title: Photoaffinity labeling of rat steroid 5 alpha-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-5 alpha-androstane-17 beta-carboxamide ([3H]-4MABP) has been synthesized as a photoaffinity probe of the steroid-binding domain of rat steroid 5 alpha-reductase isozyme-1 (5 alpha R-1). Reversible binding of the probe to 5 alpha R-1 in microsomal preparations yielded a reversible dissociation constant (Kd) 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 (Ki approximately 3 nM) with respect to testosterone. SDS-PAGE analysis of microsomal, detergent-solubilized, and (6.5%) polyethylene glycol-precipitated fractions of 5 alpha R-I 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 5 alpha R-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 Kd for its photo insertion were approximately 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-5 alpha-androstane-17 beta-carboxamide or the 3-carboxysteroid SKF-105111 resulted in partial protection of 5 alpha R-1 from the probe, whereas minimal incorporation of radioactivity was observed in the absence of NADPH or in the presence of NADP+. The results indicate that [3H]4MABP is an effective probe of the steroid (D-ring) binding domain of 5 alpha R-1.
authors: Department of Biochemistry, VA Medical Center, Lexington, KY 40506, USA.
authors: M F
authors: MF
authors: Taylor
authors: A K
authors: AK
authors: Bhattacharyya
authors: K
authors: K
authors: Rajagopalan
authors: R
authors: R
authors: Hiipakka
authors: S
authors: S
authors: Liao
authors: D C
authors: DC
authors: Collins
mesh_terms: D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000737:chemistry*; D000345:Affinity Labels; D000731:Androstanes / Q000737:chemistry*; D000818:Animals; D001378:Azasteroids / Q000737:chemistry*; D005260:Female; D007527:Isoenzymes / Q000737:chemistry*; D015335:Molecular Probes; D010777:Photochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000345:Affinity Labels; D000731:Androstanes; D001378:Azasteroids; D007527:Isoenzymes; D015335:Molecular Probes; C102561:N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5-androstane-17-carboxamide; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase