EXPRESSION OF RAT STEROID 5-ALPHA-REDUCTASE (ISOZYME-1) IN SPODOPTERA-FRUGIPERDA, SF21, INSECT CELLS - EXPRESSION OF RAT STEROID 5-ALPHA-REDUCTASE

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reducti on of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this s tudy, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enz ymatic activity when the infected cells were harvested on day 3 of pos t-transfection. The K-m values for NADPH and T were 17 eta M and 2.7 m u M, respectively. Inhibition of the recombinant r5 alpha R1 by N,N di ethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta- carboxamide (4M A) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fractio n, and the maximum velocity for the recombinant r5 alpha R1 in this fr action was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a singl e immunoreactive band at 26 kDa, which corresponds to the molecular we ight of r5 alpha R1. Photoaffinity labeling by [2'32P]-2-azido-NAD P([2'-P-32]2N(3)-NAD P+) and [1,2(3)H] N-4(benzylbenzoyl)-3-oxo-4-aza-4 -methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showe d a labeled protein band at 26 kDa. (C) 1997 Elsevier Science Inc.