ANDROGEN RECEPTORS IN PROLACTIN PRODUCING PITUITARY TUMOURS IN RATS

1977 ◽  
Vol 86 (2) ◽  
pp. 288-298 ◽  
Author(s):  
Arne Attramadal ◽  
Oddvar Naess ◽  
Egil Haug ◽  
Vidar Hansson ◽  
Ken Purvis
Keyword(s):  
Androgen Receptor ◽  
Binding Sites ◽  
Androgen Receptors ◽  
Sedimentation Coefficient ◽  
Ventral Prostate ◽  
Scatchard Analysis ◽  
Receptor Complex ◽  
Pituitary Tumours ◽  
High Affinity ◽  
Androgen Binding

ABSTRACT The androgen receptor system in prolactin secreting oestrogen induced pituitary tumours has been studied. The tumour cytosol was found to contain specific androgen receptors binding [3H]5α-dihydrotestosterone (DHT) and [3H] testosterone (T) with high affinity and low capacity. Scatchard analysis of the saturation data for T revealed one class of high affinity binding sites. The equilibrium constant of dissociation (Kd) was ∼ 4 × 10−10 m and the number of binding sites was calculated to be 12.8 femtomoles/mg protein. The sedimentation coefficient of the androgen receptor complex in low salt sucrose gradients was ∼ 7 S, the electrophoretic mobility (RF) in 3.25 % polyacrylamide gels ∼ 0.5 and the isoelectric point 5.8. The protein nature of the receptor was indicated by the finding that protease, but not DNase and RNase, eliminated androgen binding. Furthermore, the receptor was thermolabile and functionally dependent on free SH-groups since androgen binding was eliminated by heating 45°C for 30 min) and treatment with p-chloromercuriphenyl sulphonate (1 mm). Steroid specificity was tested in vitro by examining the competing efficiency of different unlabelled steroids for the binding of [3H]T. The affinity of DHT for the receptor was approximately twice that of testosterone while the binding affinity of oestradiol-17β and progesterone was very low. Cortisol had no affinity for the androgen receptor. The dissociation of the androgen receptor complex was very slow at 0°C (t ½ > 48 h). Thus, the characteristics of the cytoplasmic androgen receptors of the prolactin producing pituitary tumours are very similar to those of the androgen receptors earlier demonstrated in the anterior pituitary, hypothalamus, ventral prostate, epididymis and testis. The presence of specific androgen receptors in prolactin producing pituitary tumours indicates that androgen is involved in the regulation of synthesis and release of prolactin.

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland

1986 ◽  
Vol 108 (2) ◽  
pp. 267-273 ◽  
Author(s):  
S. Kyakumoto ◽  
R. Kurokawa ◽  
Y. Ohara-Nemoto ◽  
M. Ota
Keyword(s):  
Androgen Receptor ◽  
Nuclear Receptors ◽  
Binding Sites ◽  
Androgen Receptors ◽  
Dissociation Constants ◽  
Scatchard Analysis ◽  
Male And Female ◽  
Short Period ◽  
Almost All ◽  
Androgen Binding

ABSTRACT Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 °C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5′-phosphate (5 mmol/l) from nuclear fractions with 93–95% efficiency. The exchange of the bound steroids occurred by 24–48 h at 0 °C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0·8 and 0·9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females. J. Endocr. (1986) 108, 267–273

Extraction of androgen–receptor complexes from regions of rat ventral prostate nuclei sensitive or resistant to nucleases

1983 ◽  
Vol 99 (1) ◽  
pp. 51-61 ◽  
Author(s):  
P. Davies
Keyword(s):  
Androgen Receptor ◽  
Binding Sites ◽  
Micrococcal Nuclease ◽  
Ventral Prostate ◽  
Receptor Complex ◽  
Sedimentation Analysis ◽  
Receptor Complexes ◽  
Rat Ventral Prostate ◽  
Profile Characteristic ◽  
Androgen Binding

Rat ventral prostate nuclei contain androgen-binding sites which are susceptible or resistant to excision by endonucleolytic action. Those which were susceptible were associated both with oligonucleosomal and subnucleosomal particles. The sedimentation profile characteristic of a nuclear androgen-receptor complex could be obtained by exhaustive nucleolytic digestion or by treatment of fractions with KCl (0·6 mol/l). Androgen-binding sites resistant to DNAase I were also resistant to KCl, whereas those sites resistant to micrococcal nuclease were partially extractable with KCl. Nuclease-resistant sites could be extracted with heparin (10 mg/ml). Androgen–receptor complexes obtained from nuclease-sensitive or nuclease-resistant regions by extraction with KCl or heparin were indistinguishable by routine sedimentation analysis.

ANDROGEN AND OESTROGEN BINDING IN CYTOSOLS OF HUMAN OVARIAN TUMOURS

1981 ◽  
Vol 90 (3) ◽  
pp. 421-431 ◽  
Author(s):  
T. C. HAMILTON ◽  
P. DAVIES ◽  
K. GRIFFITHS
Keyword(s):  
Progesterone Receptor ◽  
Binding Sites ◽  
Sedimentation Coefficient ◽  
Oestrogen Receptors ◽  
Histopathological Classification ◽  
High Affinity ◽  
Ovarian Tumours ◽  
Specific Retention ◽  
Androgen Binding ◽  
Binding Component

The specific retention of androgens and oestrogens by cytoplasmic components of human ovarian tumours was investigated. High-affinity, low-capacity binding of androgens was observed in 88% of tumour specimens and oestrogen binding in 32%. Retention of oestrogens did not occur in the absence of androgen binding. The androgen-binding component, of sedimentation coefficient 7·5–8·5S, showed specificity for 5α-dihydrotestosterone and 17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one (R1881). In some instances, competition for R1881-binding sites indicated the presence of progesterone receptor-like binding. The data presented suggest strongly the existence of androgen and oestrogen receptors in some ovarian tumours and may be relevant to histopathological classification and therapeutic rationales.

SPECIFIC HIGH-AFFINITY OESTRADIOL BINDING IN RAT VENTRAL PROSTATE

1980 ◽  
Vol 87 (2) ◽  
pp. 285-292 ◽  
Author(s):  
M. GINSBURG ◽  
I. JUNG-TESTAS ◽  
E. E. BAULIEU
Keyword(s):  
Binding Sites ◽  
Ventral Prostate ◽  
Scatchard Analysis ◽  
Endocrine Control ◽  
High Affinity ◽  
Rat Ventral Prostate ◽  
Low Salt ◽  
Equilibrium Dissociation ◽  
Intact Rats ◽  
Binding Component

The presence of a specific saturable oestradiol-binding component was demonstrated in cytosol from rat ventral prostate. Centrifugation of cytosol, previously incubated with [3H]oestradiol at 0 °C, on low salt glycerol—Tris gradients revealed two oestradiol-binding systems with sedimentation coefficients of 8S and 4S. Excess unlabelled dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) did not compete with the oestradiol binding, whereas excess unlabelled oestradiol or diethylstilboestrol abolished the 8S and 4S peaks. The oestradiol binding to these components could not be detected after proteolytic treatment. Scatchard analysis of saturable oestradiol binding in cytosol of prostates from intact rats and from rats 14 days after orchidectomy indicated that the equilibrium dissociation constant (KDeq) was about 10−10 mol/l at 0 °C, and the concentrations of high-affinity binding sites were approximately 10 fmol oestradiol bound/mg protein. Lower concentrations of oestradiol binding (approximately 2 fmol/mg protein) were found in cytosols from prostates obtained 2 and 4 days after castration. The transient decrease of oestradiol binding was not due to the presence in prostate cytosol of a factor that inactivated the oestradiol receptor. It is proposed that the oestradiol receptor in the cytosol from ventral prostate tissue of the rat is under endocrine control.

Androgen-binding proteins in sheep epididymis: characterization of a cytoplasmic androgen receptor in the ram epididymis

1984 ◽  
Vol 103 (3) ◽  
pp. 273-279 ◽  
Author(s):  
S. Carreau ◽  
M. A. Drosdowsky ◽  
M. Courot
Keyword(s):  
Androgen Receptor ◽  
Binding Sites ◽  
Sucrose Gradient ◽  
Proteolytic Enzymes ◽  
Molecular Weights ◽  
High Affinity ◽  
Isoelectric Points ◽  
Extracellular Transport ◽  
Androgen Binding

ABSTRACT An androgen receptor (Rc) was demonstrated in caput, corpus and cauda epididymal cytosols of the ram. This receptor had a high affinity for 5α-dihydrotestosterone (Kd = 5·2 × 10−9 mol/l) and could be distinguished from the androgen-binding protein (ABP) by several characteristics. On polyacrylamidegel electrophoresis, Rc had a mobility of 0·37 and ABP 0·61; Rc sedimented in the 9S region of a linear sucrose gradient whereas ABP migrated in the 4·3S region; the molecular weights were 192 000 and 90 000 for Rc and ABP; their isoelectric points were 5·7 and 4·8–5·0; they were proteinaceous components since they were destroyed by proteolytic enzymes and heating (50 °C for Rc and 60 °C for ABP); they exhibited different half-times of dissociation: 20 h at 0 °C for Rc and 6 min for ABP, which is in agreement with their respective physiological roles, intra- and extracellular transport of androgens. The content of Rc-binding sites in caput epididymis was 18, in corpus 4 and in cauda 22 fmol/mg protein. J. Endocr. (1984) 103, 273–279

Evidence for an androgen receptor in porcine Leydig cells

1987 ◽  
Vol 115 (1) ◽  
pp. 119-124 ◽  
Author(s):  
Veli Isomaa ◽  
Mauri Orava ◽  
Reijo Vihko
Keyword(s):  
Androgen Receptor ◽  
Binding Sites ◽  
Leydig Cells ◽  
Androgen Receptors ◽  
Cultured Cells ◽  
Binding Specificity ◽  
Culture Conditions ◽  
High Affinity ◽  
The Mean ◽  
Steroid Binding

Abstract. Cytosol and nuclear androgen receptor concentrations were measured in freshly prepared and cultured Leydig cells of immature pig testis with exchange assays using [3H]methyltrienolone as labelled ligand. Androgen receptors in Leydig cells had high affinity for [3H]methyltrienolone and steroid binding specificity typical of an androgen receptor. The mean receptor concentrations were 76 fmol/mg protein and 210 fmol/mg DNA for cytosol and nuclei, respectively. In sucrose gradients, cytosol androgen receptors sedimented in the 4 S region. The cells maintained androgen receptors under culture conditions. Exposure of cultured cells to [3H]methyltrienolone (10 nmol/l) resulted in accumulation of androgen receptors in the nuclei with maximal uptake by 1 h. We conclude that methyltrienolone binding sites with characteristics of androgen receptors were identified in both cytosol and nuclei of porcine Leydig cells.

scholarly journals Binding of androgens to a nuclear-envelope fraction from the rat ventral prostate

1980 ◽  
Vol 186 (3) ◽  
pp. 641-647 ◽  
Author(s):  
Y A Lefebvre ◽  
Z Novosad
Keyword(s):  
Nuclear Envelope ◽  
Binding Sites ◽  
Specific Binding ◽  
Ventral Prostate ◽  
Scatchard Analysis ◽  
Rat Ventral Prostate ◽  
Membrane Isolation ◽  
Triton X ◽  
Androgen Binding ◽  
Plasma Membrane Isolation

