Ecdysone 20-monooxygenase in a cricket, Gryllus bimaculatus (Ensifera, Gryllidae)?characterization of the microsomal midgut steroid hydroxylase in adult females

Abstract

Ecdysone 20-monooxygenase, the enzyme system which converts ecdysone into 20-hydroxyecdysone, was characterized in the midgut of 4-day-old female adult Gryllus bimaculatus using an in vitro radioassay. Differential centrifugation and sucrose gradient centrifugation revealed that ecdysone 20-monooxygenase activity is associated with the microsomal fractions. The 20-monooxygenase was found to be most active in potassium phosphate buffer, pH 7.8, at an osmolarity of 100 mOsm and at 39 °C assay temperature. The conversion of ecdysone into 20-hydroxyecdysone was linear over an incubation period of 12 min and with respect to a protein concentration of 3 mg·ml−1. K+ and Na+ (10−3–10−1M), Ca2+ (2.3 mM), and EDTA (1–5 mM) did not affect monooxygenase activity, whereas Mg2+ (2.3–10 mM) slightly inhibited enzyme activity. The enzyme complex has an apparent Km for ecdysone of 3.7·10−7M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent Ki of 4·10−6M. The cytochrome P-450 nature of the steroid hydroxylase was shown by its obligate requirement for NADPH and its inhibition by carbon monoxide, metyrapone, and p-chloromercuribenzoate, but not by cyanide. The insect systemic growth disruptor, azadirachtin, was found to inhibit ecdysone 20-monooxygenase activity with a I50 of 8·10−4M. From the CO-difference spectrum, a cytochrome P-450 content of 285 pmol·mg protein−1 was calculated for midgut microsomes of 4-day-old females.