Abstract
Glutamate decarboxylase (GAD) is an enzyme that catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA). We previously reported that rice has two distinct isoforms of GAD (i.e., OsGAD1 and OsGAD2). The former carries a C-terminal calmodulin-binding domain (CaMBD) that is common in counterparts from dicotyledonous plants, but the latter does not (Akama et al., 2001).
In order to explore a role of the C-terminal peptide of these two GAD isoforms more in detail, the genes that lack the C-terminal were introduced in E.coli expression vector pET32a to thus purify recombinant GAD proteins (i.e., OsGAD1Δand OsGAD2Δ) with a nickel chelate affinity chromatography. An in vitro enzyme analysis with these recombinant proteins as well as wild-type GAD proteins indicated that OsGAD1Δ possesses a high and constant activity, irrespective of the presence of Ca2+/CaM and OsGAD2Δ displays five times higher activity than wild-type OsGAD2 protein.
