Structure and subunit dissociation of the mouse glucocorticoid receptor: rapid analysis using vertical tube rotor...

The structure and subunit dissociation of the glucocorticoid receptor from the mouse AtT-20 pituitary tumor cell line was analyzed on sucrose gradients using a Beckman VTi 80 vertical tube rotor. This technique afforded a very rapid analysis (65 min) of the variously sedimenting forms compared to swinging-bucket rotor sucrose gradients, which take 16 h to run. Thus, it was possible to detect and study the molybdatestabilized, oligomeric, untransformed receptor (9.1 S) in the presence of 0.3 M KCl. Under similar conditions using the swinging-bucket rotor, only the monomeric, transformed species (3.8 S) was observed. That is, artifactual subunit dissociation was minimized using the vertical tube rotor, allowing the study of the receptor structure in a more native state. Further studies demonstrated that Sephadex LH-20 chromatography causes receptor transformation. Thus, dextran-charcoal adsorption is preferred for the removal of unbound hormone under certain circumstances. Finally, using vertical tube rotor sucrose gradients, it was determined that the transformation of the mouse AtT-20 glucocorticoid receptor involves a conversion of the oligomeric, 9.1 S, untransformed species to a 5.2 S, transformed moiety. This suggests that the 5.2 S, intermediate transformed species may be the physiologically relevant form of this gene regulatory protein.