A nuclear-envelope fraction was isolated from the rat ventral prostate which is virtually free of DNA and contains little RNA or plasma membrane. Isolation of this nuclear-envelope fraction after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More binding of dihydrotestosterone is observed after incubations at 22 degrees C for 17 h than at 4 degrees C for 17 h or at 22 degrees C for 60 min. Scatchard analysis revealed a class of binding sites with KD 8.4 nM. Dihydrotesterone and testosterone were almost equally effective as competitors of labelled dihydrotestosterone binding on the purified nuclear-envelope fraction, whereas diethylstilboestrol was less effective and dexamethasone did not compete well. When the outer membrane of the nuclei was removed with Triton X-100, a 24% decrease in specific binding of androgens was observed. Castration 24 h before preparation of nuclei resulted in loss of the androgen binding to the membrane.

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study

1991 ◽  
Vol 69 (12) ◽  
pp. 809-820 ◽  
Author(s):  
William Goumakos ◽  
Jean-Pierre Laussac ◽  
Bibudhendra Sarkar
Keyword(s):  
Serum Albumin ◽  
Human Serum ◽  
Binding Sites ◽  
Metal Ion ◽  
Equilibrium Dialysis ◽  
Scatchard Analysis ◽  
Serum Albumins ◽  
High Affinity ◽  
Nmr Study ◽  
Nmr Techniques

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 ± 0.09 (log K = 5.3 ± 0.6) and 1.07 ± 0.12 (log K = 6.4 ± 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 ± 0.19 (log K = 5.1 ± 0.8), and 1.06 ± 0.15 (log K = 6.0 ± 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.Key words: albumin, human serum, dog serum, cadmium, zinc, copper, NMR, equilibrium dialysis, binding.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Abstract Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

Hormone regulation of the rodent Harderian gland: binding properties of the androgen receptor in the male golden hamster

1987 ◽  
Vol 112 (1) ◽  
pp. 3-8 ◽  
Author(s):  
F. Vilchis ◽  
A. Hernandez ◽  
A. E. Perez ◽  
G. Perez-Palacios
Keyword(s):  
Androgen Receptor ◽  
Binding Capacity ◽  
Sedimentation Coefficient ◽  
Harderian Gland ◽  
Hormone Regulation ◽  
Apparent Dissociation Constant ◽  
Receptor Complexes ◽  
Male Golden Hamster ◽  
Sucrose Gradient Ultracentrifugation ◽  
Androgen Binding

ABSTRACT Studies were conducted in castrated golden hamsters to assess whether sexual dimorphism and sensitivity to sex steroid hormones in the rodent Harderian gland are mediated by an interaction of androgens with specific intracellular receptors. Physical properties, binding kinetics and stereospecificity of the androgen receptor were analysed using [3H]mibolerone as the radioligand. The presence of [3H]mibolerone–androgen receptor complexes with a sedimentation coefficient of 7–8S was demonstrated in Harderian gland cytosol by a linear sucrose gradient ultracentrifugation technique using a vertical rotor. Kinetic analysis revealed an androgen-binding site with an apparent dissociation constant of 0·3±0·07 (s.d.) nmol/l and a saturation binding capacity of 113±15 fmol/mg protein. Displacement studies indicated that unlabelled mibolerone, methyltrienolone, 5α-dihydrotestosterone and testosterone were efficient competitors for the androgen-binding sites, while progesterone, 17β-oestradiol, dexamethasone, dehydroepiandrosterone, ethiocholanolone and 5α-16-androsten-3-one were not. Experiments in long-term castrated animals revealed that the Harderian gland androgen receptor concentration and sedimentation coefficient remained unmodified. The results of these studies were interpreted as demonstrating the presence of a specific high-affinity intracellular androgen receptor in the male hamster Harderian gland. J. Endocr. (1987) 112, 3–8

Sign in / Sign up

Export Citation Format

Share Document