Figure 3
Determination of optimal temperature, pH range, and metal cation requirements for CmsA activity. A reaction mixture (20 µl) containing 20 mM NaCl, 6 mM KCl, 0.1% glycerol, 5 mM MnCl2, 10 mM α-Man-pNP (substrate), 10 mM GDP-Man (donor), and 3.7 µg purified CmsA was incubated at 30 °C for 9 h. (A) Effect of temperature on CmsA enzymatic activity in 100 mM MOPS-NaOH (pH 7.0). A value of 100% corresponds to the incorporation of 2.21 × 10−1 nmol (α-Man-(1 → 2)-α-Man-pNP)/min/mg at 40 °C. (B) Effect of pH on CmsA enzymatic activity in 100 mM MES-NaOH (circles), 100 mM MOPS-NaOH (triangles), or 100 mM Tris-HCl (squares). A value of 100% corresponds to the incorporation of 1.32 × 10−1 nmol (α-Man-(1 → 2)-α-Man-pNP)/min/mg in 100 mM Tris-HCl at pH 8.0. (C) Metal cation requirements for CmsA activity. Reaction mixtures were incubated with 5 mM EDTA or various divalent metals (each 5 mM). A value of 100% corresponds to the incorporation of 6.84 × 10−1 nmol (α-Man-(1 → 2)-α-Man-pNP)/min/mg in 5 mM manganese. All data are presented as mean ± SD (n = 3 independent experiments).
