Defective conformational response in a selectively trypsinized [formula omitted] studied with tryptophan fluorescence

Abstract

1. 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. 2. The protein fluorescence intensity of the K + form (E 2K) of both control and invalid (Na + + K +)-ATPase is 2–3% higher than that of the Na + form (E 1Na). By varying the NaCl concentration we found evidence for different fluorescence intensities of the two phosphoenzymes; E 2P has the same fluorescence intensity as E 2K and the intensity of E 1P is similar to that of E 1Na. The fraction of phosphoenzyme present as E 2P 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. 3. Titration of the fluorescence responses of the invalid (Na + + K +)-ATPase shows that the tryptic split alters the poise of the equilibria between the cation-bound conformations, E 1Na and E 2K, and between the phosphoforms, E 1P and E 2P, in the direction of the E 1 forms. 4. 4. Vanadate binds to the Mg 2+-bound form of E 2K 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. 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 (E 1) and K + forms (E 2) of the protein are coupled to the catalytic and transport reactions of the (Na + + K +)-pump .