Activation of a Recombinant Petunia Glutamate Decarboxylase by Calcium/Calmodulin or by a Monoclonal Antibody Which Recognizes the Calmodulin Binding Domain

Abstract

To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0 -7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (V max ‫؍‬ 30 mol of CO 2 min ؊1 mg of protein ؊1 ) by the addition of exogenous calmodulin (K 0.5 ‫؍‬ 15 nM) in the presence of calcium (K 0.5 ‫؍‬ 0.8 M). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (K m ‫؍‬ 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulinbinding domain, a monoclonal antibody or calcium/ calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.

Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform

“…This may reflect differences among organisms with respect to the cellular levels of NADK substrates. The high affinity of Arabidopsis NADK for CaM (S 0.5 of 2.2 nM) is consistent with reports on other plant NADKs (Harmon et al, 1984;Lee et al, 1997;Delumeau et al, 2000) and other plant CaM-binding proteins (Snedden et al, 1996;Bouche et al, 2002;Zhang et al, 2002;Chandok et al, 2003). Similarly, the S 0.5 value for Ca 21 (0.8 mM) suggests stimuli that elevate Ca 21 from resting levels in vivo may elicit a CaM-modulated NADK response.…”

The Auxin-induced Maize Gene ZmSAUR2 Encodes a Short-lived Nuclear Protein Expressed in Elongating Tissues

“…For transcription factors of the basic-helix-loop-helix group it was reported that Ca 2ϩ /CaM binding directly inhibits their DNA binding (56). Also for the Arabidopsis transcription factors TGA3 and DL10, an interaction with CaM was shown (57,58). Therefore, one may speculate that SAURs represent an important integrative element of the cross-talk between the Ca 2ϩ /CaM messenger system and the auxin signal transduction.…”

[gamma]-Aminobutyric Acid Stimulates Ethylene Biosynthesis in Sunflower

“…Glutamate is then decarboxylated to GABA by GAD. GAD has recently been identified as a Ca2+ / calmodulin-dependent enzyme (Baum et al, 1993;Snedden et al, 1996). Regulation of GAD is under developmental control (Chen et al, 1994) and has recently been proposed to play an important role in normal plant growth and development and fruit ripening (Gallego et al, 1995).…”