Competitive interactions between corticosterone and 11-dehydrocorticosterone in binding to receptors in fetal mouse tissues

The binding of ³H-corticosterone and ³H-11-dehydrocorticosterone to receptors in cytosol and nucleus was examined in fetal mouse brain and placenta using Sephadex gel filtration or charcoal to separate bound and unbound steroid. In the cytosol, competitive displacement of each steroid by the other was observed. The binding was unaffected by RNase, DNase, dithiothreitol or N-ethyl maleimide but was diminished by Pronase. Nuclei were isolated by hypotonic shock using dilute MgCl₂ and the steroid receptor-complexes of both steroids were obtained from the nuclear sap. Receptor-complexes of both steroids were observed in brain and placental tissues. Competitive displacement of each steroid by the other was also observed in nuclear binding. Both 11-dehydrocorticosterone and 11-deoxycorticosterone bound to a chromatin fraction as did the hormone corticosterone. Identity of the steroids was established by using chromatography and co-crystallization techniques. This work raises the possibility that in the fetal mouse, 11-dehydrocorticosterone, previously considered biologically inactive and an abundant metabolite in fetal mouse tissues, may in fact play a more positive role in regulation.     

Characterization of a unique corticosterone-binding protein in Candida albicans

This paper further characterizes a protein we have demonstrated in Candida albicans which has the ability to bind corticosterone and related steroid hormones. Fungal cells are disrupted and cytosol is incubated with [3H]corticosterone for 3 h at which time peak steady state binding is achieved. Bound hormone is separated from free using Sephadex G-50 minicolumns or dextran-coated charcoal. Binding was found to be a linear function of protein concentration. The bound hormone co-migrates with authentic corticosterone in thin layer chromatographic systems indicating no metabolism of the radioprobe. Scatchard analysis of the binding in the pseudohyphal form of C. albicans yielded values of 6.3 nM for the Kd and a binding capacity of about 650 fmol/mg of cytosol protein; both determinations are comparable to our findings in the yeast form of this organism. A series of sterols were tested for their ability to displace [3H]corticosterone from the yeast binder, and the results show that the binder is remarkably selective and stereo specific. Physical-chemical studies show the binder to be degraded at high temperatures and that binding is destroyed by trypsin and sulfhydryl blockers. The protein sediments at 4 S on sucrose gradients and does not exhibit ionic dependent aggregation. The molecular weight is estimated to be approximately 43,000 daltons by gel chromatography. We hypothesize that this intracellular protein may represent a primitive form of either the mammalian glucocorticoid receptor or the plasma corticosteroid-binding globulin.     

Interaction of testosterone, corticosterone and corticosterone binding globulin in the white-throated sparrow (Zonotrichia albicollis)

Plasma binding globulins bind steroid hormones and are thought to regulate hormone access to tissues. Mammals have both sex steroid binding globulin (SSBG) and corticosteroid binding globulin (CBG). Birds, however, have no detectable SSBG, leading to the early conclusion that birds have no plasma regulation of sex steroids. CBG, however, can bind androgens with relatively high affinity. In birds, therefore, the control of androgenic effects may be tightly regulated by glucocorticoid physiology because glucocorticoids compete with androgens for CBG binding sites. We report levels of total testosterone (T), total corticosterone, CBG, and estimated free T in the males, the more aggressive morph had higher levels of total T; female morphs did not differ. Approximately 96% of T was bound to CBG, but a lack of morph or sex-specific differences in corticosterone titers or CBG capacity caused patterns of free T to mirror those of total T. While CBG has the potential to greatly influence T availability to tissues, in this species interactions between T, CBG and corticosterone do not appear to alter general patterns of T availability to tissues.     

