Role of Ca²⁺ binding to M4 in TNAP activity.

<p>Dose-response of stimulation of AP activity (mean mA405nm/min) in steady-state (i.e. the slope measured between 60–90 min) at pH 9.8 (10 mM pNPP) for increasing [CaCl₂], added to AbM2-bound Zn²⁺-TNAP and the indicated mutants, after pre-treatment with EDTA (2 h) and loading with 20 μM Zn²⁺; correlation between calculated apparent K_ds for the functionally relevant Mg²⁺ and Ca²⁺ binding to TNAP and the three indicated TNAP mutants. Lines constructed in the absence and presence of 20 μM ZnCl₂ are as indicated; Apparent K_ds are represented with their respective SD.