Photoaffinity labeling identification of a specific binding protein for the anabolic steroids stanozolol and danazol: An oligomeric protein regulatedby age, pituitary hormones, and ethinyl estradiol

We have demonstrated previously that both rat and human liver microsomes co ntain a highly specific binding protein for the anabolic steroids stanozolo l(ST) and danazol (DA). In this study we solubilized the male rat liver ST- binding protein (STBP) and investigated the following parameters: 1) pharma cological properties, 2) hydrodynamic properties, 3) peptidic composition, 4) the effects of age and hypophysectomy, and 5) inducibility by 17 alpha-e thinyl estradiol. We found that STEP is an integral protein bound to the en doplasmic reticulum. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfon ate (CHAPS) provided its optimal solubilization without changes in its phar macological properties, i.e, high specificity for ST and danazol, between n atural steroids and ligands of low affinity glucocorticoid-binding sites or of progesterone-binding sites. Hydrodynamic properties of the STEP showed that it has a molecular mass of at least 118 kDa. SDS-PAGE of covalently la beled STEP under nonreducing conditions showed that [H-3]ST binds to a 110- kDa protein. The STEP was resolved under reducing conditions into three pep tides of 55, 31, and 22 bDa, respectively. STEP increased from immature to adult rats, and it dramatically decreased after hypophysectomy. Unlike the 22-kDa peptide, both the 55- and 31-kDa peptides drastically decreased in b oth immature and hypophysectomized rats. 17 alpha-Ethinyl estradiol adminis tration to immature or hypophysectomized rats induced the 55- and 31-kDa [H -3]STBP to a greater extent than the 22-kDa peptide. Thus, STEP appears as an oligomeric protein composed of hormone-regulated peptides. The availabil ity of solubilized STEP and the fact that it can be induced in vivo represe nt major steps toward the purification and functional significance of this unique steroid-binding protein.