The determination of the dissociation velocity constants of rabbit uterine cytosol receptor-oestrogen complexes

Abstract

The dissociation velocity constants of rabbit uterine cytosol receptor-oestrogen complexes have been measured by three methods in a cell-free system at 30°, pH 8.0. Two of the methods were based on following the release of radioactivity from the receptor-oestrogen complexes. In the third method the receptor-oestradiol complexes were prepared and incubated with free [ 3H]oestradiol under steady-state conditions and the exchange between oestradiol and [ 3H]oestradiol was measured. Dextracharcoal suspension was used in each case to separate the bound from free oestrogen. The velocity constant ( k −1) for the dissociation of the high-affinity receptor- oestradiol complex was found to be close to 1·10 −4 sec −1; the value of this constant for oestrone and oestriol was 1.5·10 −4 sec −1 and 2.4·10 −4 sec −1, respectively. The velocity constant of dissociation of the low-affinity receptor-oestrogen complex is the same for all three oestrogens; its value was found to be close to 3·10 −3 sec −1. Comparison of the three methods indicated that (1) the binding reaction between the high-affinity receptors and oestrogen molecules does not require small molecular weight cofactors which could be adsorbed by charcoal; (2) the receptor sites are not inactivated by the release of the ligand.