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E Milgrom

Publications and source records attributed to E Milgrom.

At least 199 records · Page 11Linked to original sources
PubMed1978

Factors modifying equilibrium between activated and non-activated forms of steroid-receptor complexes.

Steroid-receptor complexes formed in concentrated cytosol at low temperature, low ionic strength and neutral pH are unable to bind to nuclei. Various procedures are known to promote their 'activation'. In the present work it is shown that an increase in temperature only enhances the rate of the reaction whereas no change in the equilibrium between activated and non-activated complexes is observed. On the contrary an increase in ionic strength or pH, as well as a removal of a low-molecular-weight inhibitor, not only accelerate the reaction but also increase the concentration of activated complexes at equilibrium. Using two steroids differing 3-fold in their affinity for the receptor, no difference was seen in the effect of the bound steroid on receptor activation. When combining various activation procedures it was observed that they acted independently of each other and additively. In all cases they retained their property of either modifying only the rate of the reaction or both its rate and equilibrium. Using changes in pH, it was also possible to induce shifts in the equilibrium between activated and non-activated complexes. After activation at pH 6.5, a first equilibrium was attained. When the pH was increased to 8 the equilibrium was displaced towards higher concentrations of activated complexes. A lowering of the pH resulted in a reversal of steroid-receptor complexes from the activated to the non-activated state. To clearly establish that this was not due to irreversible damage of the receptor, which would render it unable to bind to nuclei, it was shown that the complexes which had reverted to the non-activated state were still susceptible to activation. Regulatory events may thus exist which, for a given level of hormone and receptor, modulate the concentration of activated steroid-receptor complexes.

Adrenalectomy↗
PubMed1978

Immunochemical studies of guinea pig progesterone-binding plasma protein.

An antiserum specific for guinea pig progesterone-binding plasma protein (PBP) has been prepared. Using a very sensitive immunoenzymatic assay, PBP could be detected not only in pregnant guinea pig plasma (970 microgram/ml of plasma at day 40--60 of pregnancy), but also in the plasma of fetuses (2.77 micrograms/ml), umbilical arteries (1.79 micrograms/ml), umbilical vein (2.90 micrograms/ml), and in amniotic fluid (0.47 microgram/ml). The protein was also found in low concentration in the plasma of nonpregnant females (2.10 micrograms/ml) and males (1.56 micrograms/ml). The antiserum was used to search for immunological similarities between various steroid-binding proteins. No cross reaction was found with cavian or human corticosteroid-binding globulin and human sex steroid-binding plasma protein. There was no cross reaction between guinea pig PBP and PBP of other pregnant hystricomorphs (viscacha, degu, and coypu). Moreover, no evidence was found of an interaction between guinea pig uterine progesterone receptor and the anti-PBP antiserum.

Alpha-Globulins↗
PubMed1978

Assay of unbound cortisol in plasma.

A method for measuring the unbound fraction of plasma cortisol which is suitable for clinical use is described. It is shown that unless some precise experimental conditions are fulfilled, important errors may result when determining the unbound fraction of the hormone. The assay is practical (2 ml of plasma are necessary) and reproducible (coefficient of variation: 2.7% within the same assay and 3.1% in different assays). Unbound cortisol measured at 0800 h in 86 healthy individuals was 9.7 +/- 2.6% (SD) of total cortisol and 15.0 +/- 8.5 ng/ml of plasma (temperature: 37 C). No significant difference was found between men and women or according to age. In most cases, variations of unbound plasma cortisol were more important than the variations of total plasma cortisol. This explains why unbound cortisol was a better discriminator in some pathological conditions. In Cushing's syndrome unbound cortisol was found to be increased on the average 2.9 fold whereas total cortisol was only increased by 53%. Unbound cortisol was especially high (24.7 +/- 3.8% of total cortisol and 78 +/- 18.5 ng/ml of plasma) in Cushing's syndrome due to adrenal carcinoma. In adrenal insufficiency, unbound cortisol averaged 6% of total cortisol and 1.4 ng/ml of plasma.

Addison Disease↗
PubMed1978

Characterization and assay of the progesterone receptor in rat uterine cytosol.

The synthetic progestogen R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) binds with high affinity (Ka = 8.8 X 10(8) l/mol at 0 degree C) to the progesterone receptor from rat uterine cytosol. At nanomolar concentrations, equilibrium is attained in less than 90 min. R5020 has a very low affinity for other specific steroid-binding proteins (corticosteroid-binding globulin and oestrogen receptors) present in relatively high concentrations in the urine cytosol. The affinity of the receptor for the natural hormone progesterone is remarkably low (Ka = 1 X 10(8)-1.7 X 10(8) l/mol at 0 degree C) which explains the instability of progesterone-receptor complexes. Advantage may be taken of this property to remove endogenous progesterone easily by charcoal treatment at 0 degree C, a treatment which does not modify the concentration of receptors. A method based on these characteristics is described for the assay of the total number (progesteron-bound and unbound) of receptor sites in uterine cytosol. This assay may be used in various physiological situations where endogenous progesterone is present at unknown concentrations.

Animals↗
PubMed1978

Progesterone receptors in the rat uterus: variations in cytosol and nuclei during the oestrous cycle and pregnancy.

The concentration of progesterone receptors in rat uterine cytosol and nuclei was measured during the oestrous cycle and pregnancy. The method used allowed the measurement of the total concentration of binding sites (unbound and hormone-bound). During the oestrous cycle, the concentration of receptors in the cytosol peaked at prooestrus, low concentrations were observed at oestrus and metoestrus, and in increase was seen at dioestrus. In the nuclei, maximum concentrations occurred at pro-oestrus and dioestrus. The number of receptors in the cytosol was very low during the first half of pregnancy, but the concentration increased progressively after day 15 to attain a very high level (about 26 000 binding sites/cell) on day 22. In the nuclei, the concentration of receptors was low at the beginning of pregnancy. On day 5 (day of implantation) there was a slight increase, which corresponded to a decrease in the number of cytosolic receptors and a small peak in the level of progesterone in the plasma. Maximum concentrations were attained during a 'plateau' period between days 9 and 15. Thereafter, there was very low; in some animals, probably on the verge of parturition, no receptors were detectable in the nuclei.

Animals↗
PubMed1977

Hormonal control of progesterone receptors.

Uterine progesterone receptors are under dual hormonal control. Estrogen increases the concentration of receptor through a mechanism that depends on synthesis of both RNA and protein. Progesterone decreases the concentration of its own receptor, probably by enhancing its inactivation rate. This regulation explains receptor variations during the estrous cycle. In both guinea pig and rat uteri, cytosol receptor concentration is maximal at the preovulatory period and decreases after ovulation. Nuclear receptor was measured in the rat. Its concentration is also maximal at proestrus, but the higher nuclear to cytosol receptor ratio was observed at metestrus. There is a good correlation (r = 0.78) between nuclear receptor concentration, on one hand, and the product of cytosol receptor concentration times the plasma progesterone concentration, on the other hand. Autoradiographic studies show that receptor variations during the estrous cycle occur simultaneously in all cell types of uterine horn, cervix, and vagina, which suggests that similar mechanisms control receptor concentration in all of these cells. Progesterone receptor was also measured during pregnancy in rat uterus. Cytosol receptor concentration is low at the beginning of pregnancy (approximately 6000 binding sites per cell), declines slightly on Day 5 (approximately 4000 binding sites per cell), and then increases progressively during the remainder of pregnancy to attain its highest value on Day 22 (26,000 binding sites per cell). Nuclear receptor concentration is very low on Day 3 (1200 binding sites per cell), increases slightly on Day 5 (1900 binding sites per cell), decreases on Day 6, and then increases again to attain a plateau between Days 9 and 15 (approximately 2600 binding sites per cell). Thereafter, its concentration begins to decrease rapidly. On Day 22, the mean concentration is very low (700 binding sites per cell); in some animals (probably on the verge of parturition), no nuclear receptor can be detected.

Animals↗
PubMed1977

A low molecular weight inhibitor of steroid receptor activation.

Dilution at 0 degrees of rat liver cytosol incubated with [3H]triamcinolone acetonide provoked an enhanced binding of steroid-receptor complexes to nuclei. The explanation of this phenomenon was found to be an "activation" of the complexes. Dilution acted by decreasing the concentration of a cytosol inhibitor. This reaction was irreversible at 0 degrees: once activated the complexes could not be reversed to the nonactivated state by the addition of inhibitor. The presence of hormone was necessary, since hormone-free receptor molecules could not be activated by dilution. Removal of the inhibitor did not lead to activation of all complexes: after 24 h a "plateau" was attained where 55 to 70% of the complexes were activated. The inhibitor was shown to be a low molecular weight molecule by dialysis, Sephadex G-25 chromatography, ammonium sulfate precipitation, and ultrafiltration. Thus [3H]triamcinolone acetonide-receptor complexes present in a cytosol from which the inhibitor had been removed by Sephadex G-25 chromatography became spontaneously activated at low ionic strength and at 0 degrees. The inhibitor is not a steroid (at least of usual polarity) since it cannot be extracted by methylene chloride or adsorbed by activated charcoal. It is thermostable (resists to 30 min at 100 degrees). Its removal by incubation with a cation exchange resin suggests that it may be positively charged, however it is not complexed by EDTA. This inhibitor must be distinguished from a previously described inhibitor of steroid-receptor complexes binding to nuclei. The latter compound has been shown in various systems to be responsible for an artifactual saturation of nuclear acceptor by steroid-receptor complexes. It inhibits the binding to nuclear acceptors of already activated complexes and is probably a macromolecule. It is thus different from the low molecular weight activation inhibitor described in the present paper.

Animals↗
PubMed1977

Radioautography of the uterus and vagina after [3H]progesterone injection into guinea pigs at various periods of the estrous cycle.

Radioautograms were made of uterine horns, cervix and vagina from guinea pigs injected with[3H]progesterone at various periods of the estrous cycle. The concentration of silver grains peaked at proestrus and estrus and then fell during metestrus, attaining a minimum at diestrus. These variations were not due to masking of binding sites by endogenous hormone since ovariectomy of guinea pigs at diestrus did not change this pattern. During proestrus and estrus, radioactivity was found in all parts of the endometrium and myometrium of the uterine horns. However, the muscle fibers and the cells of the stroma contained more silver grains than did the luminal and glandular epithelium. In the vagina, radioactivity was concentrated mostly in the nuclei of the basal layer cells of the stratified epithelium. During metestrus and diestrus, less radioactivity was present in these regions. These differences in labelling were observed simultaneously in all the uterine and vaginal cell types, suggesting that similar hormonal mechansims may control receptor variations in these different target cells.

Animals↗
PubMed1977

Characterization and assay of progesterone receptor in human mammary carcinoma.

