Production of monoclonal antibody against the C1 component of rat estramustine-binding protein: Immunohistochemical study of rat prostate

The monoclonal antibody against estramustine-binding protein (EMBP) was produced by immunizing a mouse with EMBP antigen purified from rat ventral prostate. On western blotting analysis this antibody recognized the EMBP C₁ component, and in an absorption test it recognized the EMBP antigen. Immunohistochemically, this antibody revealed positive staining for the ventral and dorsolateral prostate. In the epithelium of the ventral prostate, intense immunostaining was observed in the intraluminal secretory product; however, in the epithelium of the dorsolateral prostate, the staining was intense in the cytoplasm of the epithelial cells. These findings suggested differences of EMBP localization in the ventral and the dorsolateral prostate. Since the intensity of immunostaining for EMBP was decreased in the prostate of castrated rats, we considered that this antibody reflected the androgen dependency of EMBP.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

Characterization of androgen receptors after photoaffinity labelling with [3H]methyltrienolone (R1881)

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in rat prostate, in a human transplantable prostatic adenocarcinoma (PC-82) and in calf uterus. Androgen receptors preparations were partially purified either via differential chromatography on 2',5'-ADP-Sepharose (rat prostate), via anion exchange fast protein liquid chromatography (rat prostate and PC-82) or via DNA-cellulose chromatography (calf uterus). Purification factors obtained with the three different methods were: 245, 75 and 40 respectively. Photolabelling of receptor preparations was performed via irradiation with a high pressure mercury lamp either before or after partial purification. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of approx 95 kD. The covalent attachment of [3H]R1881 to the 95 kD protein could be completely suppressed by a 200-fold molar excess of dihydrotestosterone. In rat prostate cytosol an androgen receptor with a molecular mass of approx 50 kD could be photoaffinity labelled with R1881. A similar size was found for the androgen receptor in the human prostatic adenocarcinoma. Our results show that photoaffinity labelling of androgen receptors with [3H]R1881 as ligand can be applied for characterization of partial purified androgen receptor preparations.     

5alpha-Dihydrotestosterone binding protein in rat ventral prostate; purification, nuclear incorporation, and subnuclear localization.

Treatment of cytosol from the rat ventral prostate with cold acetone (-20 degrees C) evoked a 8 approximately 10-fold increase in the binding capacity with 5alpha-dihydrotestosterone (DHT). Starting from the extract of acetone-dried prostate cytosol, some 400 approximately 600-fold purification of the DHT-binding protein complex was acieved by (NH4)2504 fractionation, DEAE-cellulose chromatography and gel- filtration with Sephadex G-200. The purified 3H-DHT-binding protein complex was incorporated into the nuclei from the ventral prostate in a temperature dependent manner. The similar incorporation was also observed in nuclei from the liver and the kidney...     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

Reduction of prostatic binding protein-messenger ribonucleic acid sequences in rat prostate by castration

Messenger RNA coding for the three subunits of prostatic binding protein was isolated from polysomal RNA of rat ventral prostate by oligo (dT)-cellulose affinity chromatography and purified by repeated sedimentations through sucrose gradients under denaturing conditions. The purified mRNA migrated as a 9S peak in sucrose gradient centrifugation and hybridized with its cDNA within 2 log Rot units. In a cell-free reticulocyte lysate system, the mRNA directed the synthesis of three polypeptides of 12000, 9000, and 8000 daltons. These translation products were identified as the subunits of prostatic binding protein by immunoreaction with antibodies to this protein. Quantitation of prostatic binding protein-mRNA sequences in normal and castrated rats by hybridization with the cDNA probe showed that 3-day castration reduced the prostatic binding protein-mRNA sequences to less than 2% of the normal level. Similar hybridization was performed by using the cDNA to determine the level of prostatic binding protein coding sequences in polysomal poly(A) RNA following castration. The results showed a first-order rate constant of 3.92 X 10-2 h-1 for reduction of prostatic binding protein-mRNA sequences in polysomes. The period of castration required to reduce the level of these sequences to 50% of the normal level was calculated to be 17.6 h.     

Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect.

The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40--60% within 20--40 h after castration. This effect is reversed very rapidly within 15--30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate.     

Purification and characterization of a steroid-binding sialoglycoprotein from rat ventral prostate

An androgen-dependent sialoglycoprotein was purified from the secretion of rat ventral prostate by chromatofocusing and DEAE-Sepharose column chromatography. It showed a native molecular weight of 47,000 and consisted of two dissimilar subunits with molecular weights of 20,000 and 18,000. However, each subunit contained a common peptide with molecular weight of 16,000. It also contained 442 +/- 62 micrograms sialic acids per milligram protein and bound pregnenolone with a binding affinity of 1.2 microM-1. Its amino acid composition was similar to those of other known prostatic steroid-binding proteins. Hence, we propose that it is the sialylated form of rat prostatic steroid-binding protein.     

