©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of a Recombinant Petunia Glutamate Decarboxylase by Calcium/Calmodulin or by a Monoclonal Antibody Which Recognizes the Calmodulin Binding Domain (*)

(Received for publication, September 22, 1995; and in revised form, November 14, 1995)

Wayne A. Snedden (§) Nataly Koutsia Gideon Baum (¶) Hillel Fromm (**)

From the Department of Plant Genetics, Weizmann Institute of Science, 76100 Rehovot, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 µmol of CO(2) min mg of protein) by the addition of exogenous calmodulin (K(0.5) = 15 nM) in the presence of calcium (K(0.5) = 0.8 µM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (K = 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 calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.


INTRODUCTION

Calcium ions play a central role in intracellular signal transduction pathways in eukaryotes. The response to various stimuli is mediated by calcium-binding proteins such as calmodulin (CaM) (^1)which translate a transient calcium signal into a variety of cellular processes. CaM is a highly conserved protein, which upon binding calcium, undergoes a conformational change and is able to recognize and activate a diverse array ofspecific target proteins (for review, see (1) and (2) ). Previously, we cloned a cDNA for a novel 58-kDa CaM-binding protein by screening a petunia petal cDNA expression library with recombinant S-labeled CaM and identified it as glutamate decarboxylase (GAD)(3) . Plant GAD bears greater sequence similarity to bacterial than to animal GAD (3) and appears to be unique among GADs in its ability to bind CaM. A detailed molecular analysis of the CaM binding domain of petunia GAD identified a 26-amino acid region near the carboxyl terminus with specific residues involved in CaM binding (4) . Although the CaM binding domain of petunia GAD displays features similar to those of many animal CaM-binding proteins, it also possesses unique characteristics(4) .

GAD catalyzes the decarboxylation of glutamate to yield CO(2) and -aminobutyrate (GABA). GABA is a ubiquitous nonprotein amino acid whose role as an inhibitory neurotransmitter in animals is well known. Several forms of animal GAD have been described previously(5, 6) , and GAD appears as an autoantigen in insulin-dependent diabetes mellitus and in the rare neurophysiological disorder, stiff-man syndrome(7, 8) . In contrast, the function of GAD and GABA in plants remains unclear(9, 10) . Interestingly, GABA accumulates rapidly in plants in response to a variety of environmental stimuli including hypoxia, temperature shock, water stress, and mechanical manipulation (11, 12, 13, 14, 15, 16) . Prior to the knowledge that plant GAD is a Ca/CaM-binding protein, it was suggested that GAD activity under stress may help resist cytosolic acidosis(15, 17, 18) , given the fact that the decarboxylation of glutamate is a proton-consuming reaction and because plant GAD exhibits an acidic pH optimum(19, 20) . Yet, recent results suggest that a decrease in cytosolic pH does not seem to be a prerequisite for GAD activation (15) . Moreover, many of the same stresses that induce GABA production also cause increases in cytosolic calcium levels(21, 22, 23) . It is therefore conceivable that GABA production in plants is regulated at least in part via Ca/CaM. Consistent with this hypothesis, we and others recently demonstrated a 1.5-9-fold stimulation of activity by Ca/CaM using partially purified plant GAD(4, 24, 25) . However, interpretation of these studies remains limited because of the possible presence of contaminating CaM (25) and different forms of GAD(26) .

Expression of recombinant CaM-binding proteins in bacteria offers the advantages of a typically high yield in the absence of endogenous CaM, other plant CaM-binding proteins, or other putative GAD isozymes. In our initial studies on recombinant GAD, we experienced difficulty in obtaining the 58-kDa recombinant GAD in a soluble form(3) . Consequently, in the present study, our objective was to purify the soluble 58-kDa recombinant CaM-binding petunia GAD and examine its Ca/CaM responsiveness in vitro.


