ER 125 CNRS and Unité de Recherches sur le Métabolisme Moléculaire et la Physiopathologie der Stéroides de l''INSERM, Département de Chimie Biologique, Faculté de Médecine de Bicêtre, 78 rue du Général Ledere, 94270 BicêtreFrance
Abstract:
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 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees 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.