Purification and characterization of glutamate decarboxylase from rice germ

Glutamate decarboxylase (EC 4.1.1.15, GAD) is a pyridoxal 5'-phosphate (PLP) dependent enzyme, which catalyses the irreversible alpha-decarboxylation Of L-glutamic acid to gamma-aminobutyric acid. GAD was purified 186-fold from rice germ using a combination of ammonium sulfate fractionation, DEAE-Sepharose FF ion exchange chromatography, Superdex-200 gel filtration chromatography, and Glu-Sepharose CL 4B affinity chromatography. The purified preparation showed a single peak on SE-HPLC with an approximate molecular mass of 78 kDa and a single band on SDS-PAGE with a subunit Mr of 40 kDa. This indicated that the GAD from rice germ existed as a dimer of homological subunits. Rice germ GAD has an optimum pH range between 5.5 and 5.8, and an optimum temperature at 40 degrees C. K-m values for glutamic acid and PLP were determined at 32.3 mM and 1.7 mu m, respectively. Chemicals reagents such as HgCl2, KI and AgNo3 decreased the enzyme activity by 68.5%, 44.9% and 32.4%, respectively, but 500 mu M of CaCl2 at the optimum pH could increase the activity by 145%. (c) 2006 Elsevier Ltd. All rights reserved.