Fig 3. In vitro GST-CBU1241 activity assays for MDH, LDH or ME function.

(A) MDH activity was exhibited by 2 μg GST-CBU1241 and 0.013 units commercial pig heart mitochondria MDH, whereas 2 μg GST alone exhibited no activity in the MDH standard assay containing 2 mM OAA and 0.5 mM NADH in PBS pH 7.4. (B) LDH activity was not observed for 2 μg GST-CBU1241 or 2 μg GST in the LDH standard assay containing 2 mM pyruvate and 0.5 mM NADH in PBS pH 7.4. Conversely, 0.1 unit of commercial LDH enzyme had measurable activity. (C) Enzyme activity relative to pH 7.4 over a range of pHs, measured using MDH standard assay containing 1 μg GST-CBU1241. Activity was measured using increase or decrease of light absorbance at 340 nm, correlating to NADH oxidation and NAD⁺ reduction respectively during enzyme activity. Error bars represent standard deviation from the mean from at least three independent experiments.