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目的:表达M_2AChR-G_(ila)融合蛋白,检测不同配体或镁离子对M_2AChR与G_(ila)间相互作用的影响.方法:两步PCR建立M_2AChR-G_(ila)融合DNA,并在Sf9细胞内表达.[~3H]QNB和[~(35)S]GTPγS结合实验检测M_2AChR-G_(ila)融合蛋白功能.结果:M_2AChR-G_(ila)的表达水平为(20.12±1.14)nmol·g~(-1)protein.不同配体的存在使融合蛋白中G_(ila)与GDP的亲和力 发生变化.乙酰胆碱,卡巴胆碱,McN-A-343,毛果芸香碱和阿托品存在以及无配体存在时,GDP的IC_(50)值(95％可信区间)分别是178(148-214)μmol/L,158 (126—199)μmol/L,66(56—78)μmol/L,62(55-72)μmol/L,5.0(4.6-5.5)μmol/L和15.9(14.3-17.6)μmol/L.在卡巴胆碱的作用下,C_(ila)随Mg~(2+)浓度增加而减小对GDP的亲和力,而阿托品使G_(ila)不受Mg~(2+)浓度的影响,始终保持对GDP的高亲和性.结论:杆状病毒-Sf9细胞系统表达的M_2AChR-G_(ila)具备M受体配体结合特性和组分间的偶联功能.M_2AChR-G_(ila)对GDP的亲和性决定于M_2受体配体的性质,分析GDP亲和力的大小有利于筛选和鉴别M_2亚型特异性药物.Mg~(2+)离子是M_2AChR-G_(ila)与低亲和性GDP结合必需的阳离子.
Objective: To express M_2AChR-G_ (ila) fusion protein and detect the effect of different ligands or magnesium ions on the interaction between M_2AChR and G_ (ila) .Methods: M_2AChR-G_ (ila) fusion DNA was established by two- (~3H) QNB and [~ (35) S] GTPγS binding assay.Results: The expression level of M_2AChR-G_ (ila) was (20.12 ± 1.14) nmol · g - (-1) protein. The presence of different ligands changed the affinity of G_ (ila) to GDP in the fusion protein.The presence of acetylcholine, carbachol, McN-A-343, pilocarpine and atropine, , And the IC 50 values (95% confidence interval) of GDP were 178 (148-214) μmol / L, 158 (126-199) μmol / L, 66 (56-78) μmol / L, -72) μmol / L, 5.0 (4.6-5.5) μmol / L and 15.9 (14.3-17.6) μmol / L. Under the action of carbachol, C ila increased with the increase of Mg 2+ concentration Small affinity for GDP, whereas atropine kept G_ (ila) independent of Mg 2+ concentration and maintained high affinity for GDP.Conclusion: The expression of M_2AChR-G_ ila) possesses the binding properties of M receptor ligands and the coupling function among the components. The affinity of M_2AChR-G_ (ila) for GDP The properties of M_2 receptor ligands and the analysis of the affinity of GDP are favorable for the screening and identification of M_2 subtype specific drugs.Mg 2+ ions are essential for the binding of M_2AChR-G_ (ila) to low-affinity GDP cation.