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

Abstract

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.

The effect of experimental conditions on the detection of spermine in cell extracts and tissues

“…Polyamines are strong bases and form hydrogen bonds with ionic sites such as those found on proteins and nucleotides (DNA, RNA, tRNA) at physiological pH (18,19). Total protein content in prostatic fluid averages 15 mg/ml (15), however, the affinity of binding may differ between different proteins for example, it has been mentioned that spermine has a high affinity for spermine binding protein (SBP), a secretory product of the prostate (20). The binding to high molecular weight proteins reduces the value of T 2 and increases the broadening of the MR signals.…”

Prostatic Ductal System in Rats: Regional Variation in Morphological and Functional Activities1

“…Generation of polyclonal antisera for cathepsiil D and spermine binding protein was described earlier [8,9]. Purified prostatic binding protein [ l o ] was used to raise polyclonal antibody in rabbits [ 111.…”

Genes regulated by androgen in the rat ventral prostate

“…This analysis has generated 200-300 bases of sequence information for each of the fragments, which allowed us to determine the identity of 11 up-regulated genes and 1 down-regulated gene (Table 3). Six of these genes are known to be up-regulated by androgen in the rat prostate; these encode prostate-binding protein C2A (21), spermine-binding protein (19), probasin (26), cystatin-related protein 1 (22), cystatin-related protein 2 (22), and prostatein C3 (17). The other five genes were not previously known to be up-regulated by androgen in the prostate; these encode calreticulin (43), farnesyl pyrophosphate synthetase (44), LDL-R (45), adrenomedullin (46), and histo-blood group A transferase (47).…”

“…Genes that are up-regulated by androgen in the prostate include prostate-specific antigen (PSA) (15,16), prostatein C3 (17), sex-limited protein (slp) (18), spermine-binding protein (19,20), prostate-binding protein C2A (21), cystatin-related protein 2 (22,23), calreticulin (24), and probasin (25,26). Androgen ablation-induced genes include clusterin͞sulfate glycoprotein-2 (SGP-2)͞testosterone repressed prostate mRNA-2 (TRPM-2) (27,28), transforming growth factor ␤ (29), rat ventral prostate gene 1 (RVP.1) (30), glutathione S-transferase (31), c-myc (32), c-fos (33), and matrix carboxyglutamic acid protein (30).…”