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

Abstract

SUMMARY1. Interactions ofNa, 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.2. 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-075 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 (KO.5) for K stimulation of ATP-ADP exchange and Na,K-ATPase activity were indistinguishable from each other both at 20 mmand 120 mM-Na. All Na and Na + K-dependent exchanges were completely abolished by 10-4 M-ouabain.3. 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.4. 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 ofMg, 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.5. 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 1 PHY 375ATP-ADP EXCHANGE AND Na,K-ATPase E2PNa-E1PNa transformation. At high ionic strength, with higher concentrations of Na alone and of Na + K, Mg was also a strong inhibitor of ATP-ADP exchange; in this case the possible mechanism is less clear, although K ions could, to some extent, counteract this Mg effect. 6. The present results are consistent with Na inhibition of ATP-ADP exchange and ATPase activity by stabilizing the E2P conformation. This stabilization is selective in that it is easier to overcome in the forward direction (towards ATP hydrolysis) than in the backward directions (leading to ATP synthesis). These results seem to be best explained on the basis that K stimulation of ouabain...

Magnesium Deficiency in Critical Illness

“…This enzyme uses the energy derived from ATP hydrolysis to actively pump sodium and potassium across the plasma membrane against their respective concentration gradients to maintain the physiologically normal intracellular concentrations of these cations. Cyclic binding and release of Mg 2+ occur between the enzyme complex and the intracellular space during the sodium and potassium exchange [90]. During Mg depletion, intracellular sodium and calcium increase, and Mg 2+ and potassium decrease [91].…”

Breakdown of Na+/K+‐exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+‐ATPase activity.

“…In the absence of K + , extracellular Na + affects in a similar way several total and partial reactions of the Na + /K + ‐ATPase. Thus, the efflux of Na + [13], the Na + ‐ATPase activity [14–16], the ATP‐ADP exchange [15–17], and the breakdown of phosphoenzyme formed from ATP [15] all start high at or near zero external Na + , become inhibited as [Na + ] o is raised to about 5 m m , and are stimulated again as [Na + ] o is raised further. The idea put forward [15] was that, at intermediate concentrations, Na + o blocked the enzyme in the E 2 P form inhibiting both dephosphorylation and conversion into E 1 P. On the other hand, it has been proposed that external Na + effects are ‘allosteric’ in the sense that they modify the cation equilibrium on the intracellular surface of the cell without binding to extracellular transport sites [18].…”

“…Finally, there is the question of which extracellular sites are involved in this Na + stabilization of E 2 P. It is possible that there are extracellular inhibitory Na + sites with intermediate affinity [15]. Alternatively, as we first proposed [16,26], external Na + sites other than those for transport may not be required. Indeed, it is possible to simulate external Na + inhibition with intermediate affinity (a) assuming that only the E 2 P forms that are either free of Na + or saturated with it (three Na + ions for ATP–ADP exchange and two for the overall hydrolytic cycle) are catalytically competent [26] or (b) by adjusting different dissociation constants for Na + binding to the transporting sites in E 2 P, which leads to the same situation [27].…”