[3H]Pregn-4-ene-3,20-dione ([3H]progesterone)-receptor complexes from human mammary carcinoma were found to be stabilized in the presence of glycerol. The dissociation rate constant was lowered and the equilibrium dissociation constant was decreased (KD=3 nM in the absence of glycerol and 1.1 nM in the presence of 30% glycerol), whereas no clear-cut effect on the association rate was observed and no change occurred in the concentration of binding sites. Cortisol was found to compete with [3H]progesterone only at concentrations higher than 1 muM. This made it possible to distinguish [3H]progesterone binding to the receptor from binding to corticosteroid-binding globulin. Synthetic progestins [6-chloro-17-acetoxypregna-4,6-diene-3,20-dione (chlromadinone acetate), 17alpha-ethinyl, 17-hydroxyestr-4-en-3-one (norethisterone), and 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione (R5020)] were found to have a high affinity for the receptor, whereas 5alpha-pregnane-3,20-dione had an affinity about one-half that of progesterone itself 5beta-Pregnane-3,20-dione, 17alpha-hydroxypregn-4-ene-3,20-dione (estradiol), 11beta,21-dihydroxy-pregn-4-ene-3,20-dione (corticosterone), estra-1,3,5(10)-triene-3,17beta-diol, and 17beta-hydroxyandrost-4-en-3-one (testosterone) were weak inhibitors of [3H]progesterone binding. Sedimentation on glycerol gradients showed different patterns in different tumors; i.e., [3H]progesterone specific binding having the characteristics of receptor was found either in the 8 S region, in the 4.5 S region, or in both. Activated progesterone-receptor complex from human mammary carcinoma cytosol was shown to bind to human DNA. An assay of the receptor based on these binding properties is described. This assay measures the total concentration of cytosol receptor since it makes possible the exchange of endogenous hormone for excess added [3H]progesterone. Of 55 biopsies examined by this method, 35 (64%) had a concentration of progesterone receptor-binding sites higher than 10 fmoles/mg protein. There was a positive correlation between the amounts of estrogen and progesterone receptors.

20-alpha-Dihydroprogesterone↗
PubMed1976

Interaction of rat-liver glucocorticoid receptor with DNA.

The complex of [3H]dexamethasone and rat liver receptor binds to rat liver DNA. This interaction takes place only in the presence of hormone and is enhanced by 'activation'. No evidence of saturatability can be obtained with concentrations of steroid-receptor complexes corresponding to those observed physiologically in the intact liver cell. The binding is inhibited by high ionic strength and by millimolar concentrations of divalent cations. No species specificity has been observed: the complex binds equally well to prokaryotic and eukaryotic DNA'S. There was no difference between binding to native and denatured DNA. In comparable conditions twice as much [3H]dexamethasone-receptor complexes were bound by DNA than by rat liver nuclei. Thus, the interaction of steroid-receptor complexes with DNA probably does not correspond to the recognition of a few very specific sequences. It is however possible that this interaction is actually operating in vivo in the intact cell.

Animals↗
PubMed1976

Chromatographic separation on phosphocellulose of activated and nonactivated forms of steroid-receptor complex. Purification of the activated complex.

Steroid-receptor complexes formed at low temperature and ionic strength are unable to bind to target cell nuclei. After a temporary exposure to high ionic strength and/or temperature they become activated (i.e., able to bind to nuclei). However, there exists an equilibrium between activated and nonactivated complexes; thus, mixtures of both populations are obtained. In this paper it is shown that activated [3H]triamcinolone acetonide-rat liver receptor complexes bind strongly to phosphocellulose, whereas nonactivated complexes do not. Thus, it is chromatographically possible to isolate these two populations of complexes. The experimental conditions of the separation have been established. The most important feature is that upon prolonged exposure to phosphocellulose, nonactivated complexes become progressively activated. The separation on phosphocellulose has at least three potential applications. A first application is the possibility of measuring the concentration of activated complexes in incubates. However, when activated complexes were titrated with rat liver nuclei in excess or assayed through binding to phosphocellulose, slightly different results were obtained. This discrepancy was due on one hand to the difficulty of obtaining binding of all the activated complexes and on the other hand to the second activation of some of the complexes during their exposure to phosphocellulose. A second application was the possibility of obtaining a homogeneous population of activated complexes. This was actually achieved, since the complexes eluted from phosphocellulose were demonstrated to be 90--100% activated. The use of such homogeneous preparations simplifies considerably studies on binding of steroid-receptor complexes to nuclear acceptors (nuclei, chromatin, DNA). A third application is the use of phosphocellulose for the purification of receptor. Cytosol containing nonactivated complexes was filtered through phosphocellulose the complexes present in the breakthrough of the column were then activated and bound to phosphocellulose in a second chromatography. Advantage was also taken of the "amphoteric" behavior of the receptor that binds to both anionic (phosphocellulose) and cationic (diethylaminoethylcellulose) resins. Purification (940fold) with 24% yield could be obtained in preparations taking less than 2 days. The partially purified receptor was a heavy aggregate (greater than 12 S) that could be dissociated into 4S subunits by exposure to 0.3 M K C1. It has kept its property of interacting with nuclear acceptor. Preliminary experiments have shown that this technique could be of general application for steroid hormone receptors: activation enhanced binding to phosphocellulose of progesterone, aldosterone, and estradiol receptors.

