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.
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.
Luzardo
O P
OP
Toxicology Section, Center of Health Sciences and Faculty of Veterinary, University of Las Palmas de Gran Canaria, Canary Islands, Spain.
Machín
R P
RP
Díaz-Chico
B N
BN
Fernández
L
L
D000375:Aging / Q000502:physiology*; D045930:Anabolic Agents / Q000493:pharmacokinetics*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003613:Danazol / Q000493:pharmacokinetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004965:Estrogen Antagonists / Q000493:pharmacokinetics*; D004997:Ethinyl Estradiol / Q000494:pharmacology*; D007016:Hypophysectomy; D008099:Liver / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008862:Microsomes, Liver / Q000378:metabolism; D019920:Photoaffinity Labels; D010907:Pituitary Hormones / Q000502:physiology*; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013197:Stanozolol / Q000493:pharmacokinetics*; D013347:Subcellular Fractions / Q000378:metabolism
D045930:Anabolic Agents; D002352:Carrier Proteins; D004965:Estrogen Antagonists; D008565:Membrane Proteins; D019920:Photoaffinity Labels; D010907:Pituitary Hormones; D004997:Ethinyl Estradiol; D013197:Stanozolol; D003613:Danazol