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苦皮藤素V是一种对昆虫具有毒杀活性的化合物,从植物苦皮藤(Celastrus angulatus Max)中分离出来。目前,已发现苦皮藤素V可与粘虫中肠液泡型ATP酶(V-ATPase)的H、B和a亚基结合,但是其具体作用机理还尚不清楚。本研究将大肠杆菌(Escherichia coli)中表达得到的东方粘虫中肠V-ATPase A亚基突变体TSCA和V-ATPase B亚基包涵体洗涤、溶解后进行复性,获得可溶性AB亚基复合物后采用亲和层析纯化。将纯化好的AB亚基复合物测定H~+K~+-ATPase活性,证明其有ATP水解活性。随后,测定苦皮藤素V对复合物ATPase的抑制活性,发现加入苦皮藤素后,复合物ATPase活性降低。因此,其可能是通过抑制了AB亚基复合物的ATPase活性,从而产生了杀虫效果,证明AB亚基复合物为苦皮藤素V的潜在靶点之一。这为了解苦皮藤素与VATPase相互作用机制打下了基础,也为进一步开发新型杀虫药物奠定了基础。
Celangulin V is a compound that is toxic to insects and is isolated from the plant Celastrus angulatus Max. At present, Celangulin V has been found to bind to the H, B and a subunits of the armyworm midgut vacuolar ATPase (V-ATPase), but its exact mechanism of action is not yet clear. In this study, inclusion bodies of the TSCA and V-ATPase B subunits of the midgut midguts of the armyworm, Mythimna separate, were isolated from Escherichia coli, lysed and then refolded to obtain soluble AB subunit complex After purification by affinity chromatography. The purified AB subunit complex was assayed for H ~ + K ~ + -ATPase activity, demonstrating that it has ATP hydrolysis activity. Subsequently, the inhibitory activity of celangulin V on the complex ATPase was determined and it was found that the complex ATPase activity was decreased after addition of celangulin. Therefore, it may be through the inhibition of the AB subunit complex ATPase activity, resulting in insecticidal effect, AB subunit complex that one of the potential targets of celangulin V. This laid the foundation for understanding the interaction mechanism between cephalosporin and VATPase and laid the foundation for the further development of new insecticide.