Interaction of the hepatic glucocorticoid-receptor complex with Affigel blue.

Unlike the unactivated glucocorticoid-receptor complex, the thermally activated glucocorticoid-receptor complex was able to bind to Affigel blue (a matrix previously shown to bind proteins containing a dinucleotide fold region) under low ionic conditions (0.05 M K C1). Glucocorticoid-receptor complex binding capacity to Affigel blue was enhanced by increasing salt concentration. Optimal binding was obtained at 0.15 M K C1 and remained at a plateau level up to 0.4 M K C1. In contrast to Affigel blue binding, glucocorticoid-receptor complex binding to nuclei was optimum at low ionic strength buffer, declined at 0.15 M K C1 and became negligible at 0.4 M K C1. Interestingly, at physiological ionic strength (0.15 M K C1) both nuclei and Affigel blue bound to the glucocorticoid-receptor complex with almost identical capacity. Glucocorticoid-receptor complexes incubated 45 min at 25 degrees C (activation conditions) in the presence of 10 mM molybdate were unable to bind to Affigel blue (or isolated nuclei) as expected. The results obtained suggest that Affigel blue mimics isolated nuclei in the binding of activated glucocorticoid-receptor complexes under physiological (0.15 M K C1) conditions. In addition, Affigel blue may provide a rapid and easy method to study glucocorticoid-receptor complex activation and interaction with nuclear acceptor sites.