High Affinity Protein For Stanozolol

J Steroid Biochem Mol Biol. 2003 Dec;87(4-5):253-64.

Photoaffinity labeling identification of thyroid hormone-regulated glucocorticoid-binding peptides in rat liver endoplasmic reticulum: an oligomeric protein with high affinity for 16beta-hydroxylated Stanozolol.

Betancor-Hernandez E, Perez-Machin R, Henriquez-Hernandez L, Mateos-Diaz C, Novoa-Mogollon J, Fernandez-Perez L.

Department of Clinical Sciences, Health Sciences Center, University of Las Palmas de Gran Canaria, and Instituto Canario de Investigacion del Cancer, P.O. Box 550, 35080 Canary Islands, Spain.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic actions of the17alpha-alkylated Testosterone derivative (17alpha-AA) stanozolol (ST). We have previously reported that male rat liver endoplasmic reticulum contains two steroid-binding sites associated with high molecular mass oligomeric proteins: (1) the ST-binding protein (STBP); and (2) the low-affinity glucocorticoid-binding protein (LAGS). To further explore the role of LAGS on the mechanism of action of ST, we have now studied: (1) the interaction of ST and its hydroxylated metabolites with solubilized LAGS and the cytosolic glucocorticoid receptor (GR); and (2) the effects of hormones on the capability of STBP to bind ST. We found that, unlike 17alpha-methyltestosterone, neither ST nor its hydroxylated metabolites bind to GR. However, the 16beta-hydroxylation of ST significantly increases the capability of LAGS to bind ST. Interestingly, 3'-hydroxylation of ST abrogates the capability of LAGS to bind ST. ST (k(i)=30 nM) and 16beta-hydroxystanozolol (k(i)=13 nM) bind with high affinity to LAGS, and are capable of accelerating the rate of dissociation of previously bound dexamethasone from the LAGS. STBP and LAGS are strongly induced by ethinylestradiol. However, unlike STBP, LAGS is regulated by thyroid hormones and growth hormone, which proves that these steroid-binding activities are associated with different binding sites. These findings seem to suggest a novel mechanism for ST whereby membrane-associated glucocorticoid-binding activity is targeted by the 16beta-hydroxylated metabolite of ST. ST and its 16beta-hydroxylated metabolite modulate glucocorticoid activity in the liver through negative allosteric modulation of LAGS, with the result of this interaction an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

Endocrinology. 2000 Sep;141(9):3377-87.

Photoaffinity labeling identification of a specific binding protein for the anabolic steroids stanozolol and danazol: an oligomeric protein regulated by age, pituitary hormones, and ethinyl estradiol.

Luzardo OP, Machin RP, Diaz-Chico BN, Fernandez L.

Toxicology Section, Center of Health Sciences and Faculty of Veterinary, University of Las Palmas de Gran Canaria, Canary Islands, Spain.

We have demonstrated previously that both rat and human liver microsomes contain a highly specific binding protein for the anabolic steroids stanozolol (ST) and danazol (DA). In this study we solubilized the male rat liver ST-binding protein (STBP) and investigated the following parameters: 1) pharmacological properties, 2) hydrodynamic properties, 3) peptidic composition, 4) the effects of age and hypophysectomy, and 5) inducibility by 17alpha-ethinyl estradiol. We found that STBP is an integral protein bound to the endoplasmic reticulum. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) provided its optimal solubilization without changes in its pharmacological properties, i.e. high specificity for ST and danazol, between natural steroids and ligands of low affinity glucocorticoid-binding sites or of progesterone-binding sites. Hydrodynamic properties of the STBP showed that it has a molecular mass of at least 118 kDa. SDS-PAGE of covalently labeled STBP under nonreducing conditions showed that [3H]ST binds to a 110-kDa protein. The STBP was resolved under reducing conditions into three peptides of 55, 31, and 22 kDa, respectively. STBP 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 both immature and hypophysectomized rats. 17alpha-Ethinyl estradiol administration to immature or hypophysectomized rats induced the 55- and 31-kDa [3H]STBP to a greater extent than the 22-kDa peptide. Thus, STBP appears as an oligomeric protein composed of hormone-regulated peptides. The availability of solubilized STBP and the fact that it can be induced in vivo represent major steps toward the purification and functional significance of this unique steroid-binding protein.