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• W2020207491 abstract "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 (K m = 38 ± 10 μm) at a 3-fold greater steady-state affinity than with Mg-GTP (K m = 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. 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 (K m = 38 ± 10 μm) at a 3-fold greater steady-state affinity than with Mg-GTP (K m = 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. Tissue transglutaminase (tTG) 1The abbreviations used are: tTG, tissue transglutaminase; TGase, transglutaminase; ATPγS, adenosine 5′-O-(thiotriphosphate); GTPγS, guanosine 5′-O-(thiotriphosphate); PAGE, polyacrylamide gel electrophoresis; GMP-PCP, adenosine 5′-(β,γ-methylenetriphosphate). 1The abbreviations used are: tTG, tissue transglutaminase; TGase, transglutaminase; ATPγS, adenosine 5′-O-(thiotriphosphate); GTPγS, guanosine 5′-O-(thiotriphosphate); PAGE, polyacrylamide gel electrophoresis; GMP-PCP, adenosine 5′-(β,γ-methylenetriphosphate). exhibits two distinct enzymatic activities (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (493) Google Scholar): 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 (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (493) Google Scholar, 3Melino G. Annicchiarico P.M. Piredda L. Candi E. Gentile V. Davis P.J.A. Piacentini M. Mol. Cell. Biol. 1994; 14: 6584-6596Crossref PubMed Scopus (254) Google Scholar, 4Byrd J.C. Lichti U. J. Biol. Chem. 1987; 262: 11699-11705Abstract Full Text PDF PubMed Google Scholar, 5Cai D. Ben T. Deluca L.M. Biochem. Biophys. Res. Commun. 1991; 175: 1119-1124Crossref PubMed Scopus (42) Google Scholar, 6Gentile V. Thomazy V. Piacentini M. Fesus L. Davis P.J.A. J. Cell Biol. 1992; 119: 463-474Crossref PubMed Scopus (229) Google Scholar, 7Johnson T.S. Knight C.R.L. El-Alaoui S. Mian S. Rees R.C. Gentile V. Davis P.J.A. Griffin M. Oncogene. 1994; 9: 2935-2942PubMed Google Scholar) and a GTP binding and hydrolysis activity that is involved in signal transduction and that plays a role in cell cycle progression (8Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (529) Google Scholar, 9Mian S. El-Alaoui S. Lowry J. Gentile V. Davis P.J.A. Griffin M. FEBS Lett. 1995; 370: 27-31Crossref PubMed Scopus (87) Google Scholar). 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 (10Lee K.N. Shelly A.A. Birckbichler P.J. Patterson Jr., M.K. Fraji B.M. Takeuchi Y. Carter H.A. Biochim. Biophys. Acta. 1993; 1202: 1-6Crossref PubMed Scopus (77) Google Scholar). A putative calcium-binding site in human tTG required for TGase activity is located between Ser-430 and His-441 (11Gentile V. Saydak M. Chiocca E.A. Akande N. Birckbichler P.J. Lee K.N. Stein J.P. Davies P.J.A. J. Biol. Chem. 1991; 266: 478-483Abstract Full Text PDF PubMed Google Scholar). Magnesium ions are required for GTP and ATP hydrolysis (10Lee K.N. Shelly A.A. Birckbichler P.J. Patterson Jr., M.K. Fraji B.M. Takeuchi Y. Carter H.A. Biochim. Biophys. Acta. 1993; 1202: 1-6Crossref PubMed Scopus (77) Google Scholar), 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 (12Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar). A single GTP-binding site was subsequently reported for human erythrocyte tTG, and this binding caused a reduction in affinity for calcium ions (13Bergamini C.M. FEBS Lett. 1988; 239: 255-258Crossref PubMed Scopus (74) Google Scholar, 14Bergamini C.M. Signorini M. Biochem. J. 1993; 291: 37-39Crossref PubMed Scopus (30) Google Scholar). Recent studies from our laboratory demonstrated that the GTP- and ATP-binding domains are located in the N-terminal 185 amino acid residues (15Lai T.-S. Slaughter T.F. Koropchak C.M. Haroon Z.A. Greenberg C.S. J. Biol. Chem. 1996; 271: 31191-31195Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). 