From the Departments of Medicine,
§ Anesthesiology, and ¶ Pathology, Duke University
Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 ± 10 µM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 ± 35 µM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 µM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 µM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.
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INTRODUCTION |
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Tissue transglutaminase (tTG)1 exhibits two distinct enzymatic activities (1, 2): a calcium-dependent transglutaminase (TGase) activity that plays an important role in protein cross-linking and the regulation of apoptosis, cell morphology, cell adhesion, and tumor growth and metastasis (1-7) and a GTP binding and hydrolysis activity that is involved in signal transduction and that plays a role in cell cycle progression (8, 9). The biochemical factors modulating these divergent activities remain poorly defined.
The TGase activity function requires the active-site cysteine 277, whereas the GTPase function does not, suggesting the presence of different catalytic sites (10). A putative calcium-binding site in human tTG required for TGase activity is located between Ser-430 and His-441 (11). Magnesium ions are required for GTP and ATP hydrolysis (10), and the location of this site(s) is unknown.
We previously reported that GTP was reversibly bound to guinea pig tTG and inhibited TGase activity by inducing a conformational change that could be reversed by calcium ions (12). A single GTP-binding site was subsequently reported for human erythrocyte tTG, and this binding caused a reduction in affinity for calcium ions (13, 14). Recent studies from our laboratory demonstrated that the GTP- and ATP-binding domains are located in the N-terminal 185 amino acid residues (15).
Tissue transglutaminase is found in several distinct compartments in
cells and tissues. Inside cells, tTG appears in the cytoplasm, although
a small fraction (4-20%) associates with the membrane fraction (16).
The cell-surface membrane form associates with the
1-adrenergic receptor to mediate a G-protein signal
transduction pathway (8). In addition, an intact N-terminal fragment
with increased GTP binding capacity is bound to the nuclear pore,
although its function has not been elucidated (17). The majority of tTG is found in the cytoplasm, where it could interact with membrane, cytoplasmic, and cytoskeletal proteins. The recent finding that intracellular TGase activity is increased during apoptosis coupled with
the observation that the protein is associated with the apoptotic envelope (18) suggest that a mechanism exists to control the activity
of this enzyme during various physiological and pathological processes.
In support of this hypothesis, we recently reported that
sphingosylphosphocholine is able to reduce the Ca2+
requirement to activate TGase activity (19).
In this study, we found that Mg-GTP (or Mg-ATP) is the true substrate for GTP (or ATP) hydrolysis activity. Mg-ATP induces a conformational change in tTG that inhibits GTPase activity but that has no effect on TGase activity. In contrast, Mg-GTP binding induces a different conformation that inhibits TGase activity, but not ATPase activity. These results suggest that local concentrations of Mg-nucleotide complexes can modulate the enzymatic activities of tTG.
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EXPERIMENTAL PROCEDURES |
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Materials--
Sodium salts of ATPS, ATP, ADP, AMP, GTP
S,
GTP, GDP, and GMP and the MgCl2 stock solution (1 M) were purchased from Sigma. [3H]Putrescine
dihydrochloride (35.5 Ci/mmol) and [
-32P]GTP and
[
-32P]ATP (30 Ci/mmol) were purchased from NEN Life
Science Products. Monoclonal antibody against guinea pig liver
transglutaminase (CUB 7401) was kindly provided by Dr. P. Birckbichler (20). All ATP, ADP, AMP, GTP, GDP, and GMP solutions were
prepared in 50 mM Tris-Cl, pH 7.0, and stored in aliquots
at
80 °C. Restriction enzymes, T4 DNA ligase, LB medium, and yeast
extract were obtained from Life Technologies, Inc. All other reagents
used in this investigation were purchased from Sigma unless stated
otherwise.
Assembly of Human tTG cDNA and Expression and Purification of
Recombinant tTG--
The assembly of full-length human tTG cDNA
and the purification of glutathione S-transferase-tTG fusion
protein were as described (15). The purified glutathione
S-transferase-tTG fusion protein was cleaved with factor Xa
(1%, w/w; Hematologic Technologies Inc., Essex Junction, VT) overnight
at 4 °C and re-applied to glutathione resin to remove the
glutathione S-transferase protein. The cleaved tTG migrated
as a single band on a Coomassie Blue-stained gel with identical
electrophoretic mobility to the tTG in human EAhy926 cells (21).
