DEPENDENCE OF DIVALENT METAL-IONS ON PHOSPHOTRANSFERASE ACTIVITY OF OSSEOUS PLATE ALKALINE-PHOSPHATASE

Kinetic evidence for the role of divalent metal ions in the phosphotra nsferase activity of polidocanol-solubilized alkaline phosphatase from osseous plate is reported. Ethylenediamine tetreacetate, 1,10-phenant hrolin, and Chelex-100 were used to prepare metal-depleted alkaline ph osphatase. 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 rec overed upon addition of metal ions. On the other hand, only 20% of tra nsferase activity was restored with 0.1 mu M 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%. Indepen dently of the phosphate acceptor used, the transferase activity of the metal-depleted alkaline phosphatase was fully restored by 8 mu M ZnCl 2 plus 5 mM MgCl2. In the presence of diethanolamine as phosphate acce ptor, manganese, cobalt, and calcium ions did nor stimulate the transf erase activity. However, manganese and cobalt-enzyme catalyzed the tra nsfer of phosphate to glycerol and glucose. (C) 1997 Elsevier Science Inc.