Reversible change in light scattering following formation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide.

Abstract

An increase in light scattering (3.5 +/- 0.2%) was observed when pig kidney Na+,K+-ATPase preparations modified with N-[p-(2-benzimidazolyl)phenyl] maleimide were phosphorylated by ATP in the presence of 2 M Na+ with Mg2+ to form ADP-sensitive phosphoenzyme (E1P), which had a negative fluorescence intensity (-1.5 +/- 0.3%). Addition of K+ or ouabain to E1P reduced the light scattering to the original level observed in the absence of ATP. Stopped flow measurements showed that the fluorescence change accompanying the E1P formation (t1/2 = 0.1 s) occurred preceding the light-scattering change (t1/2 = 1 s). Oligomycin affected the rate of the scattering increase little, but it diminished the effect of K+ on E1P to reduce the light scattering and increase the fluorescence. The addition of 2 M Na+ to K+-sensitive phosphoenzyme (E2P) immediately decreased the fluorescence (t1/2 = 0.02 s) to form E1P which was followed by a slow increase in the light scattering (t1/2 = 0.25 s). Oligomycin reduced both rates of the above changes accompanying the transition of E2P to E1P. The data suggest the sequential appearance of species of E1P that precede E2P formation during the hydrolysis of ATP.