Purification of calmodulin from rice bran and activation of glutamate decarboxylase by Ca2+/calmodulin

BACKGROUND: γ-Aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). In plants, it was verified that the production of GABA is regulated, in part, via Ca2+/calmodulin (CaM). Our preliminary studies showed that rice bran GAD is probably also a Ca2+/CaM dependent enzyme; hence, in the current investigation, we purified calmodulin from rice bran, and studied the effect of the Ca2+/calmodulin complex on the activity of rice bran GAD in vitro. RESULTS: CaM was purified to homogeneity from the rice bran by a combined protocol involving TCA precipitation, heat treatment, and hydrophobic interaction chromatography, with the purification fold and recovery of 851.7 and 55.6%, respectively. This protein had similar amino acid composition as the CaMs from other higher plants. The rice bran GAD was found to be quite sensitive to the Ca2+/CaM complex at pH 7.0, and addition of exogenous EGTA or TFP efficiently inhibited the stimulatory effect of Ca2+/CaM complex. At a separate concentration of Ca2+ and CaM of 200 µmol L−1 and 150 nmol L−1, the rice bran GAD was significantly enhanced 3-fold. Moreover, upon binding Ca2+, CaM underwent a conformational change that facilitated a more obvious emergency of phenylalanine and tyrosine residues. CONCLUSION: This investigation provided preliminary information for the development of a GABA-based, cost-effective rice bran GAD-related functional food. Copyright © 2010 Society of Chemical Industry