GLUCOCORTICOID RECEPTOR IN HUMAN-EMBRYO FIBROBLASTS .1. KINETIC AND PHYSICOCHEMICAL PROPERTIES

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1-3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 × 105 M-1 × min-1 and the dissociation rate constant was 4.6 × 10-5 min-1 at 0°C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5-9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone-receptor complexes was observed. © 1979.