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

We have previously shown that the photoactive 4-azasteroid, [1,2 H-3]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha-androstan-17 beta-carboxamide is an effective probe of rat steroid Scr-reductase (isozyme-l) (5 alpha R-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5aR-1 activity were ultraviolet (UV)-photolyzed with [H-3]-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 5 alpha R-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 5 alpha R-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an similar to 12-fold increase in the K-m for testosterone, whereas the K-m for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered. (C) 1999 Elsevier Science Inc. All rights reserved.