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.

Binding of androgens in 5α-reductase-deficient human genital skin fibroblasts: inhibition by progesterone and its metabolites

1982 ◽  
Vol 94 (3) ◽  
pp. 415-427 ◽  
Author(s):  
M. B. Hodgins
Keyword(s):  
Androgen Receptor ◽  
Androgen Receptors ◽  
Cultured Cells ◽  
Reductase Activity ◽  
External Genitalia ◽  
Skin Fibroblasts ◽  
Urogenital Sinus ◽  
Receptor Complexes ◽  
High Affinity ◽  
Receptor Sites

Binding of [3H]testosterone and 5α-dihydro[3H]testosterone ([3H]DHT) to specific androgen-receptor sites of 5α-reductase-deficient human genital skin fibroblasts (five cell-lines) was studied in the intact cultured cells at 37 °C. Under the conditions of the experiments, conversion of [3H]testosterone into [3H]DHT was negligible. Both steroids bound to the same set of high-affinity saturable sites in cytoplasmic and nuclear fractions of the cells. Unlabelled testosterone, DHT and methyltrienolone competed effectively with the labelled steroids. Progesterone and oestradiol were weaker competitors; cortisol did not compete. The dissociation constant (Kd) for high-affinity complexes with [3H]testosterone (0·44 ± 0·035 nmol/l) was higher than that for [3H]DHT complexes (0·20 ± 0·090 nmol/l). Unlabelled DHT was more effective than unlabelled testosterone in competing with either radioactive steroid. Complexes of [3H]DHT and receptor dissociated more slowly than [3H]testosterone-receptor complexes and [3H]DHT bound more extensively to low-affinity non-saturable sites in fibroblasts. As judged by competition with the radioactive androgens, progesterone bound to the androgen receptor with a Kd of about 7 nmol/l. 5α-Pregnane-3,20-dione had an approximately fivefold lower affinity than progesterone for androgen receptors; 3α/β- or 20α-reduction lowered its affinity further. It is suggested that in 5α-reductase deficiency in man, progesterone in amniotic fluid and blood could effectively inhibit testosterone binding to androgen receptors in the male embryonic external genitalia. One function of the high levels of 5α-reductase activity normally found in embryonic external genitalia and urogenital sinus may be to protect these tissues from the potentially antiandrogenic action of progesterone.

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

scholarly journals Assay of Procarboxypeptidase U, a Novel Determinant of the Fibrinolytic Cascade, in Human Plasma

1999 ◽  
Vol 45 (6) ◽  
pp. 807-813 ◽  
Author(s):  
Katinka A Schatteman ◽  
Filip J Goossens ◽  
Simon S Scharpé ◽  
Hugo M Neels ◽  
Dirk F Hendriks
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Hippuric Acid ◽  
Active Form ◽  
Reference Interval ◽  
Healthy Individuals ◽  
High Affinity ◽  
Lysine Residues ◽  
The Mean

Abstract Background: Procarboxypeptidase U (proCPU) is a novel proenzyme found in human plasma. The active form, carboxypeptidase U (CPU; EC 3.4.17.20), retards the rate of fibrinolysis through its ability to cleave C-terminal lysine residues on fibrin partially degraded by plasmin. This reduces the number of high-affinity plasminogen-binding sites on fibrin. Methods: We developed an assay to determine the proCPU concentration in human plasma. The assay involved quantitative conversion of proCPU to active CPU by thrombin-thrombomodulin, a very efficient activator of proCPU, followed by determination of the enzymatic activity of CPU with the substrate hippuryl-l-arginine, using an HPLC-assisted determination of the released hippuric acid. Using this method, we established a reference interval based on 490 healthy individuals. Results: The mean proCPU concentration, determined after activation of the zymogen in diluted plasma and expressed as CPU activity, was 964 U/L, with a SD of 155 U/L. The population showed a gaussian distribution. However, we noticed important differences related to age and the use of hormone preparations. Conclusions: The sensitivity and precision of the method make it suitable for routine clinical determinations and as a reference procedure.

TRIIODOTHYRONINE AND THYROXINE NUCLEAR RECEPTORS IN LYMPHOCYTES FROM NORMAL, HYPER- AND HYPOTHYROID SUBJECTS

1977 ◽  
Vol 85 (1) ◽  
pp. 44-54 ◽  
Author(s):  
Th. Lemarchand-Béraud ◽  
A.-Ch. Holm ◽  
B. R. Scazziga
Keyword(s):  
Nuclear Receptors ◽  
Binding Sites ◽  
Intact Cell ◽  
Affinity Constant ◽  
Limited Capacity ◽  
Intact Cells ◽  
High Affinity ◽  
The Mean ◽  
Hypothyroid Patients ◽  
Hyperthyroid Patients

ABSTRACT In an investigation of thyroxine (T4) and triiodothyronine (T3) receptors in humans, the lymphocyte was chosen as the target cell. This study was performed to elucidate whether T3 and T4 bind to different receptors, if T4 is bound only after conversion into T3, and whether there is any modification of the receptors in hyper- and hypothyroidism. Lymphocytes were found to possess a high-affinity, limited-capacity binding sites for both T4 and T3. The mean equilibrium affinity constant (Ka) was 2.28 · 1010 ± 0.21 m−1 for T3, and 0.98 · 1010 ± 0.16 m−1 for T4. The mean number of saturable binding sites was 115 for T3, and 102 for T4. The binding capacities and affinities also determined in the lymphocyte nuclei isolated after incubation of the intact cell, were similar to those observed in the intact cells. In competition experiments, labelled T4 was as readily displaced by T3 as by T4 itself, whereas labelled T3 was displaced only by a 40 times higher concentration of T4 than T3. These observations suggest identical receptors for the two hormones and a binding of T4 as such, provided it is not in competition with T3. In lymphocytes from hyperthyroid patients, receptor affinities and numbers remained unchanged. In lymphocytes from hypothyroid patients, the affinity was normal, but the mean number of T3 binding sites was increased to 310 (P < 0.001), to return to normal after a few months of treatment.

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

Androgen regulation of the androgen receptor of the quail uropygial gland: application of a [3H]mibolerone exchange assay

1986 ◽  
Vol 109 (3) ◽  
pp. 299-306 ◽  
Author(s):  
Y. Amet ◽  
J.-H. Abalain ◽  
S. di Stefano ◽  
J.-Y. Daniel ◽  
K. Tea ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Binding Sites ◽  
Androgen Receptors ◽  
Sodium Molybdate ◽  
Uropygial Gland ◽  
Sebaceous Glands ◽  
Androgen Action ◽  
Androgen Regulation ◽  
Exchange Technique

ABSTRACT An exchange assay for androgen receptors in the quail uropygial gland using [3H]mibolerone was established. The most efficient exchange conditions were 3 days of incubation at 15 °C. Under these conditions, androgen receptors were stable in the presence of sodium molybdate, and the exchange of [3H]mibolerone with endogenous testosterone bound to cytosolic or nuclear androgen receptors was maximal. Less than 5% of [3H]mibolerone-binding sites occurred in the extracted nuclear pellets. Using this exchange technique, it was shown that androgen receptors in the uropygial gland of photostimulated male quail or castrated quail treated with testosterone were activated and that their concentrations in both cytosolic and nuclear fractions were increased. These results confirm the androgen dependency of the quail uropygial gland, and show that it is an organ which can be used as a model for the study of androgen action in sebaceous glands. J. Endocr. (1986) 109, 299–306

BINDING OF METHYLTRIENOLONE (R1881) TO A PROGESTERONE RECEPTOR-LIKE COMPONENT OF HUMAN PROSTATIC CYTOSOL

1977 ◽  
Vol 74 (2) ◽  
pp. 281-289 ◽  
Author(s):  
R. A. COWAN ◽  
SHEILA K. COWAN ◽  
J. K. GRANT
Keyword(s):  
Androgen Receptor ◽  
Progesterone Receptor ◽  
Binding Sites ◽  
High Affinity ◽  
Rat Prostate

The synthetic steroid methyltrienolone (R1881, 17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one) binds with high affinity to protein in cytosols prepared from human hyperplastic prostate. R1881 also binds to the androgen receptor of rat prostate and the progesterone receptor of rabbit uterus. Other steroids compete with R1881 for unoccupied binding sites in the human prostatic cytosols in a manner similar to that observed with the rabbit uterine progesterone receptor, rather than the rat prostatic androgen receptor. The progesterone receptor-like binding sites are a feature of the prostatic stroma rather than the epithelium.

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