Assay of PDHc from E. coli and Pig Heart

The expressing plasmid pMal-C2x-PDHc-El was transformed into E. coli stain TB1 and inoculated in Luria-Bertani (LB) broth containing 2 % glucose and 30 mg/ml ampicillin at 37 °C until reaching a cell density to A600 of 0.6-0.8. Then cells were 

The inhibitory potencies of test compounds against PDHc El were measured with pig PDHc enzyme in vitro. TTP and pyruvate were purchased from Sigma. Pyruvate Dehydrogenase from porcine heart is purchased from Sigma (CAS 9014-20-4). 

The inhibitory activities of test compounds were measured by the enzymatic reaction. PDHc-El activity was measured by monitoring the absorbance at 566 nm wave length using a microplate reader (BioTek Synergy2, USA). The total final volume of 100 pL reaction mixture contained 50 mM K3PO4 (pH 7.0), 1.0 mM MgCla, 0.2 mM thiamine pyrophosphate (TPP), 0.5 mM thiazolyl blue (MTT), 

5 mM Phenazine dimethyl sulfate (PMS), 2.0 mM sodium pymvate (as substrate), 

5 oL pymvate dehydrogenase (PDHc enzyme), and buffered aqueous glycerol solution and different concentrations of inhibitor. The reaction mixmres were incubated for 3 min at 30 °C, then different concentrations of inhibitor was added to the initial reaction. The 50 % inhibition concentration (ICso) of test compounds was estimated by nonlinear least-squares fitting of the data using the logarithmic regression curves. All experiments were repeated for three times. 

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