Low molecular weight component of androgen receptor in cytosols from benign hypertrophic human prostate treated with high KCl solution

Cytosol of the benign hypertrophic human prostate was prepared in a low salt medium and then the concentration of salt was increased to 0.4 M with KCl (0.4 M KCl-cytosol). This preparation showed a high affinity binding to R 1881 and the binding was specific for androgens. These results suggest that the binding of the preparation to R 1881 was due mainly to the cytosolic androgen receptor. The R 1881 binding component in the 0.4 M KCl-cytosol was sedimented at 3S by sucrose density gradient centrifugation. The small sedimentation coefficient of the binder seems to be due to the high concentration of salt and not to degradation by proteolytic enzymes in the preparation. The molecular weight, Stokes radius and frictional ratio of this binding component were 32,000, 25.9 A and 1.24, respectively.     

An androgen-dependent pilosebaceous tumor spontaneously developed in the Japanese house musk shrew Suncus murinus

Following administration of [3H]testosterone to castrated male Japanese house musk shrews (Suncus murinus), radioactive metabolites were detected in sidegland nuclei and the major one of them was dihydrotestosterone (DHT). The androgen binding capacity of the cytoplasmic fraction of sideglands was measured in vitro by the use of [3H]R1881 as a ligand. The binding showed a high affinity for R1881 (Kd = 6.2 X 10(-10) M) and a low capacity (Bmax = 22 fmol/mg protein). Sucrose density gradient centrifugation brought about a peak of [3H]R1881 in the 7S region in low ionic strength buffer. Their characteristics as described above are consistent with those of other androgen target organs. A cutaneous pilosebaceous tumor, which spontaneously developed on the sidegland of old male S. murinus, was transplanted to nude athymic mice. It grew in males only and failed to grow in females and castrated males. A specific androgen binding was found in this tumor (Kd = 7.8 X 10(-10) M, Bmax = 100 fmol/mg protein). Therefore, this transplantable pilosebaceous tumor is androgen-dependent and can be utilized as a new suitable model in the study of the mechanism of androgen on tumor development.     

The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor

Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H]R1881, with high affinity (Kd = 0.8 +/- 0.3 nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H]R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.     

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).     

Up-regulation of androgen receptor binding in male rat fat pad adipose precursor cells exposed to testosterone: study in a whole cell assay system

Binding of androgens to adipocytes has previously been evaluated using cytosol fractions without taking into account nuclear binding, although the latter is suggested to be close to the physiological site of action. In the present study, performed in differentiated fat pad adipose precursor cells, we describe a simple, reliable and reproducible androgen binding assay in a system with intact cells. Tritiated and unlabeled methyltrienolone (R1881) were used to define specific and unspecific androgen binding. Triamcinolone acetonide was added to prevent the binding of R1881 to other types of receptors. Differentiated adipose precursor cells contain a homogeneous class of high affinity androgen binding sites, and binding is saturable and reversible. Binding apparently occurs at one site, with a Kd in the range of physiological androgen concentration (about 4 nM). Competition studies indicate that the receptor is specific for R1881, testosterone and dihydrotestosterone, which have approximately the same affinity, while progesterone, estradiol and dexamethasone show much lower affinity. Androgen binding was markedly enhanced after cellular exposure to R1881 and testosterone but not dihydrotestosterone, and this increase was dependent on protein synthesis, suggesting the formation of new receptors by these androgens. In conclusion, fully differentiated adipocytes contain a specific, high affinity receptor, the density of which is dependent on androgens.     

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.     

Androgen receptor binding to nuclear matrix in vitro and its inhibition by 8S androgen receptor promoting factor

