PURIFICATION AND CHARACTERIZATION OF HEMOLYMPH 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 1 8%). 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 ch aracterised by reverse-phase and adsorption HPLC, chemical transformat ion into ecdysone 2, S-acetonide, and mass spectrometry. The condition s for optimal activity were determined. The enzyme requires NADPH or N ADH as cofactor and K-m values for NADPH and NADH were determined to b e 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, reve aled that it exhibits maximal activity at low 3-dehydroecdysone substr ate concentrations, with a drastic inhibition of activity at higher co ncentrations (>5 mu M). The results suggest that the 3-dehydroecdysone 3 beta-reductase has a high-affinity (low K-m) binding site for 3-deh ydroecdysone substrate, together with a lower-affinity inhibition site . The 3 beta-reductase enzyme was purified to homogeneity using a comb ination of poly(ethylene glycol) 6000 precipitation and successive FPL C fractionation on Mono-Qt phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular m ass of 36 kDa by SDS/PAGE and gel-filtration chromatography. Furthermo re, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, su pporting a role for the enzyme in development.