PubMed Health⌕ Search

Biomedical subjects

A M Traish

Publications and source records attributed to A M Traish.

At least 73 records · Page 4Linked to original sources
PubMed1987

Prostatic epidermal growth factor receptors and their regulation by androgens.

Prostatic membranes contain high affinity [dissociation constant (KD) = 1.16 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I]iodo-EGF is specific since it is displaced by excess EGF but not by insulin, fibroblast growth factor, platelet-derived growth factor, or multiplication-stimulating activity. Affinity labeling with [125I]iodo-EGF and subsequent cross-linking with disuccinimidyl suberate demonstrated the specific binding of [125I]iodo-EGF to a macromolecule with a mol wt of 170,000. Castration of mature rats resulted in a 3- to 6-fold increase in [125I]iodo-EGF binding, while treatment of 7-day castrated rats with 5 alpha-dihydrotestosterone decreased the number of binding sites. Administration of estrogen or progesterone produced a slight decrease in EGF binding sites but not nearly to the extent observed with 5 alpha-dihydrotestosterone, suggesting that the observed effect is androgen specific. These results demonstrate that rat prostate contains specific binding sites for EGF and that their level is modulated by androgens.

Androgens↗
PubMed1986

Binding of 7 alpha, 17 alpha-dimethyl-19-nortestosterone (mibolerone) to androgen and progesterone receptors in human and animal tissues.

In rat uterus and prostate, 7 alpha, 17 alpha-dimethyl-19-nortestosterone (DMNT) binds to the androgen receptor specifically and with high affinity. However, this steroid does not bind to glucocorticoid receptors, since it does not displace binding of [3H]triamcinolone acetonide in calf thymus cytosol. In calf uterine and human breast tumor cytosols DMNT binds to the androgen and progesterone receptors, since binding of [3H] DMNT is displaced by unlabeled 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3, 20-dione triamcinolone acetonide, and 5 alpha-dihydrotestosterone (DHT). Conversely, binding of [3H]16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione is effectively competed for by unlabeled DMNT but not by DHT. The observed differences in binding of [3H]DMNT to rat and calf uterine cytosols suggest the species specificity of progesterone receptors. Unlike DHT, DMNT has no appreciable binding to human sex-steroid binding globulin. These findings suggest DMNT as a suitable ligand for measurement and characterization of androgen receptors in rat and human prostate.

Animals↗
PubMed1985

A new exchange procedure for the quantitation of prostatic androgen receptor complexes formed in vivo.

A procedure is described for the measurement of rat prostatic androgen receptor saturated in vivo with non-radioactive androgen. While NaSCN alone induces irreversible dissociation (denaturation) of androgen from the receptor, the combination of this chaotropic salt (0.15 M) with sucrose (15%) and sodium molybdate (10 mM) allows the exchange of R DHT with [3H]DHT at 0 degrees C with only minimal receptor denaturation. The validity of the present exchange assay is based on the following: a similar quantity of androgen receptor was detected when binding was measured directly after in vivo treatment with radioactive androgen or indirectly by [3H]DHT exchange after treatment with non-radioactive androgen. Steroid specificity, sedimentation analysis and equilibrium association constants indicated that this exchange assay labels the androgen receptor without interference from other prostatic steroid binding proteins. With this method it is now possible to quantitate not only prostatic androgen receptors bound to androgens in vitro but also hormone-receptor complexes formed in intact animals under the influence of endogenous androgen.

Animals↗
PubMed1985

Resolution of non-activated and activated androgen receptors based on differences in their hydrodynamic properties.