Protein concentrations were quantitated using the Bradford method (22)
(Bio-Rad). Preliminary studies demonstrated that recombinant tTG
preferentially cross-linked the A-chains of fibrinogen to form high
molecular weight
-chain polymers and bound to a fibronectin-coated
microtiter plate in a concentration-dependent manner. Based
on the SDS-PAGE profile, the fibrin cross-linking pattern, fibronectin
binding, TGase activity, and GTP hydrolysis (GTPase) activities, the
affinity-purified recombinant human tTG demonstrated properties similar
to those of tTG purified from other sources.
Preparation of Mg-Nucleotide Complexes--
The concentrations
of Mg-nucleotide complexes were prepared according to the procedures
described by Morrison (23) and O'Sullivan and Smithers (24). Briefly,
the maintenance of a 1-2 mM excess of free
Mg2+ over the total nucleotide (GTP or ATP) concentration
ensures that the proportion of ATP (or GTP) present as
Mg-ATP2 (or Mg-GTP2
) is maximized and
remains constant over a large range of ATP (or GTP) concentrations (23,
24). Calculation of the actual concentration of the various free
Mg2+ ions, free GTP or ATP, and Mg-GTP or Mg-ATP complexes
was made using the computer program developed by Bers et al.
(25). Unless otherwise specified, all reactions containing
Mg-nucleotide complexes had 1 mM Mg2+ in excess
over the total nucleotide concentration to maximize the formation of
Mg-nucleotide complexes (23-25).
Transglutaminase Assay--
TGase activity was determined by
quantitating the incorporation of [3H]putrescine (26) or
5-biotinamidopentylamine into N,N-dimethylcasein as described previously (27). For inhibition of TGase activity by
different Mg-nucleotide complexes, tTG (0.1 µg/ml) was incubated with
different concentrations of Mg-GTP, Mg-GDP, Mg-GMP, Mg-ATP, Mg-ADP, or
Mg-AMP in the presence of 1 mM CaCl2 and 2 mM excess Mg2+, and TGase activity was measured
in triplicate after a 40-min incubation at 37 °C using the
5-biotinamidopentylamine incorporation assay.
[-32P]GTP or [
-32P]ATP
Hydrolysis Assay--
The assay was performed according to the
procedure described (15), with some modifications. For determination of
Mg-GTP (or Mg-ATP) as a substrate for GTP (or ATP) hydrolysis, the
reaction mixture (50 µl) contained 60 mM Tris-Cl, pH 7.6, 1 mM dithiothreitol, 2 µCi of [
-32P]GTP
(or ATP) (30 Ci/mmol), 250 µM unlabeled GTP (or ATP), and 0-3.2 mM Mg2+. For GTPase competitive
inhibition studies (see Fig. 2A), the reaction mixture was
as described above, except that 2 mM Mg2+, 250 µM labeled and unlabeled GTP, and 0-100 µM
Mg-ATP, Mg-ADP, or Mg-AMP were used. For ATPase competitive inhibition
studies (see Fig. 2B), the reaction mixture contained 40 µM labeled and unlabeled ATP and 0-1000 µM
Mg-GTP, Mg-GDP, or Mg-GMP. The reactions were initiated by the addition
of tTG and allowed to proceed at 37 °C for 30 min. The reaction was
terminated by the addition of 750 µl of 50 mM ice-cold
monobasic sodium phosphate containing 5% activated charcoal. After
centrifugation for 2 min at 12,000 rpm in Sorvall microcentrifuge
(Microspin 24S), 400 µl of the supernatant was used for determination
of Pi release by scintillation counting. All the rates of
GTP or ATP hydrolysis reported in this study are initial velocity.
Kinetic constants were determined by serial dilution of Mg-GTP and
Mg-ATP from 12 to 400 µM and from 8 to 200 µM, respectively. Kinetic analysis of the data was performed by the Lineweaver-Burk method (see Ref. 28). Results are
representative of at least two duplicate experiments.
Trypsin Proteolysis of Recombinant tTG-- Purified recombinant tTG (1 µg) was incubated with 0.1 µg of trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated and high pressure liquid chromatography-purified; Calbiochem) in the presence of 5 mM CaCl2, 5 mM MgCl2, 6 µM free GTP, 6 µM free ATP, 6-50 µM Mg-GTP, 6-50 µM Mg-GDP, 6-50 µM Mg-ATP, or 6-50 µM Mg-ADP and incubated at 37 °C for 1 h. The reaction was stopped by the addition of SDS-PAGE loading buffer. Samples were separated by SDS-PAGE and analyzed by immunoblotting using monoclonal antibody CUB 7401 against human tTG (20).
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RESULTS |
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Identification of the Mg-Nucleotide Complex as the Substrate for Hydrolysis-- It is not known if the requirement for Mg2+ in the tTG-mediated GTPase/ATPase reaction reflects the need for a magnesium-complexed enzyme or a magnesium-GTP/ATP substrate. To address this question, the effect of increasing concentrations of magnesium ions on the rate of GTP hydrolysis was determined in the presence of 250 µM GTP (Fig. 1). If the role of Mg2+ is to provide the enzyme with a Mg-GTP substrate, then the ability of Mg2+ to stimulate GTP hydrolysis should be directly proportional to the formation of a Mg-GTP complex. As shown in Fig. 1A, GTPase activity increased and plateaued at 600 µM Mg2+. The increase in GTP hydrolysis activity was proportional to the formation of a Mg-GTP complex (Fig. 1A), suggesting that Mg-GTP was the substrate in this hydrolysis reaction. At 250 µM Mg2+, which correlates with the appearance of free Mg2+ in solution (Fig. 1B), GTP hydrolysis activity was only 71% of the maximum activity. Thus, free Mg2+ has an additional activator effect on GTPase activity (Fig. 1B). Similar experiments performed with ATP demonstrated that Mg-ATP was also a substrate for hydrolysis.
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Separate Nucleotide-binding Sites for Mg-GTP and Mg-ATP Hydrolysis-- Since tTG can hydrolyze ATP as well as GTP, we investigated which is the preferred substrate. Fig. 2A illustrates the effect of increasing concentrations of Mg-ATP, Mg-ADP, and Mg-AMP on GTP hydrolysis. In the presence of 250 µM GTP, Mg-ATP was able to inhibit GTPase activity (IC50 = 24 µM). Substitution of Mg-ATP with Mg-ADP resulted in a more potent inhibition of GTPase activity (IC50 = 5 µM) (Fig. 2A). Preincubation of tTG with Mg-ADP on ice for up to 2 h did not further increase this inhibitory effect. Fig. 2B examines the effect of increasing concentrations of Mg-GTP, Mg-GDP, and Mg-GMP on ATP hydrolysis. In the presence of 40 µM Mg-ATP, up to 1 mM Mg-GTP or Mg-GMP inhibited only 20% of ATPase activity, whereas 1 mM Mg-GDP inhibited 50% of ATPase activity (Fig. 2B). Preincubation of tTG with Mg-GTP or Mg-GDP on ice for up to 1 h did not enhance the inhibition.
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Effect of the Mg-Nucleotide Complex on the Modulation of the TGase
Activity of tTG--
Previous studies using nucleotides as inhibitors
of TGase activity did not consider the role of Mg2+ in this
process (29). Mg2+ exists mainly as a Mg-nucleotide complex
inside cells (30) and is required for nucleotide hydrolysis by tTG. We
therefore examined the effect of Mg-nucleotide complexes on the
modulation of the TGase activity of tTG. In a transglutaminase reaction
containing 1 mM Ca2+, we found that Mg-GTPS,
Mg-GTP, and Mg-GDP were equipotent inhibitors, with IC50
values of 9 µM (Fig.
3A), and that Mg-GMP was less
inhibitory, with an IC50 of 400 µM.
Ca-GTP
S, Ca-GTP, Ca-GDP, and Ca-GMP were found to inhibit TGase
activity, with IC50 values of 4, 4, 4, and 100 µM, respectively.
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Analysis of tTG Conformational Changes by Trypsin Proteolysis-- We evaluated the possibility that different conformations were induced by divalent cations, free GTP, free ATP, Mg-GTP, Mg-GDP, Mg-ATP, and Mg-ADP. Trypsin digestion patterns of tTG bound to divalent cations and nucleotide complexes were analyzed by SDS-PAGE and immunoblotting using a monoclonal antibody to tTG. The presence of 1 mM Ca2+ preserved a 50-kDa fragment of tTG (Fig. 4, lane 1). However, when 5 mM Mg2+ was present, the protein was susceptible to proteolysis, and the epitope was completely degraded (Fig. 4, lane 2). Mg-GTP (6 µM) protected tTG from degradation by trypsin (Fig. 4, lane 3). The same pattern was also observed in the presence of 6 µM free GTP or GDP or 6-50 µM Mg-GTP or Mg-GDP. The addition of 5 mM Ca2+ in the presence of 6 µM GTP or ATP converted tTG to the Ca2+-protected conformation (Fig. 4, lanes 4 and 5, respectively). In addition, the epitope was completely degraded in the presence of 6 µM Mg-ATP (Fig. 4, lane 6). A similar pattern was also observed in the presence of 6 µM free ATP or ADP or 6-50 µM Mg-ATP or Mg-ADP.
