Experimental Triple Mixing Rapid Filtiation Apparatus

用N- [p-（2-苯并咪唑基）苯基]马来酰亚胺（BIPM-酶）修饰的猪肾Na，K-ATP酶制备物与0.7 μ M N-（4-磺丁基）-4- [4-（p-二戊基-氨基苯基）丁二烯基]吡啶鎓内盐（RH 421）孵育。在各种反应中间体积累的条件下，所得的酶制剂改变了各自的相对荧光强度，在2 M Na^+和4 mM Mg^<2+>存在的情况下，向酶制剂中加入ATP，诱导RH 421荧光迅速下降（25/s），随后缓慢形成磷酸酶，BIPM荧光下降（5-6/s）。在存在16 mM Na^+和1 mM Ca^<2+>的情况下，向制备物中加入ATP，诱导RH 421（44/s）和BIPM（26/s）的荧光依次降低，随后BIPM荧光和磷酸酶的荧光缓慢增加（约6/s），RH 421荧光缓慢增加（1.7/s）。RH 421似乎在BIPM荧光降低之前、ADP敏感性磷酸酶形成之前和K^+敏感性磷酸酶形成之后感知ATP依赖性分子事件。

Pig Kidney Na, K-ATPase preparations modified with N- [p- (2-benzimidazolyl) phenyl] maleimide (BIPM-enzyme) were incubated with 0.7muM N- (4-sulfobutyl) -4- [4- (p-dipentyl-aminophenyl) butadienyl] pyridinium, inner salt (RH421). The resulting enzyme preparations changed each relative fluorescence intensity under conditions of accumulation of various reaction intermediates.Addition of ATP to the enzyme preparations in the presence of 2M Na^+ and 4 mM Mg^<2+> induced a rapid decrease in RH421 fluorescence (25/s) follwed by slow phosphoenzyme formation and decrease in BIPM fluorescence (5-6/s). The addition of ATP to the preparation in the presence of 16 mM Na^+ and 1 mM Ca^<2+> induced sequential decreases in fluorescence of RH421 (44/s) and BIPM(26/s), which were followed by slow increases in both BIPM fluorescence and phosphoenzyme (around 6/s) and slower increase in RH421 fluorescence (1.7/s). RH421 seemed to sense ATP dependent molecular events preceding BIPM fluorescence decrease prior ADP sensitive phosphoenzyme formation and following K^+ -sensitive phosphoenzyme formation.