Biochemical characterization of glucocorticoid receptors of rat testis

Abstract

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 KC1. Activation of the complex at 25°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 KC1. 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 μg/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 10mM molybdate. Steroid unbound receptors were inactivated at 25°C and 4°C with a T 1 2 of 2 h and 24 h, respectively. Ten mM molybdate slightly protected the unbound testis receptor at 25°C. However, molybdate, dithiothreitol, and molybdate plus dithiothreitol were unable to either enhance or reactivate [ 3H]dexamethasone binding of unbound receptors at 4°C or 25°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.