Kinetic characteristics of ATP hydrolysis by a detergent-solubilized alkaline phosphatase from rat osseous plate.
Polidocanol-solubilized alkaline phosphatase was purified to homogeneity with 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 represented by biphasic curves with V = 94.3 U/mg, K0.5 = 17.2 microM, and n = 1.8 and V = 430.3 U/mg, K0.5 = 3.2 mM, and n = 3.2 for high- and low-affinity sites, respectively. In the presence of saturating concentrations of Mg2+ or Ca2+ ions, the hydrolysis of ATP also followed biphasic curves. However, the specific activity increased to as much as 1,000 U/mg, whereas the K0.5 and n values remained almost unchanged. In the presence of nonsaturating concentrations of metal ions, the hydrolysis of ATP was similar to that observed in the absence of these ions, but with a marked decrease in K0.5 values. At pH 7.5, the enzyme also hydrolyzed ATP with K0.5 = 8.1 microM and V = 719.8 U/mg. Apparently, alkaline phosphatase was able to hydrolyze ATP in vivo, 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.
Demenis
M A
MA
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brasil.
Leone
F A
FA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D003902:Detergents; D006132:Growth Plate / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D051381:Rats
D003902:Detergents; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium