Photoaffinity labeling of rat liver microsomal steroid 5α-reductase by 2-azido-NADP

Abstract

Preincubation of female rat liver microsomal preparations with [2′- 32P]2N 3-NADP + followed by photolysis with UV lighjt (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 14–197 Kd. Labeling of a 26 Kd band, the apparent molecular weight of 5α-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP + (100μM) completely inhibited the incorporation of [2′- 32P]2N 3-NADP + and preserved the enzyme activity, whereas excess NAD + (100μM) failed to protect 5α-reductase (5αR) activity. Similar results were obtained with the detergent-solubilized form of 5αR. Polyethylene glycol (PEG) fractionation of detergent-solubilized with the detergent-solubilized preparations of 5αR showed that all the 5αR 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]2N 3-NADP + resulted in ∼ 2-fold increase in 32P labeling of the 5αR band. Increasing photolysis time and concentration of the [2′- 32P]2N 3-NADP + indicated that the half-life for photoincorporation and the apparent K d were 1.0 min and 2 μM, respectively. Theser results suggest that 2N 3-NADP + is an effective probe of the NADP(H) binding site of 5αR, and is a useful marker during purification of the enzyme.