Bruno Moncharmont

Estrogen receptors in cultured rabbit articular chondrocytes: Influence of age

Primary cultures of pubertal and prepubertal rabbit articular cartilage cells were performed. Total homogenates or cell extracts were used to determine the specific binding of 17 beta-estradiol. A comparative study was undertaken with tissue minces homogenized without enzymatic treatment. Scatchard analysis of cell or tissue extracts revealed the presence of a high-affinity receptor with Kd values of 0.55 +/- 0.16 nM and 0.12 +/- 0.03 nM in prepubertal and pubertal rabbit chondrocytes respectively. A significant difference in the affinity of estrogen receptor for its ligand as a function of age was observed. In contrast there was no significant difference in the number of binding sites expressed as fmol per mg DNA between the two age groups. The ligand binding specificity was as expected for an estrogen receptor and the sedimentation coefficient was 3.2 S when analyzed by ultracentrifugation on sucrose density gradient in presence of 0.4 M KCl and 8.1 S in low salt conditions. The binding sites, labeled with [125I]estradiol, were specifically immunoprecipitated by a monoclonal antibody to the estrogen receptor (JS34/32).

Phosphorylation of calf uterus 17β-estradiol receptor by endogenous Ca2+-stimulated kinase activating the hormone binding of the receptor

Abstract Direct evidence is presented that uterus 17 β -estradiol receptor is phosphorylated in , vitro by an endogenous kinase. Nuclear phosphatase, cytosol Ca 2+ -stimulated kinase (the former inactivating and the latter reactivating the hormone binding of the 17 β -estradiol receptor) and receptor were purified from calf uterus. 17 β -estradiol binding was inactivated by phosphatase, then reactivated by kinase in the presence of [ γ - 32 P] ATP, Ca 2+ and calmodulin, and the receptor was examined by various methods. The results of gel electrophoresis in non denaturating and denaturating conditions, and of centrifugation through sucrose gradients of receptor preincubated with monoclonal antibodies showed that the receptor is phosphorylated.

Binding of monoclonal antibodies to the nuclear estrogen receptor in intact nuclei

A monoclonal antibody to calf uterus cytoplasmic estrogen receptor shows a specifically displaceable and saturable binding to intact nuclei of mouse uterus after estradiol stimulation. The binding is complete after 3 hr at 0 degree C. The binding of the antibody correlates with the exchangeable estradiol binding activity of the nuclei over a 4-hr time course following in vivo injection of 17 beta-estradiol.

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.

Monoclonal Antibodies to Steroid Receptors

The original idea of the interaction of toxins and hormones with specific recognition sites on the cell was proposed at the beginning of this century by Paul Ehrlich (1900). Furthermore, he proposed that such sites were responsible for the physiological sensitivity and selectivity of the target cell. It was only two decades ago, however, that the existence of a specific site of interaction in the cells of target tissue for a steroid hormone, estradiol, was discovered (Glascock and Hoekstra, 1959; Jensen and Jacobson, 1962). In the following years this receptor was characterized (Toft and Gorski, 1966; Puca and Bresciani, 1969) and receptors for other steroid hormones were discovered. A receptor for androgen was observed in the rat ventral prostate (Baulieu and Jung, 1970; Fang et al., 1969; Mainwaring, 1969) and the presence of progesterone receptor established in guinea pig uterus (Milgrom et al., 1970) and chick oviduct (O’Malley et al., 1970). A receptor for the glucocorticoid hormones was described in thymus cells (Munck and Wira, 1971) and hepatoma cells (Baxter and Tomkins, 1971) and for the mineral-ocorticoids in the toad bladder (Sharp et al., 1966). Specific receptors for 1,25-dihydroxyvitamin D (Brumbaugh et al., 1975) and for the insect hormone ecdysone (Yund et al., 1978) were also discovered.