Immunochemical characterization of the androgen-dependent spermine-binding protein of the rat ventral prostate.

A solid-phase radioimmunoassay was developed to measure the level of the androgen-dependent spermine-binding protein (SBP) in the cytosol fraction of the rat ventral prostate during endocrine manipulation. The concentration of SBP and immunologically cross-reacting material (CRM) in the ventral prostate was at least 5000 times higher than the level of CRM detected in rat serum or cytosol from other rat tissues. Cytosol from the ventral prostate of intact rats was separated by DEAE-cellulose chromatography into three major fractions of CRM. One of these fractions corresponded to the elution position of SBP. Cytosol prepared from rats 48 h after castration lacked SBP and one of the two other fractions of CRM. This loss coincided with an increase in CRM in the remaining fraction. No significant difference was detected in the total level of CRM when intact and 48 h-castrated rats were compared. Injection of rats with 5 alpha-dihydrotestosterone (DHT) immediately after castration prevented these changes in the profile of CRM. Several proteins cross-reacting with antibodies to purified SBP were detected in cytosol by using an immunoblot procedure. The highest-Mr band corresponded to SBP. The effect of short- and long-term castration and subsequent DHT treatment on CRM was studied by using the immunoblot technique. Short-term castration (2 days) led to the disappearance of CRM coinciding with SBP (Mr 35 000-38 000) and an increase in smaller forms of CRM (Mr 24 000 and 22 000). Injection of rats with DHT 2 days after castration led to the reappearance of CRM corresponding to SBP, which returned to normal levels within 4 to 5 days of treatment. Long-term castration (up to 14 days) led to a gradual disappearance of all CRM; subsequent DHT treatment led to the reappearance of all forms of CRM and normal levels were attained within 5 days. We have identified SBP and the various forms of CRM as a secretory product of the rat ventral prostate by immunohistochemical staining and by DEAE-cellulose fractionation of prostatic fluid. Prostatic fluid is rich in proteolytic activity and these proteinases may be responsible for processing SBP to small forms of CRM.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Estramustine binding in rat,baboon and human prostate measured by high pressure liquid chromatography

High pressure liquid chromatography (HPLC) was used to determine ³H-estramustine (estradiol-17β3N-bis-[2-chlorethyl] carbamate), ³H-17β-hydroxy-5α-androstan-3-one (³H-dihydrotestosterone or ³H-DHT), ³H-estradiol-17β (³H-E₂) and ³H-3β-hydroxy-5-pregnen-20-one (³H-pregnenolone) binding in 50μ1 of cytosol utilizing a column which separates proteins in the molecular weight range of 2,000 to 70,000 daltons. The rat prostate contains a protein in considerable concentration and with the highest affinity for estramustine (375,000dpm ³H-estramustine per mg. cytosol protein) among the substances tested. Operationally, we have named this protein “estramustine binding protein” (EBP), though it is very likely similar to other previously described prostatic proteins (e.g., α-protein, prostatein, prostatic binding protein). The sensitivity of the HPLC method disclosed EBP-like proteins, but in much lesser concentrations, in some of the other tissues tested. The concentration of these proteins in the human and baboon prostates was much lower (average for the baboon cranial lobe 4800dpm/mg cytosol protein, with a somewhat higher value for the caudal lobe) than that in the rat gland. The amount of the EBP-like protein was higher in prostatic cancer than in that of benign prostatic hypertrophy (BPH) (range 9350 – 25,900 vs. 2200 – 18,900 dpm/mg cytosol protein). In the human, the highest value was found in one normal prostate tested (106,000dpm/mg cytosol protein).     

Assay of androgen binding sites by exchange with methyltrienolone (R 1881).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) binds specifically to androgen receptor in rat prostate cytosol where, unlike androstanolone, it is not metabolized. By exchanging bound endogenous hormone in rat prostate cytosol with labelled R 1881, it is possible to measure total (free anc occupied) binding sites. This assay method has also been applied to the measurement of androgen receptor sites in human benign prostatic hypertrophy where R 1881 has the added advantage of not being bound by any contaminating plasma protein (sex hormone binding protein).     

Conservation of the DNA binding domain and other properties between porcine and rat glucocorticoid receptors

Activated glucocorticoid receptor protein (GCR) was partially purified from porcine liver cytosol by sequential chromatography on phosphocellulose and DNA-cellulose using a modification of a protocol developed for purification of rat GCR. This partially purified preparation, when separated by SDS-polyacrylamide gel electrophoresis and immunoblotted, indicated that a Mr = 94,000 protein band cross-reacts with a monoclonal antibody against rat GCR. A nitrocellulose filter binding assay showed that both the partially purified porcine and rat GCRs interact specifically with a cloned synthetic 24 base pair deoxyoligonucleotide containing the GCR binding sequence in the first intron of the human growth hormone (hGH) gene. This specific protein-DNA interaction is blocked by a single base pair change in the binding site. All three putative domains of the GCR molecule: the steroid binding, immunoreactive, and DNA binding have been conserved between two divergent species.     

Aromatase activity in microsomes from rat ventral prostate and Dunning R3327H rat prostatic adenocarcinoma

We have measured aromatase activity in microsomes obtained from rat ventral prostate, using the 3H2O release method as described by Weisz. Production of 3H2O from 1 beta-[3H]androstenedione correlated with estrogen production measured by RIA and by TLC. The assay was optimized for incubation time and protein concentration, and used to determine the aromatase activity of ventral prostate microsomes from rats of varying age. Aromatase activity per mg microsomal protein increased from an average of 4 pmol/mg protein X h in 3-month old rats to 68 pmol/mg protein X h in 8-month old rats. Aromatase activity was also measured in microsomes from the Dunning R3327H rat prostatic adenocarcinoma, and was increased in tumors removed 225 days after implantation compared to tumors removed 141 days after implantation. Tumors removed 225 days after implantation from rats which had been treated with DES for 14 days displayed increased aromatase activity compared to untreated tumors. The presence of aromatase activity in the rat ventral prostate and rat prostatic adenocarcinoma would allow regulation of estrogen levels independent of circulating estrogen. Thus, in situ changes in estrogen production with age may contribute to the development of prostatic disease.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Two molecular forms of thymidine kinase in the cytosol of regenerating rat liver.

Two forms (Peak A and Peak B) of thymidine kinase [EC 2.7.1.75] from regenerating rat liver cytosol were resolved and partially purified by Deae-cellulose chromatography. Both fractions were identical with respect to their substrate requirement, pH optima, metal requirements, and molecular weight, as judged by their sedimentation in sucrose density gradient centrifugation. Peak B differed from Peak A in heat sensitivity, inhibition by dCTP and Km for thymidine and ATP. Peak B enzyme was the only enzyme found in normal adult liver and Peak A enzyme was the form increasing predominantly in regenerating liver.     

Rat epidermal growth factors: purification and tissue content

Rat epidermal growth factor (rEGF) was isolated from the submaxillary gland of male rat by reversed phase high performance liquid chromatography and ion exchange chromatography. The binding of purified rEGF to human carcinoma cells (A-431) and its tritiated thymidine uptake on rat epidermal fibroblast cells (FR) were almost the same as those of purified commercially available mouse EGF (mEGF). Antisera to rEGF was raised in rabbits and a radioimmunoassay (RIA) system was established. The assay range of the RIA was about 1.0 to 100 ng/ml. The within assay coefficients of variation were 5 to 10%, while the between assay coefficients of variation were 5 to 13%. The tissue content of rEGF of male rats (10 weeks old) was examined. As a result, the submaxillary gland was found to contain a very high concentration of rEGF (214 micrograms/g wet tissue) as predicted, and digestive tissues, stomach, intestine and duodenum contained 2.49, 3.57, 9.44 ng/g wet tissue, respectively. The amounts in prostate and seminal vesicle were relatively high, being 65.6 and 2,268 ng/g wet tissue, respectively. The amount in the submaxillary gland increased markedly after 7 weeks of age. These results suggest that EGF is an important factor in gonadal function.     

Analysis of an estrogen binding macromolecule in human pancreas by radioimmunoassay

A radioimmunoassay for quantitation of the human pancreatic estrogen binding protein (hEBP) was developed using polyclonal rabbit hEBP antiserum and iodinated purified hEBP. Parallel dose-response curves were obtained when serial dilutions of human serum and of cytosols obtained from human pancreas, prostate and colon were analyzed simultaneously with serial dilutions of purified hEBP standard. Very high levels of hEBP (500-1000 mg/kg wet weight) were found in normal pancreas. High as well as medium levels were found in pancreatic carcinoma tissue and medium values (0.1-1 mg/kg wet weight) in prostate, colon and ovarian tissue. Other tissues and serum from healthy volunteers showed low values, usually below 0.1 mg/kg. When serial dilutions of rat pancreatic cytosol were analyzed in the hEBP assay, [125I]hEBP was displaced by the rat preparation, but the dose-response curves were not parallel to the standard curves, indicating similarity but non-identity between the estrogen binding proteins in human and in rat pancreas.