Rat uterine progesterone receptor analyzed by [3H]R5020 photoaffinity labeling: evidence that the A and B subunits are not equimolar

The hormone-binding components of the rat uterine progesterone receptor were investigated by the methods of [3H]R5020 photoaffinity labeling and sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis. Two specifically labeled peaks were observed at mol wt of 85,600 +/- 1,200 and 109,600 +/- 1,200 (n = 31), resembling the A and B progesterone receptor components previously described in other systems. However, in contrast to the equimolar ratio reported in other systems, the level of subunit A observed was consistently greater than that of B (A/B ratio = 3.2 +/- 0.3; n = 31). The unusual A/B ratio prompted a complete validation of the photolabeling procedure in this system. Although the levels of specific binding increased, there was no change in the A/B ratio with varying [3H]R5020 concentrations (5-80 nM) or with time of UV exposure (0.5 min to 3 h). Although adsorption to hydroxylapatite indicated that specific [3H]R5020 binding was reduced by 72.0 +/- 6.4% within 5 min of UV exposure, relabeling the irradiated preparations with [3H]R5020 resulted in little change in specific [3H]R5020 binding. TLC analysis of [3H]R5020 (Rf = 0.48 +/- 0.01; n = 4) after irradiation demonstrated rapid photolysis resulting in a 94.3 +/- 2.5% (n = 3) loss of authentic [3H]R5020 within 5 min. After photolysis, at least two new tritiated products were recovered with Rf values of 0.20 +/- 0.03 and 0.72 +/- 0.02. Analysis by adsorption to hydroxylapatite indicated that the photolysis products competed for specific [3H]R5020-binding sites in cytosol with only 10-fold lower relative binding activity than authentic R5020. Thus, these compounds probably account for the increase in specific photolabeling of the A and B peaks achieved when UV exposure is prolonged from 5 to 30 min. (Abstract Truncated)