title: LC/MS/MS identification of 20-hydroxyecdysone in a scorpion (Liocheles australasiae) and its binding affinity to in vitro-translated molting hormone receptors.
abstract: Recent advances in mass spectrometry (MS) technology have facilitated the detection and quantification of minor components in organisms and the environment. In this study, we successfully identified 20-hydroxyecdysone (20E) in first instar nymphs (7 days after hatching) of the scorpion Liocheles australasiae, using tandem mass spectrometry combined with high-performance liquid chromatography (LC/MS/MS). This substance was not found in adults after the fifth stage. Other possible molting hormone candidates such as makisterone A (MaA) and ponasterone A (PoA), both of which are reported to be the molting hormones of a few arthropod species, were not detected in this scorpion. The ligand-receptor binding of 20E and its analogs was quantitatively evaluated against the in vitro-translated molting hormone receptor, the heterodimer of ecdysone receptor (EcR) and the retinoid X receptor (RXR) of L. australasiae (LaEcR/LaRXR). The concentrations of ecdysone (E), MaA, 20E, and PoA that are required to inhibit 50% of [(3)H]PoA binding to the LaEcR/LaRXR complex were determined to be 1.9, 0.69, 0.05, and 0.017 μM, respectively. The activity profiles of these 4 ecdysteroids are consistent with those obtained for the molting hormone receptors of several insects. The binding of a non-steroidal E agonist, tebufenozide, to EcR was not observed even at high concentrations, indicating that the structure of the ligand-binding pocket of LaEcR is not favorable for interaction with tebufenozide.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Miyashita
authors.forename: Kaori
authors.initials: K
authors.lastname: Matsushita
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Nakamura
authors.forename: Sho
authors.initials: S
authors.lastname: Akahane
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Nakagawa
authors.forename: Hisashi
authors.initials: H
authors.lastname: Miyagawa
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D004441:Ecdysterone / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006458:Hemolymph / Q000378:metabolism; D009758:Nymph / Q000737:chemistry / Q000378:metabolism; D012605:Scorpions / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D004441:Ecdysterone