Purification and characterization of formate dehydrogenase from Arabidopsis thaliana

Formate dehydrogenase (FDH, EC 1.2.1.2.) has been purified from leaves of Arabidopsis thaliana by a procedure involving extraction of mitochondria and subfractionation with ultrasonication, followed by column chromatography with DEAE-cellulose, Sephadex G-200, and 5′-AMP-agarose. The specific activity of the most highly purified FDH preparation was 1.91 μmol NADH/min/mg protein. The yield was 1.9 % and purification was 87-fold as compared to the mitochondrial extract. The final enzyme preparations appeared to be homogeneous by native and SDS-PAGE. The relative molecular mass was estimated at 78 kDa by gel filtration. The subunit molecular mass was 42 kDa as estimated by SDS-PAGE. Further specification was obtained by MALDI/TOF Mass Spectrometric detection of the monomer at 39.2 kDa and the dimer at 78.4 kDa. Based on these results, the enzyme was assumed to comprise two identical subunits. Western blotting showed cross-reactivity with the antibody raised against the FDH from potato. The Arabidopsis FDH was specific for its natural substrate, formate. No enzyme catalyzed activity was detected with methanol, ethanol, formaldehyde, malate, acetate, pyruvate, oxalate, ethylene glycol, acetone, or citrate. The enzyme had a broad pH optimum of catalytic activity in the neutral pH range. The Michaelis constants for formate and NAD at pH 7.5 and 25 °C were 1.4 mmol/L and 34 μmol/L, respectively. The N-terminal 25 amino acid sequence of the Arabidopsis FDH showed 72 % identity to that of FDH from potato, barley or rice. Polyclonal antibodies raised against FDH purified from Arabidopsis were obtained and were shown to be specific to the enzyme.