Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles.
Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.
D000818:Animals; D002794:Choline / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D008094:Lithium / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine; D013861:Thiocyanates / Q000378:metabolism*
D005452:Fluoresceins; D013861:Thiocyanates; D008094:Lithium; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D002794:Choline; D011188:Potassium