Binding of the Photoaffinity Ligand 17β-Hydroxy-4,6- Androstadien-3-One to Rat Androgen-Binding Protein: Comparison with the Binding of 17β-Hydroxy-5α- Androstan-3-One*

Abstract

17β-Hydroxy-4,6-androstadien-3-one (δ-testosterone), because of its αβ,unsaturated carbonyl group, appeared, on a theoretical basis, to be a potential photoaffinity label for rat androgen-binding protein (ABP). However, before we attempted to use this compound as an affinity label, we first determined if it would bind to ABP and compared its binding parameters with those of 17β-hydroxy-5α-androstan-3-one (5α- DHT), the most potent natural ligand for ABP. We have demonstrated, using polyacrylamide gel electrophoresis, sucrose gradient ultracentrifugation, and a charcoal assay method, that both [³H]δ⁶-testosterone and [³H]5α-DHT bind to a low capacity component in rat epididymal cytosol having the same relative mobility and sedimentation coefficient as ABP. Competitive binding studies, performed using a charcoal adsorption procedure, indicated that the relative affinities of 10 unlabeled steroids for ABP are essentially iden ical regardless of whether [³H]δ⁶testosterone or [³H]5α-DHT was used as the ligand. We have also shown that unlabeled δ⁶-testosterone and unlabeled 5α- DHT inhibited labeled ligand binding to ABP in a parallel manner. These data strongly suggested that Δ⁶-testosterone and 5α-DHT bind to the same site on ABP. This suggestion was confirmed by kinetic analysis of the binding data. This analysis showed that Δ⁶-testosterone and 5α-DHT are competitive inhibitors of androgen binding to ABP. Although both steroids bind to the same site, their binding kinetics are different. The dissociation half-time of [³HΔ⁶-testosterone from ABP is 0.8 ± 0.2 min, while that of [³H]5α-DHT is 4.5 ± 0.4 min. Scatchard analysis of the binding reactions indicated that [³H]δ⁶-testosterone has a lower affinity for ABP (K_d = 1.15 ± 0.08 × 10^-8 M) han does [³H]5α-DHT (K_d = 6.08 ± 0.62 × 10^-9 M). Fewer binding sites were detected in cytosol when [³H]δ-testosterone was the ligand (0.27 ± 0.06 × 10^-12 mol/mg cytosol protein) than when [³H]5α-DHT was used (0.66 ± 0.14 × 1O^-12 mol/mg protein). These quantitative differences are probably, at least in part, attributable to the more rapid dissociation of δ⁶-testosterone from ABP. These data indicated the potential utility of δ⁶- testosterone as a photoaffinity label for ABP.