The enzyme 5-alpha-reductase (5alphaR) catalyzes the reduction of testosterone to 5-alpha-dihydrotestosterone (5alpha-DNT) and has been implicated in numerous androgen related disorders such as benign prostatic hyperplasia, prostate cancer and male pattern baldness. Two isozymes have been identified in humans and rats (5alphaR-l and 5alphaR- 2) that can be distinguished on the basis of their ph optima, affinities for steroid substrate and inhibition by 4-azasteroids. The primary goals of the this research are to identify the NADP(H) and the steroid A and D ring binding domains of rat 5alphaR-1 using Photoaffinity probes. The long term goals are to perform site-directed mutagenesis studies such that specific amino acid residues that influence cofactor and steroid binding and catalysis may be identified in 5alphaR- 1. [2'-32P]2-azido-NADP+ has been synthesized to probe the NADP(H) binding domain of rat 5alphaR-1, and we have identified a 11 amino acid peptide corresponding to the NADP+ (Adenine) binding domain. Photoreactive (diazo, azidophenyl and benzophenone) radiolabeled (125I, 3H) analogs of 4-methyl-4-azasteroids will be synthesized and used as specific steroid A and D ring probes of 5alphaR. We have obtained high levels of expression of rat 5alphaR-1 in Sf21 insect cells using a baculovirus expression system. Partially purified 5alphaR-l fractions from natural or recombinant sources will be prelabeled with the probes and further purified by preparative electrophoretic methods. These preparations will be subject to proteolytic digestion, and the photolabeled peptides purified and sequenced. Using RCPCR techniques, we propose to carry out site-directed mutagenesis studies on 5alphaR-1 using this system.
| Wang, M; Bhattacharyya, A K; Taylor, M F et al. (1999) Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5alpha-reductase (isozyme-1) I: analysis of aromatic and hydroxylated amino acid residues. Steroids 64:356-62 |