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 (16Korner G. Schneider D.E. Purdon M.A. Bjornsson T.D. Biochem. J. 1989; 262: 633-641Crossref PubMed Scopus (39) Google Scholar). The cell-surface membrane form associates with the α1-adrenergic receptor to mediate a G-protein signal transduction pathway (8Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (529) Google Scholar). 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 (17Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; : 15863-15871Crossref PubMed Scopus (85) Google Scholar). 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 (18Fesus L. Zsuzsa S. Uray I. J. Cell. Biochem. Suppl. 1995; 22: 151-161Crossref PubMed Scopus (74) Google Scholar) 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 (19Lai T.-S. Bielawska A. Peoples K.A. Hannun Y.A. Greenberg C.S. J. Biol. Chem. 1997; 272: 16295-16300Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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. Sodium salts of ATPγS, ATP, ADP, AMP, GTPγS, GTP, GDP, and GMP and the MgCl2 stock solution (1m) were purchased from Sigma. [3H]Putrescine dihydrochloride (35.5 Ci/mmol) and [γ-32P]GTP and [γ-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 (20Birckbichler P.J. Upchurch H.F. Patterson Jr., M.K. Conway E. Hybridoma. 1985; 4: 179-186Crossref PubMed Scopus (59) Google Scholar). 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. The assembly of full-length human tTG cDNA and the purification of glutathione S-transferase-tTG fusion protein were as described (15Lai T.-S. Slaughter T.F. Koropchak C.M. Haroon Z.A. Greenberg C.S. J. Biol. Chem. 1996; 271: 31191-31195Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The purified glutathioneS-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 (21Edgell C.-J.S. McDonald C.C. Graham J.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737Crossref PubMed Scopus (1358) Google Scholar). Protein concentrations were quantitated using the Bradford method (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar) (Bio-Rad). Preliminary studies demonstrated that recombinant tTG preferentially cross-linked the Aα-chains of fibrinogen to form high molecular weight α-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. The concentrations of Mg-nucleotide complexes were prepared according to the procedures described by Morrison (23Morrison J.F. Methods Enzymol. 1979; 63: 257-294Crossref PubMed Scopus (80) Google Scholar) and O'Sullivan and Smithers (24O'Sullivan W.J. Smithers G.W. Methods Enzymol. 1979; 63: 294-337Crossref PubMed Scopus (188) Google Scholar). 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 (23Morrison J.F. Methods Enzymol. 1979; 63: 257-294Crossref PubMed Scopus (80) Google Scholar,24O'Sullivan W.J. Smithers G.W. Methods Enzymol. 1979; 63: 294-337Crossref PubMed Scopus (188) Google Scholar). 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.(25Bers D.M. Patton C.W. Nuccitelli R. Methods Cell Biol. 1994; 40: 4-28Google Scholar). 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 (23Morrison J.F. Methods Enzymol. 1979; 63: 257-294Crossref PubMed Scopus (80) Google Scholar, 24O'Sullivan W.J. Smithers G.W. Methods Enzymol. 1979; 63: 294-337Crossref PubMed Scopus (188) Google Scholar, 25Bers D.M. Patton C.W. Nuccitelli R. Methods Cell Biol. 1994; 40: 4-28Google Scholar). TGase activity was determined by quantitating the incorporation of [3H]putrescine (26Miraglia C.C. Greenberg C.S. Anal. Biochem. 1985; 144: 165-171Crossref PubMed Scopus (28) Google Scholar) or 5-biotinamidopentylamine into N,N′-dimethylcasein as described previously (27Slaughter T.F. Achyuthan K.E. Lai T.S. Greenberg C.S. Anal. Biochem. 1992; 205: 166-171Crossref PubMed Scopus (156) Google Scholar). 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. The assay was performed according to the procedure described (15Lai T.