identification of a guanosine triphosphate-binding site on guinea pig

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc

Vol. 262, No. 4, Issue of February 5, pp. 190-1906 1987 Printed in il.S.A.

Identification of a Guanosine Triphosphate-binding Site on Guinea Pig Liver Transglutaminase ROLE OF GTP AND CALCIUM IONS IN MODULATING ACTIVITY*

(Received for publication, May 6, 1986)

Komandoor E. AchyuthanS and Charles S. GreenbergSpll From the Departments of $Medicine and §Pathology, Duke University Medical Center, Durham, North Carolina 27710

Guanosine 5“triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-7-23 also inhibited enzyme activity (GTP-7-S > GTP > GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaClz partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIII, activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase ad- sorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine ~ ’ - [Y-~’P] triphosphate, which was inhibited by the presence of GTP or CaClZ. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Pro- teolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.

Transglutaminases (R-glutaminyl-peptide:amine-y-gluta- myltransferase, EC 2.3.2.13) are calcium-dependent enzymes which catalyze the formation of covalent bonds between peptide-bound glutamyl residues and either the lysyl group of a protein or the primary amino group of polyamines (for a review see Najjar and Lorand, 1984). Transglutaminase-catalyzed bonding between peptide-bound glutamines and polyamines has been found to occur intracellularly and extracel- lularly (Folk and Finlayson, 1977), and the enzyme has been demonstrated in most cells and tissues. Although several cofactors have been shown to affect transglutaminase function, the regulation of transglutaminase activity remains poorly understood (Folk, 1980). Folk et al. (1967) demonstrated that the presence of calcium ions induced a conformational change in purified guinea pig liver transglutaminase that promoted its enzymatic activity. Binding of the glutamine substrate to the enzyme was subsequently found to induce a

* This research was supported in part by National Institutes of Health Grant HL-32342 and Grant 1591 from the Council for Tobacco Research, New York. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed.

conformational change in the active site of the enzyme which also stimulated its catalytic activity (Gross and Folk, 1973). When added to cells grown in culture, phorbol esters, retinoic acid, and extracellular calcium induce transglutaminase activity in epidermal cells (Yuspa et al., 1983). Similarly, cystamine was found to inhibit intracellular transglutaminase activity in WI-38 human lung cells (Birckbichler et al., 19811, while the presence of retinoic acid increased transglutaminase activity in several cell lines, including Chinese hamster ovary cells (Scott et al., 1982), human erythroleukemia HL-60 cells (Davies et al., 1985) and mouse epidermal cells (Lichti et aL, 1985).

Recent studies have implicated nucleotide triphosphates as potential factors in regulating intracellular transglutaminase activity. Loewy et al. (1981a, 1981b) reported an increase in the formation of intracellular (7-glutamy1)-e-lysyl cross-links when ATP was added to extracts from tissue-cultured embry- onic chick heart myofibrils or from cultures of the slime mold, Physarium polycephulum. Human erythrocyte transglutaminase is stabilized by the presence of ATP during purification of the enzyme but not required for stability once purified (Brenner and Finn Wold, 1978). Cohen et al. (1980) reported that Factor XIIIa-catalyzed incorporation of amines into platelet, and muscle actin was inhibited by ATP, ADP, GTP, and CTP but was not modified by cyclic AMP or AMP.

Several investigators have suggested mechanisms for the induction of intracellular transglutaminase activity. Mur- taugh et al. (1983) concluded that an increase in macrophage transglutaminase activity was due to an increase in enzyme synthesis. Lichti et al. (1985) found that the increase in transglutaminase activity in mouse epidermal cells correlated with the synthesis of yet a second enzyme, while Birckbichler et at. (1985) have postulated that post-translational modifi- cation of the enzyme leads to changes in tissue transglutaminase activity.

The purpose of this study was to investigate the effects of guanine nucleotides on transglutaminase activity. We have found that guanosine 5’-triphosphate (GTP) is a reversible and noncompetitive inhibitor of guinea pig liver transglutaminase function. Specific binding of GTP to transglutaminase was demonstrated using GTP-agarose affinity chromatography, trypsin proteolysis, and photoaffinity-labeling tech- niques.

