Expression of rat steroid 5α-reductase (isozyme-1) in Spodoptera Frugiperda, SF21, insect cells: Expression of rat steroid 5α-reductase

• Published in: Steroids 1997-04, Vol.62 (4), p.373-378
• Main Authors: Taylor, Matthew F., Wang, Min, Bhattacharyya, Anjan K., Chiang, Nan, Tai, Hsin-Hsiung, Collins, Delwood C.
• Format: Article
• Language: English
• Subjects: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase - genetics; 3-Oxo-5-alpha-Steroid 4-Dehydrogenase - metabolism; 5α-reductase; Affinity Labels - metabolism; Androstanes - metabolism; Animals; Azasteroids - metabolism; Azides - metabolism; baculovirus; Biological and medical sciences; Cloning, Molecular; expression; Fundamental and applied biological sciences. Psychology; Kinetics; NADP - analogs & derivatives; NADP - metabolism; Nucleopolyhedrovirus; Rats; Sf 21 insect cells; Spodoptera; Steroid hormones. Cholecalciferol derivatives; Testosterone - metabolism; Transfection; Vertebrates: endocrinology
• ISSN: 0039-128X; 1878-5867
• DOI: 10.1016/S0039-128X(96)00254-1

The enzyme steroid 5α-reductase (5αR) catalyzes the reduction of testosterone (T) to 5α-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5αR type 1 isozyme (r5αR 1). The full length of r5αR1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda. Sf21, insect cells. The expressed recombinant r5α-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 μM and 2.7 μM, respectively. Inhibition of the recombinant r5αR1 by N,N diethyl-4-aza-4-methyl-3-oxo-5α-androstane-17β-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 r5αR1 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 r5αR1. Photoaffinity labeling by [2′-32P]-2-azido-NAD P + ([2′- 32P]2N 3-NAD P +) and [1,2 3 H] N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5α androstane-17β-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.