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 reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, 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, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.
Taylor
M F
MF
VA Medical Center, Lexington, Kentucky 40506, USA.
Wang
M
M
Bhattacharyya
A K
AK
Chiang
N
N
Tai
H H
HH
Collins
D C
DC
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000731:Androstanes / Q000378:metabolism; D000818:Animals; D001378:Azasteroids / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D003001:Cloning, Molecular; D007700:Kinetics; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism; D017924:Nucleopolyhedroviruses; D051381:Rats; D018411:Spodoptera; D013739:Testosterone / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D000731:Androstanes; D001378:Azasteroids; D001386:Azides; C102561:N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5-androstane-17-carboxamide; C092743:nicotinamide-2-azidoadenine-dinucleotide phosphate; D013739:Testosterone; D009249:NADP; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase