Abstract
Inactivation of ecdysteroids (insect moulting hormones), such as ecdysone, occurs in Lepidopteran midgut cytosol by ecdysone oxidase to give 3-dehydroecdysone (3DE), which can either be irreversibly reduced to 3-epiecdysone(3α-hydroxy) by 3DE 3α-reductase, or reduced back to ecdysone by 3DE 3β-reductase. The ecdysteroids can also be inactivated by phosphorylation. Fractionation of the midgut cytosol from Spodoptera littoralis by DEAE-cellulose chromatography followed by Mono-Q FPLC enabled ecdysone oxidase, ecdysteroid 2-phosphotransferase, as well as at least two 3DE 3α-reductases and two 3α-reductases to be separated. Following chromatography, the 3α-reductases possessed slightly more NADPH- than NADH-linked activity, whereas the activities of the enzymes in dialysed crude cytosol were much higher with NADPH as cofactor. However, for the 3β-reductases, the NADH-dependent activity was much greater than the NADPH-linked activity both in the dialysed crude cytosol and following chromatography. The activity 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 3β-reductase, which showed a greater than 3-fold activation. The K m values for ecdysteroid substrates (ecdysone series) determined for all the dialysed enzymes following DEAE-cellulose chromatography were within the range 20–230 μM. TheV max value for ecdysone oxidase was appreciably lower than those of the NADH- and NADPH-linked 3DE 3α-reductase enzymes, suggesting that formation of 3-dehydroecdysone may be rate-limiting in ecdysteroid epimerization. The physiological significance of the higher V max values observed for both 3DE 3α- and 3β-reductases with the less favoured cosubstrate is unclear, but may reflect the NADPH/NADH ratio in the midgut cytosol, a factor which may represent a mechanism by which the enzymes are controlled.
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