Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. I. Exchange and hydrolysis kinetics at millimolar nucleotide concentrations.
Parallel measurements in steady-state of ATP hydrolysis rate (vhydr) and the simultaneous reverse reaction, i.e., the ADP-ATP exchange rate (vexch), allowed the determination of a kinetic parameter, KE, containing only the four rate constants needed to characterize the enzyme intermediates involved in the sequence (Formula: see text). In order to compare the properties of these enzyme intermediates under different sets of conditions, KE was measured at varying K+ and Na+ concentrations in the presence of millimolar concentrations of ATP, ADP and MgATP, using an enzyme preparation that was partially purified from bovine brain. (1) In the presence of Na+ (150 mM), K+ (20-150 mM) was found to increase the exchange rate and decrease the ATP hydrolysis rate at steady-state. As a result, KE increased at increasing K+. However, the value of KE found by extrapolation to K+ = 0 was 7-times lower than the value actually measured in the absence of K+. This finding indicates that one of the intermediates, EATP or EP, or both, when formed in the presence of Na+ alone, are different from the corresponding intermediate(s) formed in the presence of Na+ + K+ (at millimolar substrate concentration). (2) In the presence of 150 mM K+, Na+ (5-30 mM) was found to increase the ADP/ATP exchange as well as the ATP hydrolysis rate at steady-state. The ratio of the two rates was constant. This finding, when interpreted in terms of KE, indicates that Na+ does not have to leave the enzyme for ATP release to be accelerated by K+ in the backward reaction. This also is in opposition to the usual versions of the Albers-Post model, which does not have simultaneous presence of Na+ and K+.
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Institute of Biophysics, University of Aarhus, Denmark.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D027682:Cation Transport Proteins; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D027682:Cation Transport Proteins; D009711:Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D000254:Sodium-Potassium-Exchanging ATPase