9S binding protein for androgens and progesterone.

Abstract

A steroid binding protein fraction with a sedimentation coefficient of approximately 9 S (molecular weight -200,000) has been identified in 105,000 X g supernatants of several androgen-responsive organs. Highest concentrations were found in epididymis and testis, but small amounts were detected in prostate, seminal vesicle, kidney, submandibular gland, and lung. The 9S protein binds [3H~dihydrotestosterone (17B-hydroxy-5a-androstan-3-one) and [3H~progesterone (4-pregnene-3,20-dione) with equilibrium binding constants of approximately 105 M-' and 106 M-1, respectively. The concentration of 9S binding sites in epididymis is approximately 10-11 mol/mg of supernatant protein, which is at least 105 times greater than the concentration of androgen receptor. 9S binding protein appears to be a nonsecretory, intracellular protein and has properties different from the androgen receptor. It is unretarded on DEAE-Sephadex chromatography at pH 8.0, and its sedimentation rate on sucrose gradients is not altered at high ionic strength (0.4 M KCI). Like the androgen receptor, its binding activity, which is maximal between pH 7 and 9.5, is heat labile, decreased by sulfhydryl reagents, and enhanced by 2-mercaptoethanol. It is suggested that because of its high concentration and low affinity, 9S binding protein may function in the intracellular accumulation or compartmentalization of androgens or progesterone.Androgen-dependent organs accumulate androgens from blood against a concentration gradient. This ability has been explained in part by the presence of high-affinity receptor proteins, which specifically bind and translocate androgen to the cell nucleus (1-5). Because of the small number of receptors in these target cells, however, it is unlikely that binding to this protein alone can fully account for their androgen accumulating capacity. Moreover, saturation of receptor sites with unlabeled androgen does not appreciably diminish target tissue accumulation of radioactive androgen (5). For these reasons it seems that additional mechanisms take part in promoting androgen accumulation, possibly membrane-associated active uptake or binding to high-capacity intracellular proteins. Experiments described in this report demonstrate the presence of a 9S protein fraction with a high binding capacity for dihydrotestosterone (1713-hydroxy-5a-androstan-3-one) and progesterone (4-pregnene-3,20-dione Preparation of 105,000 X g Supernatants. Supernatants (105,000 X g) were prepared as described (6) from tissues derived from Sprague-Dawley rats that were either castrated 24-48 hr before they were killed or hypophysectomized (Hormone Assay Lab, Chicago, IL) at age 60 days, 30 days before they were killed. Rats were killed by decapitation; the tissues were excised and placed in saline (40), blotted, and weighed. Tissues were minced and homogenized with a Thomas glass-Teflon homogenizer (decapsulated testes) or with an Ultra Turrax (other tissues) in three or four volumes of buffer containing 10% (wt/vol) glycerol/i mM EDTA/1 mM 2-mer...

Hsp90: Chaperoning signal transduction

“…Ligand binding is a prerequisite for the specific interaction of SHRs with DNA. Sedimentation data indicated that untransformed SHRs are part of large multiprotein complexes (Wilson et al, 1977; cf. Pratt and Toft, 1997).…”

Sex steroid-binding protein in a subline of dunning R 3327 prostatic adenocarcinoma of the rat.

“…Wilson et al (1977), which sedimented at 9S and bound to dihydrotestosterone with an equilibium binding constant of approximately 10-5 M, was distributed in androgen-target tissues of rats. Androgen binding protein produced by Sertoli cells of testis (Hansson et al, 1975) is another sex steroid-binding protein, whose molecular weight is about 90,000 (Larrea et al, 1981).…”

Hsp70 and Hsp90—a relay team for protein folding

“…Summary of known Hsp90 client proteins (taken, extended and modified from Picard 2002) Protein Reference Transcription Androgen receptor Joab et al 1984;Veldscholte et al 1992 Aryl hydrocarbon (Ah) receptor Denis et al 1988;Perdew 1988 Ecdysone receptor Arbeitman and Hogness 2000 Estrogen receptor Joab et al 1984;Sabbah et al 1989 Glucocorticoid receptor Ben Or 1989 Heme activator protein (Hap1) Zhang et al 1998HSF-1 Ali et al 1998 Hypoxia-inducible factor-1a Minet et al 1999Mineralocorticoid receptor Rafestin-Oblin et al 1989 MTG8 myeloid leukemia protein Komori et al 1999p53 Blagosklonny et al 1996 Progesterone receptor Catelli et al 1985;Schuh et al 1985 Toft and Gorski (Toft and Gorski 1966) demonstrated that estradiol added to the cytosol of rat uteri was incorporated in a large 9S complex. Subsequently, ultracentrifugation experiments indicated that ligand-free steroid hormone receptors (SHRs) are part of large multiprotein complexes (Pratt and Toft 1997;Wilson et al 1977). This represents the first direct demonstration of hormone binding to a receptor protein in a cell-free system.…”