Ecdysterone Biosynthesis: A Microsomal Cytochrome-P-450-Linked Ecdysone 20-Monooxygenase from Tissues of the African Migratory Locust

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Abstract
Ecdysone 20-monooxygenase, an enzyme which converts ecdysone to ecdysterone (the major molting hormone of insects) was characterized in cell-free preparations of tissues from African migratory locust [Locusta migratoria migratorioides]. The reaction product was identified as ecdysterone on the basis of several microchemical derivatization and chromatographic methods. Ecdysone 20-monooxygenase activity was located primarily in the microsomal fraction which carried NADPH cytochrome c reductase and cytochrome P-450, as shown by sucrose density gradient centrifugation. Optimal conditions for the ecdysone 20-monooxygenase assay were determined. The enzyme has a Km for ecdysone of 2.7 .times. 10-7 M and was competitively inhibited by ecdysterone (Ki = 7.5 .times. 10-7 M). Ecdysone 20-monooxygenase was a typical cytochrome P-450 linked monooxygenase: the reaction required O2 and was inhibited by CO, an effect partially reversed by white light. The enzyme was effectively inhibited by several specific monooxygenase inhibitors and by sulfhydryl reagents, but not by cyanide ions. Ecdysone elicited a type I difference spectrum when added to oxidized microsomes. NADPH acted as preferential electron donor. The transfer of reducing equivalents proceeded through NADPH cytochrome c (P-450) reductase: ecdysone 20-monooxygenase was inhibited by cytochrome c. Both NADPH cytochrome c reductase and ecdysone 20-monooxygenase were inhibited by NADP+ and showed a similar Km for NADPH. The Malpighian tubules had the highest specific activity of ecdysone 20-monooxygenase, while the fat body contained most of the cytochrome P-450 and NADPH cytochrome c reductase.