Some physicochemical characteristics of photoaffinity-labeled rabbit androgen-binding protein

Photoaffinity-labeled androgen-binding protein (ABP), present in cytosol prepared from the caput epididymidis of intact sexually mature rabbits, was produced using 17 beta-hydroxy-[1,2-3H]4,6-androstadien-3-one (delta 6-testosterone). Photo-labeled ABP could not be distinguished from unlabeled ABP by gel filtration chromatography, electrophoresis on nondenaturing polyacrylamide gels, or sucrose gradient ultracentrifugation. Rabbit ABP was found to have a sedimentation coefficient of 4.6S and a Stokes radium of approximately 45A. Based on these parameters, its native molecular weight was calculated to be approximately 86,300. A model of ABP as a prolate ellipsoid having an axial ratio of 10 is consistent with its frictional ratio of 1.555. When photolabeled rabbit ABP was examined on polyacrylamide gels containing sodium dodecyl sulfate, two androgen-specific peaks of approximately 52,000 and approximately 48,000 daltons were detected. Both peaks contained approximately the same amount of radioactivity. When photolabeled ABP was treated with the cross-linking reagent disuccinimidyl suberate before electrophoresis on gels containing sodium dodecyl sulfate, an additional androgen-specific peak corresponding to approximately 100,000 daltons was obtained. We interpret this peak to represent dimers of the lower molecular weight species. The 52,000- and 48,000-dalton subunits were observed regardless of whether the proteins were treated with 2-mercaptoethanol, indicating that the monomers are not linked by disulfide bonds.