Radioligand assay for 5 -3 -hydroxysteroids. I. 3 -Hydroxy-5-androstene-17-one and its 3-sulfate

A specific, sensitive and reliable radioligand assay for plasma dehydroepiandrosterone (DHA) and its sulfate has been developed. Antisera were obtained by immunizing rabbits with a DHA-17-albumin conjugate. DHA was separated from cross-reacting Δ⁵-steroids by thin layer chromatography. DHA-sulfate was solvolyzed prior to chromatography. Separation of antibody bound and free steroid was achieved with γ-globulin-dextran-coated charcoal. The standard curve was linear on a logit-log plot from 0.1 to 10 ng.     

Distribution of free and membrane-bound ribosomes after corticosterone administration in perfused rat liver

Contrasting results have been reported on the effect of steroid hormones on the interaction between ribosomes and endoplasmic reticulum in rat liver. Exposure to high gravitational forces for a long time was found necessary to obtain a constant ratio of free to membrane-bound ribosomes from the post-nuclear supernatant. Using these isolation conditions, rat liver from fasted controls and from fasted, adrenalectomized rats contain both about 45% membranebound ribosomes. Addition of corticosterone to the livers from adrenalectomized rats did not increase the pool of bound ribosomes more than in the control livers; 55% was found in both. Corticosterone had therefore no effect on the pool sizes of free and membrane-bound ribosomes in perfused rat livers.     

Rates of dissociation of steroid and thyroid hormones from human serum albumin

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure rates of dissociation of steroid and thyroid hormones from human serum albumin. Modification of a previously described assay allowed measurements at 1-s intervals. Nevertheless, this still permitted only minimum estimates of the dissociation rate constants. The hormones studied were thyroxine, 3,5,3'-triiodothyronine, cortisol, corticosterone, testosterone, dihydrotestosterone, estradiol, progesterone, and aldosterone. The apparent dissociation rate constant of the thyroxine-albumin complex at 37 degrees C was 1.3 +/- 0.2 s-1 (t 1/2, 0.5 s). The apparent dissociation rate constants of the other hormone-albumin complexes at 37 degrees C generally exceeded 2 s-1 (t 1/2 less than 0.35 s). Apparent dissociation rate constants at 4 degrees C were only slightly lower. These findings indicate that steroid and thyroid hormones dissociate from albumin rapidly compared with the 1-s capillary transit times that characterize many tissues.     

Rapid,high temperature exchange assay for the hepatic glucocorticoid receptor

Summary Liver cytosol from adrenalectomized rats was prebound for 2 hr at 4°C with unlabeled 10^–5 M corticosterone. After treatment of cytosol with dextran-coated charcoal to remove free steroid, samples were incubated at 15–25°C in the presence of 10 mM molybdate plus 5 mM dithiothreitol (followed by a 60 min incubation at 4°C). Essentially, complete exchange of [³H] dexamethasone for receptor-bound unlabeled steroid was observed after 120 min at 15°C, and near complete (80–95%) exchange occurred within 60 min at 25°C using these conditions. However in control, 5 mM dithiothreitol (alone) and 10 mM molybdate (alone) treated samples, less than 50% exchange was found. Using a similar protocol, only partial exchange was found in brain and kidney cytosols, suggesting at least partial specificity for the hepatic system. We have used this rapid, high temperature exchange assay to study the regulation of hepatic cytoplasmic glucocorticoid receptors under some experimental conditions.     

A highly specific radioimmunoassay for progesterone using antibodies covalently linked to arylamine-glass particles

Antibody was produced in sheep following the administration of the antigen, progesterone-11α-succinyl-BSA. After treatment with Rivanol, the protein of the albumin-free antiserum was covalently bound to arylamine glass particles using glutar-aldehyde as the coupling agent. This antibody-glass preparation was used for measurement of plasma progesterone by radioimmunoassay. Progesterone was extracted from human plasma with petroleum ether. Of those Steroids which are petroleum ether extractable, only pregnenolone exhibited minimal cross-reactivity. Immobilization of the antibody by attachment to an insoluble support allows the separation of free from antibody-bound steroid without the use of absorbents, such as florisil and charcoal.     

