On the possible role of the phosphorylated intermediate in the reaction mechanism of (Na+−K+)-ATPase

Abstract 1. Phosphorylation and dephosphorylation of (Na + −K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) by [γ- 32 P]ATP was examined using highly specific NaI-treated preparations from pig-brain microsomes. 2. The phosphorylated enzyme consisted of an essentially single chemical species. The incorporated phosphate was relatively stable in an acidic medium (pH 1–2), and was easily released from the enzyme by treatment with hydroxylamine at a neutral pH. 3. The chemical nature of the phosphate bond was similar under the following alternative conditions: (a) whether the labeling was stopped by HClO 4 or dodecyl sulfate; (b) whether K + was present or not in addition to Na + ; or (c) whether the labeling was performed at 37° or 0°. 4. Longer incubation of the enzyme preparation with [γ- 32 P]ATP resulted in another form of incorporation which was hydroxylamine-resistant. 5. The high specificity of the ATP-hydrolyzing reaction of (Na + −K + )-ATPase to Na + and K + was partially lost at 0°, while the specific response of the phosphorylated enzyme to Na + and K + was still maintained. 6. Based on these results, possible relationships of the phosphorylated (Na + −K + )-ATPase to the ATP-hydrolyzing reaction of the enzyme are discussed.