人神经母细胞瘤株SH-SY5Y中calcineurin比活性测定的最佳条件

OPTIMIZING CONDITIONS FOR CALCINEURIN ACTIVITY IN SH-SY5Y CELL EXTRACTS

研究了在人神经母细胞瘤株SH-SY5Y中calcineirin比活性测定的最佳条件.以RⅡ为底物,在测活缓冲液中,Ca2+和岗田酸(okadaic acid,OA)的加入是必须的,这与在脑组织中的测定是不一样的,同时进一步确定了需要加入的OA是100 nmol.L-1.在细胞破碎液中,calcineurin比活性的测定值是以缓冲液Ⅰ(含Ca2+与OA)中测得CN比活性数值减去缓冲液Ⅱ(含EGTA与OA)中所测比活性数值之差来表示.

Effects of Ca^2＋ and OA on calcineurin （CN） activity in SH-SY5Y cell extracts were investigated with RⅡ phosphopeptide as substrate. Data showed that adding Ca^2＋ to assay buffer Ⅰ was necessary to activate CN in SH-SY5Y cell extracts, which was unlike extracts from brain. Further, it was necessary to add OA to assay buffers to inhibit PP1 and PP2A in SH-SY5Y cell extracts, with an optimal OA concentration of 100 nmol.L^-1, which was also unlike the brain extracts. In conclusion, calcineurin phosphatase activity in SH-SY5Y cell extracts should be calculated as the difference in protein phosphatase activity in buffer Ⅰ （cantains Ca^2＋ and OA） and buffer Ⅱ （cantains EGTAand OA）＂.