Evidences for a Sulfhydryl Group in the ATP‐Binding Site of (Na + + K + )‐Activated ATPase

5,5′‐Dithio‐bis(2‐nitrobenzoate) inhibited (Na + + K + )‐activated ATPase by affecting the Na + ‐dependent phosphorylation reaction. ATP and ADP but not ITP protected the enzyme at low concentrations against the inactivation. The ATP analogues 6‐mercaptopurine riboside‐5′‐triphosphate and S ‐(2,4‐dinitrophenyl)‐6‐mercaptopurine riboside‐5′‐triphosphate inactivated (Na + + K + )‐ATPase whereas the respective monophosphates left the enzyme unaffected. S ‐(2,4‐Dinitrophenyl)‐6‐mercaptopurine triphosphate inactivated the partial reactions of (Na + + K + )‐ATPase, i.e. the ADP‐binding capacity and the Na + ‐dependent phosphorylation reaction in parallel with the overall enzymatic activity. ATP protected against the inactivation of (Na + + K + )‐ATPase by S ‐(2,4‐dinitrophenyl)‐6‐mercaptopurine triphosphate. Dithiothreitol hindered the inactivation of (Na + + K + )‐ATPase by S ‐(2,4‐dinitrophenyl)‐6‐mercaptopurine riboside‐5′‐triphosphate and 6‐mercaptopurine riboside‐5′‐triphosphate. Treatment with dithiothreitol restored the activity of (Na + + K + )‐ATPase which had been inactivated by 6‐mercaptopurine riboside‐5′‐triphosphate but it did not restore the activity of that enzyme which was pretreated with S ‐(2,4‐dinitrophenyl)‐6‐mercaptopurine riboside‐5′‐triphosphate. S ‐(2,4‐Dinitrophenyl)‐6‐mercaptopurine riboside‐5′‐triphosphate inactivated the associated enzymic activites of K + ‐activated acetylphosphatase and K + ‐dependent p ‐nitrophenylphosphatase but more slowly than (Na + + K + )‐activated ATPase. It is concluded, that S ‐(2,4‐dinitrophenyl)‐6‐mercaptopurine riboside‐5′‐triphosphate and 6‐mercaptopurine riboside‐5′‐triphosphate exert their action on (Na + + K + )‐ATPase by reacting with a sulfhydryl group in the ATP‐binding site of the enzyme.