Interaction of Na+, K+ and ATP with the Na,K-ATPase

P. J. GARRAHAN; R. C. ROSSI; A. F. REGA

ANN. N. Y. ACADEMY SCIEN.

The New York Academy of Sciences

Año: 1982 vol. 402 p. 239 - 252

1749-6632

Since the initial observations by Post and coworkers, a large amount of information has accumulated indicating that hydrolysis of ATP by the (Na,K)- ATPase proceeds through a series of steps involving the formation and hydrolysis of phosphoenzymes. This information has led to a detailed reaction scheme for the hydrolysis of ATP and for the associated active transport of Na+ and K+. The main experimental support for the currently accepted reaction scheme comes from studies on partial reactions. Few attempts have been made to see to what extent this scheme is able to predict quantitatively the steady-state kinetic behavior of the (Na,K)-ATPase. Studies of this kind are important since agreement between theoretical predictions and experimental results is one of the necessary requirements for the validity of any reaction scheme. We will report here experiments on the interaction of ATP, K+, Na+, and Mg2+ with the (Na,K)-ATPase during steady-state ATP hydrolysis. We will try to show that comparison of the experimental results with theoretical kinetic equations seems to indicate that the behavior of the system is in some respects difficult to reconcile with the predictions of the currently held view about the role of Na+, K+, Mg2+, and ATP during hydrolysis of ATP by the (Na,K)-ATPase. The experiments reported here were performed using a partially purified (Na,K)-ATPase isolated from the outer medulla of dog kidney following Jdrgensen?s procedure ?C.?4 ATPase activity was estimated from the initial rate of release of 32P (Pi) from [gamma-32P]ATP. Incubations were carried out at 37oC, adjusting the length of incubation and the concentration of enzyme so that no more than 10% of the ATP underwent enzymatic hydrolysis. In all media, pH was kept at 7.4 with 20 mM imidazol-HC1 and total salt concentration at 150 mM by mixing adequate amounts of NaCl, KCI, and choline C1. All media contained 1% (w/v) of bovine serum albumin.