-S. Slaughter T.F. Koropchak C.M. Haroon Z.A. Greenberg C.S. J. Biol. Chem. 1996; 271: 31191-31195Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), 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 [γ-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. 2 A), the reaction mixture was as described above, except that 2 mm Mg2+, 250 μm labeled and unlabeled GTP, and 0–100 μmMg-ATP, Mg-ADP, or Mg-AMP were used. For ATPase competitive inhibition studies (see Fig. 2 B), the reaction mixture contained 40 μm labeled and unlabeled ATP and 0–1000 μmMg-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. 28Eisenthal R. Danson M.J. Enzyme Assays. Oxford University Press, Oxford1993: 277-316Google Scholar). Results are representative of at least two duplicate experiments. 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 mmMgCl2, 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 (20Birckbichler P.J. Upchurch H.F. Patterson Jr., M.K. Conway E. Hybridoma. 1985; 4: 179-186Crossref PubMed Scopus (59) Google Scholar). 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.1 A, 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.1 A), 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. 1 B), GTP hydrolysis activity was only 71% of the maximum activity. Thus, free Mg2+ has an additional activator effect on GTPase activity (Fig. 1 B). Similar experiments performed with ATP demonstrated that Mg-ATP was also a substrate for hydrolysis. In the reaction containing predominately Mg-GTP (or Mg-ATP; see “Preparation of Mg-Nucleotide Complexes”), the apparentK m for Mg-GTP was 130 ± 35 μm, with a k cat of 0.06 ± 0.01 min−1. The apparent K m for Mg-ATP was 38 ± 10 μm, with a k cat of 0.08 ± 0.02 min−1. When Ca2+ (at 0–4 mm) was substituted for Mg2+, GTP or ATP hydrolysis activity was 10% of that obtained in the presence of the optimum Mg2+ concentration. When the Ca2+ concentration was increased to 8–16 mm, GTPase or ATPase activity was only 20–40% of that obtained in the presence of the optimum Mg2+ concentration. The addition of 0–16 mm Ca2+ to a GTPase or ATPase reaction containing the optimum Mg2+ concentration did not significantly affect hydrolysis activity. Since tTG can hydrolyze ATP as well as GTP, we investigated which is the preferred substrate. Fig.2 A 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. 2 A). Preincubation of tTG with Mg-ADP on ice for up to 2 h did not further increase this inhibitory effect. Fig. 2 B 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.2 B). Preincubation of tTG with Mg-GTP or Mg-GDP on ice for up to 1 h did not enhance the inhibition. Fig. 2 C illustrates the Lineweaver-Burk plot of GTP hydrolysis in the presence or absence of Mg-ATP to examine the mechanism of inhibition. The kinetic patterns gave lines intersecting the abscissa. Therefore, Mg-ATP was acting as a noncompetitive inhibitor of GTP hydrolysis, with aK i of 22 ± 4 μm (Fig.2 C). We also found that Mg-ADP acted as a noncompetitive inhibitor of GTP hydrolysis. In addition, Mg-GDP and Mg-ADP acted as competitive inhibitors in GTPase and ATPase reactions, respectively, further supporting the presence of separate binding sites for Mg-GTP and Mg-ATP. We also performed a TGase reaction to confirm that there were separate binding sites for Mg-GTP and Mg-ATP. If there were separate binding sites for Mg-GTP and Mg-ATP, preincubation of tTG with Mg-ATP or Mg-ADP should not affect the ability of Mg-GTP to inhibit TGase activity. Indeed, we found that preincubation of tTG with Mg-ATP or Mg-ADP did not change the IC50 values for Mg-GTP. Previous studies using nucleotides as inhibitors of TGase activity did not consider the role of Mg2+ in this process (29Takeuchi Y. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. FEBS Lett. 1992; 307: 177-180Crossref PubMed Scopus (32) Google Scholar). Mg2+ exists mainly as a Mg-nucleotide complex inside cells (30Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1290) Google Scholar) 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-GTPγS, Mg-GTP, and Mg-GDP were equipotent inhibitors, with IC50values of 9 μm (Fig.3 A), and that Mg-GMP was less inhibitory, with an IC50 of 400 μm. Ca-GTPγS, Ca-GTP, Ca-GDP, and Ca-GMP were found to inhibit TGase activity, with IC50 values of 4, 4, 4, and 100 μm, respectively. To determine whether hydrolysis of GTP was required for the inhibition of TGase activity, we preincubated tTG with GTP in the presence of 1 mm excess Mg2+ or Ca2+ at 37 °C for 30 min. We found that preincubation did not increase the potency of inhibition, demonstrating that hydrolysis of GTP was not required for inhibition. Furthermore, we found that GTP, GTPγS, and GMP-PCP inhibited TGase activity with the same IC50 value (4 μm). Therefore, the nonhydrolyzable analogs, GTPγS and GMP-PCP, inhibit the TGase reaction with the same potency as GTP. Hydrolysis of GTP is not required to inhibit TGase activity. We previously reported that GTP was a more potent inhibitor of TGase activity than GDP and that GMP had no effect (12Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar). In these earlier experiments using the [3H]putrescine incorporation assay, the substrate (N,N′-dimethylcasein) was in solution (12Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar), whereas in the experiments described in this study,N,N′-dimethylcasein was bound to the microtiter plate. Therefore, experiments were performed using the [3H]putrescine incorporation assay to determine whether concentrations of N,N′-dimethylcasein in solution altered the ability of GTP/GDP to inhibit TGase activity. Indeed, we found that the IC50 values for GTP and GDP were similar when the concentration of N,N′-dimethylcasein in the [3H]putrescine assay was reduced from 25 to 1 μm. In contrast to the effects of Mg-GTP and Mg-GDP, various Mg-adenine nucleotide complexes did not significantly inhibit the TGase activity of recombinant human tTG (Fig. 3 B) or guinea pig liver tTG. In a transglutaminase reaction containing 1 mmCa2+, free ATP and ADP were able to inhibit TGase activity, with IC50 values of 400 μm and 1 mm, respectively. However, this inhibition was completely reversed in the reaction by increasing the Ca2+concentration from 1 to 3 mm, suggesting that the inhibition by ATP was a chelation effect (data not shown). 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 and5, 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. tTG is present in several different tissues and cellular compartments (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (493) Google Scholar, 16Korner G. Schneider D.E. Purdon M.A. Bjornsson T.D. Biochem. J. 1989; 262: 633-641Crossref PubMed Scopus (39) Google Scholar, 17Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; : 15863-15871Crossref PubMed Scopus (85) Google Scholar). In the extracellular environment, tTG plays a role in extracellular matrix assembly, cell adhesion, and wound healing (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (493) Google Scholar). Inside cells, tTG is associated with the cell-surface membrane and plays an important role in signal transduction and nuclear pore assembly (8Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (529) Google Scholar, 17Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; : 15863-15871Crossref PubMed Scopus (85) Google Scholar). 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 (9Mian S. El-Alaoui S. Lowry J. Gentile V. Davis P.J.A. Griffin M. FEBS Lett. 1995; 370: 27-31Crossref PubMed Scopus (87) Google Scholar, 18Fesus L. Zsuzsa S. Uray I. J. Cell. Biochem. Suppl. 1995; 22: 151-161Crossref PubMed Scopus (74) Google Scholar). 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 (31Carafoli E. Annu. Rev. Biochem. 1987; 56: 395-433Crossref PubMed Scopus (1770) Google Scholar, 32Paterson C.R. Essentials of Human Biochemistry. Churchill Livingstone, Edinburgh, Scotland1983: 237-248Google Scholar). 