Development of an [3H] glucocorticoid exchange assay in rat liver cytosol.

An exchange assay for the assessment of glucocorticoid binding sites both free and steroid bound, in rat liver cytosol has been developed. The procedure, which is straight forward and noncumbersome involves the utilization of two sulfhydryl interacting agents, p-hydroxymercuribenzoate and dithiothreitol. The former in low concentration dissociates the steroid from the glucocorticoid receptor complex in relatively short time. The latter regenerates, quite rapidly and with good yield, the glucocorticoid binding activity of the receptor treated with the mercurial compound. The high recovery (99%) of the hormone binding activity of the receptor may be due to the few steps involved in the procedure. The exchange assay when applied to a physiological experimental situation was found to be valid and gave a true measure of total receptor content in rat hepatic cytosolic preparations.