Effects of mono and divalent cations on total and partial reactions catalysed by pig kidney Na,K-ATPase

Abstract

Interactions of Na, K, Ca and Mg ions with partially purified Na,K-ATPase from pig kidneys were investigated using as a tool simultaneous determinations of adenosine 5'-triphosphate (ATP)-adenosine 5'-diphosphate (ADP) exchange and ATPase activity catalysed by the enzyme. In the presence of 120 mM-NaCl and 0.5 mM-MgCl2, the effects 12 mM-KCl on ATP-ADP exchange depended on the ATP and ADP concentrations: stimulation (about 10-fold) with 3 mM-ATP-0.75 mM-ADP, no effect with 0.04 mM-ATP-0.01 mM-ADP and inhibition when ATP and ADP concentrations were 0.003 mM and 0.001 mM respectively. The apparent affinities (K0.5) for K stimulation of ATP-ADP exchange and Na,K-ATPase activity were indistinguishable from each other both at 20 mM- and 120 mM-Na. All Na and Na+K-dependent exchanges were completely abolished by 10(-4) M-ouabain. In the absence of K, intermediate Na concentrations were inhibitory of ouabain-sensitive ATP-ADP exchange and ATPase activity. This Na inhibition was more pronounced at low than at high ionic strength and was more noticeable in the exchange reaction. K ions antagonized these Na effects; this K-Na antagonism was more effective on ATPase than on the ATP-ADP exchange. Ca ions, in the absence of Mg, could sustain ATP-ADP exchange. Qualitatively the behaviour of the exchange reaction was similar to that observed with Mg. Quantitatively the rates were always lower with Ca. In the presence of Mg, at low ionic strength and at concentrations which stimulated phosphatase activity, Ca ions were not able to counteract Na inhibition of ATPase and ATP-ADP exchange or to stimulate ATPase activity in the presence of Na. This is another indication of the different reactivity of the K sites involved in phosphatase and ATPase activities as well as of those responsible for the release of Na inhibition of ATPase and exchange reaction. In K-free solutions containing 0.5 mM-Na and low ionic strength, Mg was a powerful inhibitor of ouabain-sensitive ATPase and ATP-ADP exchange; the curves relating remaining activities to Mg concentrations were identical for both reactions. When Na was increased to 5 mM, ATP-ADP exchange became more sensitive and ATPase more resistant to Mg inhibition. These data are consistent with (i) a Na-Mg antagonism at the phosphorylation step, and (ii) a Na-Mg synergism in blocking the E2PNa-E1PNa transformation.(ABSTRACT TRUNCATED AT 400 WORDS)