Covalent immobilization of the estrogen receptor to a cationic N-hydroxysuccinimide ester derivative of agarose

Covalent immobilization of the soluble estrogen receptor from a rabbit uterus to N-hydroxysuccinimide ester derivative of agarose is shown. At first, the condition for the immobilization reaction was examined. The non-immobilized receptor was extracted with 0.4 M NaCl-containing medium. Sixty seven to 80% of the input receptor were immobilized within 30 min at 0 degrees C in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4). The immobilized [3H]estradiol(3,17 beta-dihydroxy-1,3,5(10)-estratriene)-receptor complex was stable for at least 24 h. The optimum pH for immobilization was 7.4. Ca2+ or Na+ ions in the reaction media decreased the yields in immobilization of the receptor to the reagent with an electrostatically positive spacer arm. Next, influences of immobilization on the receptor were examined. The dissociation rate of [3H]estradiol from the immobilized receptor was a little slower than that from the native receptor. The estrogen-free immobilized receptor was saturated by incubating with 10 nM [3H]estradiol for 10 h at 0 degrees C in 0.1 M HEPES (pH 7.4). From Scatchard plot analysis, it was found that the hormone binding affinity in the immobilized receptor decreased to approximately one-fourth of that in the native receptor.     

Ligand-binding properties of estrogen receptor proteins after interaction with surface-immobilized Zn(II) ions: evidence for localized surface interactions and minimal conformational changes

The site- or domain-specific immobilization of steroid receptor proteins with preserved structure and function would facilitate the identification and purification of receptor-associated regulatory components and nucleic acids. We have demonstrated previously that restricted surface regions of the estrogen receptor protein contain high affinity binding sites for immobilized Zn(II) ions. Possible conformational changes in receptor at the stationary phase immobilized metal ion interface were evaluated by monitoring alterations in the equilibrium dissociation constant (Kd) for [3H]estradiol. Soluble estrogen receptor proteins (unliganded) present in immature calf uterine cytosol were immobilized via surface-exposed Zn(II)-binding sites to beads of agarose derivatized with iminodiacetate (IDA)-Zn(II) ions. The IDA-Zn(II) bound receptor was incubated with increasing concentrations of [3H]estradiol (0.01-20 nM) in the presence and absence of unlabeled competitor (diethylstilbestrol) to determine the level of specific hormone binding. Steroid-binding experiments were performed in parallel with identical aliquots of soluble receptor. Analyses of the equilibrium binding data revealed the presence of a single class of high-affinity (Kd = 2.44 +/- 1.5 nM, n = 10) steroid-binding sites which were only marginally affected by receptor immobilization via surface-exposed Zn(II) bindings sites (Kd = 2.58 +/- 0.56 nM, n = 4). These data are consistent with the location of surface accessible Zn(II) binding site(s) on the receptor at or near the DNA binding domain which, upon occupancy, do not influence the steroid binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Analysis of progesterone receptor in the quail oviduct. Correlation between plasmatic estradiol and cytoplasmic progesterone receptor concentrations

Specific binding sites for [3H]-progesterone are found in the cytosol fraction of the oviduct of castrated, immature and developing quails. The optimal conditions to accurately measure the total cytoplasmic concentration of this progesterone receptor are described. The dissociation constant (KD) at 0 degrees C is 3.6 +/- 0.6 x 10(-9) M (mean +/- SE) for [3H]-P and the concentration of binding sites is 13.4 +/- 2 pmol/mg DNA in immature animals. This binding capacity is not altered even 2 weeks after ovariectomy. During sexual development, although the dissociation constant remains unchanged, the number of binding sites increases to 74.5 +/- 1.6 pmol/mg DNA just before the beginning of the laying cycle. The concentration of cytoplasmic P receptor is under the inductive influence of estradiol. In castrated quails, estradiol 17 beta (E2) perfusion through the portal vein at a rate below or equal to 2 ng/min for 24 hr does not increase plasmatic E2 concentration and consequently does not change [3H]-P binding sites concentration in the oviduct. While E2 perfusion rate exceeds the metabolizing capacity of the liver (6.8 ng/min), both plasmatic E2 level and oviductal P receptor concentration are increased. When E2 is perfused through the jugular vein, plasmatic E2 level increases with the dose of E2 but P receptor concentration only increases when E2 perfusion rate reaches to 2.0 ng/min for 24 h.     

11 beta-chloromethyl-[3H]estradiol-17 beta: a very high affinity, reversible ligand for the estrogen receptor

It has been suggested that binding of 11 beta-chloromethyl estradiol (11 beta-CME2) to the estrogen receptor is irreversible, since its complex with receptor fails to undergo exchange with estradiol (E2). To investigate this behavior directly, 11 beta-CME2 was prepared in high specific activity, tritium-labeled form: The binding of [3H]11 beta-CME2 to the estrogen receptor from lamb and rat uterus and MCF-7 human breast cancer cells was shown to be fully reversible; the 11 beta-CME2 complex with receptor, as well as that of a structural analog 11 beta-ethyl estradiol, however, do not dissociate or exchange with [3H]E2 over a 22 h period at 25 degrees C. By competitive or direct binding assays, the affinity of 11 beta-CME2 for the estrogen receptor can be estimated to be as much as 10- to 30-fold higher than that of E2. The complexes of estrogen receptor from MCF-7 cells with [3H]11 beta-CME2 and [3H]E2 show identical velocity sedimentation profiles on sucrose gradients, under conditions when the receptor is either a monomer of a dimer. Because of its very high affinity and unusual dissociation kinetics, [3H]11 beta-CME2 should be a very useful ligand for studies of estrogen receptor dynamics and in the assay of estrogen receptor concentrations in tumors and tissues.     

Interaction of the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.     

Continuous estrogen exposure in the rat does not induce loss of uterine estrogen receptor

Cytosol estrogen receptor (ERc) and nuclear estrogen receptor (ERN) levels were investigated in rat uteri under different conditions of hormonal exposure. The amount of directly assayable receptor was closely related to the serum concentration of 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2). A double injection technique was established to maintain serum levels of [3H]E2 which were sufficient to saturate receptor sites. Under these conditions, stable ERC and ERN levels are maintained throughout the study period. 30% of the total ER remains cytoplasmic in localization despite continuous hormonal exposure. Properties of ERC and ERN after 6 h of continuous hormonal exposure were investigated and found to be different from receptors found in these subcellular compartments 30 min after hormone injections. ERC from uteri 30 min after injection showed a faster sedimentation coefficient than ERC prepared 6 h after hormone treatment. ERC after 6 h of hormonal exposure showed a reduction of binding to calf thymus DNA adsorbed on cellulose in a cell-free system. ERC 30 min after [3H]E2 treatment had a biphasic dissociation pattern consistent with two different receptor populations, whereas uterine ERC obtained after 6 h of in vivo exposure to estradiol showed virtually no dissociation at 22 and 28 degrees C. In contrast to ERC, ERN 6 h after hormone injection sedimented faster than ERN obtained 30 min after treatment. KCl extractable ERN obtained either at 30 min or 6 h posthormone treatment showed biphasic dissociation kinetics at 22 and 28 degrees C, whereas KCl nonextractable ERN showed virtually no dissociation. Virtually all of the specifically bound ligand in cytosol and nuclear preparations was proven to be authentic E2. We conclude that total cellular receptor is quantitatively conserved during 6 h of continuous hormonal treatment. Nuclear receptor loss is not a requisite for receptor-mediated steroid function, although important time-dependent changes in receptor properties in both cytoplasmic and nuclear compartments do occur.     