The partially purified 4.5S [3H]dihydrotestosterone receptor binds to nuclear matrix isolated from rat Dunning prostate tumor with properties similar to those reported for androgen receptor binding in intact nuclei [Colvard, D.S., & Wilson, E.M. (1984) Biochemistry (preceding paper in this issue)] in that it requires Zn2+ and mercaptoethanol, is saturable, and is temperature dependent and of high affinity (Ka approximately 10(13) M-1). On a milligrams of DNA equivalent basis, the extent of matrix binding of androgen receptor (700 fmol of receptor bound/mg of matrix protein) is similar to that of intact nuclei, corresponding to approximately 1400 sites/nucleus. Association rate constants (ka) for 4.5S androgen receptor binding to matrix at 0, 15, and 25 degrees C are 2.7 X 10(5), 1.2 X 10(6), and 2.4 X 10(6) M-1 min-1, respectively, indicating an energy of activation of 15 kcal/mol. Up to 50% of matrix-bound receptor is extractable in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 5 mM pyridoxal 5'-phosphate. A protein fraction designated 8S androgen receptor promoting factor that promotes conversion of the 4.5S androgen receptor to 8 S [Colvard, D. S., & Wilson, E. M. (1981) Endocrinology (Baltimore) 109, 496-504] has been further purified and found to inhibit the binding of the 4.5S androgen receptor to isolated nuclei and nuclear matrix in a concentration-dependent manner. The results support the hypothesis that the 8S steroid receptor is a complex of the activated 4.5S androgen receptor with a non-steroid binding protein that renders the receptor incapable of binding in nuclei.     

Characterization studies of a rat hepatic cytosolic androgen-binding protein

A rat hepatic cytosolic [3H]methyltrienolone (R1881) binding protein was studied under various conditions. This protein was also compared with the male-specific high capacity--low affinity estrogen-binding protein derived from the same cytosolic fraction. Analysis of the R1881 binding protein in adult (60-85 days old) male rat liver cytosol indicated the presence of a high affinity--low capacity binding site (Kd = 0.3 nM; Bmax = 5.9 fmol/mg) and a lower affinity--higher capacity component (Kd = 10.4 nM; Bmax = 131 fmol/mg). The latter component was eliminated by addition of triamcinolone or cortisol to the assay mixture. Steroid binding to the high affinity R1881 site was specific for testosterone, dihydrotestosterone, androstenedione, and mibolerone, with a moderate specificity to cyproterone acetate, flutamide hydroxide, and estradiol. Saturation studies indicated that these steroids were binding to the same or a similar high affinity component except for flutamide hydroxide which produced nonsaturable displacement. The high affinity site had no specificity for progesterone, diethylstilbestrol, or cortisol. Like the high capacity--low affinity protein, this protein was not present in the immature, adult, or 10-day ovariectomized adult female. However, unlike the high capacity--low affinity protein, it was present in low quantities in the immature male. In addition, castration of the adult for 18 h, 4 days, or 10 days or hypophysectomy for 10-17 days did not have a significant effect on the high affinity component compared with the controls. Testosterone administration to these animals did not alter this protein binding. These studies indicate that a specific, high affinity--low capacity androgen-binding protein exists in rat hepatic cytosol. Furthermore, this protein shows age and sex dependency, but its presence is not affected by altering gonadal or hypophyseal factors in the adult male.     

Purification and characterization of the untransformed androgen receptor in rat prostate

The isolation and characterization of the untransformed form of androgen receptors has not yet been successful, owing to their inherent lability as well as to their ready proteolysis. In this study, we have stabilized rat prostate androgen receptors by sodium molybdate and by rapid filtration on phosphocellulose. Proteases were inhibited by bacitracin, aprotinin, leupeptin and PMSF. Under these conditions the untransformed complex was purified approx 3000-fold, corresponding to 18% yield, by differential chromatography on DEAE cellulose and phosphocellulose gels. The partially purified receptor has the same ionic characteristics as the original untransformed receptor of crude cytosol; in addition, it possesses a Stokes' radius of 75 A, as determined by Sephacryl S-300 gel filtration, a sedimentation coefficient of 8.8S, a calculated molecular weight of 275 kDa and a friction coefficient of 1.6. The [3H]R1881 receptor complex was specific to androgens since unlabelled R1881 and dihydrotestosterone were able to completely displace bound [3H]R1881, whereas estradiol, cortisol, and triamcinolone acetonide did not compete. The purified complex was a multimer dissociable by 0.6 M KCl, resulting in a form migrating in the 4S area on sucrose density gradient. After treatment with 0.5% formaldehyde, three forms were obtained, migrating in the areas of 8-9, 5-6 and 3-4S respectively, of a sucrose density gradient containing 0.6 M KCl. This is the first step towards the purification to homogeneity of the untransformed androgen receptor.     

