Stabilization of Glucocorticoid Receptors.

The stabilization of glucocorticoid receptors in rat liver cytosol by molybdate and an endogenous heat-stable factor has been examined. Molybdate stabilizes receptors to inactivation caused by temperature, salt, ammonium sulfate precipitation, gel filtration and low concentrations of detergent. Since these are not all enzymatically-mediated processes, it is proposed that molybdate stabilizes the glucocorticoid receptor through a reversible interaction with the receptor itself, and not through its well-known ability to act as a phosphatase inhibitor. Although the mechanism of inhibition is not known, molybdate may interact with sulfhydryl and /or phosphate groups on the receptor protein or on some accessory binding factor. To assay the activity of the heat-stable factor, rat liver cytosol was washed and filtered using an Amicon UM 10 membrane to remove low M(,r) stabilizing components. The filtered cytosol binding capacity is more rapidly inactivated than unfiltered cytosol at both 0(DEGREES) and 20(DEGREES)C and addition of the heat-stable factor inhibits the rapid inactivation. The factor also inhibits transformation of steroid-bound receptors in filtered cytosol. Both the receptor stabilizing and the transformation inhibiting assays have been used to quantitate factor activity. Incubation with a variety of hydrolases, alkaline phosphatase or N-ethylmaleimide does not affect factor activity. The heat-stable activity is not due to the presence of nucleotides, inorganic phosphate or pyridoxal 5'-phosphate, and the activity is not adsorbed to charcoal. Receptor stabilizing activity is widely distributed and has been found in several tissues in the rat and in liver from avians, amphibians and mammals. It is also present in primitive eukaryotes such as yeast. The heat-stable factor has been purified 200-fold with respect to Lowry-reactive material and the M(,r) is estimated to be between 200 and 700 daltons. The major peak of receptor stabilizing activity and transformation inhibiting activity coelutes on both Sephadex G-10 and Dowex 1 chromatography, suggesting that the same heat-stable factor (or factors) are responsible for both activities. Preliminary evidence is presented suggesting that the heat-stable factor may also inhibit protein dephosphorylation using ('32)P-labeled protein as substrate. "Ph.D.BiologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/158672/1/8204694.pd