Photoaffinity labeling of rat liver microsomal steroid 5 alpha-reductase by 2-azido-NADP+.
Preincubation of female rat liver microsomal preparations with [2'-32P]2N3-NADP+ followed by photolysis with UV light (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32P into at least 3 major protein bands in the molecular weight range of 14-97 Kd. Labeling of a 26 kD band, the apparent molecular weight of 5 alpha-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP+ (100 microM) completely inhibited the incorporation of [2'-32P]2N3-NADP+ and preserved the enzyme activity, whereas excess NAD+ (100 microM) failed to protect 5 alpha-reductase (5 alpha R) activity. Similar results were obtained with the detergent-solubilized form of 5 alpha R. Polyethylene glycol (PEG) fractionation of detergent-solubilized preparations of 5 alpha R showed that all the 5 alpha R activity could be recovered in the 6.5% pellet with a 3-4-fold increase in the specific activity. photolysis of this fraction with [2'-32P]2N3-NADP+ resulted in approximately 2-fold increase in 32P labeling of the 5 alpha R band. Increasing photolysis time and concentration of the [2'-32P]2N3-NADP+ indicated that the half-life for photoincorporation and the apparent Kd were 1.0 min and 2 microM, respectively. These results suggest that 2N3-NADP+ is an effective probe of the NADP(H) binding site of 5 alpha R, and is a useful marker during purification of the enzyme.
Bhattacharyya
A K
AK
Veterans Affairs Medical Center, Lexington, Kentucky.
Chavan
A J
AJ
Shuffett
M
M
Haley
B E
BE
Collins
D C
DC
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism* / Q000528:radiation effects; D058891:5-alpha Reductase Inhibitors; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D005260:Female; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D051381:Rats; D017207:Rats, Sprague-Dawley; D014466:Ultraviolet Rays
D058891:5-alpha Reductase Inhibitors; D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; C092743:nicotinamide-2-azidoadenine-dinucleotide phosphate; D009249:NADP; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase