Studies on the partial reactions catalyzed by the (Na 2+ + L +-activated ATPase. I. Effects of simple anions and nucleoside triphosphates on the alkali-cation specificity of the p-nitrophenylphosphatase

Abstract

1. 1. The K +-dependent p-nitrophenylphosphatase activity of the (Na 2+ + K +)-activated. ATPase complex is stimulated by the addition of Na + + ATP. Since oligomycin blocks the (Na + + ATP)-stimulation, but not the K +-dependent activity, the existence of a “K +-sensitive sit” and a “Na +-sensitive site” is indicated. The object of this work was to learn more about these sites through kinetic studies. 2. 2. The “K +-sensitive site” responded to Li +, Rb +, and Cs +; but the “Na +-sensitive site” showed absolute specificity for Na +. 3. 3. The order of cation specificity of the “K +-sensitive site” (K + = Rb + > Cs + > Li +), and the absolute specificity of the “Na +-sensitive site” remained constant under conditions ( e.g., change in the major anion of the assay medium) which had been used to demonstrate the changing specificity of another alkali-cation-activated enzyme (AMP deaminase). 4. 4. The “ +-sensitive site” could be demonstrated only in the presence of certain nucleoside triphosphates (ATP, ITP and CTP). Nucleoside diphosphates, nucleoside monophosphates, pyrophosphate and orthophosphate had no activating effects in the presence of Na +. 5. 5. A variety of simple anions were found to have inhibitory effects on the enzyme. Because of this, and due to the impurity of the enzyme, the complex kinetic data were of little value for mechanistic interpretations. 6. 6. Na + inhibited the enzyme both in the presence and absence of an activator cation. From the kinetic data it was not possible to determine if this inhibition was exerted at the same “Na +-sensitive site” that is involved in the (Na + + ATP)-activation of the enzyme.