Efficacy of five methods for the bound-free separation of gluco- and mineralocorticoids from type I,II and III receptors found in HEPES- and TRIS-buffered mouse brain cytosol.

We compared the efficacy of G-25 and LH-20 column chromatography, dextran-coated charcoal adsorption, and DEAE-cellulose and glass fiber filter disc assays to separate unbound steroids from three classes of brain cytosolic receptors prepared in HEPES and TRIS buffers and labeled selectively as follows: Type I = [³H]aldosterone + unlabeled RU26988, Type II = [³H]triamcinolone acetomide and Type III = [³H]corticosterone + unlabeled Prorenone and RU26988. Prorenone and RU26988 were added to reduce unwanted [³H]steroid binding to Type I and Type II receptors, respectively. In each case total, non-specific and specific binding and free steroid were compared individually. No single assay was found to be best for all three receptor classes, but both buffers and most assays could be used with appropriate correction factors. Variations between the results with different assays suggest fundamental differences between the three classes of adrenosteroid receptors and their ligands.     

High affinity progesterone binding sites of human uterine microsomal membranes

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.     

竹炭固定化微生物对水中壬基酚的降解效率

竹炭是一种优质生物质炭,不仅比表面积大,孔隙发达,而且机械强度高,是微生物固定化载体的最佳选择之一.本文采用正交试验确定了竹炭固定化微生物的最佳制备条件,对比了竹炭固定菌和游离菌对水中类雌激素壬基酚的降解效果,并考察了竹炭固定菌的重复利用性.结果表明:固定化后降解菌大量地附着在竹炭表面及内部孔隙中,其最佳制备条件为温度30℃、pH=7、竹炭粒径35目.壬基酚的降解符合一级动力学方程,在不同的壬基酚初始浓度下(30、50、80、100 mg·L^-1),竹炭固定菌对壬基酚的7 d降解率分别为100%、75.3%、67.3%和78.7%,显著优于游离菌(54.2%、51.5%、30.6%和23.5%).经过8轮重复利用后,竹炭固定菌对壬基酚的降解率仍可达到36.5%,而此时游离菌的降解率仅为8.9%,说明竹炭固定菌具有长期可重复利用性,在去除废水有机污染物中具有较好的工程应用前景.     

Increase of ionic strength for charcoal separation of B/F fractions in radioimmunoassays

The increase in protein adsorption by charcoal as ionic strength increases (salting-out adsorption), was used to separate the bound and free fractions of glucagon, insulin, hGH, hLH and hPRL in the radioimmunoassay. The hormones were labelled with 125I and to express the immunocomplex, gamma-globulin was labelled with 125I. The charcoal used to produce the separation was suspended in magnesium sulfate 3 M (charcoal-SO4Mg). The optimum amount of charcoal and the final concentration of magnesium sulfate determined for each hormone were: glucagon (charcoal 5 mg/tube, 0.125 M); insulin (charcoal 5 mg/tube, 0.131 M); hGH (charcoal 40 mg/tube, 0. 447 M); hLH (charcoal 40 mg/tube, 0.447 M) and hPRL (charcoal 60 mg/tube, 0.321 M). The serum concentration was 1/20 for all hormones, excepting glucagon, where 1/10 was used. The stability of the immunocomplex was studied and it was shown that, under suitable conditions, increased ionic strength does not cause the dissociation of the bound fraction.     

An exchange assay for the cytoplasmic glucocorticoid receptor in the liver of the rat

Cytosolic receptor for glucocorticoids can exist in either the free or bound form; assays now in use measure only the free form. In order to assay the total glucocorticoid receptor content of rat liver, free plus bound, we have developed an exchange assay wherein specifically bound [³H]dexamethasone is shown to be a valid measure of receptor in the presence of high concentrations of corticosterone. The exchange between [³H]dexamethasone and corticosterone is able to proceed because, under the conditions of the assay, corticosterone is almost completely metabolized.     

