Na,K-ATPase in artificial lipid vesicles. Comparison of Na,K and Na-only pumping mode.
Na,K-ATPase from rabbit kidney outer medulla was reconstituted in large unilamellar lipid vesicles by detergent dialysis. Vesicles prepared in the presence or absence of potassium allowed to study two different transport modes: the (physiological) Na,K-mode in buffers containing Na+ and K+ and the Na-only mode in buffers containing Na+ but no K+. The ATP hydrolysis activity was obtained by determination of the liberated inorganic phosphate, Pi, and the inward directed Na+ flux was measured by 22Na-tracer flux. Electrogenic transport properties were studied using the membrane potential sensitive fluorescence-dye oxonol VI. The ratio upsilon(Na,K)/upsilon(Na) of the turnover rates in the Na,K-mode and in the Na-only mode is 6.6 +/- 2.0 under otherwise identical conditions and nonlimiting Na+ concentrations. Strong evidence is found that the Na-only mode exhibits a stoichiometry of 3Na+cyt/2Na+ext/1ATP, i.e. the extracellular (= intravesicular) Na+ has a potassium-like effect. In the Na-only mode one high-affinity binding side for ATP (KM congruent to 50 nM) was found, in the Na,K-mode a high- and low-affinity binding side with equilibrium dissociation constants, KM, of 60 nM and 13 microM, respectively. The sensitivity against the noncompetitively inhibiting ADP (KI = 6 microM) is higher by a factor of 20 in the Na-only mode compared to the Na,K-mode. From the temperature dependence of the pumping activity in both transport modes, activation energies of 160 kJ/mol for the Na,K-mode and 110 kJ/mol for the Na-only mode were determined.
Apell
H J
HJ
Department of Biology, University of Konstanz, F.R.G.
Häring
V
V
Roudna
M
M
D000818:Animals; D001693:Biological Transport, Active; D003593:Cytoplasm / Q000378:metabolism; D007679:Kidney Medulla / Q000201:enzymology / Q000378:metabolism; D008564:Membrane Potentials; D008567:Membranes, Artificial; D010710:Phosphates / Q000032:analysis; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism*; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D008567:Membranes, Artificial; D010710:Phosphates; D015221:Potassium Channels; D015222:Sodium Channels; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium