Purification of ecdysone oxidase and 3-dehydroecdysone 3α-reductase from the cotton leafworm, Spodoptera littoralis

Abstract

The 3-epimerization of ecdysteroids (insect moulting hormones) is an inactivation pathway of the hormones that has been reported to occur in midgut cytosol of Lepidoptera. The pathway involves ecdysone oxidase-catalysed conversion of ecdysone into 3-dehydroecdysone, which is then irreversibly reduced to 3-epiecdysone by 3DE 3α-reductase. In this study, ecdysone oxidase and 3DE 3α-reductase from the cotton leafworm, S. littoralis, have been purified by extensive chromatography together with electrophoresis on native gels. Gel filtration suggested that the native ecdysone oxidase might be a trimer with apparent molecular mass of approximately 190 kDa, since the apparent molecular mass of the oxidase subunit was determined to be 64 kDa by SDS-PAGE. Two forms of 3DE 3α-reductase were observed during the purification, the 26 kDa form reductase has been purified to homogeneity and the second form of the reductase identified as a 51 kDa protein. The former reductase may be a trimer with apparent molecular mass of 76 kDa, whilst the latter was suggested to be a monomer by gel filtration. Chromatographic behaviour suggested that the 26 kDa form of the reductase has a lower pI value and a higher degree of hydrophobicity than that of the 51 kDa reductase. Substrate specificity and the tissue distribution of these enzymes are discussed.