用偶氮胂Ⅲ指示剂检测心肌肌质网Ca^(2+)主动转运

Measurement of ATP-dependent Ca^(2+) transport by cardiac sarcoplasmic reticulum with arsenazo Ⅲ as a Ca^(2+) indicator

采用金属染包指示剂偶氮胂Ⅲ(arsenazoⅢ,ASⅢ)作为Ca^(2+)指示剂,用双波长分光光度计(675～685nm)配对波长检测心肌肌质网(SR)依赖ATP的Ca^(2+)摄取量。根据本实验室条件建立了SR摄取Ca^(2+)的测定方法。本法操作简便,灵敏度高,能够迅速选择性测定SR的Ca(2+)摄取量。正常大鼠心肌肌质网依赖ATP的Ca(2+)摄取量平均为20.3±1.2ng Ca^(2+)/(mg蛋白·min)。

ATP-dependent Ca^(2+)uptake by cardiac sarcoplasmic reticulum was measured with arsenazoⅢ (ASⅢ) as a Ca^(2+) indicator. This was performed by using a dual wavelength spectrophotometer (675-685nm) with the reaction mixture which should contain 5-20μmol Ca^(2+), 0.2-1.0mg sarcoplasmic reticulum protein, 30-50μmol ASⅢ, and most important, 10-15mmol Mg^(2+) to prevent Ca^(2+) chelation following ATP addition. According to the present condition in our laboratory, the optimal concentration of ATP is 200 μmol and the optimal temperature of the re- action is 22-30℃. The technique is simple and highly sensitive for measuring ATP-dependent Ca^(2+) transport in biological systems.