Analysis of the steroid binding domain of rat steroid 5alpha-reductase (isozyme-1): the steroid D-ring binding domain of 5alpha-reductase.
We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5alpha-androst an-17beta-carboxamide is an effective probe of rat steroid 5alpha-reductase (isozyme-1) (5alphaR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5alphaR-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 5alphaR-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 5alphaR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an approximately 12-fold increase in the Km for testosterone, whereas the Km for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.
VA Medical Center and Department of OB/GYN, University of Kentucky, Lexington 40536, USA. akbhat1@pop.uky.edu
A K
AK
Bhattacharyya
M
M
Wang
K
K
Rajagopalan
M F
MF
Taylor
R
R
Hiipakka
S
S
Liao
D C
DC
Collins
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000731:Androstanes / Q000737:chemistry; D000818:Animals; D001378:Azasteroids / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007700:Kinetics; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D013256:Steroids / Q000378:metabolism*
D000731:Androstanes; D001378:Azasteroids; D017931:DNA Primers; D015335:Molecular Probes; C102561:N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5-androstane-17-carboxamide; D010446:Peptide Fragments; D013256:Steroids; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase