Conformational change accompanying transition of ADP-sensitive phosphoenzyme to potassium-sensitive phosphoenzyme of (Na+,K+)-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide.
The addition of Mg2+ or ATP to (Na+,K+)-ATPase (EC 3.6.1.3) of pig kidney modified with a sulfhydryl fluorescent reagent N-[p-(2-benzimidazolyl)phenyl]maleimide simply reduced fluorescence in the presence of Na+; however, the addition of both ligands to the enzyme induced a reversible dynamic change. The direction of the change was dependent on the concentration of Na+ present. These dynamic changes in fluorescence intensity both in the presence of low and high concentrations of Na+ can be repeated by the re-addition of ATP but not by ADP. Addition of ouabain under the former condition stabilized the fluorescence at the highest level, but the addition of ouabain under the latter condition increased the fluorescence from the lowest to the highest level. The phosphoenzyme formed under the former condition was sensitive to K+ and insensitive to ADP while the phosphoenzyme formed under the latter condition was sensitive to ADP and insensitive to K+. The data indicate that the positive and negative fluorescence changes were induced by the formation of K+-sensitive phosphoenzyme and ADP-sensitive phosphoenzyme, respectively. N-Ethylmaleimide treatment partially inhibited the positive change without affecting the negative change. These data also indicate that the transition of ADP-sensitive phosphoenzyme to K+-sensitive phosphoenzyme accompanied the largest fluorescence intensity change which was examined during the hydrolysis of ATP. The data obtained from the tryptophan fluorescence of both the native and the modified enzyme suggest that the micro-environments of the tryptophan and the sulfhydryl residues are similar in the state of K+-sensitive phosphoenzyme but different in the state of ADP-sensitive phosphoenzyme.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D002794:Choline / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology*; D010042:Ouabain / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013552:Swine
D008301:Maleimides; D010750:Phosphoproteins; D013439:Sulfhydryl Reagents; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D010042:Ouabain; D000244:Adenosine Diphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D002794:Choline; D011188:Potassium