Effect of calcium ions on rat osseous plate alkaline phosphatase activity

Abstract

Rat osseous plate alkaline phosphatase is a metalloenzyme with two binding sites for Zn 2+ (sites I and III) and one for Mg 2+ (site II). This enzyme is stimulated synergistically by Zn 2+ and Mg 2+ (Ciancaglini et al., 1992) and also by Mn 2+ (Leone et al., 1995) and Co 2+ (Ciancaglini et al., 1995). This study was aimed to investigate the modulation of enzyme activity by Ca 2+. In the absence of Zn 2+ and Mg 2+, Ca 2+ had no effects on the activity of Chelex-treated, Polidocanol-solubilized enzyme. However, in the presence of 10 μM MgCl 2, increasing concentration of Ca 2+ were inhibitory, suggesting the displacement of Mg 2+ from the magnesium-reconstituted enzyme. For calcium-reconstituted enzyme, Zn 2+ concentrations up to 0.1 μM were stimulatory, increasing specific activity from 130 U/mg to about 240 U/mg with a K 0.5 = 8.5 nM. Above 0.1 μM Zn 2+ exerted a strong inhibotory effect and concentrations of Ca 2+ up to 1 mM were not enough to counteract this inhibition, indicating that Ca 2+ was easily displaced by Zn 2+. At fixed concentrations of Ca 2+, increasing concentrations of Mg 2+ increased the enzyme specific activity from 472 U/mg to about 547 U/mg, but K 0.5 values were significantly affected (from 4.4 μM to 38.0 μM). The synergistic effects observed for the activity of Ca 2+ plus magnesium-reconstituted enzyme, suggested that these two ions bind to the different sites. A model to explain the effect of Ca 2+ on the activity of the enzyme is presented.