A simple spectrophotometric method for the assay of steroid 5
α-reductase (5
α-SR) was developed in which 5
α-dihydrotestosterone (5
α-DHT) and 5
α-androstane-3
α,17
β-diol (5
α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5
α-SR, were measured by enzymatic cycling using 3
α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5
α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5
α-DHT and 5
α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5
α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5
α-SR.
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