ACTIVATION OF A RECOMBINANT PETUNIA GLUTAMATE-DECARBOXYLASE BY CALCIUM CALMODULIN OR BY A MONOCLONAL-ANTIBODY WHICH RECOGNIZES THE CALMODULIN-BINDING DOMAIN/

To date, only plants have been shown to possess a form of glutamate de carboxylase (GAD) that binds calmodulin, In the present study, a recom binant calmodulin-binding 58-kDa petunia GAD produced in Escherichia c oli was purified to homogeneity using calmodulin-affinity chromatograp hy, and its responsiveness to calcium and calmodulin was examined in v itro, At pH 7.0-7.5, the purified recombinant enzyme was essentially i nactive in the absence of calcium and calmodulin, but it could be stim ulated to high levels of activity (V-max = 30 mu mol of CO2 min(-1) mg of protein(-1)) by the addition of exogenous calmodulin (K-0.5 = 15 n M) in the presence of calcium (K-0.5 = 0.8 mu M). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displ ayed hyperbolic kinetics at pH 7.3 (K-m = 8.2 mM). A monoclonal antibo dy directed against the carboxyl-terminal region, which contains the c almodulin-binding domain of GAD, was able to fully activate GAD in a d ose-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 activ ity of the purified 58-kDa GAD polypeptide is essentially calcium/calm odulin-dependent at physiological pH. Furthermore, activation of GAD b y two different proteins that interact with the calmodulin binding dom ain, a monoclonal antibody or calcium/ calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.