Ecdysone 20‐monooxygenase in eggs of the silkworm, Bombyx mori: Enzymatic properties and developmental changes

Abstract

Ecdysone 20-monooxygenase in eggs of the silkworm Bombyx mori was characterized in relation to embryonic development. First, subcellular fractions were prepared by means of differential centrifugation, and analyzed using marker enzymes and antibodies against NADPH-cytochrome P450 reductase. It was demonstrated that most ecdysone 20-monooxygenase activity was associated with microsomes, and that there was little or no intrinsic mitochondrial ecdysone 20-monooxygenase. Next, conditions for the measurement of ecdysone 20-monooxygenase activity were established for the microsomal fraction, and changes in the enzyme activity were measured in diapause eggs and non-diapause eggs during early embryogenesis. It was demonstrated that enzyme activity in diapause eggs remained at a low level, while that in the non-diapause eggs increased from the gastrula stage. The increase in egg ecdysone 20-monooxygenase activity was prevented by actinomycin D and alpha-amanitin, suggesting that gene transcription is required for eliciting an increase in ecdysone 20-monooxygenase activity.

Molecular cloning of NADPH‐cytochrome P450 oxidoreductase from silkworm eggs

“…By examining the effect of actinomycin D or α‐amanitin on 20‐hydroxyecdysone biosynthesis in the silkworm egg, we have shown previously that gene transcription was required for the induction of ecdysone 20‐hydroxylase activity [8]. Our present study of ecdysone 20‐hydroxylation in silkworm egg microsomes suggests that the activity depends upon the presence of both ecdysone 20‐hydroxylase P450 and P450 reductase, and changes during the embryogenesis.…”

“…In silkworm eggs, ecdysone 20‐hydroxylation activity was associated with the microsomes [8], suggesting the involvement of the microsomal P450 system in this activity. All the microsomal P450‐mediated hydroxylation reactions are dependent upon the presence of the P450 reductase that supplies reducing equivalents from NADPH to the P450.…”

“…Ecdysone, 10 pmol, mixed with 0.2 µCi [ 3 H]ecdysone (Dupont/NEN Research Products), was placed in a 1.5‐mL Eppendorf tube, and the solvent was evaporated under nitrogen flow. Microsomes in 90 µL of 0.1 m sodium phosphate buffer, pH 7.5, containing 0.2 m m dithiothreitol and 0.2% BSA were added to the tube, which was kept on ice [8]. The tube was pre‐incubated at 35 °C for 3 min, and then the reaction was initiated by the addition of 10 µL 1.5 m m NADPH.…”

“…We previously reported that the content of 20‐hydroxyecdysone in the diapause egg remains low, whereas that in the nondiapause egg increases significantly during development [4–6], and suggested that characterization of the ecdysone 20‐hydroxylation reaction would be important in understanding silkworm embryogenesis [7]. Our subsequent studies have demonstrated that most of the 20‐hydroxylation activity in the three‐day‐old nondiapause egg is associated with the microsomes [8], suggesting that the microsomal cytochrome P450‐dependent hydroxylation system is involved in this metabolism.…”

Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum

“…A deficiency of 20‐hydroxyecdysteroid induces embryonic diapause, leading to the cessation of embryonic development at the late gastrula stage (Sonobe and Odake , Sonobe et al ). 20‐Hydroxyecdysteroid is known to be formed by de novo synthesis and dephosphorylation of 20‐hydroxyecdysteroid 22‐phosphate and ecdysteroid 22‐phosphate, which are synthesized by an ATP:ecdysteroid phosphotransferase:ecdysteroid 22‐kinase (Horike and Sonobe , Sonobe et al , Makka and Sonobe ). Therefore, the reciprocal conversion of 20‐hydroxyecdysteroid and ecdysteroid 22‐phosphates by ecdysteroid kinase and ecdysteroid‐phosphate phosphatase plays an important role in the homeostasis of 20‐hydroxyecdysteroid in eggs.…”