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-beta-(4-dimethylaminophenyl)-17a-hydroxy-17 beta-(3- hydroxypropyl)-13 alpha-methyl-4,9-gonadien-3-one]). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [3H]ZK98299 with affinity approximately equal to that of [3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0 degrees C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [3H]ZK98299 and [3H]triamcinolone acetonide binding. While [3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide (NEM), [3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 degrees C) and receptor transforming (23 degrees C) conditions, and exhibited a faster rate of dissociation at 23 degrees C when compared with [3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

Catechol modification and covalent immobilization of catalase on titania submicrospheres

In this article, chemical modification with catecholic derivative in solution and subsequent immobilization of catalase (CAT) on titania submicrospheres (450–500 nm) were described. Catalase was first reacted with 3-(3,4-dihydroxyphenyl) propionic acid activated via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry. The above chemically modified CAT bearing catechol groups was then covalently bound to the surface of titania through the facile chelation reaction between the catechol groups and titania. The immobilized CAT retained 60% catalytic activity with a high loading capacity of 500 mg/g titania. Meanwhile, the immobilized CAT displayed enhanced operational stability, thermal stability and storage stability compared with native, modified CAT counterparts. In repeated batches of decomposition of hydrogen peroxide, after 10 and 19 cycles, the immobilized CAT maintained about 90% and 75% of its initial activity, respectively.     

Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.     

Similarities and differences of the binding of estradiol and 4-hydroxytamoxifen (an antiestrogen) in the chick oviduct cytosol

The binding characteristics of [³H]estradiol and 4-[³H]hydroxytamoxifen (a powerful estradiol antagonist) in the chick oviduct cytosol was analyzed by sucrose gradient centrifugation and dissociation kinetics experiments at 28°C. Heating the cytoplasmic estradiol-estrogen receptor complexes led to the ‘transformation’ of the receptor; as with the estrogen receptor in other target tissues and species, the transformed receptor sedimented in the 5 S region of sucrose gradients containing 0.4 M KCI and had a slower rate of dissociation of bound estradiol. Upon heating, the cytoplasmic 4-hydroxytamoxifen complexes also appeared to undergo similar changes in their physical states as analyzed by sedimentation rates and dissociation kinetics, and we conclude that antiestrogen can transform the receptor. Sodium molybdate inhibited the temperature mediated changes with both estrogen and antiestrogen complexes. Slight but consistent differences in the sedimentation coefficient and rate of ligand dissociation were observed between the complexes formed by estradiol and 4-hydroxytamoxifen but the relevance to opposite biological activities remains unknown.     

Effect of chemical perturbation with NaSCN on receptor-estradiol interaction. A new exchange assay at low temperature

When 0.5 M sodium thiocyanate is added to uterine cytosol previously labeled with excess [3H]-17 beta-estradiol, no change can be detected in the steady-state cytosol concentration of [3H]estradiol-receptor complex for at least 20 h at 4 degrees C. However, the rate of exchange of bound estradiol in the presence of NaSCN was found to be substantially higher than that in the absence of the chaotropic salt. In the presence of NaSCN, the dissociation rate of the complex increases about 10-fold (K-1 SCN = 1.10 x 10(-2) min-1 vs. K-1 = 1.07 X 10 (-3)min-1) while the rate of association increases about 2-fold (K1 SCN = 1.2 X 10(7) min-1M-1 vs.K1= 7.4 X 10(6) min-1 M-1). The Kd changes 6.4-fold (Kd SCN = 9 X 10(-10) M vs. Kd = 1.4 x 10(-10 M) with no decrease in the number of binding sites as shown by Scatchard plots of saturation experiments. This effect of NaSCN can be exploited to assay preformed estrogen-receptor complex by exchange with [3H]estradiol at low temperature. When the sample containing preformed complex is incubated overnight (16 h) at 4 degrees C with excess [3H]estradiol in the presence of 0.5 M NaSCN, there is a quantitative exchange of nonlabeled for estradiol without loss of binding sites. Hormonal steroids other than estrogens do not interfere, and the exchange estradiol is bound with high affinity. Precision, accuracy, and linearity of the method are highly satisfactory.     

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.     

Aflatoxin inhibition of glucocorticoid finding capacity of rat liver nuclei.

The effect of aflatoxin B1 on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [3H]desamethasone-cytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B1. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 26 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B1 dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant. Administration of [3H]dexamethasone to alfatoxin B1 -treated rats produced a similar pattern of glucocorticoid binding distribution in vivo to that observed in vitro. No differences in [3H]dexamethasone-cytoplasmic receptor binding between control and alfatoxin B1 -treated rats were found, whereas nuclear [3H]dexanthasone binding was reduced 34% by alfatoxin B1 -treatment.     

