【摘要】 以32P标记的RII多肽为底物,研究了在脑提取物中钙调神经磷酸酶(CN)活性的测定过程中,CN活性依赖的一些离子或蛋白及测活条件对CN活性的影响,对脑提取物中CN活性的测定方法进行了优化.实验表明,脑提取物中的Ca2+足以激活CN活性,在测活反应体系中不需再补加Ca2+,但补加钙调素(CaM)是必须的,其在反应体系中的最适浓度是0.13μmol.L-1;抑制反应体系中的CN活性的最适EGTA浓度为0.16 mmol.L-1;对于脑提取物中的CN活性,Mn2+是比Mg2+更有效的激活剂,其最适终浓度为0.5 mmol.L-1;脑匀浆过程中蛋白酶抑制剂的加入有助于在90min内保持CN的活性.更多还原

【Abstract】 In the course of measurement of Calcineurin(CN) activity in brain extracts,the effects on CN activity of some important ion metals and proteins,as well as some conditions of measurement of activity are investigated with RII phosphopeptide substrate.The method of measurement of CN activity in brain extracts is optimized.The results show that Ca2+ is sufficient to activate CN in brain extracts and it is unnecessary to add Ca2+ to assay buffer.However,it is necessary to add CaM to assay buffer to activate CN in brain extracts and the optimal concentration is 0.13 μmol·L-1.The optimal concentration of added EGTA is 0.16 mmol·L-1 to inhibit CN activity in brain extracts effectively.Mn2+ is a more effective activator than Mg2+ on CN activity in brain extracts,and the optimal final concentration of Mn2+ is 0.5 mmol·L-1 in assay buffer.In addition,the protease inhibitors added in the course of preparation of brain extracts are helpful for keeping CN activity.更多还原