Androphilic Proteins Research Articles

Grade and histochemical R-1881 binding in prostatic cancer.

Comparison of the grades classified by Gleason with R-1881 binding which was attributable to total androphilic proteins in tissues was performed in 42 untreated and 13 relapsed prostatic cancers. Increasing malignancy correlated with loss of staining on R-1881 binding when examined both in primary pattern and in individual tumor cell clusters. In relapsed cancer, however, there was no R-1881 binding irrespective of any grades.

Studies of the human testis. XIX

Human testosterone-oestradiol-binding globulin (hTeBG) was purified from pregnancy serum by ammonium sulphate precipitation, preparative flatbed electrofocusing, Concanavalin A-Sepharose affinity chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex chromatography and preparative polyacrylamide gel electrophoresis. The yield was 0.3 mg of hTeBG with a specific acitivity of 1.1 nmoles DHT bound per mg. An antiserum to TeBG was raised in rabbits. Anti-hTeBG IgG was separated from rabbit TeBG by DEAE-Affi-Gel-Blue chromatography. Anti-hTeBG was titrated using protein A-Sepharose which quantitatively binds IgG and therefore bound [3H]DHT-hTeBG-anti-TeBG complexes. The androgen binding components from human testis were separated on Concanavalin A-Sepharose columns into excluded and retained fractions. The antibody bound both testis fractions with titration curves which paralleled that of TeBG, indicating that these androphilic proteins share common immunodeterminants with hTe-BG. The possibility that these testicular proteins are identical in amino acid sequence to TeBG and differ only in carbohydrate content will require further verification. Finally, these results indicate that antibodies to TeBG can be used to study human testicular androgen-binding protein.

Androgen receptor content in cytosol from non-tumoral human kidney and its relation to steroid hormone environment.

The content of cytoplasmic androgen receptors (Bmax) was analyzed in non-tumoral renal tissue of 12 men and 15 postmenopausal women using a synthetic androgen (methyltrienolone) as ligand and a method of dextran-coated charcoal. The Bmax in both sexes was compared, establishing correlations between it and circulating levels of testosterone, dehydroepiandrosterone sulfate, androstenedione, and estradiol to find out the possible influence of the hormonal environment on androgen receptors in the human kidney. No differences in Bmax were observed between males (46.3 +/- 24 fmol/g of tissue) and females (45.4 +/- 26 fmol/g), in spite of the significantly greater (p less than 0.01) levels of circulating testosterone in the former group. No significant linear correlations existed between any of the steroids analyzed and the Bmax. These results demonstrate the existence of androgenic receptors in non-tumoral human kidney and indicate that its content is not regulated by circulating levels of testosterone. The concentrations of the principal extratesticular androgens and estradiol do not seem to have a quantitative influence on these androphilic proteins either.

Histochemical observation of R 1881-binding protein in human prostatic cancer.

A conjugate of fluorescein isothiocyanate and bovine serum albumin bound with R 1881-3-carboxymethyloxime was used to visualize the location of androphilic proteins in tissues from patients with prostatic cancer. The fluorescence was located in the carcinoma cells but not in the stroma. Although intensity of the staining was varied in fluorescence-positive cases, almost all carcinoma cells were stained. On the contrary, positively stained cells were scarcely observed in fluorescence-negative cancer. Among 46 cases of untreated prostatic cancer examined, 76% were fluorescence-positive. 7 cases out of 9 who had relapsed from endocrine therapy showed negative fluorescence. It was concluded that the fluorescence in the tissue sections of the untreated prostatic cancer were well correlated with the histologic grade, and that fluorescence-positive cases had responded well to the endocrine therapy, as judged 6 months after the start of treatment.

Androphilic proteins in the human prostate.

Dihydrotestosterone-binding protein in cytosols from human prostates was different from the R1881- and R5020-binding proteins. The R1881- and R5020-binding proteins in the cytosol were very similar and most of the binding sites for R1881 were capable of binding R5020. However, nuclear extracts showed equal binding to dihydrotestosterone and R1881, but not to R5020, suggesting that there might not be a progestin receptor in the human prostate. Maximum binding sites to dihydrotestosterone, R1881, and R5020 in cytosols were very similar among "normal" and pathological prostates except that dihydrotestosterone binding was higher in "normal" prostates than in the pathological ones. Histochemically, R1881-binding was observed in the epithelial cells and in malignant cells, but not in the stroma.

Properties of androphilic proteins in cytosols of human benign prostatic hypertrophy.

Androphilic proteins in the cytosol from the human benign prostatic hypertrophy are separated into two fractions by Sephadex chromatography; void volume fraction and IgG fraction which was eluted near the site of hIgG. In the present study, properties of these two androphilic proteins were compared. Association constants of these proteins were in the order of 10(9) M-1. However, the binding capacity of the former was smaller than that of the latter. These two androphilic proteins well bound to nuclei, and the high-affinity and saturable binding to nuclei was observed in the 3H-dihydrotestosterone-IgG fraction complex, while binding of 3H-dihydrotestosterone-void volume fraction complex to nuclei was low affinity and unsaturable. The binding of the complexes to chromatin seems to be of low affinity and nonsaturable. These androphilic proteins did not bind to calf thymus DNA. Salt extractability of the bound void volume fraction after incubation with nuclei was not different from that of the bound IgG fraction. It was observed that the chromatographic behavior of the androphilic protein in IgG fraction was changed after incubation with nuclei.