Decomposition of 11-deoxycorticosterone and corticosterone in soda-lime glass.

When methanolic solutions of tritiated 11-deoxycorticosterone or corticosterone (0.1 to 300 ng) were evaporated to dryness with a stream of nitrogen in soda-lime test tubes only 8-24% of the radioactivity was recovered as the parent steroid. Evaporation in borosilicate test tubes led to a recovery of 90% or more. With ethyl acetate as solvent no decomposition occured in soda-lime test tubes.     

The effect of exposure to charcoal and ion-exchange chromatography on the dissociation rate of estrogen from the nuclear estrogen receptor of hen oviduct

The method of initiating dissociation of 3H-estradiol from the nuclear estrogen receptor of hen oviduct was found to have a profound effect on the dissociation rate. Likewise, prior exposure to charcoal or partial purification by ion-exchange chromatography had an effect on the dissociation rate. When the reaction was initiated by isotopic dilution with the addition of 1 microM unlabeled estradiol, dissociation of the complexes was rapid (t 1/2 approximately 3 min). When the reaction was initiated by the addition of charcoal to adsorb free steroid, the dissociation of the complexes proceeded slowly (t 1/2 approximately 30 min). Partial purification of the receptors by DEAE-Sephacel chromatography or 15 min exposure to charcoal at 0 degree C prior to initiation of the dissociation reaction by isotopic dilution produced a form of the receptor that exhibited an intermediate dissociation rate (t 1/2 approximately 10 min). The partially purified receptor that exhibited an intermediate dissociation rate was reconverted to the rapidly dissociating form in a reconstitution experiment. These data raise the possibility of a nuclear substance that regulates the rates of estrogen dissociation.     

A comparison of the fatty acid esters of estradiol and corticosterone synthesized by tissues of the rat

The biosynthesis of the fatty acid esters of the corticoid (corticosterone) and estrogen (estradiol) was compared in parallel incubations of corticosterone and estradiol with several tissues of the rat. The fatty acid composition of the esters of the two steroids was characterized in mammary and uterine tissue. In both of these tissues, the esters of estradiol were extremely heterogeneous. To the contrary, in the same tissues only one predominant ester of corticosterone, corticosterone-21-oleate, was formed. It comprised 70-80% of the total. The oleate ester of estradiol accounted for only 20% of the esters of this estrogen. In addition, fatty acid esters of an A-ring reduced metabolite of corticosterone, 5 beta-dihydrocorticosterone, was also identified. Its fatty acid composition is identical to that of corticosterone. In other experiments the fatty acid esters of both steroids were isolated from several tissues and quantified. When the amount of steroidal ester formed was compared, there was over a 100-fold difference among the various tissues in the ratio of estradiol to corticosterone ester synthesized. Thus, the rate of synthesis of the fatty acid esters of each class of steroid varies dramatically from tissue to tissue, and their fatty acid composition differs markedly as well. If the same enzyme synthesized both the estrogen and corticoid esters, then it would be expected that the relative amount of both esters synthesized in various tissues should be constant and likewise that their composition should be the same. Since neither occurred, these results suggest that the enzyme which produces the C-17 fatty acid esters of the estrogens may be different from the one which synthesizes the C-21 esters of the corticoids. The existence of separate enzyme systems for the synthesis of the fatty acid esters of these steroid hormones opens the possibility of specific physiological controls of each of these unusual steroidal metabolites.     

