IUBMB Life, 49: 113 – 119, 2000 Copyright c ° 2000 IUBMB 1521-6543/00 $12.00 + .00 Original Research Article Kinetic Characteristics of ATP Hydrolysis by a Detergent-Solubilized Alkaline Phosphatase From Rat Osseous Plate Marlene A. Demenis and Francisco A. Leone Departamento de Qu´õ mica, Faculdade de Filoso a, Ci ˆ encias e Letras de Ribeir ˜ ao Preto, Universidade de S ˜ ao Paulo, 14040-901 Ribeir ˜ ao Preto, SP, Brasil Summary Polidocanol-solubilized alkaline phosphatase was puri ed to ho- mogeneity with a speci c activity of 822.3 U/mg. In the absence of Mg 2+ and Ca 2+ 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, K 0.5 = 17.2 ¹M, and n = 1.8 and V = 430.3 U/mg, K 0.5 = 3.2 mM, and n = 3.2 for high- and low-af nity sites, respectively. In the presence of saturating concentrations of Mg 2+ or Ca 2+ ions, the hydrolysis of ATP also followed biphasic curves. However, the speci c 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 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 K 0.5 values. At pH 7.5, the enzyme also hy- drolyzed ATP with K 0.5 = 8.1 ¹M 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-af nity binding site for ATP at alkaline pH. IUBMB Life, 49: 113 – 119, 2000 Keywords Alkaline phosphatase; ATP; divalent ions; polidocanol; rat osseous plate. INTRODUCTION Biological calci cation and bone formation are complex pro- cesses mediated by the concerted action of physicochemical and biochemical factors that lead to deposition of a mineral phase into a speci c organic matrix. According to several authors, matrix vesicles are the focuses where calci cation of cartilage Received 14 September 1999; accepted 9 December 1999. Address correspondence to Francisco A. Leone. Fax: +55 16 633 8151; E-mail: fdaleone@ffclrp.usp.br and bone starts, but the exact mechanism of initiation of cal- ci cation is not fully understood (for review, see 1). Since the pioneering work of Robison (2), who rst associated alkaline phosphatase activity with calci cation, several workers have in- vestigated the multiple activities of this enzyme. However, de- spite circumstantial evidence found for each postulated role, no unique function has thus far been attributed to this enzyme (see 1). Alkaline phosphatases from cartilage and bone are ectopro- teins anchored to the matrix vesicle membrane by glycosylphos- phatidylinositol (3 –5), but they are not the only membrane- bound enzymes relevant to the calci cation process (6, 7 ). Recent reports emphasize the signi cance of the hydrolysis of ATP and pyrophosphate by alkaline phosphatase from cartilage and bone, given that these two enzymatic activities are believed to be involved in the removal of inhibitors of mineral formation, providing a phosphate pool for nascent mineral, or both (1, 3, 8 – 10). Furthermore, ATP hydrolysis could support the energy- dependent transport of calcium into matrix vesicles (11, 12). An intrinsic ATPase activity present in rat osseous plate (13) and in matrix vesicles from rachitic rats (14– 16) seems also to participate in the deposition of calcium and phosphate (1, 5, 7, 14, 15). However, although many studies suggest that ATP could initiate matrix vesicle – mediated calci cation, it is not known whether alkaline phosphatase or a speci c ATPase is responsible for the ATP-dependent calci cation, because both enzymes are able to hydrolyze ATP (17 ). We have been using a model system to study an alkaline phosphatase from developing rat osseous cartilage to clarify its possible role during endochondral ossi cation (1). Our stud- ies show that this enzyme is a glycosylphosphatidylinositol- anchored metalloprotein, consisting of two apparently identi- cal subunits of 65 kDa each that require zinc and magnesium ions for maximum activity and show multifunctional activities (1, 18 – 22). 113