Fig. 2. Effects of pH, Mg²⁺ and Ca²⁺ on MP phosphorylation. (A) Effect of pH. The cell wall preparation was dialyzed against 0.1 M citrate buffer pH 4.0–5.5, HEPES pH 6.0–7.5, Tris–HCl pH 8.0–9.0 or borate pH 9.5–10.5 supplemented in each case with 5 mM MgCl₂. Each dialyzed sample was then assayed for its ability to phosphorylate MP. Inset (from left to right): MP phosphorylated at pH 5.0, 8.0 and 11.0, respectively. (B) Effect of MgCl₂. MP phosphorylation was assayed in the pH 8.0 reaction buffer supplemented with the indicated concentrations of MgCl₂. (C) Effect of CaCl₂. MP phosphorylation was assayed in the pH 8.0 reaction buffer supplemented with the indicated concentrations of CaCl₂. For a positive control, 5 mM MgCl₂ was used. The degree of MP phosphorylation was quantified based on analysis of the gel area corresponding to the phosphorylated protein band, using a Molecular Dynamics phosphorimager.