Effects of copper on cytoplasmic and nuclear binding of 17 beta-estradiol in the rat uterus

Interference of Cu++ with the initial events in estrogen action was tested by determining Cu++ effects on estradiol-receptor interactions. When immature rat uteri were incubated in vitro with [3H] estradiol ([3H]E2), steroid was bound in cytoplasmic fractions and rapidly accumulated in the nuclear fraction in a manner which was dependent upon time and hormone concentration. Uteri which were preincubated with 2 X 10(-4) M CuCl2 for 40-60 min and then exposed to [3H]E2 were found to have a 30-50% decrease in the amount of steroid bound in the cytoplasmic and nuclear fractions. When copper-treated uteri were exposed to [3H]E2 for variable times, the quantity of steroid bound in the cytoplasmic fraction was markedly depressed and the rate of nuclear accumulation of [3H]E2 was significantly decreased. These results show that Cu++ can inhibit [3H]E2 binding to tissue cytoplasmic receptors in vitro and thereby interfere with hormone delivery to target cell nuclei.     

Studies on the mechanism of secretion of rat adrenal steroids in vitro

The possibility that in the rat adrenal cortex synthesis and secretion of steroid hormones may be distinct. processes has been studied using in vitro methods.The uptake and subsequent release of radioactive corticosterone and 18-OH-DOC added to incubation media showed little difference between the two steroids. Both were taken up rapidly by the tissue, and after exchange of medium, 50% was released within 15 min. The properties of muscle and adrenal tissue were qualitatively similar in these respects, and were consistent with steroid movement by diffusion.Different results were obtained when steroids formed from the tissues' endogenous precursor supply were studied. Following incubation for 2h tissue and medium steroid cohtent were measured, and tissue/medium ratios of steroid were used as an index of secretion. With varying volumes of incubation medium (1.4–10 ml), secretion of corticosterone was related to volume, although synthesis was constant. With 18-hydroxydeoxycorticosterone (18-OH-DOC) on the other hand neither synthesis nor secretion was affected by medium volume.The effects of ouabain (10⁻⁴ M) on synthesis and secretion were also studied, using a 5 ml volume of medium. Under these conditions, tissue levels of corticosterone were very low, while 18-OH-DOC was extractable from tissues in amounts up to 30% of the total synthesised. In capsules, steroid synthesis was decreased by ouabain, or by a lower potassium medium (3.6 mM) or both together. Secretion of corticosterone was not affected by these treatements, whereas secretion of 18-OH-DOC and aldosterone was greatly inhibited: maximally 50% of the synthesised 18-oxygenated steroid was retained within the tissue. These treatments also affected inner zone function, ouabain inhibited steroid synthesis, but the low potassium medium did not. Secretion of corticosterone was unaffected, whereas secretion of 18-OH-DOC was again inhibited both by low potassium medium and by ouabain. Maximally as in the capsules, 50% of the synthesised 18-OH-DOC was retained within the tissue.The results suggest that the secretion of 18-OH-DOC formed from endogenous precursors by rat adrenal tissue is controlled by a mechanism involving a ouabain sensitive process in the cell membrane. Corticosterone is released by a different mechanism, quite possibly passive diffusion.     

Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.     

A method for the isolation of lipid droplet fractions from decapsulated rat adrenals

Ultrastructural and cell fractionation studies implicate lipid droplets in the storage of cholesterol and in the secretion of steroids. To evaluate the role of the lipid droplet in steroidogenesis, a discontinuous gradient centrifugation method has been developed for the isolation of both lipid droplet and non-lipid fractions from decapsulated rat adrenal homogenates. Steroids were extracted from the fractions with chloroform/methanol; the cholesterol ester, cholesterol and corticosterone in each extract were purified using a single chromatogram and the purified steroid and sterols were assayed fluorometrically. The lipid droplet fraction contained 85% of the esterified cholesterol and 32% of the free cholesterol found in whole gland extracts. Although adrenal lipid droplet fractions isolated from non-stimulated control animals contained 65–79% of the total corticosterone assayed in extracts of the whole gland, $\text{in vivo}$ injections of ACTH did not increase corticosterone 1n this fraction. On the other hand, the corticosterone measured in non-lipid fraction extracts increased significantly following ACTH treatment. These results suggest that the synthesis/release mechanism for corticosterone is not associated with the lipid droplets but may involve specific components in the non-lipid fraction. The function of lipid droplet corticosterone is unknown.