EXPERIMENTAL PROCEDURES

Expression and Purification of the Recombinant GAD

Expression of the full-length petunia GAD in Escherichia coli strain BL21(DE3)pLysS using a pET12 expression vector (Novagen) was performed essentially as described previously(3) , except that a 100-ml culture of bacterial cells was routinely used, and expression time was 6 h at 30 °C. Proteins were extracted as described previously(3) , with the exceptions that the extraction buffer (10 ml/100 ml of bacterial culture) consisted of 50 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, 10% glycerol, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride (PMSF). Following extraction, soluble protein was immediately frozen in liquid nitrogen and stored at -70 °C. To a 4-ml aliquot of thawed extract, CaCl(2) and PMSF were added to final concentrations of 10 and 1 mM, respectively. The sample was passed through a 0.45-µm filter and then loaded onto a CaM-agarose (Sigma) affinity column (approximately 300 µl bed volume) pre-equilibrated with CaM binding buffer (50 mM Tris-HCl, pH 7.5, 1 mM CaCl(2), 150 mM NaCl, 10% glycerol, and 1 mM freshly prepared PMSF). The first column-volume of effluent was reloaded onto the column and then effluent containing non-adsorbed proteins was collected. The column was washed with 20 column-volumes of washing buffer lacking added calcium (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM PMSF) in order to remove weakly adsorbed bacterial proteins. Adsorbed GAD was eluted with elution buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EGTA, 10% glycerol, 1 mM PMSF), and fractions were either assayed immediately for GAD activity or were frozen in liquid nitrogen and stored at -70 °C. From a 100-ml culture of transformed bacteria, typically about 50 µg of purified soluble GAD were recovered. Purity and intactness of the protein were assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining or by Western blot analysis with a monoclonal anti-GAD antibody (designated as mAb-GAD-107.1)

In Vitro Assay of GAD Activity

GAD assays were performed using a radiometric method based upon L-[1-^14C]Glu-dependent or L-[U-^14C]Gludependent ^14CO(2) production(25) . Reactions were performed using 15 times 100-mm disposable culture tubes containing a CO(2) trap of 0.5 ml of freshly prepared 0.1 M NaOH. Unless indicated otherwise, the reaction media consisted of 100 mM 1,3-bis-Tris-HCl buffer (Sigma), pH 7.0, or 100 mM 1,3-bis-Tris-propane-HCl buffer (Sigma), pH 7.3, containing 1 mM dithiothreitol (Sigma), 5 mML-glutamate containing L-[1-^14C]glutamate or L-[U-^14C]glutamate (0.1 µCi/ml, Amersham Corp.), 0.1 mM pyridoxal phosphate, and 10% glycerol (v/v) in a final volume of 1 ml. Calcium (as CaCl(2)) and bovine brain CaM (Sigma) were included in the reaction mixture at concentrations indicated in figure legends. In experiments where the effect of calcium concentration on GAD activity was examined, estimations of free calcium in the presence of EDTA or 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`tetracetic acid (at concentrations indicated in the figure legends) were obtained using a computer program(27) . Otherwise, calcium concentrations refer to total calcium added to a reaction. For the analysis of GAD responsiveness to Ca/CaM as a function of pH, an overlapping system of separate 100 mM buffers (with the reaction media as described above) was used: pyridine-HCl (pH 4.5, 5.0, 5.5, 6.0), 1,3-bis-Tris-HCl (pH 5.5, 6.0, 6.5, 7.0), and bis-Tris-propane (pH 6.5, 7.0, 7.5, 8.0). All reactions were initiated by the addition of purified recombinant GAD (0.3-1.3 µg protein/reaction). Tubes were sealed with rubber stoppers and incubated in a shaking water bath at 30 °C for 15 min. Reactions were terminated by the injection of 0.1 ml of 6 M HCl through the stopper into the reaction medium and incubated at room temperature for at least 2 h to ensure complete evolution of CO(2) and absorption by the hydroxide trap before the ^14C content of the CO(2) trap was determined using liquid scintillation counting (counting efficiency >95%). Protein determinations were performed using a Bradford (28) reagent (Bio-Rad). Activity was also determined (where indicated) using a radiometric assay for the conversion of [^14C]glutamate to [^14C]GABA (3) . The reaction mixture contained 100 mM 1,3-bis-Tris-propane-HCl, pH 7.3 (Sigma), 5 mM glutamate containing L-[U-^14C] glutamic acid (0.5 µCi/ml, Amersham Corp.), 0.2 mM pyridoxal phosphate, 1 mM dithiothreitol, 10% glycerol (v/v), and 0.4 µg of purified recombinant GAD in a total volume of 200 µl. Reactions were performed at 30 °C for 60 min. Reactions were terminated by adding 350 µl of chloroform and 800 µl of methanol to the reaction mixture. Amino acids were extracted and analyzed by thin-layer chromatography and autoradiography as described previously(3) .

Monoclonal Antibody Preparation and Screening

Proteins were extracted as described previously (29) from leaves of transgenic tobacco plants overexpressing the 58-kDa petunia GAD. Calcium chloride was added to a final concentration of 2 mM, and the samples were centrifuged at 4 °C, 20,000 times g for 30 min. The supernatant was loaded onto a CaM-agarose column and affinity-chromatography was performed as described above. Eluted proteins were separated by SDS-PAGE, and the major band, that had the same gel mobility as the petunia GAD and that was detected by the anti-petunia-GAD polyclonal antibodies(3) , was cut out of the gel and electroeluted from the acrylamide slice with an Elutrap apparatus (Schleicher & Schuell) into SDS-PAGE running buffer. Mouse immunizations and screening for positive hybridomas were performed in the Monoclonal and Polyclonal Antibody Services of the Weizmann Institute. Hybridomas were screened first by enzyme-linked immunosorbent assay using the same antigen as used for mouse immunizations. Positive hybridomas were further tested on Western blots against different recombinant GAD proteins. Specificity of the monoclonal antibodies was determined using either an insoluble recombinant GAD (3) or a recombinant GAD lacking 27 amino acid residues from the carboxyl terminus(4) . In addition, antibodies were tested against fusion proteins of glutathione S-transferase with the GAD carboxyl-terminal region (amino acids 469-500) containing the CaM binding domain(3) , or with deleted forms of the CaM binding domain (4) . One monoclonal antibody (designated as mAb-GAD-430.8) recognized a region corresponding to amino acids 475-492 within the CaM binding domain. Another monoclonal antibody (designated as mAb-GAD-107.1) recognized an epitope outside of the CaM binding domain. Both antibodies recognized the recombinant as well as the plant 58-kDa GAD. Ascitic fluids containing each monoclonal antibody were prepared by injection of the corresponding hybridomas into the peritoneal cavity of pristaned (2,6,10,14-tetramethylathylpentadecane) (Aldrich) CD(2) mice (10^7 cells/mouse).


RESULTS

Purification of the Recombinant 58-kDa Petunia GAD

The recombinant GAD was expressed in bacteria for about 6 h at 30 °C after induction by isopropyl-1-thio-beta-D-galactopyranoside. A simple, single-step CaM affinity chromatographic method was used to purify the recombinant GAD from the soluble fraction of bacteria. Washing of the CaM affinity column in the absence of added calcium proved useful in removing weakly adsorbed bacterial proteins. SDS-PAGE analysis demonstrated that recombinant GAD is one of the most abundant proteins present in the total E. coli soluble extract, and it was essentially homogeneous after elution from the CaM affinity column (Fig. 1A). Western blotting confirmed that the recombinant GAD was completely bound by the CaM affinity column and that the purified protein was intact (Fig. 1B). The specific activities of GAD in eluted fractions (cf. Fig. 1) was typically about 12-fold higher than in total extracts. GAD activity increased linearly as a function of either time (for at least 15 min) or protein concentration, and an analysis by thin-layer chromatography confirmed that [^14C]GABA was the only other reaction product (data not shown). Previously we demonstrated that under our experimental conditions, E. coli GAD does not bind CaM nor display detectable activity(3) .


Figure 1: Purification of a recombinant petunia GAD by calmodulin-affinity chromatography. Recombinant petunia GAD was prepared as described under ``Experimental Procedures.'' A sample of total E. coli soluble protein (about 1 mg) was loaded onto a CaM-agarose column, and aliquots of total soluble protein (Total, 7 µg of protein), column-effluent (Effluent, 7 µg of protein), and EGTA-eluted fractions (Eluted fractions, 0.3 µg of protein) were separated on SDS-PAGE and either Coomassie stained (panel A) or transferred to a nitrocellulose membrane, and the presence of GAD was detected with an anti-GAD monoclonal antibody (mAb-107.1; 1:20,000 dilution of ascitis, about 4 times 10 µg/ml of protein) (panel B). The arrow indicates the position of the recombinant GAD (Rec. GAD). The lane marked kDa contains molecular mass protein markers.



Recombinant GAD Is Inactive in the Absence of Calcium and Calmodulin at Physiological pH

When purified GAD was assayed at physiological pH (7.0-7.5) in the absence of calcium and CaM, activity was less than 1.5% of that observed under saturating CaM concentrations (Fig. 2A). Addition of CaM or calcium alone was insufficient to activate GAD, whereas in the presence of both calcium and CaM, GAD displayed high specific activity (Fig. 2A). In addition, GAD was activated by two different isoforms of recombinant petunia calmodulin (GenBank accession numbers M80836 and M80832, respectively) (data not shown).


Figure 2: Response of GAD activity to calcium and calmodulin. Panel A, GAD activity was measured at pH 7.0 as described under ``Experimental Procedures'' in the absence of calcium and calmodulin (Control), in the presence of 500 µM free calcium (+Calcium), in the presence of 50 nM calmodulin without calcium (+CaM), or in the presence of 500 µM free calcium and 50 nM calmodulin (+Ca/CaM). EDTA was present in each reaction at a final concentration of 500 µM. Panel B, profile of GAD activity at pH 7.3 in response to increasing calmodulin (CaM) concentration in the presence of 1.0 mM added calcium. Panel C, profile of GAD activity at pH 7.3 in response to increasing free calcium concentration in the presence of 50 nM calmodulin. Reactions in C were carried out in the presence of 1 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`tetracetic acid and 0.05 mM EGTA (filled circles) or in the presence of 0.55 mM EDTA (crosses). In both A and C, estimates of free calcium were determined as described under ``Experimental Procedures.'' Values represent mean and standard error of three replicate reactions.



The effects of increasing exogenous CaM concentrations on GAD activity at pH 7.3 in the presence of 1 mM calcium was also investigated. Stimulation of GAD activity was most pronounced at CaM concentrations between 3 and 50 nM with half-maximal activation at about 15 nM (Fig. 2B). When GAD activity at pH 7.3 was examined as a function of free calcium concentration in the presence of saturating CaM (50 nM), a steep rise in activity was observed at about 0.5 µM free calcium with half-maximal activity estimated at approximately 0.8 µM free calcium (Fig. 2C).

pH-Dependent Responsiveness of Recombinant GAD to Ca/CaM

GAD activity was examined over the pH range of 4.5-8.0 using an overlapping system of buffers in either the presence of 1 mM added calcium and 50 nM CaM or in the absence of Ca/CaM. When Ca/CaM was not included in the assays, GAD displayed a sharp response to pH with maximal activity observed between pH 5.0 and 6.0, consistent with previous estimations of the pH optimum for plant GAD(10) . In the presence of Ca/CaM, GAD activity was similar to that observed in the absence of Ca/CaM except at pH values around neutrality. Only a slight stimulation of GAD activity by Ca/CaM (1.2-1.6-fold) was observed between pH 4.5-6.5 (Fig. 3). In contrast, between pH 7.0 and 7.5, GAD activity was virtually Ca/CaM-dependent with a peak in response observed at pH 7.5. Between pH 7.5 and 8.0, GAD activity and responsiveness to Ca/CaM dropped steeply. Plant GAD is cytosolic(30, 31) , and estimates of cytosolic pH in plants range from pH 7.0 to 7.5(32) . Consequently, subsequent analysis of recombinant GAD in this study was performed at pH 7.3.


Figure 3: pH-dependent stimulation of GAD activity by calcium and calmodulin. GAD activity was measured as described under ``Experimental Procedures'' using a system of overlapping buffers (100 mM) to cover a range of pH from 4.5 to 8.0: pyridine-HCl (pH 4.5, 5.5, 6.0), bis-Tris-HCl (pH 5.5, 6.0, 6.5, 7.0), and bis-Tris-propane-HCl (pH 6.5, 7.0, 7.5, 8.0). Activity was examined either in the presence of 1 mM added calcium and 50 nM calmodulin (solid line, filled circles) or in the absence of calcium and calmodulin (solid line, filled squares). Values represent the mean of three replicate reactions at each pH value shown or the mean of six values from two sets of three replicates at overlapping pH values. The ratio of GAD activity observed in the presence of Ca/CaM to the activity observed in the absence of Ca/CaM (i.e. stimulation by Ca/CaM) is also presented (dashed line, crosses), where each value represents the mean and standard error of the ratio of activity observed for each pH value.



Kinetic Analysis of Recombinant GAD

The activity of the purified recombinant GAD as a function of substrate concentration at pH 7.3 in the presence of 1 mM added calcium and 50 nM CaM followed Michaelis-Menten kinetics. Estimates of K(m) and V(max) were 8.2 mM and 30.6 µmol CO(2) min mg of protein, respectively, as determined from Lineweaver-Burk plot analysis (Fig. 4). Analysis using half-reciprocal or EadieHofstie plots gave similar values (data not shown). Calcium- and CaM-independent GAD activity was consistently less than 1.5% of the activity observed in the presence of Ca/CaM over the range of substrate concentrations examined (data not shown).


Figure 4: Double-reciprocal plot of GAD activity in the presence of calcium and calmodulin. GAD activity was measured as described under ``Experimental Procedures'' at pH 7.3 in the presence of 1.0 mM added calcium and 50 nM calmodulin at different concentrations of substrate (L-Glu). Inset, the same data presented using a Michaelis-Menten plot. Values represent mean and standard error of three replicate reactions.



Calcium- and Calmodulin-independent Activation of GAD by a Monoclonal Antibody Recognizing the Calmodulin-Binding Domain

The CaM binding domain presented in Fig. 5represents a region (petunia GAD amino acids 470-495), which contains residues previously determined to be involved in CaM binding by at least one of three criteria: chemical cross-linking studies, CaM affinity chromatography, or S-labeled CaM overlay assays(4) . To further elucidate the role of the CaM binding domain in regulating GAD activity, we examined whether a monoclonal antibody, which recognizes an epitope within an 18-residue span of this domain (mAb-430.8; Fig. 5), could activate GAD in the absence of Ca/CaM. Indeed, GAD was activated by this antibody, and this activation was independent of Ca/CaM (Fig. 6A), was dose-dependent (Fig. 6B), was not inhibited by 0.5 mM EDTA (Fig. 6A), and was comparable in magnitude with that induced by saturating Ca/CaM alone (Fig. 6A). In addition, incubation of GAD with both Ca/CaM and this antibody (mAb-430.8 + Ca/CaM) yielded similar activation as was observed in the separate treatments, indicating there was no synergism between these two factors (Fig. 6A). No effect on GAD activity was observed in the presence of an antibody, which recognizes an epitope outside of the CaM binding domain (mAb-107.1) (Fig. 5) even at concentrations that were 20-fold higher (Fig. 6A) than used for immunodetection of GAD by Western blotting (Fig. 1B) or enzyme-linked immunosorbent assay (not shown). The addition of exogenous Ca/CaM together with this antibody (mAb-107.1 + Ca/CaM) resulted in full activation of GAD (Fig. 6A). In addition, a nonrelevant monoclonal antibody, which recognizes plant CaM (mAb-17.28), was used as a control and did not affect GAD activity (Fig. 6A).


Figure 5: Diagram of GAD showing the calmodulin-binding domain and anti-GAD monoclonal antibody recognition regions. The numbers above the diagram refer to amino acid residues as described previously(3, 4) . The location of the calmodulin-binding domain, including a critical tryptophan residue (amino acid 485), was determined previously(4) . The diagram shows a region (amino acids 1-469) containing an epitope recognized by an anti-GAD monoclonal antibody designated mAb-107.1, and the carboxyl-terminal region (amino acids 469-500), which contains the calmodulin-binding domain (amino acids 470-495), and a domain (amino acids 475-492) recognized by a different anti-GAD monoclonal antibody (mAb-430.8). Determination of monoclonal antibody specificity was performed as described under ``Experimental Procedures.''




Figure 6: Activation of GAD by a monoclonal antibody, which recognizes the calmodulin-binding domain. Panel A, GAD activity was examined at pH 7.3 in the absence of calcium and calmodulin (Control), in the presence of 1.0 mM added calcium, and 50 nM calmodulin (+Ca/CaM), with or without anti-GAD monoclonal antibodies (cf. Fig. 5) as indicated (8-9 µg/ml of protein), in the presence or absence of 500 µM EDTA as indicated. An anti-calmodulin monoclonal antibody (mAb-17.28: 10.5 µg/ml) was also used as a control. Panel B, GAD activity was examined at pH 7.3 in the presence of 500 µM EDTA as a function of the concentration of antibody mAb-430.8. In B, values are expressed as a percentage of the activity observed in the presence of saturating calcium and calmodulin (100%). In both A and B, reactions were conducted as described under ``Experimental Procedures,'' and values represent mean and standard error of three replicate reactions.




DISCUSSION

Previous work showing in vitro CaM stimulation of plant GAD activity utilized partially purified preparations and thus could not unequivocally exclude the involvement of other proteins in this stimulation(4, 24, 25) , including contaminating CaM or other putative subunits of a GAD complex. Moreover, in these earlier studies calmodulin-independent GAD activity ranged from 10% to as high as 50% of Ca/CaM-stimulated GAD activity(4, 24, 25) . In the present study, we purified a soluble recombinant petunia GAD from E. coli cells and studied its activation by Ca/CaM. Our study did not address the question of whether GAD functions as a multi- or monomeric enzyme under these conditions. It is noteworthy, however, that previous findings suggest that GAD purified from plants is a multimeric enzyme(19, 26, 33) .

Prior to the knowledge of GAD regulation by Ca/CaM, studies of GAD from different plant species demonstrated that the optimal pH for GAD activity in vitro is about 5.8 and that at pH 7.0 the activity ranges from 12 to 30% of maximal activity(11, 19, 20) . GAD in plants has been shown to be a cytosolic enzyme(30, 31) , and in addition, it was often proposed that activation of GAD is a consequence of cytosolic acidification. Our studies, while confirming the fact that GAD exhibits maximal activity in vitro at acidic pH, establish that over a pH range of 7.0-7.5, which corresponds to the cytosolic pH of a typical plant cell(32) , GAD activity is essentially dependent upon Ca/calmodulin. Similar to our findings, the CaM activation of myosin light chain kinase(34) , phosphodiesterase(35) , and phosphorylase kinase (36) has also been demonstrated to be pH-responsive. GAD activity becomes nearly CaM-independent below pH 6.5 (Fig. 3). However, plant cytosolic pH is carefully maintained at levels slightly above neutrality(32) , and nonlethal perturbations generally do not cause a decline of more than 0.5 pH units(15, 37) . Taken together, our results imply that an acidic pH is not essential for GAD activation. Therefore, GAD may be activated in plants by signals that do not involve pH changes.

The concentration of CaM required for half-maximal stimulation of the purified recombinant GAD (K(0.5) about 15 nM, Fig. 2B) is similar to values published for other CaM-dependent enzymes (38, 39, 40, 41) and is lower than a previous estimation using partially pure soybean GAD (K(0.5) = 25 nM)(25) . In the presence of saturating CaM, recombinant GAD was inactive at calcium levels estimated to represent resting physiological values (0.01-0.1 µM calcium) (42) but was activated at calcium concentrations greater than about 0.3 µM. External stimuli induce calcium fluxes in plant cells(42) , and thus GAD activity may be regulated by calcium fluxes in vivo via CaM activation. Three lines of evidence imply that calcium influences in vitro GAD activity only via CaM: (i) no effect on GAD activity by calcium alone was observed (Fig. 2A); (ii) in the presence of CaM, the response of GAD activity to increasing levels of calcium was sigmoidal (on a logarithmic scale) (Fig. 2C), as would be expected for a strictly CaM-mediated process; (iii) antibody-dependent activation of recombinant GAD was observed in the absence of calcium (i.e. with 0.5 mM EDTA) and CaM (Fig. 6B). However, it is possible that calcium could be involved in other aspects of GAD regulation in vivo. For example, several studies have demonstrated that calcium ions influence the ability of one form of brain GAD to interact with intracellular membranes(6, 43) .

Our estimate of K(m) of the purified recombinant GAD for glutamate (8.2 mM, Fig. 4) at pH 7.3 in the presence of Ca/CaM is similar to that reported for partially-purified soybean GAD (9 mM) (25) assayed under similar conditions and also to K(m) estimates of other plant GADs examined at acidic pH(10) . This suggests that the K(m) of plant GAD may not be as sensitive to pH as E. coli GAD(44) . The specific activity of the purified Ca/CaM-activated recombinant GAD (V(max) 30 µmol min mg of protein; Fig. 4) is higher than values reported for GAD purified from plants (19, 45) and is most similar to the reported values for E. coli GAD (44) and brain GADs(46) .

The ability of recombinant GAD to be fully activated by a monoclonal antibody recognizing an epitope within the CaM binding domain is reminiscent of a previous report using the CaM-regulated myosin light chain kinase(47) . Activation of the kinase by a monoclonal antibody that recognizes the CaM binding domain appears to result from a disruption of autoinhibition(47) . Autoinhibition by CaM binding domains seems to be a common feature of CaM-regulated proteins (for review, see (2) and (48) ). Several models describing this phenomenon suggest that the CaM binding and autoinhibitory domains either overlap or are at least in close proximity to one another(49, 50, 51) . It is possible that the C-terminal region of GAD, which contains the CaM binding domain, may be involved in suppressing GAD activity in the absence of Ca/CaM. Our results suggest that CaM binding induces a conformational change necessary for GAD activation. The monoclonal antibody may mimic the action Ca/CaM in this respect, or it may induce a different conformational change that results in GAD activation. Further research is needed to characterize these phenomena.

Our data support the idea that plant GAD is CaM-regulated but do not exclude the possibility that other mechanisms of post-translational regulation may be involved in vivo. Phosphorylation of brain GAD by protein kinase A results in inhibition of activity, which can be reversed by the calcium-dependent phosphatase calcineurin (52) . Similarly, the CaM binding domain of many proteins is often a target for reversible serine/threonine phosphorylation(53, 54, 55) . Current models suggest that the introduction of this negative charge hinders CaM (an acidic protein) binding and serves to inactivate CaM targets(2, 48) . It is not known whether a similar mechanism occurs in plants.

To our knowledge, plant GAD may be unique among eukaryotic GADs in having evolved as a CaM-regulated enzyme. If so, it implies that some adaptive advantage is associated with the regulation of GABA synthesis by calcium signaling in plants. The ability of Ca/CaM to activate recombinant GAD is consistent with reports on the rapid induction of GABA synthesis in plants under various stress conditions (11, 12, 13, 14, 15, 16) as many of these stresses are known to increase the level of cytosolic calcium ( (42) and references therein). However, the role of GABA accumulation under stress remains unresolved.

In summary, our results demonstrate that the catalytic activity of the 58-kDa recombinant petunia GAD is essentially dependent upon Ca/CaM at physiological pH. Furthermore, activation of GAD by a monoclonal antibody that recognizes the CaM binding domain suggests that one function of this domain may be to suppress GAD activity in the absence of a calcium signal and that the role of Ca/CaM is to relieve this suppression. Further studies are required to elucidate the regulatory features of the carboxyl-terminal region of GAD containing the CaM binding domain.


FOOTNOTES

*
This work was supported by Grant IS-2132-92 (to H. F.) from the United States-Israel Binational Agricultural Research and Development Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Holds a Feinberg Postdoctoral Fellowship.

Recipient of a Levi Eshkol Graduate Fellowship from the Ministry of Science and the Arts, Israel.

**
Incumbent of the Abraham and Jenny Fialkow Career Development Chair in Biology. To whom correspondence should be addressed. Fax: 972-89-344181; :LPFROMM{at}Weizmann.Weizmann.AC.IL.

(^1)
The abbreviations used are: CaM, calmodulin; GABA, -aminobutyric acid; GAD, glutamate decarboxylase; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonylfluoride; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.


ACKNOWLEDGEMENTS

We thank Drs. Gideon Fleminger and Roni Seger for critical reading of the manuscript.


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