Kinetic and thermodynamic evidence of a molybdate interaction with glucocorticoid receptor in calf thymus

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Abstract
The effect of molybdate on the kinetic and thermodynamic properties of the dexamethasone-receptor interaction was studied in calf thymus cytosol. In the presence of molybdate both the equilibrium binding studies and the association and dissociation experiments reveal a significantly lower affinity of the receptor for [3H]dexamethasone. At 0°C the equilibrium dissociation constant increases from 0.8 nM to 1.8 nM, the association rate constant shifts from 1.5 × 108 M−1 h−1 to 0.2 × 108 M−1 h−1, whereas the rate of dissociation of the untransformed receptor increases from 0.04 h−1 to 1.1 h−1 in the molybdate-containing buffer. All these effects appear dependent on the concentration of molybdate but the dissociation of the transformed receptor (0.01 h−1) is unaffected. The enthalpy for the association, ΔH+, increases at least twofold whereas the entropy, both for the association (ΔS*=−25 to +104 J K−1 mol−1) and for the equilibrium (ΔS°=−100 to +38 J K−1 mol−1), is markedly influenced by the presence of molybdate. Taken all together these data suggest that molybdate interacts with the receptor molecule turning it into a form that displays low affinity for steroid, in addition to the well-documented incapacity to transform itself. This fact leads us to think that both the binding and the transformation are the expression of conformational modifications involving molybdate-sensitive groups.