Analysis of the steroid binding domain of rat steroid 5α-reductase (Isozyme-1): The steroid D-ring binding domain of 5α-reductase

Abstract

We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5α-androstan-17β-carboxamide is an effective probe of rat steroid 5α-reductase (isozyme-1) (5αR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5αR-1 activity were ultraviolet (UV)-photolyzed with [ 3H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5αR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55–56 min. Rechromatography of this fraction using a modified gradient (elution 54–55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15–18 of the 5αR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an ∼12-fold increase in the K m for testosterone, whereas the K m for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.