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Purification of calmodulin from rice bran and activation of glutamate decarboxylase by Ca 2+ /calmodulin
Author(s) -
Wang Li,
Liu Min,
Guo Lv Ying,
Zhang Hui
Publication year - 2010
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.3866
Subject(s) - calmodulin , chemistry , glutamate decarboxylase , biochemistry , calcium , enzyme , organic chemistry
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 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 . 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 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 µmol 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. 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