Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Insect Biochem Mol Biol. 2011 Dec;41(12):932-7. doi: 10.1016/j.ibmb.2011.09.002. Epub 2011 Sep 22.
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.
近年来，质谱（MS）技术的进步促进了生物体和环境中微量成分的检测和定量。在这项研究中，我们成功地鉴定了澳大利亚蝎子第一龄若虫（孵化后 7 天）中的 20-羟基蜕皮酮（20E），使用串联质谱法结合高效液相色谱（LC/MS/MS）。在第五阶段之后，在成虫中没有发现这种物质。其他可能的蜕皮激素候选物，如 makisterone A（MaA）和 ponasterone A（PoA），据报道它们是几种节肢动物的蜕皮激素，在这种蝎子中也没有检测到。20E 及其类似物与体外翻译的蜕皮激素受体，即澳大利亚蝎子的蜕皮激素受体（EcR）和视黄酸 X 受体（RXR）的异二聚体（LaEcR/LaRXR）的配体-受体结合进行了定量评估。抑制 50%[（3）H]PoA 与 LaEcR/LaRXR 复合物结合所需的蜕皮激素（E）、MaA、20E 和 PoA 的浓度分别为 1.9、0.69、0.05 和 0.017 μM。这 4 种蜕皮甾酮的活性谱与几种昆虫的蜕皮激素受体获得的活性谱一致。非甾体 E 激动剂 tebufenozide 与 EcR 的结合甚至在高浓度下也未观察到，表明 LaEcR 的配体结合口袋的结构不利于与 tebufenozide 相互作用。
