SAPK/JNK活性的非放射性测定方法与初步应用

A Nonradioactive Assay for the Activity of the Stress Activated Protein Kinase

目的 建立SAPK/JNK活性的非放射性测定方法。方法 采用“免疫沉淀法”提取SAPK ,根据SAPK可以使底物c Jun(Ser6 3和Ser73)磷酸化的原理 ,以N 末端融合有c Jun的谷胱甘肽Seharosebeads从细胞裂解液中“亲合分离”SAPK ;然后进行体外磷酸化 ;以c Jun的磷酸化特异性抗体进行蛋白质免疫印迹杂交 ;最后以“化学发光法”测定磷酸化底物c Jun来反映细胞内SAPK的活性。结果 以 2 5～ 10 0 μmol/LCdCl2处理豚鼠肾上腺皮质细胞 2小时 ,细胞内SAPK活性随染毒剂量增加而轻度增加。结论 该法测定细胞内SAPK活性 ,方法灵敏、特异性强。

Objective To establish a nonradioactive assay for determining activity of stress activated protein kinase.Methods A new non radioactive assay for measuring SAPK activity was established as follow:SPAK was pulled down from cell lysates using c Jun fusion protein beads.The kinase reaction was carried out in the presence of cold ATP.The level of c Jun phosphorylation was specifically determined with western immunoblotting of phospho c Jun antibody and chemiluminescent detection system.In this detection system,the activity of SAPK was indirectly reflected by the level of c Jun phosphorylation which was the substract of phosphorylation.Results As the primarily cultured fasciculata glomerulosa(FG) cells of males guinea pig were incubated with 25μmol/L～100μmol/L cadmium chloride for two hours,the activity of SAPK was showed to have a mildly elevation as the dosage increased.Conclusions The protocol for activity assay is an effective method with the features of highly sensitivity and specificity