K + stimulation of ADP/ATP exchange catalyzed by the ( ifNa + + K +)-dependent ATPase

Abstract

A ( ifNa + + K +)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparation from rat brain catalyzed a ( ifNa +-dependent ADP/ATP exchange reaction. With millimolar concentrations of ATP and ADP, successively higher concentrations of MgCl 2 inhibited the exchange reaction. Examination of this inhibition in terms of the concentrations of free Mg 2+ and Mg - nucleotide complexes indicated that inhibition resulted from either Mg 2+ or Mg · nucleotide occupying the low-affinity substrate sites of the enzyme ( Km for Mg · ATP about 0.5 mM); earlier studies assigned catalysis of the exchange reaction to the high-affinity substrate sites ( K m for Mg · ATP about 1μM). When inhibition could be attributed to occupancy of the low-affinity sites by Mg · ATP, K + stimulated ADP/ATP exchange. The sites through which K + relieved inhibition due to Mg · ATP appeared to be the moderate-affinity α-sites for K +, as indicated by the concentration dependence, the effect of dimethyl sulfoxide on apparent affinity for K +, and by the inability of Li +to substitute for K +. The observation fit an erlier formulation of antagonism between K + at the α-sites and Mg · ATP at the low-affinity substrates sites. Under conditions in which inhibition of ADP/ATP exchange could be attributed instead to Mg 2+ occupying the low-affinity substrate sites, K + produced little or no stimulation, in accord with earlier studies showing that K + at the α-sites is a poor antagonist toward Mg 2+. With micromolar concentration of ATP and ADP, K + inhibited the exchange reaction, and competition toward Na + at the Na +-sites was apparent.