Demenis M A, Leone F A
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brasil.
IUBMB Life. 2000 Feb;49(2):113-9. doi: 10.1080/15216540050022421.
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.
聚多卡醇增溶的碱性磷酸酶被纯化至同质，比活性为822.3 U/mg。在不存在Mg2+和Ca2+离子且pH为9.4的条件下，该酶以双相曲线的方式水解ATP，高亲和力位点的V = 94.3 U/mg，K0.5 = 17.2 microM，n = 1.8，低亲和力位点的V = 430.3 U/mg，K0.5 = 3.2 mM，n = 3.2。在存在饱和浓度的Mg2+或Ca2+离子时，ATP的水解也遵循双相曲线。然而，比活性增加至高达1000 U/mg，而K0.5和n值几乎保持不变。在存在非饱和浓度的金属离子时，ATP的水解与在不存在这些离子时观察到的情况相似，但K0.5值显著降低。在pH 7.5时，该酶也水解ATP，K0.5 = 8.1 microM，V = 719.8 U/mg。显然，碱性磷酸酶在体内能够在pH 7.5或pH 9.4时水解ATP。这些数据有助于了解骨骼碱性磷酸酶的生物学特性，并表明该酶在碱性pH下可能具有ATP的高亲和力结合位点。
