In vitro 6xHis-CBU0823 activity assays for ME (A), LDH (B) and MDH (C) function, including the influence of varied pH (D), and substituted cofactors (E and F).
(A) ME activity was observed for 20 μg 6xHis-CBU0823 in the ME standard assay containing 3 mM malate, 5 mM NAD+, 1 mM MnCl2. 28.8 μg 6xHis-oMLE and 6xHis-mock purification negative control had no activity. (B) LDH activity was not detected for 20 μg 6xHis-CBU0823 in the LDH standard assay containing 2 mM pyruvate and 0.5 mM NADH, with or without the addition of MnCl2. 28.8 μg 6xHis-oMLE and 6xHis-mock purification also failed to show activity, while 0.1 units commercial LDH enzyme had measurable performance in standard conditions. (C) MDH activity was measured for 20 μg 6xHis-CBU0823 in the MDH standard assay containing 2 mM OAA, 0.5 mM NADH, with improved action on addition of 1 mM MnCl2. 0.013 units commercial pig heart mitochondrial MDH and 1 μg GST-CBU1241 had efficient activity in the assay and 6xHis-mock purification had no detectable activity. (D) Enzyme activity relative to pH 7.4 over a range of pHs, measured using ME standard assay containing 2 μg 6xHis-CBU0823. Activity was measured using increase or decrease of light absorbance at 340 nm, correlating to NADH oxidation and NAD+ reduction respectively during enzyme activity. (E) Removal of MnCl2 and/or substitution with NADP+ cofactor from the standard ME assay reduced 6xHis-CBU0823 activity. Conditions with significantly less activity are indicated. (F) Replacement of the MnCl2 cofactor with 1mM CaCl2, CuCl2, MgCl2, or ZnSO4 achieved varying levels of 6xHis-CBU0823 function. Those with significantly lower cofactor facility compared to 1 mM MnCl2 on ordinary one-way ANOVA are indicated (p < 0.05). Error bars represent standard deviation around the mean from at least three independent experiments.
