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Free keywords:
ATPase, (Na++K+); Pump current; Black lipid membrane; caged ATP; (Pig kidney)
Abstract:
Purified (Na++K+)-ATPase from pig kidney was attached to black lipid membranes and ATP-induced electric currents were measured as described previously by Fendler et al. ((1985) EMBO J. 4, 3079–3085). An ATP concentration jump was produced by an ultraviolet-light flash converting non-hydrolysable caged ATP to ATP. In the presence of Na+ and Mg2+ this resulted in a transient current signal. The pump current was not only ATP dependent, but also was influenced by the ATP/caged ATP ratio. It was concluded that caged ATP binds to the enzyme (and hence inhibits the signal) with a Ki of approx. 30 μM, which was confirmed by enzymatic activity studies. An ATP affinity of approx. 2 μM was determined. The addition of the protonophore 1799 and the Me+/H+ exchanger monensin made the bilayer conductive leading to a stationary pump current. The stationary current was strongly increased by the addition of K+ with a K05 of 700 μM. Even in the absence of K+ a stationary current could be measured, which showed two Na+-affinities: a high-affinity (K05 <1mM) and a low-affinity (K05 <2mM). In order to explain the sustained electrogenic Na+ transport during the Na+-ATPase activity, it is proposed, that Na+ can replace K+ in dephophorylating the enzyme, but binds about 1000-times weaker than K+. The ATP requirement of the Na+-ATPase was the same (K05 <2 μM) with regard to the peak currents and the stationary currents. However, for the the stationary currents required more ATP. The results are discussed on the basis of the Albers-Post scheme.
