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目的:考察大鼠CTP:磷酸胆碱胞苷转移酶β(CCTβ)在脑内的分布及在昆虫细胞中的表达以研究其活性和特征.方法:用地高辛标记CCTβ探针对大鼠脑切片进行原位杂交;利用杆状病毒表达系统在粉纹夜蛾细胞中表达CCTβ;利用酶测活实验和代谢标记实验,研究表达蛋白的活性和特征,及对细胞磷脂酰胆碱(PC)合成的影响.结果:(1)CCTβ在海马的齿状回、CA1、CA2和CA4区有明显分布.(2)Tn细胞中表达的CCTβ含量是天然脑内的1×10~4倍,它的表达加快了细胞的PC合成.(3)软脂酰磷酸胆碱以及一些离子会抑制CCTβ活性;dCTP是CCTβ另一最适底物.结论:CCTβ在大鼠脑内有与精氨酸加压素片段(4—8)相似的分布.
OBJECTIVE: To investigate the distribution of rat CTP: CCTβ in brain and its expression in insect cells in order to study the activity and characteristics of CCTβ.Methods: (CCTβ) was expressed in Helicoverpa armigera by using the baculovirus expression system. The activity and characteristics of the expressed protein were studied by enzyme assay and metabolic labeling. (2) The expression of CCTβ in Tn cells was 1 × 10 ~ 4 times of that in natural brain, and it was significantly higher than that in CA1, CA2 and CA4 regions of hippocampus (3) Choline palmitate and some ions could inhibit the activity of CCTβ; dCTP was another most suitable substrate for CCTβ.Conclusion: CCTβin rat brain has the same effect as arginine plus Hypothesis fragments (4-8) similar distribution.