Animals↗
PubMed1976

Mechanism and kinetics of the thermal activation of glucocorticoid hormone receptor complex.

Steroid-receptor complexes formed at low temperature and ionic strength do not bind to nuclei or chromatin. After a temporary exposure to high temperature, or ionic strength, or both, a fraction of them becomes activated (able to bind to nuclei). An assay of the activated form of the complex based upon titration with nuclei in excess was established. This assay was used to perform kinetic and equilibrium studies of the thermal activation of glucocorticoid-receptor complex in order to elucidate its mechanism. It was found that the reaction is of apparent first order and yields a monomolecular product. It thus probably consists of a conformational change in the steroid-receptor complex. The rate of activation is 1.37 +/- 0.06 X 10(-3) S-1 at 25 degrees. The free energy of thermodynamic activation (The word activation is used here in its usual thermodynamic meaning and not in the sense of receptor modification) of this reaction is greater than G = 21.3 Kcal. The corresponding enthalpy and entropy are respectively greater than H = 31.4 kcal and greater than S = 4 cal/degree. These positive and high values of greater than H and greater than S are very similar to those described for denaturation reactions of proteins suggesting that breakage of some noncovalent bonds could take place during activation. The reaction proceeds until approximately 60% of the complexes are activated. It was shown that this corresponds to an equilibrium between activated and nonactivated forms and not to the presence of a population of complexes unable to undergo activation. This equilibrium is not modified by temperature variations between 10 degrees and 30 degrees. It is possible to activate over 80% of the complexes when the activation is performed in the presence of excess acceptor, thus shifting the equilibrium. A similar situation is probably observed in situ in cells since 90% of the complexes are found in the nuclei when liver slices are incubated with hormone.

Animals↗
PubMed1976

Interaction of uteroglobin with progesterone, 5alphapregnane-3,20-dione and estrogens.

Uteroglobin was obtained from 5 day pregnant rabbits and purified to homogeneity by Sephadex G 75 and DEAE-cellulose chromatographies. Progesterone binding to uteroglobin was decreased by lyophilization and enhanced by SH-reducing agents. Dithiothreitol was more effective than dithioerythritol, and beta-mercaptoethanol was only active at 25 to 100 mM concentrations. SH-blocking agents (iodoacetate, iodoacetamide, phydroxymercuribenzoate and, dithiobisnitrobenzoic acid) inhibited binding. In the absence of SH-reducing agents only one in every 500 uteroglobin molecules bound the hormone, whereas under optimal conditions (20 mM dithiothreitol) one in every two molecules bound progesterone. There was no significant difference in equilibrium dissociation constants under these two conditions. Uteroglobin had a relatively high affinity for progesterone (KD=4.1 X 10(-7)M) but a threefold higher affinity for 5alpha-pregnane-3,20-dione (KD=1.3 X 10(-7)M). Estradiol was bound but non-specifically with a very low affinity, and its binding was not enhanced by SH-reducing agents. Hormonal specificity of binding to uteroglobin was different from that of binding to rabbit uterine progesterone receptor. Various synthetic progestagens (chlormadinone acetate, norethisterone, R5020) were bound to the latter but not to the former protein. Diethylstilbestrol had some affinity (15% of that of progesterone) for uteroglobin and no affinity for the progesterone receptor. Uteroglobin incubated in the presence or absence of cofactors (NADH and NADPH) with or without dithiothreitol did not metabolize progesterone.

Androgens↗