Stopped flow measurement of conformational change induced by phosphorylation in (Na+,K+)-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide.
The addition of ATP to (Na+,K+)-ATPase (EC 3.6.1.3) of pig kidney modified with N-[p-(2-benzimidazolyl)phenyl]maleimide induced three different modes of fluorescence change: positive, biphasic, and negative, which were observed, respectively, in the presence of Mg2+ with 16 or 160, 320 or 640, and 1500 mM Na+. At 16-320 mM Na+, the extent of positive change was almost saturated at 0.4 microM ATP, but the rate of the change was significantly accelerated by much higher concentrations of ATP. In the presence of 640 mM Na+, the change showed an initial small decrease in fluorescence (t 1/2 = 15 ms), followed by an increase (t 1/2 = 145 ms). The amount of phosphoenzyme increased rapidly and gave a steady level (t 1/2 = 38 ms) after the addition of ATP to give 12.2 microM. Comparison of the relative fluorescence intensity and the amount of phosphoenzyme at a given time permitted modeling the amount of ADP-sensitive phosphoenzyme and K+-sensitive phosphoenzyme present. A simulation gave a good fit to the smoothed data. These data show directly that ATP is hydrolyzed through ADP-sensitive phosphoenzyme and K+-sensitive phosphoenzyme in the presence of Mg2+ and Na+.
D000818:Animals; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D010766:Phosphorylation; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D008301:Maleimides; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D000254:Sodium-Potassium-Exchanging ATPase