Experimental Triple Mixing Rapid Filtiation Apparatus

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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.