Herbert H. Wotiz

GABRILOVE, J L M.D.; SHARMA, D C Ph.D.; WOTIZ, H H Ph.D.; DORFMAN, R I Ph.D. Author Information

Prostatic membranes contain high affinity [dissociation constant (KD) = 1.16 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I]iodo-EGF is specific since it is displaced by excess EGF but not by insulin, fibroblast growth factor, platelet-derived growth factor, or multiplication-stimulating activity. Affinity labeling with [125I]iodo-EGF and subsequent cross-linking with disuccinimidyl suberate demonstrated the specific binding of [125I]iodo-EGF to a macromolecule with a mol wt of 170,000. Castration of mature rats resulted in a 3- to 6-fold increase in [125I]iodo-EGF binding, while treatment of 7-day castrated rats with 5 alpha-dihydrotestosterone decreased the number of binding sites. Administration of estrogen or progesterone produced a slight decrease in EGF binding sites but not nearly to the extent observed with 5 alpha-dihydrotestosterone, suggesting that the observed effect is androgen specific. These results demonstrate that rat prostate contains specific binding sites for EGF and that their level is modulated by androgens.

SummaryThe ovaries of the sea urchin, Strongylocentrotus franciscanus, and the pecten, Pecten hericius, were extracted for steroids. Estrogenic activity equivalent to 10 μg or less per kilo of wet tissue was found in each species by bioassay. The most active estrogenic substance was identified as estradiol-17β. Separations were made by counter-current distribution (29 transfers: upper phase—70% methanol; lower phase—50% chloroformic, 50% carbon tetrachloride), paper chromatography (3 systems: toluene-propylene glycol; ligroin-propylene glycol; benzene-formamide), and bioassay (Astwood method). Progesterone was also identified tentatively by column chromatography and the Hooker-Forbes assay. Other progestational substances were present, one of which may be Δ4-3-keto-pregnen-20β-ol. The occurrence of these steroids in 2 distinctly different phyla indicates a wide distribution and may be of some significance in the evolution of sex hormones.

We have synthesized three peptides with amino acid sequences corresponding to amino acids 533-547, 597-611, and 765-779 of the human progesterone receptor (hPR). These peptides were conjugated to keyhole limpet hemocyanin and injected into mice and rabbits to develop antibodies to hPR. Antibodies to the undenatured form of PR were elicited only by the peptide with amino acid sequence 533-547. Fusion of SP2/0 myeloma cells with spleen cells from mice immunized with this peptide produced several active clones. Rabbit sera from immunized animals produced one antiserum that reacted with the undenatured form of PR. One monoclonal antibody (PR-AT 4.14) and one antiserum (PR-AT533) raised against peptide-(533-547) were characterized. Binding of these antibodies to the undenatured form of PR was demonstrated by analysis of the antibody-receptor complexes on sucrose density gradients and by immunoprecipitation techniques. Binding of PR to the antibodies was inhibited by excess peptide. The antibodies did not react with estrogen, glucocorticoid, or androgen receptors, but recognized PR from human breast cancer as well as calf, rabbit, mouse, and rat uteri, indicating that this epitope was conserved among these species. Based on sucrose density gradient analysis of PR prepared and labeled in the presence of proteolysis inhibitors and sodium molybdate, the antibodies bound to a site on the intact undenatured PR, but failed to bind to partially degraded steroid-binding form of the receptor, suggesting that the antibody-binding domain is at or near a site sensitive to proteolysis.