PHOTOAFFINITY-LABELING OF RAT-LIVER MICROSOMAL STEROID 5-ALPHA-REDUCTASE BY 2-AZIDO-NADP(+)

Preincubation of female rat liver microsomal preparations with [2'-P-32]2N(3)-NADP(+) followed by photolysis with UV light (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of P-32 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 mu M) completely inhibited the incorporation of[2'-P-32]2N(3)-NADP(+) and preserved the enzyme activity, whereas excess NAD(+) (100 mu M) failed to protect 5 alpha-reductase (5 alpha R) activity. Similar results were obtained, with the dertergent-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'-P-32]2N(3)-NADP(+) resulted in similar to 2-fold increase in P-32 labeling of the 5 alpha R band. Increasing photolysis time and concentration of the [2'-P-32]2N(3)-NADP(+) indicated that the half-life for photoincorporation and the apparent K-d were 1.0 min and 2 mu M, respectively. These results suggest that 2N(3)-NADP(+) is an effective probe of tire NADP(H) binding site of 5 alpha R, and is a useful marker during purification of the enzyme.