient centrifugation; however, the maximum permissible concentration in 0.1 M during Sephadex G-100 and G-200 chromatography. When NaSCN 0.1 M is added to low-salt cytosol (approximately 7 mg of protein/ml); (1) age-dependent aggregation of receptor is inhibited; (2) peaks of estrogen-binding activity in sucrose gradients and on Sephadex chromatography are sharp; (3) instead of the usual larger molecular states ("8S") found in low salt, most of estrogen receptor is under the following form: 4.1S; Stokes radius, 36 A; mol wt 61 000; flfo, 1.25; homogeneous at electrofocusing, with isoelectric point at 6.0. When cytosol containing NaSCN 0.1 M is diluted down to 2-3 mg of protein/ml or, only for sucrose gradients, NaSCN concentration is increased to 0.4-0.5 M, the 61000 dalton species decreases, being substituted, without loss of bound estradiol-t, by the following estrogen-binding entity: 28S; Stokes radius, 28 A; mol wt 32 000; flfo, 1.44. In the presence of NaSCN, KCl up to 0.4 M does not affect in a significant manner the molecular properties of the above forms. When NaSCN is dialyzed out, most receptor reverts to a 8-9S state. When cytosol is preincubated with Ca2+ (4 mM) and KCl (0.4 M) before addition of NaSCN, the above picture is modified only in the following aspects: (1) Sephadex chromatography peaks are broader and slightly but reproducibly shifted toward higher elution volumes; (2) the electrofocusing pattern consists of a two-peak heterogeneous band shifted toward higher pH (isoelectric points 6.4 and 6.6); (3) upon dialysis of NaSCN there is little or no reversion to faster sedimenting states. These modifications appear to depend on limited proteolytic attack of receptor by Ca2+ -activated receptor transforming factor (RTF), not on binding of Ca2+ to receptor. Present data suggest that the 4.1S entity is a dimer resulting from side-by-side pairing of 2.8S subunits. Molecular dimension of larger receptor forms purified from cytosol are consistent with the hypothesis that under native conditions in vivo dimers are coupled end-by-end into tetrameric structures with two stronger (between subunits) and two weaker (between dimers) bonding regions, and that tetramers may further self-associate. While NaSCN reversibly releases native dimers and subunits by direct impairment of intersubunit bonds, Ca2+ activated RTF irreversibly and specifically releases slightly modified, about 60000 mol wt dimers, by preferential proteolytic attack of the weaker bonding regions and indirect destruction of involved bonds. In vivo, this effect of RTF may be instrumental in mobilization and nuclear penetration of receptor-estradiol complex. Heteroassociation of receptor with other proteins of cytosol is not excluded by the above hypothesis.
[show abstract][hide abstract] ABSTRACT: The specific interaction between 17 beta-estradiol-receptor complex and nuclear acceptors was analyzed by immobilizing various nuclear proteins to CNBr-activated agarose. The specific, high affinity sites identified in a fraction of basic proteins that can be solubilized from purified nuclei of calf uterus (Puca, G.A., Sica, V., and Nola. E (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 979-983) were chromatographed on Sephadex G-100 columns. Elution of the acceptor activity depends on the pH and ionic strength of the buffer used. With 5 mM HCl, however, a peak of acceptor activity with a molecular weight of about 70,000 was partially dissociated from the other basic nuclear proteins. The high affinity binding of the receptor to the acceptor proteins was estradiol-, but not progesterone-, cortisone-, or testosterone-dependent; it was very sensitive to ionic strength and showed a physiological pH optimum. Low affinity binding, such as that seen between receptor and histone, showed no estradiol dependence and little ionic strength and pH sensitivity. Native or heat-denatured DNA strongly modified the receptor-acceptor interaction, reducing the number of binding sites of acceptor for the receptor without changing the high affinity of the interaction. Heating of the acceptor protein before its covalent linkage to agarose considerably increased the affinity of the resulting agarose derivative. Free sulfhydryl groups of the receptor but not of the acceptor molecule play an important role in the acceptor-receptor interaction. When receptor and acceptor preparations were incubated in solution, the resulting complex was included on a Sephadex G-100 column and it eluted from DEAE-cellulose columns at lower ionic strength than the receptor alone. Even though not absolutely specific, these two properties allowed determination of the molecular weight (85,000) of the acceptor protein at neutral pH and more nearly physiological ionic strength. The apparent KD of the acceptor-receptor interaction was determined to be 2 x 10(-10) M at O degrees. Apparently similar, high affinity binding sites for estradiol receptors are also present in nuclei of other tissues.
Journal of Biological Chemistry 09/1975; 250(16):6452-9. · 4.65 Impact Factor