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机构地区:[1]沈阳医学院基础医学院形态中心实验室,辽宁沈阳110034 [2]沈阳医学院奉天医院手外科 [3]沈阳医学院基础医学院生物化学教研室
出 处:《沈阳医学院学报》2009年第4期203-207,共5页Journal of Shenyang Medical College
摘 要:目的:探讨G蛋白β、γ亚基在调节G蛋白偶联受体激酶活性的重要作用和G蛋白β、γ亚基影响M2受体磷酸化新的作用机制。方法:利用四个柱层析分离G蛋白α亚基与G蛋白β、γ亚基;将纯化的G蛋白β、γ亚基、GRK-2,[γ-P32]标记的ATP与mAChR2,共同保温,聚丙烯酰胺凝胶电泳,凝胶片干燥后放射性自显影检测M2受体磷酸化结果。结果:G蛋白β、γ亚基在没有激动剂存在的情况下明显增强M2受体的磷酸化。氨甲酰胆碱明显增强M2受体的磷酸化,阿托品或肝素(GRK2抑制剂)完全阻断M2受体的磷酸化。M2受体的磷酸化是依赖激活剂如氨甲酰胆碱作用发生的,这种依赖关系呈明显的剂量关系。结论:G蛋白β、γ亚基是通过上调GRK2活性增强M2受体的磷酸化的。说明Gβγ同Gα一样均可引起效应蛋白的激活,在细胞信号转导中起同样重要作用,共同介导一系列的生物学效应。Objective: To evaluate the role of G βγ subunits on GRK2-mediated phosphorylation of G-protein coupled receptors M2 muscarinie acetylcholine receptor (M2 receptor). Methods: DEAE-Sepharose FF, Sephacryl S-300 HR and Phenyl-Sepharose CL-4B was purfied. Finally G 137 subunits were purified from G protein by using GTP and Phenyl-Sepharose CL-4B were used to separate Gα subunit. Purified G βγsubunits was incubated with purified M2 receptor along with GRK2, and [ γ-P^32 ] ATP. The antagonist atropine, the GRK2 inhibitor heparin on the phosphorylation of M2 receptor phosphorylation GRK2 were tested. Proteins were separated by electrophoresis, gels were dried and autoradiograms developed. Bands containing receptors were excised and counted by liquid scintillation. Results: The G protein βγsubunits evidently increased the phosphorylation of M2 receptor without carbachol mediated by GRK2. The agonist carbachol evidently increased the phosphorylation of M2 receptor, both atropine and heparin completely blocked M2 receptor phosphorylation. Conclusion: These results indicated that G protein βγsubunits could regulates the activity of GRK2. It may increases the activity of GRK2. It told us that G protein βγ subunits has an important effects on some protein moleculars, the same as Gα subunit. G protein βγ subunits has an important role in the signal transduction of the cells.
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