The Role of Sulfhydryl Groups in the Binding of Glucocorticoids by Cytoplasmic Receptors of Lung and Other Mammalian Tissues1

Abstract

We examined the levels and stability of glucocorticoid receptor activity in cytosol preparations of rat lung and other tissues. [3H]Dexamethasome binding capacity at 2 C in lung cytosol decreases with a t1/2 of 40 min in the absence of steroid or a sulfhydryl compound. This rapid inactivation of unbound receptor is prevented and reversed by addition of a sulfhydryl compound (t1/2 = 22h); maximal binding occurs with 2 mM dithiothreitol (DTT), or with 20-25 mM mercaptoethanol, thioglycerol or glutathione. Binding activity is also stabilized by formation of the receptor-steroid complex; dissociation of the complex occurs at the same rate (t1/2 = 18.6h) in both the presence and absence of DTT. In the presence of DTT, cytosol of adult rat lung specifically binds 0.82 pmol of [3H]dexamethasone per mg protein (average 19,000 sites per cell) with an equilibrium dissociation contant of 2.5 nM at 2 C. At 37 C, dispersed lung cells show specific nuclear binding of hormone. DTT also increased dexamethasone binding activity in cytosol prepared from lung of adult hamster, adult mouse, fetal monkey and 11 of 15 tissues of adult rat. There was a minimal effect of DTT in reactions using cytosol of fetal rat lung, fetal and newborn human lung, rabbit lung, and liver, kidney, heart and testis of adult rat. Liver contains a heat-stable factor which mimics that stabilizing effect of DTT. The variable requirement for DTT may reflect in part tissue concentrations of endogenous sulfhydryl compounds. Our findings indicate that glucocorticoid binding activity of lung and many other tissues decays rapidly in vitro due to oxidation of receptor sulfhydryl groups. Maintenance of these groups in the reduced form by endogenous tissue factors, addition of sulfhydryl compounds, or binding of glucocorticoid stabilizes receptor and allows its detection in lung and other tissues previously found to contain little if any activity.