Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. II. Exchange and hydrolysis kinetics at micromolar nucleotide concentrations.
The ATP hydrolysis rate and the ADP-ATP exchange rate of (Na+ + K+)-ATPase from ox brain were measured at 10 microM Mg2+free and at micromolar concentrations of free ATP and ADP. (1) In the absence of K+, substrate inhibition of the hydrolysis rate was observed. It disappeared at low Na+ and diminished at increasing concentrations of ADP. This was interpreted in terms of free ATP binding to E1P. In support of this interpretation, free ATP was found to competitively inhibit ADP-ATP exchange. (2) In the presence of K+, substrate activation of the hydrolysis rate was observed. Increasing (microM) concentrations of ADP did not give rise to competitive inhibition in contrast to the situation in the absence of K+ (cf. 1, above). This was interpreted to show that at micromolar substrate, some low-affinity, high-turnover Na+ + K+ activity is possible, provided the Mg2+ concentration is low. (3) While small concentrations of K+ increased the hydrolysis rate (cf. 2) they decreased the rate of ADP-ATP exchange. To elucidate this phenomenon, parallel measurements of exchange and hydrolysis rates were performed over a wide range of ATP and ADP concentrations, with and without K+. If, in the presence and absence of K+, ADP (and ATP competing) are binding to the same phosphorylated intermediate for the backward reaction, it places quantitative restrictions on the ratio of rate constants with and without K+. The results did not conform to these restrictions, and the discrepancy is taken as evidence for the necessity for a bicyclic scheme for the action of the (Na+ + K+)-ATPase. (4) An earlier statement concerning the nature of the phosphoenzyme obtained in the presence of Na+ and K+ is amended.
Plesner
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Institute of Biophysics, University of Aarhus, Denmark.
Plesner
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IW
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D027682:Cation Transport Proteins; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D027682:Cation Transport Proteins; D009711:Nucleotides; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium