Purification and characterization of S-adenosyl-L-methionine nicotinic acid-N-methyltransferase from leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean ( Glycine max L.) leaves with a recovery of 4%. The purification procedure included ammonium sulfate ( 45 - 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration ( Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K-m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 muM, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 - 45 degreesC. High concentration of dithiothreitol ( 10 mM) and glycerol ( 20 %) stabilize the enzyme during purification and storage. Hg2+ strongly inhibits enzyme activity.