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

Summary

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⁺ (10⁻³–10⁻¹ M), 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 K_m for ecdysone of 3.7·10⁻⁷ M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent K_i of 4·10⁻⁶ M. 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 8·10⁻⁴ M. 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.