The ( Na+ + K+)-dependent ATPase: mode of inhibition of ADP/ATP exchange activity by MgCl 2

Abstract

Na +-dependent ADP/ATP exchange activity, of a (Na + + K +)-dependent ATPase preparation from eel electric organ, was measured in terms of the incorporation of 14C into ATP during incubations with unlabeled ATP and [ 14C]ADP. Estimates of initial rates of exchange were possible by keeping changes in nucleotide concentrations, from both exchange and extraneous hydrolytic processes, to less than 10%. Under these conditions, increases in MgCl 2 concentration, from 0.2 to 3 mM, generally inhibited this exchange activity. The concentrations of free Mg 2+, Mg · ATP, and Mg · ADP present, with a range of MgCl 2, ATP, and ADP concentrations, were calculated from measured dissociation constants. Inhibition was associated with Mg · ATP as well as with Mg 2+, at concentrations from 0.4 to 1 mM (Mg · ADP, in the same concentration range, probably inhibited also). The affinity of the enzyme for these inhibitors is in fair correspondence with demonstrated affinities for Mg 2+, Mg · ATP, and Mg · ADP at low affinity substrate sites, measured kinetically. These observations are considered in terms of a dimeric enzyme with high and low affinity substrates sites: ADP/ATP exchange being catalyzed at the high affinity sites, with inhibition occurring through occupancy by Mg 2+, Mg · ATP, or Mg · ADP, of the low affinity sites, thereby pulling the reaction process away from those steps involved in exchange.