Stability of receptor complexes in the rat ventral prostate and seminal vesicle bound to androgens: thermodynamic study

To examine the behaviour of the receptor-acceptor system of androgen of different biopotencies, we compared the stability of receptor complexes of dihydrotestosterone (DHT), methyltrienolone (R1881) and testosterone (Test) in cytosols, nuclei and nuclear extracts from ventral prostate and seminal vesicle of rats. Liberation of ligand from receptor complexes bound to these ligands followed the first-order kinetics. The rate constant for ligand liberation at 25 degrees C varied with the ligand. The receptor complexes bound to Test were most labile, while the receptor complexes bound to DHT were relatively stable, and intermediate stability was observed in the receptor complexes bound to R1881 under the conditions employed in the present study. Thermodynamic characteristics of the stability of the complexes were also different in these three androgens. The Arrhenius plots of the rate constant for the liberation of ligand from R1881- and DHT-receptor complexes in cytosols and nuclei showed curvilinearities, but the plots for Test-receptor complexes were almost linear. In addition, the stabilizing effect of molybdate on R1881- and DHT-receptor complexes in cytosols was observed in the range of low temperature, while the effect on Test-receptor complexes was significant at the higher temperature. The differences observed in the present study seem to be related to the difference in the biological potency of these androgens.     

Comparative binding specificity of methyltrienolone in human and rat prostate.

The binding of methyltrienolone (R 1881) in crude human hyperplastic prostate cytosol was determined by a charcoal assay. Maximum binding was observed after 2-3 h of incubation at 0 degrees C. This binding decreased steadily thereafter and reached 41% of the 2-hour values after 96 h of incubation. In human hyperplastic prostate, the binding of 3H-R 1881 was competed by low concentrations of R 1881, R 5020 and progesterone and by high concentrations of dihydrotestosterone (DHT) and 17 alpha-methyl-DHT. In the rat prostate, on the other hand, this binding was competed by low concentrations of DHT and 17 alpha-methyl-DHT and only by high concentrations of progesterone and R 5020. The apparent association constant (Ka) of R 1881 was determined in three human prostates and found to be 0.2-0.4 X 10(9) liters/mol; the number of binding sites ranged from 101 to 158 fmol per mg of protein. These findings constitute further evidence for the existence of relatively large amounts of a progesterone-binding component in human hyperplastic prostate.     

Identification of an androgen receptor in the adult chicken oviduct

The chicken oviduct androgen receptor was characterized by sucrose density gradient centrifugation, Scatchard analysis, competition studies, and affinity labeled with dihydrotestosterone 17 beta-bromoacetate. A specific 8.5 S peak was seen on 0.01 M KCl sucrose density gradients when the receptor was labeled with [3H]5 alpha-dihydrotestosterone. Specific 4.6 S peaks were seen when receptor labeled with [3H]5 alpha-dihydrotestosterone or [3H]dihydrotestosterone 17 beta-bromoacetate was analyzed on 0.3 M KCl sucrose density gradients. Scatchard analysis of [3H]5 alpha-dihydrotestosterone binding by oviduct cytosol was consistent with two binding sites. A Kd of 0.13 nM was found for the high affinity androgen receptor. Competition studies showed the following order of ligand affinity: 5 alpha-dihydrotestosterone greater than dihydrotestosterone 17 beta-bromoacetate greater than progesterone greater than estradiol. A 61.2 kDa protein was specifically covalently labeled with [3H]dihydrotestosterone 17 beta-bromoacetate. The chicken oviduct androgen receptor possesses characteristics similar to other androgen receptors, and provides a good source of androgen receptor for physicochemical studies of the native receptor protein.     

Author index

Binding of dexamethasone · receptors with isolated nuclei, DNA-cellulose and cellulose has been compared with respect to dependence on salt concentration and resistance to KCl extraction and DNAase I digestion. A solution of cytoplasmic dexamethasone-receptor complexes was prepared by the incubation of rat thymus cells with steroid at 3°C and breaking the cells by hypotonic lysis. Activation of the complexes was accomplished by warming the solution at 25°C for 15 min. Activation significantly increased the ability of dexamethasone · receptors to bind to nuclei and DNA-cellulose but not to cellulose. Dexamethasone-receptor complexes bound to nuclei at 3°C are completely resistant to extraction with 0.1 M KCl, 76% resistant to 0.2 M KCl and 20% resistant to 0.4 M KCl. Dexamethasone · receptors bound to DNA-cellulose are 45% resistant to extraction with 0.1 M and 0.2 M KCl and 29% resistant to 0.4 M KCl extraction. Cellulose-bound dexamethasone · receptors are not resistant to any of these extractions. DNAase I treatment releases 60% of the dexamethasone · receptors bound to DNA-cellulose but only 13% of those bound to nuclei, though at least 60% of the nuclear DNA is solubilized. The presence of 0.15 M KCl decreases binding of activated dexamethasone · receptors to nuclei by 73% but to DNA-cellulose by only 17%. Pretreatment of nuclei with 0.1–0.4 M KCl reduces their capacity to bind activated dexamethasone · receptors by 90% whereas similar treatment reduces the capacity of DNA-cellulose to bind dexamethasone · receptors by only 29%. Nuclei extracted with 0.1 M KCl appear to have a limited capacity to accept dexamethasone · receptors. These studies demonstrate that binding of dexamethasone · receptors to nuclei and DNA-cellulose differs by (a) the higher resistance of nuclear complexes to KCl and DNAase I treatment; (b) the much greater sensitivity of nuclei to KCl treatment.     

Zinc potentiation of androgen receptor binding to nuclei in vitro

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 microM ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl, and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 microM total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added at a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; however, in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 X 10(-13) mol of receptor sites/mg of DNA, corresponding to approximately 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10(13) M-1 was approximated. Pyridoxal 5'-phosphate (less than or equal to 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 10 mM pyridoxal 5'-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells

The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 microM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The Kd for the high affinity binding site was 2 x 10(-9) M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5 alpha-androstane-3 alpha, 17 beta-diol, testosterone, estradiol and estrone. Progesterone, 5 alpha-androstane-3 beta, 17 beta-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 +/- 7.6 fmol of bound R1881 per 10(6) cells (n = 34) or 2075 +/- 1434 fmol per mg of DNA; these values correspond to an average of 6624 +/- 4577 sites per cell. Thus, using this whole cell assay system, specific and androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.     

Activation of the rat liver androgen-receptor complex

Activation of androgen receptor in rat liver cytosol was studied in vitro. The state of activation was judged by binding of [3H] R1881-receptor complex to chromatin. High ionic strength (0.4 M KCl as a final concentration) provoked the binding of [3H] R1881-receptor complex to chromatin at 0 degrees C. At low ionic strength, activation was very slow at 0 degrees C, but was very rapid at 25 degrees C and reached the maximum at 15 min of heating.     

Androgen binding by skeletal muscle nuclei

A study was made of 3H-19-nortestosterone binding by isolated nuclei and 0.4 M KCl nuclear extract of the rat skeletal muscle. Binding specificity was ascertained by incubation in the presence of various unlabeled steroids. The Kd values were measured for nuclei and 0.4 M KCl nuclear extract (11.6 +/- 2.5 nM and 9.9 +/- 1.6 nM, respectively). The amount of binding sites was 24.1 +/- 1.7 fmol/mg DNA or 13.7 +/- 1.0 fmol/g tissue. Enzymatic treatment with pronase and DNase shows that nuclear androgen receptors are proteins. DNA was noted to have a stabilizing effect. DNase treatment of nuclei during extraction with 0.4 M KCl was shown to significantly increase the amount of specifically bound radioactivity in the extract.     

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.     

Demonstration of a nuclear androgen receptor in erythroblasts obtained by culture of human bone marrow

The new techniques of culture of bone marrow have shown that androgens and 5 beta steroids exert a direct effect on erythroid precursor cells from human or animal bone marrow. By contrast, the mechanisms of the intracellular actions of those compounds are poorly understood. Tritiated methyltrienolone (R 1881), a synthetic androgen that highly binds to androgen receptor, has been used for the study of a binding activity in nuclear extracts of cultured erythroblasts from human bone marrow. The nuclear extracts contain binding sites saturable at low concentrations of 3H-R 1881 (20-30 nM). Scatchard analysis revealed that the R 1881-nuclear complex has a dissociation constant (Kd) of 25-50 nM, and the number of binding sites was 235-370 fmoles/mg protein. The complex sedimented on 5-20% sucrose gradient in the 3.9 S region and 5 beta dihydrotestosterone compete strongly with R 1881 for binding sites. This binding component has characteristics of high affinity, low-capacity, sedimentation behaviour, and specificity commonly attributed to "androgen receptors".