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要旨

The transport activity of purified Na⁺K⁺‐ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na⁺K⁺‐ATPase from pig kidney were attached to planar lipid bilayers in a sandwich‐like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P³‐1‐(2‐nitro)phenylethyladenosine 5′‐triphosphate, caged ATP] ATP was released after illumination with u.v.‐light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H⁺, Na⁺ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and P_i the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg²⁺ together with Na⁺ and Na⁺ and K⁺. No pump current was obtained in the presence of Mg²⁺ together with K⁺. The electrical response was blocked completely by the Na⁺K⁺‐ATPase‐specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na⁺K⁺‐ATPase with a large variety of different parameters.