Dependence of divalent metal ions on phosphotransferase activity of osseous plate alkaline phosphatase.

Kinetic evidence for the role of divalent metal ions in the phosphotransferase activity of polidocanol-solubilized alkaline phosphatase from osseous plate is reported. Ethylenediamine tetreacetate, 1,10-phenanthrolin, and Chelex-100 were used to prepare metal-depleted alkaline phosphatase. Except for Chelex-100, either irreversible inactivation of the enzyme or incomplete removal of metal ions occurred. After Chelex-100 treatment, full hydrolase activity of alkaline phosphatase was recovered upon addition of metal ions. On the other hand, only 20% of transferase activity was restored with 0.1 microM ZnCl2, in the presence of 1.0 M diethanolamine as phosphate acceptor. In the presence of 0.1 mM MgCl2, the recovery of transferase activity increased to 63%. Independently of the phosphate acceptor used, the transferase activity of the metal-depleted alkaline phosphatase was fully restored by 8 microM ZnCl2 plus 5 mM MgCl2. In the presence of diethanolamine as phosphate acceptor, manganese, cobalt, and calcium ions did not stimulate the transferase activity. However, manganese and cobalt-enzyme catalyzed the transfer of phosphate to glycerol and glucose.