Purification and characterisation of haemolymph 3-dehydroecdysone 3 beta-reductase in relation to ecdysteroid biosynthesis in the cotton leafworm Spodoptera littoralis

The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littoralis was detected and analysed by HPLC-RTA. The primary product was identified as 3-dehydroeodysone (approximate to 82%), with lesser amounts of ecdysone (approximate to 18%). Interconversion of ecdysone and 3-dehydroecdysone by prothoracic glands was not detectable. 3-Dehydroecdysone 3 beta-reductase activity was demonstrated in the haemolymph. Ecdysone, the end-product, was characterised by reverse-phase and adsorption HPLC, chemical transformation into ecdysone 2, S-acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and K-m values for NADPH and NADH were determined to be 0.94 mu M and 22.8 mu M, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3-dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (>5 mu M). The results suggest that the 3-dehydroecdysone 3 beta-reductase has a high-affinity (low K-m) binding site for 3-dehydroecdysone substrate, together with a lower-affinity inhibition site. The 3 beta-reductase enzyme was purified to homogeneity using a combination of poly(ethylene glycol) 6000 precipitation and successive FPLC fractionation on Mono-Qt phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDS/PAGE and gel-filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.