Interaction of sex steroids with proteins from the soluble fraction of swine and cattle liver (a preliminary analysis)]

The interactions of [3H]estradiol, [3H]testosterone and [3H]progesterone with soluble proteins from porcine and calf liver were studied. The specific binding of [3H]progesterone and [3H]testosterone in calf liver cytosol seems to be due to serum transcortin or its intracellular precursor (analog). Contrariwise, the specific binding of [3H]progesterone observed in porcine liver cytosol was absent in the serum. This binding was characterized by slow association and dissociation dynamics, moderate affinity for the [3H]-ligand and a high binding capacity. The structural determinants of the ligands were studied by competitive inhibition of the [3H]-ligand binding. The delta 4-3-keto group in the steroid A-ring was found to be the most important determinant. An intensive metabolism of [3H]progesterone was observed during its incubation with cytosol (data from thin-layer chromatography). A 3H-metabolite (presumably, 20 beta-dihydroprogesterone) was predominant in the bound ligand fraction. The data obtained suggest that proteins of a steromodulin type are widely distributed in the mammalian liver.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

Structure and function of the Ah receptor: sulfhydryl groups required for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to cytosolic receptor from rodent livers

Cytosol from rodent liver was exposed to a variety of sulfhydryl-modifying reagents to determine if the cytosolic Ah receptor contained reactive sulfhydryl groups that were essential for preservation of the receptor's ligand binding function. At a 2 mM concentration in rat liver cytosol, all sulfhydryl-modifying reagents tested (except iodoacetamide) both blocked binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to unoccupied receptor and caused release of [3H]TCDD from receptor sites that had been labeled with [3H]TCDD before exposure to the sulfhydryl-modifying reagent. Exposure of cytosol to iodoacetamide before labeling with [3H]TCDD prevented subsequent specific binding of [3H]TCDD, but iodoacetamide was not effective at displacing previously bound [3H]TCDD from the Ah receptor. The mercurial reagents, mersalyl, mercuric chloride, and p-hydroxymercuribenzoate, were more effective at releasing bound [3H]TCDD from previously labeled sites than were alkylating agents (iodoacetamide, N-ethylmaleimide) or the disulfide compound 5,5'-dithiobis(2-nitrobenzoate). Presence of bound [3H]TCDD substantially protected the Ah receptor against loss of ligand binding function when the cytosol was exposed to sulfhydryl-modifying reagents. This may indicate that the critical sulfhydryl groups lie in or near the ligand binding site on the receptor. Subtle differences exist between the Ah receptor and the receptors for steroid hormones in response to a spectrum of sulfhydryl-modifying reagents, but the Ah receptor clearly contains a sulfhydryl group (or groups) essential for maintaining the receptor in a state in which it can bind ligands specifically and with high affinity.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Androgen and estrogen binding in rat skeletal and perineal muscles.

Specific in vitro binding of [3H]testosterone (T), 5ALPHA[3H]dihydrotestosterone (DHT), and [3H[estradiol (E2) was demonstrated in the 30 000 X g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 X 10(9) M-1, and 2.3 X 10(9) M-1 respectively) whereas DHT binding is of lower affinity (Ka = 5.0 X 10(7) M-1).] In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 X 10(9) M-1, 0.3 X 10(9) M-1, and 0.5 X 10(9) M-1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.     

Depletion of [3H]methyltrienolone cytosol binding in glucocorticoid-induced muscle atrophy

The present study was undertaken to determine cytosol binding properties of [3H]methyltrienolone, a synthetic androgen, in comparison with [3H]dexamethasone, a synthetic glucocorticoid, under conditions of glucocorticoid excess in skeletal muscle. Male hypophysectomized rats received either seven daily subcutaneous injections of cortisone acetate (CA) (100 mg X kg-1 body wt) or the vehicle, 1% carboxymethyl cellulose. Following treatment, both [3H]dexamethasone and [3H]methyltrienolone-receptor concentrations were decreased from those in vehicle-treated rats by more than 90 and 80%, respectively, in CA-treated animals. Scatchard analysis of [3H]methyltrienolone binding in muscles of vehicle-treated animals became nonlinear at high concentrations of labeled ligand and were reanalyzed by a two-component binding model. The lower affinity, higher capacity component, which was attributed to binding of methyltrienolone to a "dexamethasone" component, disappeared in muscles of CA-treated rats and Scatchard plots were linear. Receptor concentrations of the higher affinity lower capacity "methyltrienolone" component were similar in muscles of vehicle-treated and CA-treated groups. From competition studies, the high relative specificities of glucocorticoids for [3H]methyltrienolone binding in muscles of vehicle-treated animals were markedly reduced by CA treatment. In addition, the binding specificity data also showed strong competition by progesterone and methyltrienolone for [3H]dexamethasone binding and estradiol-17 beta for [3H]methyltrienolone binding. These results demonstrate that most of the [3H]methyltrienolone binding is eliminated under in vivo conditions of glucocorticoid excess. Furthermore, the competitiveness of various steroids for receptor binding suggests that rat muscle may not contain classic (ligand-specific) glucocorticoid and androgen receptors.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [³H]estradiol-17β are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [³H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4–5 S and the other had a sedimentation coefficient of 8–9 S. The two components differed from each other regarding steroid specicity and various physiocochemical parameters. [³H]-estradiol binding to the 4–5 S component was not inhibited by estrogens, 5α-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appera to be saturable and lavel was rapidly stripped from it by cahrcoal. Estradiol bindng to the 8–9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4–5 S moiety. The specific binding protein has a K_d of 3.05 · 10⁻¹⁰ M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incbuation of [³H]estradiol with mature male liver cytosol at 0–5°C polar metabolites of estradiol are produced.     

Effects of estradiol-17 beta and estriol on their binding sites in the rabbit uterus

1. Receptors for estradiol-17 beta (E2) and estriol (E3) were detected in the rabbit uterus. 2. Saturation analysis of estrogen binding sites in the cytosol showed that the dissociation constants of E2 and E3 for the high affinity binding sites were 1.8 +/- 0.5 nM and 2.3 +/- 0.3 nM, respectively, when dextran-coated charcoal was used to isolate free and bound ligands. 3. To eliminate non-specific (cross) bindings to their receptors, effects of unlabeled E2 and E3 on [3H]E3 and [3H]E2 bindings was examined. 4. [3H]E2 cytosol binding was observed to be specific for E2 and [3H]E3 cytosol binding was more specific for E3. 5. E2 priming to rabbits increased the binding sites for both E2 and E3, which was also more potent than E3 priming. 6. Moreover, the increase in E2 binding sites was greater than that in E3 binding sites. 7. These findings may suggest that there are separate binding sites for E2 and E3 in rabbit uterus and that synthesis of their binding sites is regulated by E2 but not E3.     

Characteristics of androgen binding in rat adrenal tissue using [3H]methyltrienolone (R1881) as tracer

A high level of binding of [3H]methyltrienolone (R1881 = 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) was found in cytosol prepared from adrenals of castrated male rats. Binding of [3H]R1881 was of high affinity (DK = 6.2 nM) and highly specific for androgens. The [3H]R1881 complex migrates at 7-9S on sucrose gradients in low ionic strength buffer and at 4-5S in buffer containing 0.4M KC1. All binding studies have been performed in parallel with rat ventral prostate and adrenal cytosol. The present data suggest the presence of an androgen binding component in rat adrenal tissue.     

In vivo kinetics and DNA-binding properties of the Ah receptor in the golden Syrian hamster

The in vivo long-term cytosolic-nuclear kinetics and DNA-binding properties of the Ah receptor were examined in liver from the golden Syrian hamster. For the kinetic studies, a dose of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) that has been previously shown to produce maximal and sustained hepatic enzyme induction without substantial toxicity was used. Following an intraperitoneal dose of 10 micrograms/kg of [3H]TCDD, occupied cytosolic receptor levels reached a peak within 8 h and then decreased rapidly to a level that was approximately 2% of the total receptor. Throughout the 35-day period, unoccupied cytosolic receptor represented from 65 to 80% of the total receptor content. At 8 h following dosing, less than 30% of the total amount of receptor was associated with the nuclear fraction; this percentage declined slowly to less than 5% of the total at Day 35. The half-life for the decline in detectable nuclear receptor levels was 13 days and was similar to the half-life for the decline in [3H]TCDD content of the whole liver, cytosol, and nuclear extract. The Ah receptor contained in hamster hepatic cytosol underwent a ligand-dependent transformation in vitro to two forms having affinity for DNA-Sepharose, one of which was isolated from nuclei of animals treated with [3H]TCDD in vivo. A comparison of the specific binding recovered following various analytical procedures revealed that the binding of [3H]TCDD to the form not found in nuclear extracts was more labile under certain experimental conditions. These studies indicate the heterogeneity of the Ah receptor in hamster hepatic cytosol and suggest that DNA binding in vitro and nuclear uptake in vivo occur through a ligand-dependent transformation process. The maintenance of maximal hepatic enzyme induction is, in part, a consequence of the sustained presence in the nucleus of only a small percentage of the total receptor content. The whole-tissue kinetics of TCDD appears to be a major factor regulating the long-term retention of the TCDD-receptor complex in the nucleus.     

Intracellular Ca2+ stimulates the binding to androgen receptors in platelets

In this study, we demonstrated that ADP-induced platelet aggregation activates the binding of testosterone (T) to its receptor. It is well known that binding of ADP to its receptors induced the release of Ca2+ ions from dense bodies into the cytosol of platelets. In this work, we compared the binding of testosterone or dihydrotestosterone to their receptors using cytosol obtained from ADP-treated and non-treated platelets. These experiments were repeated using EGTA (a calcium chelator) or U73122 (a phospholipase C enzymatic activity inhibitor) to the ADP-treated platelets. In addition, we also developed a competition analysis for the androgen receptors (AR) using [3H]DHT, non-radioactive T, DHT or cyproterone acetate from ADP-treated platelets cytosol. The results from this study indicate that the cytosol obtained from non-ADP-treated platelets did not show any binding to [3H]T or [3H]DHT, whereas cytosol from ADP-treated platelets binds to the radio-labeled androgens. Furthermore cytosol from ADP plus U73122-treated platelets did not show binding to [3H]T or [3H]DHT. These data suggest that intracellular Ca2+ ions stimulates the binding of androgens to their receptors in platelets cytosol. The competition analysis shows that T and DHT have high affinities for the androgen receptors with similar IC50 values, whereas cyproterone acetate shows a lower affinity. The results from these data clearly indicate the presence of androgen receptors in platelets.     

Modulation of [3H]quinpirole binding at striatal D2 dopamine receptors by a monoamine oxidaseA-like site: evidence from radioligand binding studies and D2 receptor- and MAO(A)-deficient mice.

[3H]Quinpirole is a dopamine agonist with high affinity for the D2 and D3 dopamine receptors. A variety of monoamine oxidase inhibitors (MAOIs) inhibit equilibrium binding of [3H]quinpirole binding in rat striatal membranes suggesting that MAOIs interact with a novel binding site that is labeled by [3H]quinpirole or that allosterically modulates [3H]quinpirole binding. To determine whether the D2 receptor is essential for [3H]quinpirole binding and/or modulation of [3H]quinpirole binding by MAOIs, D2 receptor-deficient mice were studied. [3H]Quinpirole binding was decreased in D2 receptor-deficient mice to 3% of that observed in wild-type controls indicating that [3H]quinpirole binding is associated with the D2 dopamine receptors. Then, in an attempt to label the site mediating the modulation of [3H]quinpirole binding, binding of the MAOI [3H]Ro 41-1049 was characterized in rat striatal membranes. [3H]Ro-41-1049 labeled a single binding site with a pharmacological profile with respect to MAOIs that was similar to both [3H]quinpirole binding (Spearman r=0.976) and MAO(A) activity. To determine whether MAO(A) plays a role in the modulation of [3H]quinpirole binding by MAOIs, MAO(A)-deficient mice were examined. In these mice, [3H]Ro-41-1049 binding was decreased to 7% of wild-type control. [3H]Spiperone binding was unaltered. Spiperone-displaceable [3H]quinpirole binding was decreased to 43% of wild-type control; however, the remaining [3H]quinpirole binding in MAO(A)-deficient animals was inhibited by Ro 41-1049 similar to wild-type. [3H]Ro-41-1049 binding was not decreased in D2 receptor-deficient mice. These data suggest that [3H]Ro-41-1049 labels multiple sites and that MAOIs modulate [3H]quinpirole binding to the D2 receptor via interactions at a novel, non-MAO binding site with MAO(A)-like pharmacology.     

