Photoaffinity labeling of rat steroid 5α-reductase (isozyme-1) by a benzophenone derivative of a 4-methyl-4-azasteroid

Abstract

[1,2- 3H]N-4(Benzylbenzoyl)-3-oxo-4-aza-4-methyl-5α-androstane-17β-carboxamide ([ 3H]-4MABP) has been synthesized as a photoaffinity probe of the steroid-binding domain of rat steroid 5α-reductase isozyme-1 (5αR-1). Reversible binding of the probe to 5αR-1 in microsomal preparations yielded a reversible dissociation constant ( K d of ∼3 nM, whereas inhibition experiments indicated that the probe had a 50% inhibition concentration of 4.4 nM and was a competitive inhibitor of the enzyme ( K i ≈ 3 nM) with respect to testosterone. SDS-PAGE analysis of microsomal, detergent-solubilized, and (6.5%) polyethylene glycol-precipitated fractions of 5αR-1 photolyzed with [ 3H]4MABP in the presence of NADPH showed that the radioactivity was incorporated into a single protein band with a mass of 26 kDa (apparent molecular weight of 5αR-1). UV photolysis was accompanied by an irreversible loss in enzyme activity, consistent with its covalent modification. Increasing the time of UV irradiation and concentration of [ 3H]4MABP indicated that the half-life and apparent K d for its photo insertion were ∼3 min and 7.5 nM, respectively. Photolysis in the presence of a 20-fold excess of N,N-diethyl-4-aza-4-methyl-3-oxo-5α-androstane-17β-carboxamide or the 3-carboxysteroid SKF-105111 resulted in partial protection of 5αR-1 from the probe, whereas minimal incorporation of radioactivity was observed in the absence of NADPH or in the presence of NADP +. The result indicate that [ 3H]4MABP is an effective probe of the steroid (D-ring) binding domain of 5αR-1.