Biochemical characterization of glucocorticoid receptors of rat testis.
Rat testis cytosolic glucocorticoid receptors were characterized by DEAE-cellulose chromatography, Sephadex G-100 columns and sucrose-density gradients. The unactivated [3H]dexamethasone-receptor complex showed two distinct peaks of macromolecular bound radioactivity on DEAE-cellulose chromatography. Peak I eluted just after the column wash, while peak II eluted at 0.28 M KCl. Activation of the complex at 25 degrees C for 45 min resulted in a significant increase in peak I with a concomitant decrease in peak II and the appearance of a third peak at 0.18 M KCl. Both the unactivated and activated [3H]dexamethasone-receptor complex, when analyzed on Sephadex G-100 columns, showed a single macromolecular bound radioactive peak having a Stokes radius of 6.5 nm. Treatment of the [3H]dexamethasone-receptor complex (6.5 nm holo-receptor) with trypsin (0.5 microgram/ml) resulted in the appearance of a smaller (2.0 nm) fragment but no intermediate sized forms of the receptor were observed. The complexes sedimented as 7-8 S (in low salt) and as 4.6 S (in high salt) forms in sucrose gradients in the presence or absence of 10 mM molybdate. Steroid unbound receptors were inactivated at 25 degrees C and 4 degrees C with a T 1/2 of 2 h and 24 h, respectively. Ten mM molybdate slightly protected the unbound testis receptor at 25 degrees C. However, molybdate, dithiothreitol, and molybdate plus dithiothreitol were unable to either enhance or reactivate [3H]dexamethasone binding of unbound receptors at 4 degrees C or 25 degrees C. Activation of testis [3H]dexamethasone-receptor complexes resulted in a 2-3 fold enhancement in subsequent binding to testis nuclei in vitro. In addition, we observed that activated [3H]dexamethasone-receptor complexes were precipitated with 30-35% ammonium sulfate, while unactivated complexes were precipitated with 30-40% ammonium sulfate.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone