| Abstrakt: |
1. It is known that extracellular Na+ ions, in low concentrations, inhibit Na+‐ATPase activity in resealed red cell ghosts and that this inhibition is reversed by high concentrations of extracellular Na+. We have attempted to elucidate these actions of extracellular Na+ by investigating the dependence on Na+ concentration of (a) ATP‐ADP exchange and Na+‐ATPase activity both in native and in N‐ethylmaleimide (NEM)‐treated (Na+ + K+)‐ATPase from pig kidney, and (b) the rate of hydrolysis of the phosphorylated kidney enzyme in the absence of K+ ions. 2. With the native enzyme, ATP‐ADP exchange and Na+‐ATPase activity showed similar responses to changes in Na+ concentration: a steep but S‐shaped rise between 0 and 2.5 mM, a slight fall (exchange) or a plateau (ATPase) between 2.5 and 10 mM, and a roughly linear rise between 10 and 150 mM. With NEM‐treated enzyme, the ATP‐ADP exchange, which was greatly accelerated, showed no sign either of inhibition at intermediate Na+ concentrations or of the reversal of that inhibition at higher concentrations. The exchange rate increased with Na+ concentration in a smooth curve and was half‐maximal at about 7 mM. 3. The effects, on ATP‐ADP exchange, of changing the concentrations of ATP, ADP and Mg have also been investigated. With both native and NEM‐treated enzyme, the interactions of ATP, ADP and Mg are complicated; they show that, for the reaction leading to ATP formation, either free ADP rather than MgADP is the substrate, or Mg2+ ions are inhibitory (or both). 4. Since NEM, in the conditions in which we have used it, is believed to act by inhibiting the conversion of an ADP‐sensitive form of the phosphoenzyme (E1P) to an ADP‐insensitive form (E2P), the absence of Na+ inhibition of ATP‐ADP exchange in NEM‐treated enzyme, together with the parallel effects of Na+ ions on the ATP‐ADP exchange activity and on the Na+‐ATPase activity of native enzyme, suggests that the inhibitory effect of external Na+ occurs after the conversion of E1P into E2P. 5. To test whether this inhibitory effect of Na+ reflected inhibition of the hydrolysis of E2P, we measured the rate of loss of incorporated 32P when enzyme, newly phosphorylated by [gamma32P]ATP, was squirted into a large volume of ice‐cold solution containing 1,2‐cyclohexylenedinitrilotetraacetic aicd (CDTA), unlabelled ATP and 0, 5 or 150 mM‐Na+. The rate of loss of radioactivity from the membranes was least at 5 mM‐Na+, about twice as great at 150 mM‐Na+, and about 5 times as great at 20 microM (final) Na+. 6. An unexpected feature of the results was that the pattern of stimulation of ATP‐ADP exchange in intact cells. If Na+ ions are absent externally, a different could be fitted better on the assumption that activation by internal Na+ occurs at two sites with equal affinities, than on the assumptions that activation occurs at a single site or at three sites with equal affinities. |
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