| Abstract: | The high-affinity triarylethylene anti-oestrogen H1285 4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between 3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with 3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. 3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. 3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with 3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of 3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for 3H]oestradiol. Because of the extremely slow dissociation of 3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of 3H]H1285-receptor complexes with those of 3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. 3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with 3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of 3H]H1285-receptor complexes and 3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of 3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for 3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor. |
