(Received for publication, September 22, 1995; and in revised form, November 14, 1995)
From the
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 = 30 µmol of CO
min
mg of protein
) by the
addition of exogenous calmodulin (K
= 15
nM) in the presence of calcium (K
= 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.
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) ()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 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.
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
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.
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).
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.
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.
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.
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 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
= 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 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
estimates of other plant GADs examined at acidic
pH(10) . This suggests that the K
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
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.