Temperature-dependent kinetic correlates of the activation of the glucocorticoid-receptor complex.

Abstract

The effects of temperature on the kinetics of activation were studied in [3H]triamcinolone acetonide[( 3H]TA)-labeled cytosol preparations from mouse whole brain. After removal of unbound [3H]TA and molybdate (which prevents activation) from the unactivated steroid-receptor complex by gel exclusion chromatography, activation was initiated by incubation at 6-30 degrees C for 0.75-24 min and then rapidly quenched at -5 degrees C with Na2MoO4 (20 mM final concentration). The loss of the 9.2S (unactivated) form of the [3H]TA-receptor complex and the concomitant formation of the 3.8S (activated) form increased dramatically with increases in the activation temperature. These hydrodynamic changes were correlated directly with rapid time- and temperature-dependent increases in the binding of [3H]TA-labeled cytosol to DNA-cellulose (DNA-C). Further analyses of these data revealed a greater than 50-fold increase in the apparent first-order rate constant for the increased binding to DNA-C as the activation temperature was increased from 6 degrees C to 30 degrees C. An Arrhenius plot of these temperature-dependent kinetic constants revealed an energy of activation of 116 kJ. These data support a proposed model for activation of the glucocorticoid-receptor complex that includes the splitting of a 297 kDa, unactivated species into a 92 kDa, activated species.