RESULTS: The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 μM (Chlorantraniliprole), 0.21 μM (Flubendiamide), and 0.57 μM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 μM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA.
CONCLUSIONS: SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.
结果:MythimnaseparataSR的形态特征具有封闭的囊泡,具有完整性和高密度。SR组分中的282种蛋白质含有RyR和SERCA。通过使用荧光分光光度计检测SR膜外的钙离子,成功建立了钙释放和摄取的测量模型。使用SR囊泡的体外测试系统发现,二酰胺杀虫剂可以剂量依赖性地激活RyR,EC50值为0.14μM(Chloantraniliprole),0.21μM(氟苯二酰胺),和0.57μM(蓝藻),分别。然而,丹曲林抑制RyR介导的钙释放,IC50值为353.9μM,表明丹曲林可以弱拮抗RyR。此外,环吡嗪酸显着降低了SERCA的酶活性和钙吸收能力。相反,CDN1163显著激活酶活性并提高SERCA的钙转运能力。
结论:SR囊泡可用于研究未知蛋白质在SR膜上的功能,以及高通量筛选靶向RyR或SERCA的高活性化合物。©2024化学工业学会。