KINETIC STUDIES ON A BRAIN MICROSOMAL ADENOSINE TRIPHOSPHATASE .2. POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY

A (Na+ + K+) dependent adenosine triphosphatase preparation from rat brain catalyzed a K+-dependent hydrolysis of p-nitrophenylphosphate. The response of the p-nitrophenylphosphate to K+ did not follow Michaelis-Menten kinetics, but gave a Lineweaver-Burk plot that curved upward at low K+ concentrations, consistent with a positive cooperative allosteric activation. Na+ inhibited by raising the K0.5 for K+ without affecting Vmax. However, in the presence of increasing concentrations of Na+ the response of the enzyme to K+ gave a Lineweaver-Burk plot that curved downward at low K+ concentrations, consistent with Na+converting the K+ activation from a positive into a negative cooperative response. This Na+-induced change in the response to K+ suggested that the Na+ inhibition was not due to simple competition with K+. Furthermore, the inhibition by Na+ similarly followed cooperative kinetics which in turn were modified by the K+ concentration. At a constant molar ratio of Na+ to K+ only Vmax was affected. Cytosine triphos phate in the absence of Na+ inhibited the p-nitrophenylphos-phate, but with Na+ it stimulated activity with an abolition of the Na+-induced negative cooperative response to K+ and a 70-fold reduction in the K0.5 for K+. ATP with Na+ also reduced the K0.5 and converted the cooperative response into a positive one. Furthermore, Na+ in the presence of low concentrations of K+ or of cytosine triphosphate (without K+) stimulated p-nitrophenylphosphate activity. These data suggest that in addition to possible competition with K+ at K+sites, Na+ could modify p-nitrophenylphosphate activity through interactions at its own sites, and that the p-nitro-phenylphosphate has coexisting regulatory sites for K+, Na+, and nucleotides. These properties of the p-nitrophenylphosphate may reflect similar features of the related (Na+ K+-dependent adenosine triphosphatase, such that interdependence between distinct Na+ and K+ activating sites may modify the enzymatic reaction and the regulation of ion movements. © 1969, American Chemical Society. All rights reserved.