The Use of a Photoaffinity Ligand to Compare Androgen-Binding Protein (ABP) Present in Rat Sertoli Cell Culture Media with ABP Present in Epididymal Cytosol*

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Abstract
The kinetic, binding, and physicochemical properties of androgen-binding protein (ABP) produced by rat Sertoli cells in culture [media ABP (mABP)] have been assessed and compared to ABP present in rat epididymal cytosol (eABP). Physicochemical studies were accomplished using ABP that had been covalently labeled using the photoaffinity probe 17β- hydroxy-[l,2-3H]4,6-androstadien-3-one ([3H]Δ6-testosterone). mABP was obtained from serum-free Sertoli cell culture media by ultrafiltration or precipitation with ammonium sulfate. mABP and eABP were photolyzed in the presence of [3H]Δ6- testosterone alone or together with nonphotoreactive 5α-dihydrotestosterone. Electrophoresis of photolabeled mABP and eABP on nondenaturing polyacrylamide gels (PAGE) showed similar mobilities. Site-specific covalent attachment of [3H]Δ6- testosterone to mABP and eABP was demonstrated by sodium dodecyl sulfate-PAGE. This analysis revealed the presence of 2 androgen-specific peaks corresponding to subunit molecular weights of approximately 50,000 and approximately 42,000. Both subunits were observed with ABP obtained by ammonium sulfate precipitation or ultrafiltration and were present in the same relative ratios when media from 1 day or 4 days in culture were assessed. Treatment of photolabeled mABP with the cross-linking reagent dimethylsuberimidate before sodium dodecyl sulfate-PAGE resulted in the appearance of 2 additional androgenspecific peaks of approximately 109,000 and 100,000 daltons, indicating that mABP, like eABP, is composed of dimers. Chromatography of photolabeled eABP and mABP on a calibrated Sephadex G-200 column indicated identical Stoke's radii (45 A). ABPs from both sources have the same native molecular weight (94,000) and frictional ratio (1.5). mABP and eABP showed similar mobilities on steady state polyacrylamide gels. The halftimes of dissociation of [3H]5α-dihydrotestosterone from them (6.30 ± 0.23 and 4.90 ± 0.47 min, respectively) were statistically identical, and the relative binding affinities of 10 unlabeled compounds for sites on mABP and eABP were comparable. In contrast, mABP exhibited a significantly (P < 0.005) lower equilibrium dissociation constant than eABP under identical assay conditions (1.98 ± 0.51 vs. 6.08 ± 0.62 nM, respectively). (Endocrinology108: 786, 1981)