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DISCUSSION |
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tTG is present in several different tissues and cellular compartments (1, 2, 16-17). In the extracellular environment, tTG plays a role in extracellular matrix assembly, cell adhesion, and wound healing (1, 2). Inside cells, tTG is associated with the cell-surface membrane and plays an important role in signal transduction and nuclear pore assembly (8, 17). The majority of tTG is present in the cytoplasm, where it can interact with a wide variety of intracellular factors, and is involved in apoptosis and cell cycle arrest (9, 18). In this study, we investigated the effects of divalent cations, nucleotides, and Mg-nucleotide complexes on the modulation of the dual enzymatic activities of tTG.
Calcium and magnesium ions are important regulators of many physiological activities in cells and tissues (31, 32). The fact that TGase activity was observed only in the presence of calcium ions, whereas optimum GTP or ATP hydrolysis occurred in the presence of magnesium ions, suggests that different domains of tTG are involved in these distinct catalytic events. Furthermore, these results suggest that at physiological intracellular and extracellular concentrations of these factors, tTG can display a distinct spectrum of activities. Results from trypsin digestion experiments demonstrate that tTG has distinct conformational states that are dependent on the relative concentrations of divalent cations and Mg-nucleotide complexes. A protease-susceptible conformation occurred when Mg2+, Mg-ATP, or Mg-ADP was present. Calcium ions alone protected a 50-kDa fragment of tTG, whereas GTP, GDP, Mg-GTP, and Mg-GDP binding made the entire tTG molecule resistant to degradation.
Magnesium ions are relatively abundant inside cells and do not antagonize TGase activity alone. However, when present in a Mg-GTP or Mg-GDP complex, the complex inhibits TGase activity. It is interesting that up to 1 mM Mg-ATP did not have a significant effect on TGase activity. Previous studies using ATP alone as an inhibitor (29) did not examine the role of the Mg-ATP complex in this process. Furthermore, the effects of free ATP on the inhibition of TGase activity might be due to a chelation effect since Ca2+ and Mg2+ completely reversed the inhibition.
The finding that Mg-ATP or Mg-ADP acts as a noncompetitive inhibitor of GTP hydrolysis suggests there are separate binding sites for Mg-GTP and Mg-ATP. This conclusion is further supported by the finding that Mg-ADP (or Mg-GDP) acts as a competitive inhibitor in ATPase (or GTPase) reactions. In addition, experiments demonstrating that preincubation of Mg-ATP or Mg-ADP with tTG did not affect the IC50 for Mg-GTP-mediated inhibition of TGase activity also suggested the presence of separate binding sites. Binding of Mg-ATP to tTG apparently induces a conformational change that inhibits GTPase activity, but not TGase activity. On the other hand, the conformation induced by binding to Mg-GTP inhibits TGase activity, but not ATPase activity. In addition, Mg-ADP is a potent inhibitor of GTPase activity, but not TGase activity, whereas Mg-GDP is a potent inhibitor of TGase activity, but not ATPase activity.
We previously reported that the peptide containing the N-terminal 185 amino acid residues of tTG is the minimal structure required for GTPase/ATPase activity (15). Therefore, the Mg-GTP- and Mg-ATP-binding sites must reside in the N-terminal 185 residues of tTG. Examination of the N-terminal 185 residues of tTG did not reveal an exact match with the conserved sequences for GTP-binding proteins such as GXXXXGK(S/T), DXXGQ, and (N/T)(K/Q)XD (33). However, two regions at 64GPAPSQEAGTK74 and 165GFIYQGSAK173 are homologous to the GXXXXGK(S/T) sequence and may act as binding sites for ATP and GTP. Further investigations are needed to address this question.
It is clear that the enzymatic activities of tTG are controlled by
local concentrations of Mg2+, Ca2+, and
nucleotides. Under physiological conditions, intracellular free calcium
ion (~107 M) and GTP (~100-150
µM) concentrations are sufficient to keep tTG in a latent
state. However, intracellular concentrations of free Mg2+
(approximately millimolar) are sufficient for tTG to express ATPase or
GTPase activity. Because GDP and ADP are the major products of the
hydrolysis reaction (15), it is conceivable that intracellular tTG is
in the Mg-ADP- or Mg-GDP-bound state. Since Mg-ADP is more abundant and
is a strong inhibitor of GTP and ATP hydrolysis, one would expect tTG
to display minimal hydrolysis activity intracellularly. In addition,
there should be no TGase activity because GDP and GTP (both free and
complexed forms) are strong inhibitors. For tTG to display TGase
activity inside cells, a cofactor must exist to dissociate Mg-GDP or
Mg-GTP and/or to reduce the Ca2+ requirement for TGase
activity. In support of this hypothesis, we recently discovered that
sphingosylphosphocholine can act as a specific cofactor in activating
TGase activity at physiological levels of Ca2+ and is able
to reverse the inhibition by GTP (19).
It is important to note that the intracellular location of tTG is
dependent upon cell types and that the distribution of tTG in the
cytoplasm or membrane in the same cell type can also be changed upon
retinoic acid treatment (34). Therefore, the enzymatic activities of
tTG are modulated by the local environment. Thus, divalent cations,
nucleotides, and Mg-nucleotide complexes are not the only factors
modulating the enzymatic activities of tTG. The local environment of
tTG should also be considered. In the case of rat liver
Gh (also a tissue-type transglutaminase), it plays an
important role in transmembrane signaling through the
-adrenergic
receptor complex (8). The GTP binding of G
h is modulated
by a 50-kDa protein called G
h. G
h
accelerates the release of GTP
S from G
h and changes
the affinity of G
h from GTP to GDP (35). Therefore,
modulation of the activity of membrane-bound tTG differs due to the
differences in the local environment and the presence of other
protein(s) or cofactor(s).
G subunits of heterotrimeric G-proteins hydrolyze GTP at
a relatively constant intrinsic rate (kcat(GTP) = 1-5 min
1). Other GTPases hydrolyze GTP quite slowly,
usually at rates
that of G
(36), but can
be stimulated by GTPase-activating proteins to hydrolyze GTP at rates
~100-fold faster than that of G
(37, 38). tTG
hydrolyzes GTP quite slowly (0.06 min
1) and may require
modification by a protein cofactor like a GTPase-activating protein to
stimulate its GTPase activity. In the case of the particulate form of
tTG, the cofactor could be membrane components or other proteins like
G
h (35).
Earlier studies, performed in the absence of Mg2+,
demonstrated that GTP was a more potent inhibitor of TGase activity
than GDP and that GMP had no effect (12). The nature and sensitivity of
the prior assay could account for the observed difference in results.
The substrate N,N-dimethylcasein remains in
solution at high concentrations in the [3H]putrescine
incorporation assay (26), whereas it was bound to a microtiter plate in
the assay used in this study (27). Immobilization of lower
concentrations of N,N
-dimethylcasein could
eliminate interference with the effective concentration of nucleoside
phosphates. This was supported by the experiments using 25-fold lower
concentrations of N,N
-dimethylcasein in the
[3H]putrescine incorporation assay. We found that the
IC50 values for GTP and GDP were similar under these
conditions of reduced substrate. When
N,N
-dimethylcasein is coated on microtiter
plates, the interference of N,N
-dimethylcasein
with GTP and GDP is minimized. Furthermore, using the
5-biotinamidopentylamine incorporation assay, the IC50
values of Mg-GTP, Mg-GDP, and Mg-GMP for guinea pig liver tTG were the
same as for recombinant human tTG, demonstrating that it is not a
species-dependent effect.
In conclusion, results from this study suggest that local concentrations of Mg-nucleotide complexes play a role in modulating the enzymatic activities of tTG. In the absence of other factors, intracellular tTG is most likely in the Mg-ADP- or Mg-GDP-bound state and displays minimal hydrolysis activity. As calcium levels increase, Mg-ADP- and Mg-GDP-bound tTG can display TGase activity. Further studies are in progress to define the magnesium-, NTP-, and calcium-binding sites of tTG by site-directed mutagenesis.
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ACKNOWLEDGEMENTS |
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We thank Drs. P. Casey and Y. A. Hannun for helpful suggestions concerning this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL 03205 (to T. F. S.) and Grants HL 28391, HL 38245, AR 39162, and HL 26309 (to C. S. G.); NCI Grant P50 CA68438 (to the Duke Specialized Program of Research Excellence in Breast Cancer); Duke Prostate Cancer Program CA69773-02; and a Focused Giving Award (Johnson & Johnson).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Duke University
Medical Center, P. O. Box 2603, Durham, NC 27710. Tel.: 919-684-6703; Fax: 919-684-4670; E-mail: green032{at}mc.duke.edu.
1
The abbreviations used are: tTG, tissue
transglutaminase; TGase, transglutaminase; ATPS, adenosine
5
-O-(thiotriphosphate); GTP
S, guanosine
5
-O-(thiotriphosphate); PAGE, polyacrylamide gel
electrophoresis; GMP-PCP, adenosine
5
-(
,
-methylenetriphosphate).
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REFERENCES |
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