On the Mechanism of Na+‐ and K+‐Stimulated Hydrolysis of Adenosine Triphosphate

The nucleotide specificity of membrane‐bound Na+‐ and K+‐activated ATPase from ox brain was compared with that of the Na+‐dependent phosphorylation reaction of ATPase protein. [32P]Phosphate is incorporated into enzyme protein from terminally‐labelled [32P]nucleotide triphosphates (ATP, ITP, GTP), a process dependent on the presence of Na+. Subsequent addition of K+ ions to the test system is followed by a rapid decrease in incorporated radioactivity. Enzyme‐bound phosphate exchanges its label rapidly with adenine nucleotides and slowly with inosine and guanosine nucleotides. ATP, ITP, and GTP are substrates of Na+‐ and K+‐activated ATPase. The ratio of the reaction rates is: ATP: ITP: GTP = 27: 2:1. An almost identical activation pattern of nucleotide triphosphate hydrolysis by Na+ ions in the presence of saturating concentrations of K+ is found with all substrates (Km= 10 mM NaCl). On the other hand, the optimal stimulation by K+ at saturating concentrations of Na+ is variable: hydrolysis of ATP is increased 10 times, that of ITP 4 times and that of GTP 1.2 times. ADP inhibits both ATP and ITP hydrolysis. Only high concentrations of IDP (50 mM) inhibit ATP hydrolysis, whereas low concentrations of IDP inhibit ITP hydrolysis. Identical nucleotide triphosphate specificities were found for Na+‐ and K+‐activated ATPase from ox brain and from human red blood cells. It is concluded that all nucleotide triphosphates investigated are hydrolyzed via the same acceptor group. The Na+‐dependent phosphorylation reaction of ATPase protein is considered as part of the overall reaction of membrane‐bound Na+‐ and K+‐activated ATPase.