Kinetic characteristics of ATP hydrolysis by a detergent-solubilized alkaline phosphatase from rat osseous plate

Polidocanol-solubilized alkaline phosphatase was purified to homogeneity wi th a specific activity of 822.3 U/mg, In the absence of Mg2+ and Ca2+ ions and at pH 9.4, the enzyme hydrolyzed ATP in a manner that could be represen ted by biphasic curves with V = 94.3 U/mg, K-0.5 = 17.2 mu M, and n = 1.8 a nd V = 430.3 U/mg, K-0.5 = 3.2 mM, and n = 3.2 for high- and low-affinity s ites, respectively. In the presence of saturating concentrations of Mg2+ or Ca2+ ions, the hydrolysis of ATP also followed biphasic curves. However, t he specific activity increased to as much as 1,000 U/mg, whereas the K-0.5 and n values remained almost unchanged. In the presence of nonsaturating co ncentrations of metal ions, the hydrolysis of ATP was similar to that obser ved in the absence of these ions, but with a marked decrease in K-0.5 value s, At pH 7.5, the enzyme also hydrolyzed ATP with K-0.5 = 8.1 mu M and V = 719.8 U/mg, Apparently, alkaline phosphatase was able to hydrolyze ATP in v ivo, either at pH 7.5 or pH 9.4, These data contribute to the knowledge of the biological properties of skeletal alkaline phosphatase and suggest that this enzyme may have a high-affinity binding site for ATP at alkaline pH.