Defective conformational response in a selectively trypsinized (Na+ + K+)-ATPase studied with tryptophan fluorescence.
1. Monitoring protein conformations of purified (Na+ + K+)-ATPase with intrinsic fluorescence we have examined if altered conformational responses accompany the defective catalytic and transport processes in selectively modified 'invalid' (Na+ + K+)-ATPase which is obtained by graded tryptic digestion of the Na+ form of the protein. 2. The protein fluorescence intensity of the K+ form (E2K) of both control and invalid (Na+ + K+)-ATPase is 2--3% higher than that of the Na+ form (E1Na). By varying the NaCl concentration we found evidence for different fluorescence intensities of the two phosphoenzymes; E2P has the same fluorescence intensity as E2K and the intensity of E1P is similar to that of E1Na. The fraction of phosphoenzyme present as E2P can therefore be determined as the amplitude of the fluorescence change accompanying phosphorylation in the absence of K+ divided by the amplitude of the full response to K+. 3. Titration of the fluorescence responses of the invalid (Na+ + K+)-ATPase shows that the tryptic split alters the noise of the equilibria between the cation-bound conformations, E1Na and E2K, and between the phosphoforms, E1P and E2P, in the direction of the E1 forms. 4. Vanadate binds to the Mg2+-bound form of E2K and prevents further changes in fluorescence intensity of the protein. The conformative responses of invalid (Na+ + K+)-ATPase are insensitive to vanadate in agreement with the reduced vanadate binding affinity of this enzyme. 5. The defective conformative response of the invalid (Na+ + K+)-ATPase in relation to its catalytic defects, reduced Na+ transport, and insensitivity to vanadate suggest that the transitions between Na+ forms (E1) and K+ forms (E2) of the protein are coupled to the catalytic and transport reactions of the (Na+ + K+)-pump.
D000818:Animals; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013552:Swine; D014357:Trypsin; D014364:Tryptophan / Q000032:analysis
D014364:Tryptophan; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium