Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain.
We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].
Bhattacharyya
A K
AK
Department of OB/GYN, University of Kentucky Lucille P. Markey Cancer Center, Lexington.
Chavan
A J
AJ
Haley
B E
BE
Taylor
M F
MF
Collins
D C
DC
D000225:Adenine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D042944:Cholestenone 5 alpha-Reductase; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010777:Photochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley
D007527:Isoenzymes; D010446:Peptide Fragments; D009249:NADP; D010088:Oxidoreductases; D042944:Cholestenone 5 alpha-Reductase; D000225:Adenine