Inhibition by various steroids on dihydrotestosterone binding to androphilic protein in human benign prostatic hypertrophy.

The effect of various steroidal compounds on the binding of the androphilic protein to dihydrotestosterone in the cytosol of tissues of human benign prostatic hypertrophy was examined. Androgens, as well as estrogens and gestagens showed an inhibitory effect on the binding. Non-steroidal anti-androgens were revealed to be weak inhibitors on the binding. Two androphilic proteins were observed in Sephadex G-200 chromatography in fractions eluted at the void volume and in fractions appearing at the site of IgG, and the rate of inhibition on the binding of both fractions by various steroids was compared. The rate of inhibition by various compounds was generally higher in the IgG fraction than in the void volume fraction. When the ligand and inhibitors were incubated with the cytosol prior to fractionation by Sephadex chromatography, rate of inhibition was lower than that obtained when the ligand and inhibitors were reacted with fractions after chromatography. Implications of the difference observed in these two experiments are not clear at this moment. The results obtained by the protamine precipitation experiment were almost the same as those by the experiment using the extract of the unfractionated acetone-dried cytosol, therefore, the protamine procedure does not seem to precipitate the tissue specific androphilic protein specifically.

Androphilic proteins in cytosols of human benign prostatic hypertrophy.

To examine the properties of androphilic proteins in human benign prostatic hypertrophy, the binding capacity and affinity of the proteins were determined after acetone-treatment, ammonium sulfate precipitation and chromatographies of DEAE and Sephadex G-200. Androphilic proteins in the extract of acetone-dried cytosol from the hypertrophic human prostate was precipitated at 30-50% saturation of ammonium sulfate. The binding of this fraction to dihydrotestosterone and testosterone was high affinity, but the binidng to estradiol-17 beta was the one of non-specific. Androphilic proteins in the 30-50% fraction were eluted from DEAE-cellulose column by buffer containing 0.05 M KCL. On Sephadex G-200 chromatography of 30-50% fraction, the androphilic proteins were observed in three peaks; one was eluted in the void volume and other two were eluted at the sites of IgG and albumin. The amount and ratio of proteins eluted in the void volume and the site of IgG from Sephadex G-200 column were variable in individual tissue samples. The chromatographic behavior of the 30-50% fraction in Sephadex G-200 was not changed significantly by introducing 0.4 M KCl in the system. Polyacrylamide gel electrophoresis was applied for further separation of the proteins.

A novel affinity column for isolation of androgen binding protein from rat epididymis.

An androgen affinity column was synthesized by covalently linking 3-oxo-17beta-hydroxy-5alpha-androstan-17alpha-(6-hexanoic acid) to cyanogen bromide activated Sepharose through a dipropyldiamine side arm. This column was designed to recover androphilic proteins from homogenates rich in nonspecific esterases. An extract of rat epididymis was adsorbed on the affinity column after partial purification by ammonium sulfate precipitation. The column was washed with 1 M KCl and the androgen binding protein eluted with 17beta-hydroxy-5alpha-androstan-3-one resulting in a 1,100-fold increase in specific activity. This protein had the same mobility on polyacrylamide gels and the same estimated molecular weight (135,000 daltons by gel filtration) as androgen binding protein in the original extract. By contrast, electrophoresis on sodium dodecyl sulfate containing gels yielded 2 bands with estimated molecular weights of 42,000 and 47,000 daltons. These observations are consistent with a subunit structure for rat epididymal androgen binding protein.

Tissue androgens and androphilic proteins in rat epididymis during sexual development

The appearance of the epididymal 8s cytoplasmic receptor in the rat during sexual development was followed and correlated with the endogenous concentrations of three biologically active androgens in the epididymis, testosterone (T), dihydrotestosterone (DHT)and 5α-androstane-3α, 17β-diol (Diol). The results indicate that receptors could not be evidenced in the 10-day-old animal in which androgens were undetectable. The 8s receptor was first detected in the 20 day-old rat, coincidently with a rise in the concentration of DHT (0,24 ng/ epididymis) while T and Diol remained too low to be measured. At the 26th day of life a peak of androgen binding activity, sedimenting at approximately 4S, was seen and probably corresponds to ABP. This pattern of two binding peaks (8 and 4S) remained constant through adulthood. The association constant (Ka) of the 8s receptor from 35-, 45- and 60-day-old rats was found to be similar. T became detectable in the epididymides of the 35 day-old rat (0.03 ng/ep.) and increased with sexual maturation. However, DHT concentrations were higher than those of T at all ages studied and Diol could not be detected at any age. These results raise the possibility that the synthesis or availability of receptors in the developing animals is under androgenic control.

364. Androgen tissue concentrations and androphilic proteins in rat epididymis during sexual development

364. Androgen tissue concentrations and androphilic proteins in rat epididymis during sexual development