Effect of ATP on the regulation of the steroid binding activity of the oestradiol receptor

We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity (Ka 3.3 x 10(8) M-1) and lower sedimentation coefficient (4 S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [gamma 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [gamma 32P]ATP and substantially affinity labelled with [3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Titratable effects of p-chloromercuriphenyl sulfonate, a thiol-attacking reagent, on glucocorticoid receptor binding

Cytosol prepared from cultured AtT-20 mouse pituitary cells or mouse liver was treated with concentrations of p-chloromercuriphenyl sulfonate (PCMPS) which reduced but did not abolish receptor-binding activity. Scatchard analysis of triamcinolone acetonide binding to the treated cytosol showed that the PCMPS effect was caused by a reduction of binding affinity with little effect on the apparent binding site concentration. The effect on affinity was dose-dependent. Binding specificity appeared unaffected since the relative abilities of triamcinolone acetonide, dexamethasone, cortisol, progesterone, and corticosterone to compete with labeled triamcinolone were similar at various PCMPS concentrations which caused a progressive reduction of detectable cytosol binding. The PCMPS effect was reversible since cytosol treated with up to 200 microM PCMPS followed by dithiothreitol 15 min later showed nearly complete recovery of binding sites (62-100%). The possibility that several sulfhydryl groups were involved in this phenomenon was further explored in experiments using AtT-20 cytosol labeled with [3H]dexamethasone-mesylate, a glucocorticoid affinity label which binds covalently to sulfhydryl groups. Chromatography of dexamethasone-mesylate labeled receptor on a sulfhydryl affinity column resulted in binding, indicating that the receptor had at least two sulfhydryl groups, one bound to the mesylate moiety of the steroid and the other capable of binding to the affinity column.     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

Estrogen receptors in the adrenal cortex of the possum (Trichosurus vulpecula)

1. Specific [3H]estradiol binding activity with characteristics of estrogen receptors was found in the cytosols and nuclear extracts of the adrenal cortex proper and special zone of the brushtail possum (Trichosurus vulpecula). 2. The specific estradiol receptor had a sedimentation coefficient on sucrose gradients of approximately 9S and a molecular weight on gel filtration of more than 200,000. The adrenal cortex cytosol binds [3H]estradiol with high affinity (Ka 5.5 X 10(9) M-1), and limited capacity (Bmax 62.7 fmol/mg cytosol prot). In competition experiments with different steroids the receptor showed a high affinity for four estrogens and a very low affinity to androgens, progesterone and cortisol. 3. There was no difference in the affinity and maximum binding capacity of the cytosols from cortex proper in male and female animals, but the binding capacity of the special zone of females was half that of cortex proper. Estradiol receptors were found in the kidney, liver, lung, testis and muscle but only in the adrenal and prostate was the binding capacity relatively high compared with the uterus. 4. The specific binding capacity of [3H]estradiol to cytosols of adrenal cortex at different stages of the estrus cycle and pregnancy was unrelated to that of the uterus. In the adrenal the receptor concentration was lowest at estrus, when uterine concentration was high, while in late pregnancy the binding of adrenal cortex and uterus cytosols was almost the same. 5. The possible physiological significance of the presence of a specific estrogen receptor in male and female possums is discussed.     

Chemical differentiation of type I and type II receptors for adrenal steroids in brain cytosol

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.