CHARACTERIZATION, FRACTIONATION AND KINETIC-PROPERTIES OF THE ENZYMESOF ECDYSTEROID 3-EPIMERIZATION AND PHOSPHORYLATION ISOLATED FROM THE MIDGUT CYTOSOL OF THE COTTON LEAFWORM, SPODOPTERA-LITTORALIS

Inactivation of ecdysteroids (insect moulting hormones), such as ecdys one, occurs in Lepidopteran midgut cytosol by ecdysone oxidase to give 3-dehydroecdysone (3DE), which can either be irreversibly reduced to 3-epiecdysone(3 alpha-hydroxy) by 3DE 3 alpha-reductase, or reduced ba ck to ecdysone by 3DE 3 beta-reductase. The ecdysteroids can also be i nactivated by phosphorylation, Fractionation of the midgut cytosol fro m Spodoptera littoralis by DEAE-cellulose chromatography followed by M ono-Q FPLC enabled ecdysone oxidase, ecdysteroid 2-phosphotransferase, as well as at least two 3DE 3 beta-reductases and two 3 alpha-reducta ses to be separated, Following chromatography, the 3 alpha-reductases possessed slightly more NADPH- than NADH-linked activity, whereas the activities of the enzymes in dialysed crude cytosol were much higher w ith NADPH as cofactor, However, for the 3 beta-reductases, the NADH-de pendent activity was much greater than the NADPH-linked activity both in the dialysed crude cytosol and following chromatography, The activi ty of the enzymes was reversibly decreased in the presence of either 0 .1 M or 0.2 M NaCl, with the exception of NADPH-linked 3 beta-reductas e, which showed a greater than 3-fold activation. The K-m values for e cdysteroid substrates (ecdysone series) determined for all the dialyse d enzymes following DEAE-cellulose chromatography were within the rang e 20-230 mu M. The V-max value for ecdysone oxidase was appreciably lo wer than those of the NADH- and NADPH-linked 3DE 3 alpha-reductase enz ymes, suggesting that formation of 3-dehydroecdysone may be rate-limit ing in ecdysteroid epimerization, The physiological significance of th e higher V-max values observed for both 3DE 3 alpha- and 3 beta-reduct ases with the less favoured cosubstrate is unclear, but may reflect th e NADPH/NADH ratio in the midgut cytosol, a factor which may represent a mechanism by which the enzymes are controlled. Copyright (C) 1996 E lsevier Science Ltd