L lactate dehydrogenase from rabbit muscles

The substrate specificity of the IAGlc synthase (7) was analyzed using the spectrophotometric method [16 (link)] as the release of UDP using a coupled enzyme assay containing pyruvate kinase (8) and lactate dehydrogenase (9) according to the following equations:


The reaction mixture in the total volume of 1.0 mL contained 50 mM HEPES–NaOH, pH 7.4, 2.5 mM MgCl₂, 2 mM PEP, 4 mM UDPG, 0.13 mM NADH/H⁺, 3 U pyruvate kinase from rabbit muscles (Sigma-Aldrich), 4 U l-lactate dehydrogenase from rabbit muscles (Sigma-Aldrich), 4.3 µg ZmIAGlc synthase and 3 mM sugar acceptor (IAA, IBA, IPA, IPyA, PAA, 2,4-D, Dicamba, Picloram or tryptophan). The change of the NADH/H⁺ absorbance was monitored at 340 nm using a Shimadzu UV-160A spectrophotometer (Shimadzu, Japan). The enzyme activity was calculated using the absorption coefficient 6220 M⁻¹ cm⁻¹ for NADH/H⁺.

The effect of IAA concentration on the IAGlc synthase (4) activity was analyzed using the spectrophotometric method [16 (link)] as the release of UDP using a coupled enzyme assay containing pyruvate kinase (5) and lactate dehydrogenase (6) according to the following equations:


The reaction mixture in the total volume of 1.0 mL contained 50 mM HEPES–NaOH, pH 7.4, 2.5 mM MgCl₂, 2 mM PEP, 4 mM UDPG, IAA (0.5–4 mM), 0.13 mM NADH/H⁺, 3 U pyruvate kinase from rabbit muscles (Sigma-Aldrich), 4 U l-lactate dehydrogenase from rabbit muscles (Sigma-Aldrich) and 0.26 U/mg IAGlc synthase. The change of the NADH/H⁺ absorbance was monitored at 340 nm using a Shimadzu UV-160A spectrophotometer (Shimadzu, Japan). The enzyme activity was calculated using the absorption coefficient 6220 M⁻¹ cm⁻¹ for NADH/H⁺. The V_max and K_M parameters were calculated using a Hanes–Woolf plot and verified by the Michaelis–Menten equation using SigmaPlot 11.0 (Systat Software). The effect of IAA–Asp on the K_M and V_max values was analyzed as described above using the reaction mixtures containing 0.5–4 mM IAA and additionally 0.1 mM IAA–Asp.