Effects of Mg, Ca, and Zn on human alkaline phosphatase activity

Objectives: One Mg, one Ca, and two Zn binding sites per subunit of the human placenta type alkaline phosphatase (ALP) were detected by analyzing its crystalline structure. It was an ticipated that other human ALPs also contain these ion binding sites, as there are few reports about the function of each of these ions on ALP activity. We examined the affinity of ALP isozymes for Mg, Ca, and Zn. Moreover, we also examined the effects of Mg, Ca, and Zn on the optimum pH for ALP activity and substrate specificity. Methods: We utilized commercial human bone, liver, placenta, an d small intestine type ALP, p-nitrophenyl phosphate (pNPP), and Na-PPi as substrates, and assayed the Pi released from the substrates using the Chifflet method. Results and discussion: 1) The ALP activity of all four isozyme s increased in Mg, Ca, and Zn concentration-dependent manner with pNPP as the substrate. Remarkably, the 50% activated concentrations (K0.5) of each ion did not depend on the type of ALP and increased as the concentration of Zn, Ca, a nd Mg increased. Zn promoted the activity with a low concentration, but decreased it with a higher concentration, suggesting that the first Zn binding site stimulates activity but the second one inhibits it. The optimum pH for ALP activity varied according to the ion present. We observed that Zn, Ca, and Mg binding sites are present in all ALP. 2) The concentration dependency of Mg, Ca, and Zn on the bone ALP activity was examined using pNPP and Na-PPi as substrates. Its activity was promoted by Mg, Ca, and Zn in a concentration dependent manner, but the concentration dependency curve was different between pNPP and Na-PPi, suggesting that the affinity for Mg, Ca, and Zn depends on the substrate. 3) The activity increased remarkably when divalent cations were present in combination with Mg, Ca, and Z n, suggesting that each ion promoted ALP activity independently. 4) The ALP activity in the presence of each ion was inhibited in a concentration-dependent manner by specific ALP inhibitors such as levamisole and tetramisole. Moreover, the inhibition did not depend on the substrate used. 5) Substrate concentration-dependency of ALP activity was measured in the presence of Mg, Ca, and Zn. The K0.5 values were 0.73 mM, 0.57 mM, and 0.53 mM for pNPP with Mg, Ca, and Zn, respectively. The K 0.5 values were 1.8 mM, 0.35 mM, and 0.15 mM for Na-PPi with Mg, Ca, and Zn, respectively. The affinity for the substrate of ALP varied according to the type of substrate but not to the type of ion. Conclusion: Mg, Ca, and Zn possibly promote ALP activity independently. Our results suggest that the affinity for the substrate and the optimum pH could change depending on the ions present. It is possible that the function of ALP changes when divalent cations and a certain substrate is present.