Giovanni Alfredo Puca

Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor

A new panel of monoclonal antibodies to the calf uterus estrogen receptor was prepared. Thirteen antibodies were characterized for their isotype and for the affinity for the antigen. These antibodies recognize the human receptor and can be used in Western blot analysis. The location of the epitopes was mapped on the antigen structure using synthetic fragments of estrogen receptor, and it was possible to group the antibodies in five groups. Many antibodies were useful for the purification of estrogen receptor from tissue extracts by immunoaffinity chromatography. The reciprocal inhibition of the antibodies for the antigen binding was measured with an immunoadsorption assay. This was maximal and symmetrical for antibody pairs within the same group, but was incomplete and, in some instances, asymmetrical between pairs of antibodies from different groups. One antibody was able to inhibit the estrogen receptor-DNA interaction, whereas two others were unable to recognize the receptor-DNA complexes. This new panel of antibodies is a useful addition to the existing tools for studying structure and function of the estrogen receptor.

Identification of specific high affinity sites for the estradiol receptor in the erythrocyte cytoskeleton

Abstract The possible relationship of the soluble, “cytosolic” estradiol receptor with complex membranous and cytoskeletal structures of the cell matrix has been studied using a model erythrocyte system. Extraction of erythrocyte ghosts with a nonionic detergent (Triton X-100) under conditions that yield a cytoskeletal matrix reveals the presence of a limited number (less than 100) of specific sites having high affinity (k d 10 −9 M) for the estradiol-receptor complex. The interation between the estradiol receptor and the cytoskeleton is critically dependent on temperature and it is improved by 25 mM KCl or NaCl and by 2.5 mM MgCl 2 . The data suggest that the estradiol receptor, which has been generally considered to be freely “soluble” in the cytoplasm, may actually be physiologically associated in an integral manner with a complex cytoskeletal network in the cell cytoplasm.

Effect of sodium molybdate on cytosolic estrogen receptor

Estrogen binding activity of crude calf uterus cytosol is rapidly destroyed in heating. The time course of inactivation at 37 degrees C shows a biphasic pattern; sodium molybdate (5-10 mM) completely blocks one of the components in the estradiol-free cytosol, while it has little effect on cytosolic receptor complexed with estradiol. Partially purified native 8S receptor loses its heat sensitivity, and, as a consequence, the molybdate effect disappears. By sucrose gradient analysis of crude cytosol it is evident that molybdate does not affect the sedimentation properties of the estradiol receptor at low temperature. However, at increasing temperatures, molybdate prevents the disappearance of the receptor peak in the crude cytosol or the formation of large, KCl-resistant, aggregates in the presence of estradiol. The partially purified native 8S receptor does not aggregate on heating; addition to it of receptor-depleted cytosol results in the recovery of heat inactivation and aggregate formation, and this is prevented by molybdate. Molybdate has no protective effect on any other inactivating agent which does not act through aggregation of receptors. A crude cytosolic preparation of the receptor which is unable to form heat-dependent aggregates does not display the fast heat inactivating component.

Estrogen receptor of calf uterus: An easy and fast purification procedure

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 MgCl 2 . 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 MgCl 2 , during the purification procedure decreases the aggregation phenomenon.

[86] Estrogen receptors

Publisher Summary Estrogen receptor proteins exist in at least three different molecular forms (8.6 S, 5.3 S, and 4.5 S) and are present in the cytoplasm of target tissues in very low amounts. Attempts to purify these relatively labile proteins by conventional methods have resulted only in modest purification and recovery has been poor. The reversible nature of the estradiol-receptor interaction together with its stereochemical specificity and high affinity, i.e., a dissociation constant about 10 -9 M at 4°, suggests that the receptor proteins may be ideally suited for purification by affinity chromatography. Although it has been reported that columns containing estradiol linked to benzyl cellulose and to polyvinyl-(N phenylenemaleimide) can remove estradiolbinding activity from solutions of uterine cytosol, it was not possible to subsequently elute specific estrogen-binding proteins from these columns.

Purification and properties of native oestradiol receptor.

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.

Interaction of calf uterus estradiol receptor with erythrocyte cytoskeleton

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 ( K d ⋍ 10 −9 M ) 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.

Highlighting chromosome loops in DNA-picked chromatin (DPC)

Growing evidence supports the concept that dynamic intra- and inter-chromosomal links between specific loci contribute to the creation of cell-type specific gene expression profiles. Therefore, analysis of the establishment of peculiar functional correlations between sites, also distant on linear DNA, that govern the transcriptional process appears to be of fundamental relevance. We propose here an experimental approach showing that 17β-estradiol-induced transcription associates to formation of loops between the promoter and termination regions of hormone-responsive genes. This strategy reveals as a tool to be also suitably used, in conjunction with automated techniques, for an extensive analysis of sites shared by multiple genes for induced expression.

[30] Purification of estrogen receptors. I

Publisher Summary The word “receptor” was originally used by Ehrlich to indicate a cell component able to recognize and interact with a specific molecule of the cellular environment and included the concept that the informational content of the incoming molecule was expressed through the interaction with its cellular receptor. This full and original meaning of the word still holds among pharmacologists. Therefore, when speaking of estrogen receptors, one must preliminarily state that this term refers to cellular components. There is no definite information yet as to whether these proteins also fulfill the second part of Ehrlichs proposition—that is, whether the estrogenic ligand acts by inducing some modification of these proteins which, in turn, enables them to activate the cell machinery to produce the final estrogen effects. Thus, estrogen receptors are used here not in the strictest sense of the word but as synonymous with high affinity, cellular estrogen-binding proteins (EB-proteins).

Three protein kinases from calf uterus. subcellular distribution and physical properties

Abstract Three protein-kinases which phosphorylate preferentially either histones (kinase a ), protamine (kinase b ) or casein (kinase c ) were separated from calf uterus cytosol. Kinase a weights 120,000, sediments at 6 S and shows an isoelectric point (I.P.) of 5.0. Kinase b weights 65,000, sediments at 4.7 S and shows an I.P. of 5.5. Kinase c weights 200,000, sediments at 7.0 S and shows an I.P. of 6.0. Only kinase a binds, and is stimulated by, 3′,5′-cyclic AMP.