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.     

Effects of molybdate on steroid receptors in intact GH1 cells. Evidence for dissociation of an intracellular 10 S receptor oligomer prior to nuclear accumulation

Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits.     

Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate.

Androgen receptors in crude and partially purified 105,000 X g supernatant fractions from rat testis, epididymis, and prostate were studied in vitro using a charcoal adsorption assay and sucrose gradient centrifugation. Androgen metabolism was eliminated during receptor purification allowing determination of the kinetics of [3H]-androgen-receptor complex formation. In all three tissues, receptors were found to have essentially identical capabilities to bind androgen, with the affinity for [3H] dihydrotestosterone being somewhat higher than for [3H] testosterone. Equilibrium dissociation constants for [3H] dihydrotestosterone and [3H] testosterone (KD = 2 to 5 X 10(-10) M) were estimated from independently determined rates of association (ka congruent to 6 X 10(7) M-1 h-1 for [3H] dihydrotestosterone and 2 X 10(8) M-1 h-1 for [3H] testosterone) and dissociation (t 1/2 congruent to 40 hr for [3H] dihydrotestosterone and 15 h [3H] testosterone). Evaluation of the effect of temperature on androgen receptor binding of [3H]testosterone allowed estimation of several thermodynamic parameters, including activation energies of association and dissociation (delta H congruent to 14 kcal/mol), the apparent free energy (delta G congruent to -12 kcal/mol), enthalpy (delta H congruent to -2.5 kcal/mol), and entropy (delta S congruent to 35 cal col-1 K-1). Optimum receptor binding occurred at a pH of 8. Receptor stability was greatly enhanced when bound with androgen. Receptor specificity for testosterone and dihydrotestosterone was demonstrated by competitive binding assays. The potent synthetic androgen, 7 alpha, 17 alpha-dimethyl-19-nortestosterone, inhibited binding of [3H] testosterone or [3H] dihydrotesterone nearly as well as testosterone and dihydrotestosterone while larger amounts of 5 alpha-androstane-3alpha, 17 beta-diol and nonandrogenic steroids were required. Sedimentation coefficients of androgen receptors in all unfractionated supernatants were 4 and 5 to 8 S. Differences in sedimentation coefficients were observed following (NH4)2SO4 precipitation which did not influence the binding properties of the receptors. These results, together with measurements of3alpha/beta-hydroxysteroid oxidoreductase activity in vitro, suggest that organ differences in receptor binding of [3H] dihydrotestosterone and [3H] testosterone in vivo result from relative differences in intracellular concentrations of these androgens rather than from differences in receptor affinities.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.     

Solubilization of the nitrendipine receptor from skeletal muscle transverse tubule membranes. Interactions with specific inhibitors of the voltage-dependent Ca2+ channel

The nitrendipine receptor associated with the voltage-dependent calcium channel from rabbit skeletal muscle transverse tubule membranes has been solubilized by detergent extraction. A highly stable solubilized receptor preparation was obtained using 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate as detergent with phospholipids or glycerol present as stabilizing agents. Binding of [3H]nitrendipine to the solubilized receptor was reversible and saturable. At 4 degrees C the equilibrium dissociation constant of the [3H]nitrendipine X receptor complex was 7 +/- 3 nM and was close to that determined from the rate constants of association (k1 = 1.3 10(5) M-1 s-1) and dissociation (k-1 = 1.10 X 10(-3) s-1) of 8.4nM. The nitrendipine concentration that gave a half-maximal inhibition of [3H]nitrendipine binding to the solubilized receptor was 10 nM, which was similar to the values for the dissociation constant determined for the radiolabelled ligand. [3H]Nitrendipine binding to its solubilized receptor was also inhibited by other antiarrythmic drugs, such as bepridil and verapamil, and enhanced by d-cis-diltiazem. Since these drugs are apparent non-competitive inhibitors of [3H]nitrendipine binding it was concluded that these different binding sites are tightly coupled. Sucrose density sedimentation of solubilized nitrendipine receptor resulted in the separation of three [3H]nitrendipine binding activities with apparent sedimentation coefficients of 11.4 S, 14.4 S and 21 S.