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 (29Takeuchi Y. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. FEBS Lett. 1992; 307: 177-180Crossref PubMed Scopus (32) Google Scholar) 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 (15Lai T.-S. Slaughter T.F. Koropchak C.M. Haroon Z.A. Greenberg C.S. J. Biol. Chem. 1996; 271: 31191-31195Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). 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 asG XXXX GK(S/T),D XX GQ, and (N/T)(K/Q)X D (33Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1990; 15: 430-434Abstract Full Text PDF PubMed Scopus (1748) Google Scholar). However, two regions at64GPAPSQEAGTK74and165GFIYQGSAK173are homologous to the G XXXX GK(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 (∼10−7m) 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 (15Lai T.-S. Slaughter T.F. Koropchak C.M. Haroon Z.A. Greenberg C.S. J. Biol. Chem. 1996; 271: 31191-31195Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), 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 (19Lai T.-S. Bielawska A. Peoples K.A. Hannun Y.A. Greenberg C.S. J. Biol. Chem. 1997; 272: 16295-16300Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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 (34Chowdhury Z.A. Barsigian C. Chalupowicz G.D. Bach T.L. Garcia-Manero G. Martinez J. Exp. Cell Res. 1997; 231: 38-49Crossref PubMed Scopus (44) Google Scholar). 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 Gαh (also a tissue-type transglutaminase), it plays an important role in transmembrane signaling through the α-adrenergic receptor complex (8Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (529) Google Scholar). The GTP binding of Gαh is modulated by a 50-kDa protein called Gβh. Gβhaccelerates the release of GTPγS from Gαh and changes the affinity of Gαh from GTP to GDP (35Baek K.J. Das T. Gray C.D. Desai S. Hwang K.C. Gacchui R. Ludwig M. Im M.-J. Biochemistry. 1996; 35: 2651-2657Crossref PubMed Scopus (34) Google Scholar). 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). Gα subunits of heterotrimeric G-proteins hydrolyze GTP at a relatively constant intrinsic rate (k cat(GTP)= 1–5 min−1). Other GTPases hydrolyze GTP quite slowly, usually at rates 11000 that of Gα (36Kaziro Y. Itoh H. Kozasa T. Nakafuku M. Satoh T. Annu. Rev. Biochem. 1991; 60: 349-400Crossref PubMed Scopus (549) Google Scholar), but can be stimulated by GTPase-activating proteins to hydrolyze GTP at rates ∼100-fold faster than that of Gα (37Martin G.A. Viskochil D. Bollag G. McCabe P.C. Crosier W.J. Haubruck H. Conroy L. Clark R. O'Connell P. Cawthon R.M. Cell. 1990; 63: 843-849Abstract Full Text PDF PubMed Scopus (737) Google Scholar, 38Gideon P. John J. Frech M. Lautwein A. Clark R. Scheffler J.E. Wittinghofer A. Mol. Cell. Biol. 1992; 12: 2050-2056Crossref PubMed Scopus (246) Google Scholar). 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 Gβh (35Baek K.J. Das T. Gray C.D. Desai S. Hwang K.C. Gacchui R. Ludwig M. Im M.-J. Biochemistry. 1996; 35: 2651-2657Crossref PubMed Scopus (34) Google Scholar). 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 (12Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar). 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 (26Miraglia C.C. Greenberg C.S. Anal. Biochem. 1985; 144: 165-171Crossref PubMed Scopus (28) Google Scholar), whereas it was bound to a microtiter plate in the assay used in this study (27Slaughter T.F. Achyuthan K.E. Lai T.S. Greenberg C.S. Anal. Biochem. 1992; 205: 166-171Crossref PubMed Scopus (156) Google Scholar). 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. WhenN,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 IC50values 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. We thank Drs. P. Casey and Y. A. Hannun for helpful suggestions concerning this manuscript." @default.
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