ECDYSONE 20-MONOOXYGENASE SYSTEMS IN A LARVAL AND AN ADULT DIPTERAN: AN OVERVIEW OF THEIR BIOCHEMISTRY, PHYSIOLOGY AND PHARMACOLOGY

Abstract

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone to 20-hydroxyecdysone, was characterized in wandering stage larvae of Drosophila melanogaster and blood fed adult female Aedes aegypti using an in vitro radioassay in conjunction with analytical thin layer chromatography. Preliminary data suggested that ecdysone 20-monooxygenase activity is present in body wall/fat body complexes, gut/Malpighian tubule complexes and ovaries of Aedes and that this enzyme activity varies in a tissue-specific fashion during the gonadotropic cycle. The 20-monooxygenase in both Drosophila and Aedes was most active in sodium phosphate buffer, pH 7.5, and exhibited a temperature optimum at 35°C. Differential centrifugation has revealed that ecdysone 20-monooxygenase activity is associated with both the mitochondrial and microsomal fractions of Aedes and Drosophila and this has been confirmed in Drosophila by sucrose step gradient centrifugation, electron microscopy and organelle-marker enzyme analysis. Substrate kinetics experiments have shown that both the Aedes and Drosophila monooxygenase systems have low apparent Kms for ecdysone in the range of 0.6–3 × 10–7M. Both Drosophila and Aedes monooxygenase systems were inhibited by 20-hydroxyecdysone. The cytochrome P-450 dependency of the Drosophila monooxygenase systems was established by cofactor requirements, inhibitor studies and photochemical action spectra determinations. The cytochrome P-450 dependency of the Aedes monooxygenase systems was suggested by their cofactor requirements and inhibitor studies.