Na+-dependent phosphorylation of the rat brain (Na+ + K+)-ATPase. Possible non-equivalent activation sites for Na+.
The steady state levels of Na+-dependent phosphoenzyme (E-P) in the (Na+ + K+)-ATPase (EC 3.6.1.3) of rat brain, obtained from a time course study of phosphoenzyme formation at 4 degrees C, were dependent on the concentration of Na+ in the reaction and were maximal in the presence of 64 mM Na+. The plot of phosphoenzyme vs. Na+ concentration gave a curve which on conversion to a double reciprocal plot (1/E-P vs. 1/Na+) gave a line with two breaks, yielding apparently three linear segments. This may be taken to indicate the presence of multiple Na+ sites for the formation of the phosphoenzyme. To test this hypothesis further, the following approach was taken. By making the assumption that the phosphoenzyme may represent bound Na+, it was possible to subject the data to rigorous multiple-site analysis by utilizing steady-state binding equations described by Klotz and Hunston (1971) (Biochemistry 10, 3065-3069), and by Scatchard (1949) (Ann. N.Y. Acad. Sci. 51, 660-672). The analysis of the data by these methods suggests that there may be three non-equivalent Na+ activation sites for the formation of Na+-dependent phosphoenzyme in the (Na+ + K+)-ATPase. The estimated intrinsic association constants (Ka) for activation by Na+ at each of the three sites were 3.4, 0.295, and 0.025 mM-1, respectively.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008433:Mathematics; D011188:Potassium / Q000494:pharmacology*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*
D012964:Sodium; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D011188:Potassium; D004121:Dimethyl Sulfoxide