Activation of the rat liver glucocorticoid-receptor complex

Abstract

The rat liver glucorcorticoid receptor has been activated using three procedures: heat, gel filtration, and dilution. With time after heat activation the steroid--receptor complex loses its capacity to bind to DNA--cellulose, while receptor activated by Sephadex G-25 and by dilution maintains DNA--cellulose binding capacity. The rates of steroid dissociation from nonactivated and activated receptor and essentially identical. However, nonactivated receptor is capable of rebinding steroid, while activated receptor has a reduced capacity to rebind steroid. The results of the gel filtration and dilution studies suggest that a low-molecular-weight factor(s) exists in rat liver cytosol which is involved in the process of activation.

Basic amino acids stimulate the activation of the glucocorticoid—receptor complex

“…As previously reported [6,8], dilution of liver cytosol with Tris buffer results in a dramatic increase in the degree and rate of activation of the glucocorticoid-receptor complex. That the level of activation is, at least in part, due to the Tris molecule is shown in table 1.…”

“…A low molecular weight inhibitor has been proposed to explain activation by dilution and gel filtration [6]. The basic amines could form a complex with such an inhibitor, thus favoring its dissociation from the unactivated receptor.…”

“…Receptor was activated by heating the cytosol for 30 min at 25'C, by a 5-fold dilution with buffer at 0°C or by the addition of reagents as indicated in the table legends. Receptor activation was assayed by measuring its ability to bind to DNA-cellulose [6] . L-Amino acids and other reagents were obtained from Sigma.…”

“…Dilution initiates slow activation [. 5,6] and evidence for the participation of a macromolecule [7] or a small molecule [6] as an inhibitor of the process has been presented. Recent evidence also indicates that the use of Tris buffer increases the level of activation much more than phosphate buffer during dilution or ion exchange chromatography [8].…”

Purification of chicken intestinal receptor for 1,25-dihydroxyvitamin D.

“…This resin has been used to study properties of an estrogen receptor (30). DNA-cellulose has been used to study and purify an extensive number of nuclear proteins and steroid hormone receptors such as the progesterone A subunit (31), the estrogen receptor (32,33), the glucocorticoid receptor (34), and the androgen receptor (35). In addition, heparin-Sepharose has been used to purify the "native" form of the estrogen receptor (21).…”