Temperature-Dependent Kinetic Correlates of the Activation of the Glucocorticoid-Receptor Complex

Publisher Website
Google Scholar
Abstract
The effects of temperature on the kinetics of activation were studied in [3H]triamcinoIone 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°C for 0.75–24 min and then rapidly quenched at –5°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 >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°C to 30°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.