Ak. Bhattacharyya et al., Analysis of the steroid binding domain of rat steroid 5 alpha-reductase (isozyme-1) - The steroid D-ring binding domain of 5 alpha-reductase, STEROIDS, 64(3), 1999, pp. 197-204
We have previously shown that the photoactive 4-azasteroid, [1,2 H-3]N-4(be
nzylbenzoyl)-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 wi
th [H-3]-4MABP and subsequently purified by 8.75% preparative sodium dodecy
l sulfate-polyacrylamide gel electrophoresis. The fractions corresponding t
o 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 liq
uid chromatography analysis, indicated that the majority of the radioactivi
ty eluted with a peak retention time of 55-56 min. Rechromatography of this
fraction using a modified gradient (elution 54-55 min), followed by sequen
ce 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 t
o 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.