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

Abstract

gamma-Aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the alpha-decarboxylation of glutamate by glutamate decarboxylase (GAD). In plants, it was verified that the production of GABA is regulated, in part, via Ca(2+)/calmodulin (CaM). Our preliminary studies showed that rice bran GAD is probably also a Ca(2+)/CaM dependent enzyme; hence, in the current investigation, we purified calmodulin from rice bran, and studied the effect of the Ca(2+)/calmodulin complex on the activity of rice bran GAD in vitro. 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 Ca(2+)/CaM complex at pH 7.0, and addition of exogenous EGTA or TFP efficiently inhibited the stimulatory effect of Ca(2+)/CaM complex. At a separate concentration of Ca(2+) and CaM of 200 micromol L(-1) and 150 nmol L(-1), the rice bran GAD was significantly enhanced 3-fold. Moreover, upon binding Ca(2+), CaM underwent a conformational change that facilitated a more obvious emergency of phenylalanine and tyrosine residues. This investigation provided preliminary information for the development of a GABA-based, cost-effective rice bran GAD-related functional food.