Effects of acylphosphatase on the ion transport mechanism of the Na+,K+-ATPase.

The aim of the present study is to investigate whether acylphosphatase, a widespread cytosolic enzyme of about 11 kDa, may affect the functional properties of the shark Na+,K+-ATPase in terms of ion transport. This research is carried out using a novel experimental method, which has been recently developed to perform concentration jumps of an arbitrary substrate at the surface of a solid supported membrane (SSM) [1,2]. The SSM consists of an alkanethiol monolayer firmly anchored to the gold surface via the sulphydryl group, with a second phospholipid monolayer on top of it. Membrane fragments containing shark Na+,K+-ATPase are adsorbed on the SSM. Upon adsorption, the ion pumps are activated by performing concentration jumps of ATP at the surface of the SSM, and the electrical currents generated by the Na+,K+-ATPase are measured under short-circuit conditions. To investigate the effect of acylphosphatase on the ion transport by the Na+,K+-ATPase, the SSM technique is employed to carry out ATP concentration jumps in the presence and in the absence of acylphosphatase, and acylphosphatase concentration jumps in presence of ATP and Na+ ions. The reported results clearly indicate that acylphosphatase can induce significant modifications in the transport mechanism of the Na+,K+-ATPase. Possible mechanisms for such an effect are discussed. [1] Pintshovius J. and Fendler K., Biophys. J., 76, 814-826 (1999) [2] Pintshovius J., Fendler K. and Bamberg E., Biophys. J., 76, 827-836 (1999)