This study shows that cytosolic androgen receptor of rat ventral prostate sediments at 10-11 S on conventional low salt sucrose density gradients (SDG), and at 4.6 S on high salt SDG, whether it is activated or not; inclusion of 10 mM Na2MoO4 in all buffers does not alter these sedimentation coefficients. In the presence of 50 mM Na2MoO4 non-activated and activated androgen receptors sediment in high salt SDG at 7-8 S and 4.6 S, respectively. Thus the presence of high concentrations of molybdate during centrifugation inhibits the KCl induced disaggregation of receptor into subunits. Similar effects are observed on Sephacryl-S200 gel filtration; in 50 mM MoO2-4 and 0.4 M KCl non-activated receptor has an estimated Stokes radius of 67 A; this value decreases to 52 A upon activation in the presence of proteolysis inhibitors; omission of molybdate during chromatography yielded 52 A and 27 A entities. Estimated mol. wts are 198,000 Daltons for the non-activated 67 A form and 98,000 Daltons for the activated 52 A receptor. Sodium molybdate (50 mM) prevents temperature (18 degrees C) and high ionic strength (0.4 M KCl) induced receptor activation. This inhibition was overcome by removing molybdate by centrifugal gel filtration, or by increasing the KCl concentration to 0.8 M. The inhibitory effects of molybdate on salt induced receptor disaggregation into activated subunits are no longer observed at pH greater than 7.4 or after chemical modification of sulfhydryl groups. Once androgen receptor has been disaggregated into its activated subunits the activated state is maintained even upon reassociation to 10-11 S aggregates in low salt. The relative concentrations of KCl and molybdate are critical; thus, 10 mM Na2MoO4/0.4 M KCl and 50 mM Na2MoO4/0.8-1.2 M KCl did not differentiate activated from non-activated androgen receptor based on their hydrodynamic properties. In the presence of 0.4 M KCl and 50 mM molybdate, however, the hydrodynamic properties of androgen receptor can be correlated with receptor activation.

Animals↗
PubMed1985

Changes in the sedimentation properties of cytosolic androgen receptors associated with activation in vitro and in vivo.

In cell-free systems androgen receptor (AR) labeled with (3H)DHT at 0 degrees C in the presence of 50mM molybdate remains unactivated (less than 3% binding to nuclei) and untransformed (7-8S on sucrose density gradients containing 0.4M KCl and 50mM molybdate). In the absence of molybdate, however, these complexes undergo activation and transformation even at 0 degrees C, albeit, very slowly. Incubation of unactivated, untransformed AR complexes at 18 degrees C, or at 0 degrees C in the presence of 0.4M KCl, greatly accelerated both activation and transformation. Activation and transformation are also associated with formation of high affinity (3H)DHT-receptor complexes as indicated by decreased rates of (3H)DHT dissociation from the receptor. Cytosolic AR complexes labeled with (3H)DHT in tissue slices at 37 degrees C, or in vivo, undergo rapid activation, transformation and nuclear translocation. The data suggest that activation and transformation of cytosolic AR in cell-free systems is associated with changes in the physicochemical properties of AR similar to those occurring upon hormone binding in intact cells and in vivo.

Animals↗
PubMed1985

Conversion of estrogen receptor from a state with low affinity for estradiol into a state of higher affinity does not require 4S to 5S dimerization.

This study was undertaken to establish whether the heat-promoted conversion of receptor-estradiol complex (RE2) from a state with fast into a state with slow E2 dissociation requires 8S/4S to 5S transformation. The calf uterine estrogen receptor labeled with [3H]E2 at 0 C (state with low affinity for E2) was immobilized on hydroxylapatite (HAP) in the absence (8S oligomer) or presence (4S monomer) of 0.4 M KCl and heated at 28 C in the presence of unlabeled diethylstilbestrol. Under these conditions, the dissociation of [3H]E2 was biphasic and occurred at rates similar to those obtained with R[3H]E2 free in cytosol. In contrast to the latter, however, the heat-promoted conversion of HAP-immobilized R[3H]E2 into a state of higher affinity for E2 was not accompanied by receptor dimerization, since the HAP eluate (0.4 M phosphate buffer) contained only the 4S monomer; upon reheating or desalting of this eluate, 4S to 5S dimerization occurred. The heat-promoted formation of 4S RE2 monomers with higher affinity for E2 was not due to monomer-HAP interactions, since even after elution from HAP, the 4S RE2 remained in the state of higher affinity for E2. Addition of pyridoxal 5'-phosphate to slow dissociating, high affinity 5S R[3H]E2 dimers free in cytosol induced rapid [3H]E2 dissociation, although the receptor remained unaltered in the transformed dimerized state. The effect of PLP was readily reversed by the addition of lysine. It is proposed that the 4S receptor monomers exist in two conformational states; upon E2 binding to the low affinity state, conformational changes result in stronger interactions between the steroid and the amino acid residues of the estrogen-binding domain; thus, the rate of E2 dissociation decreases. The formation of this 4S RE2 state with higher affinity for E2 is independent from receptor dimerization. A model is presented in which 4S to 5S transformation may lead to stabilization of 4S monomers in the conformation with higher affinity for E2.

Animals↗
PubMed1985

Interaction of estradiol and estriol with uterine estrogen receptor in vivo and in excised uteri or cell suspensions at 37 C: noncooperative estradiol binding and absence of estriol inhibition of estradiol-induced receptor activation and transformation.

The partial agonist and antagonist properties of estriol (E3) have been related to the brief nuclear retention of receptor-E3 complexes and to the lower affinity of E3 for the receptor compared to estradiol (E2). More recently, it was proposed that the partial agonist/antagonist activity of E3 may be due to its ability to eliminate positive cooperative binding of [3H]E2 to cytosolic estrogen receptor. In this model, positive cooperativity is related to receptor activation and transformation. We first examined the long term effects of E3 on E2 action in vivo. Mature ovariectomized rats were treated for 16 days with E2, E3, or mixtures of these two substances delivered through Alzet pumps at a constant hourly rate (E2, 0.04 microgram; E3, 0.4 and 0.04 microgram; E2 and E3, 0.04 and 0.4 microgram). The effects of E3 on uterine growth, induction of progesterone receptor synthesis, and activation (nuclear binding) of estrogen receptor suggest that when given continuously, E3 acts as a full agonist and does not inhibit E2 action. Furthermore, incubation of uteri at 37 C with [3H]E2 in the presence of a 1- to 20-fold molar excess of nonradioactive E3 did not alter the subcellular distribution of receptor-[3H]E2 complexes (80% nuclear and 20% cytosolic), demonstrating that E3 does not inhibit E2-induced receptor activation (i.e. the increased nuclear binding of receptor). Similarly, 4S to 5S transformation of [3H]E2-labeled estrogen receptor in intact uteri was not inhibited by E3. Equilibrium binding of [3H]E2 to uterine cell suspensions at physiological temperature (37 C) was noncooperative; nonradioactive E3 did not alter the affinity of the estrogen receptor for [3H]E2; Dixon plot analysis indicates that E3 is a purely competitive inhibitor of [3H]E2 binding. This, in conjunction with the lower affinity of the receptor for E3 than for E2, adequately explains the agonistic-antagonistic properties of E3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Estriol and estradiol interactions with the estrogen receptor in vivo and in vitro.

The cytosolic estrogen receptor (calf uterus) bound to estradiol (E2) at 0 degrees C changes from a state with fast into a state with slow E2 dissociation rates when placed at 28 degrees C. This temperature accelerated transition in receptor affinity for its ligand takes place within 10 min at 28 degrees C. Similarly, receptor bound to estriol (E3) at 0 degrees C changes, when heated, from a state with fast into a state with slow E3 dissociation. The main difference between RE2 and RE3 was that E3 dissociates from unheated 8S RE3 and heat-transformed 5S RE3 at a much faster rate than E2 from RE2 . In the mature ovariectomized rat a slow dissociating 5S receptor estrogen complex is found in nuclei 1 h after injection of [3H]E2 or [3H]E3. In vitro dissociation of these 2 estrogens from this nuclear bound receptor formed in vivo takes place at rates similar to those from heat-transformed cytosolic RE2 or RE3 complexes. Addition of pyridoxal 5'-phosphate (PLP) to the slow-dissociating heat-transformed 5S estrogen receptor complexes causes rapid dissociation of E2 or E3; this effect is dose-dependent and is not due to disruption of 5S dimers, since after PLP addition RE2 and RE3 sediment unchanged as 5S dimers. The presence of a large excess of non-radioactive 4S RE3 does not interfere with the temperature induced rapid transition of 4S R[3H]E2 complexes from the state with fast into a state with slow E2 dissociation kinetics. A model is presented to explain the temperature induced biphasic estrogen dissociation from the receptor. It is proposed that the low affinity 4S RE2 monomer undergoes a temperature and estrogen dependent conformation change, such that the ligand is "locked" into the receptor's binding site. This conformational change results in the formation of a high affinity 4S monomer from which estrogen dissociates at a slower rate. This reaction is independent from subsequent 4S to 5S dimerization (transformation). The different rates of ligand dissociation from the low and high affinity 4S receptors reflect the different interactions (hydrophobic and hydrogen bonding) of E2 and E3 with the estrogen binding domain.

Animals↗
PubMed1984

Differences in the physicochemical characteristics of androgen-receptor complexes formed in vivo and in vitro.

This study compares the physicochemical characteristics of the androgen-receptor hormone complexes formed in vitro by incubation of prostatic cytosol with tritiated 5 alpha-dihydrotestosterone (DHT) and methyltrienolone (R1881; 17 beta-hydroxy-17 alpha-methyl-4,9, 11-estra-trien-3-one) with those of hormone-receptor complexes formed in vivo upon hormone injection. [3H]DHT and [3H]R1881 had similar affinities for the androgen receptor in vitro (Kd = 0.3 nM). Dissociation of DHT at 0 C from the receptor complexes formed in vitro or in vivo was much slower than that of R1881. Furthermore, DHT and R1881 dissociated much more slowly from the cytoplasmic receptor labeled in vivo than in vitro. The sedimentation characteristics of the in vitro and in vivo formed hormone-receptor complexes were similar when analyzed on sucrose density gradients containing 400 mM KCl and 10 mM Na2MoO4. Higher concentrations (50 mM) of Na2MoO4, however, prevented the salt-induced disaggregation of the in vitro formed receptor complexes, which sedimented at 7-8S. In contrast, androgen-receptor complexes formed in vivo sedimented as 5.5S complexes, even in the presence of 50 mM molybdate. These differences were paralleled by the elution patterns from Sephacryl S-200. A further difference was found in the sensitivity of the hormone-receptor complex to the organic mercurial reagent mersalyl acid. This reagent, at 0.2 mM, induced ligand exchange of 80-90% of the in vitro formed hormone-receptor complexes, whereas it was nearly ineffective with complexes formed in vivo. Finally, the prostatic androgen receptor content 1 h after injection of radioactive steroid into castrated rats was 12-14 pmol/mg DNA, while incubation of tissue slices at 37 C yielded only 3-4 pmol receptor/mg DNA.

Animals↗
PubMed1983

The role of arginyl residues in estrogen receptor activation and transformation.

Receptor-estradiol complexes (RE2) formed at 0 degree C in hypotonic buffers bind poorly to nuclei (nonactivated state); their sedimentation coefficient in low or high salt sucrose density gradients (SDG) is 8 S or 4 S, respectively (untransformed state); estradiol dissociates from untransformed RE2 at a high rate (k-1 = 0.44 min-1). Brief heating (28 degrees C, 30 min) induces activation (increased binding of RE2 to nuclei and polyanions), transformation (formation of receptor dimers which sediment at 6 S in 0.4 M KCl/borate SDG) and RE2 transition into a state from which E2 dissociates at a lower rate (k-2 = 8 X 10(-3) min-1). We have examined the role of arginyl residues in the above changes in receptor properties. It is well established (Patthy, L., and Smith, E. L. (1975) J. Biol. Chem. 250, 557-564; 565-569) that 1,2-cyclohexanedione (1,2-CHD) is a highly specific arginine-modifying agent; in borate buffer at 28 degrees C, but not at 0 degrees C, peptide arginyls are covalently modified. RE2 complexes heated in the presence of 1,2-CHD (50 mM) bind poorly to nuclei; 1,4-cyclohexamedione and 1,2-cyclohexanediol had no effect. This reagent also prevents the temperature-induced transition of RE2 into a state with slow E2 dissociation rates although it does not interfere with heat transformation (formation of 6 S dimer). Modification of heat-activated and transformed RE2 by 1,2-CHD causes a loss in receptor binding to nuclei and alters RE2 from a state with slow into a state with fast E2 dissociation rates, although the receptor remains unaltered in the transformed 6 S state. At 0 degree C, i.e. in the absence of covalent arginyl modification, 1,2-CHD promotes dissociation of the 8 S aggregate into 4.6 S subunits which bind to nuclei to the same extent as heat-transformed control RE2. Heating of the molybdate-stabilized 8 S receptor in the presence of 1,2-CHD yields a nonactivated 8 S receptor (4.6 S on high salt SDG); removal of molybdate and unreacted 1,2-CHD by gel filtration at 0 degree C followed by exposure to high ionic strength causes 8 S to 4 S dissociation; these 4 S subunits, however, do not bind to nuclei, suggesting that their nucleotropic domain was accessible to 1,2-CHD modification while the receptor was in the aggregated 8 S state. It is proposed that the nuclear binding site of the estrogen receptor contains arginyl residues. Furthermore, a distinct set of arginyl residues appears to be related to the estrogen-binding domain; its integrity is required for the heat-induced formation and maintenance of the RE2 state with slow E2 dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1981

A new procedure for the quantitation of nuclear and cytoplasmic androgen receptors.

Experimental conditions for the measurement of prostatic androgen receptors occupied with unlabeled hormone are described. The assay allows quantitation of cytoplasmic and nuclear receptors occupied with unlabeled ligand by exchange with 17 beta-hydroxy-17 alpha-methyl [3H]estra-4,9,11-trien-3-one at 0 degrees C. The mercurial reagent mersalyl acid (0.2 mM) is used to promote dissociation of more than 90% receptor-bound steroid. The half-time of this reaction is 10-15 min. The reaction is reversible by addition of dithiothreitol or monothioglycerol. Treatment of the receptor with mersalyl and displacement of this reagent by thiol reagents does not alter the steroid binding affinity of the receptor or the sedimentation characteristics. Binding is measured by the hydroxylapatite procedure and all buffers contain 10 mM sodium molybdate. This assay circumvents many of the difficulties associated with conventional exchange assays performed either at elevated temperature or at 16 degrees C for 20-24 h, such as receptor degradation or incomplete exchange.

Animals↗
PubMed1979

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.

Animals↗
PubMed1979

Studies on the mechanism of estradiol uptake by rat uterine cells and on estradiol binding to uterine plasma membranes.

A method for the preparation of viable uterine cell suspensions is described. Using this system the kinetics of estradiol uptake were studied in order to asses whether the steroid enters uterine cells by passive diffusion (1) or protein mediated diffusion (2). The data presented show that a) the rates of estradiol entry are nonsaturable; b) temperature dependence of uptake kinetics gives a linear Arrhenius plot; c) E2-6-CMO-BSA does not inhibit 3H-E2 uptake; d) uterine plasma membranes do not contain specific estrogen binding sites. Thus, estrogen uptake occures by passive diffusion.

Animals↗