MATERIALS AND METHODS

Guanosine 5’-monophosphate (GMP, disodium salt), guanosine 5’diphosphat.e (GDP, sodium salt), guanosine 5’-triphosphate (GTP, sodium salt), guanosine 3‘:5’-monophosphate (cyclic GMP, sodium salt), polyguanylic acid ( 5 ’ ) (potassium salt, M, = 150,000), cytidine 5”triphosphate (CTP, sodium salt), guanosine 5”triphosphate-agarose (GTP-agarose), and cytidine 5”triphosphate agarose (CTP- agarose) were purchased from Sigma. GTP (Li4 salt) and the chrom-

1901

1902 GTP Binds to Guinea Pig Liver Transglutaminase

ogenic substrate benzoyl-D, L-arginine-p-nitroanilide were purchased from Behring Diagnostics. [3H]Putrescine dihydrochloride (28.8 Ci/ mmol) and ["C]methylamine (46 mCi/mmol) were purchased from New England Nuclear. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was purchased from Worthington and 8-azi- doguano~ine-5'-[y-~~P]triphosphate (8-AGTP)' (18.8 Ci/mmol) was purchased from ICN Radiochemicals, CA. Monoclonal antibody (CUB 7401) to guinea pig liver transglutaminase was generously provided by Dr. P. J. Birckbichler, (Birckbichler et al., 1985). Horse- radish peroxidase-conjugated goat antimouse immunoglobulin, and high and low molecular weight protein standards were obtained from Bio-Rad. A [I4C]methylated protein mixture (CFA.626) with molecular mass ranging from 14,300-200,000 daltons was purchased from Amersham Corp.

Transglutaminase Purification Liver Transglutaminnse-Guinea pig and rat liver transglutami-

nases were purified by DEAE-Sephacyl column chromatography as described by Connellan et al. (1971) and further purified by gel filtration on a Sephadex G-100 column. A Du Pont GF-250 HPLC gel filtration column was used in the final purification step in which the protein was eluted with 50 mM Tris-HC1 (pH 8.5) at a flow rate of 0.5 ml/min using an LKB 2150 dual pump HPLC instrument coupled to a 2151 HPLC controller, a 2152 variable wavelength monitor and 2210 chart recorder.

Platelet Factor XZZZ-Platelet Factor XI11 was purified from a human platelets sonicate using a DEAE-Sephacyl column followed by gel filtration through a Sephacryl S-200 column as previously described (Greenberg and Shuman, 1982). The platelet factor XI11 (13 pg) was activated to form Factor XIII. by incubation with a- thrombin (200 units/ml) for 10 min at 37 "C. Thrombin was inhibited by the addition of 200 p~ p-nitrophenyl-p-guanido-benzoate, and the Factor XIII, activity was assayed as described by Miraglia and Green- berg (1985).

Adult Bovine Aortic Endothelial (ABAE) Cell Transglutamime- ABAE cells were cultured to confluency (Gospodarowicz et al., 1976) and harvested by a brief (2-3 min) exposure to trypsin. Trypsin was subsequently inhibited by the addition of a large volume of culture media supplemented with 10% calf serum. The suspended cells were centrifuged, resuspended in 100 mM Tris-HC1, 50% glycerol (v/v), and 200 p~ phenylmethylsulfonyl fluoride (pH 8.5), sonicated 3 X 15 s), and stored at -70 "C until use.

Transglutaminase Assay-Transglutaminase activity was determined by quantitating the incorporation of [3H]putrescine into casein as previously described (Miraglia and Greenberg, 1985). This reaction was carried out in 0.1 ml of buffer containing 50 mM Tris- HC1 (pH 8.5), 20% (v/v) glycerol, N,N' dimethylcasein (1 mg/mL), 250 p M putrescine, 1 pci [3H]putrescine, 20 mM dithiothreitol, 1 mM calcium chloride, and the enzyme at indicated amounts. Glycerol was included in the buffer since its presence has been found to stabilize transglutaminase activity (Miraglia and Greenberg, 1985). The presence or absence of glycerol in the assay had no effect on GTP inhibition of guinea pig liver transglutaminase. Reaction mixtures were incubated for 1 h at 37 "C and reactions stopped by the addition of 0.1 ml of 50% trichloroacetic acid. The precipitate was collected on Whatman GF/C filters, washed three times with 10 ml of 5% trichloroacetic acid, and the radioactivity measured by liquid scintillation. Transglutaminase activity was expressed as nanomoles of putrescine incorporated into casein in 1 h at 37 "C. Enzyme kinetic data were analyzed by Dixon plots (Dixon, 1953). Inhibition constants were calculated under conditions in which less than 10% of the primary amine substrate had been incorporated into casein.

Affinity Chromatography

One milliliter columns of GTP-agarose (1.2 pmol of GTP) and CTP-agarose (1.2 pmol of CTP) were equilibrated with a buffer of 100 mM Tris-HC1 and 10% glycerol (pH 8.5). Sixty micrograms of guinea pig liver transglutaminase (ca. 700 pmol assuming a molecular weight of 85,000 according to Connellan et al., 1971) was loaded onto each column and washed through the columns with buffer. Fractions of 0.25 ml were collected and assayed for transglutaminase activity as well as for total protein content by the method of Bradford (1976),

' The abbreviations used are: 8-AGTP, 8-azidoguanosine 5'-[~-~'p] triphosphate; ABAE, adult bovine aortic endothelial; GTP-y-S, guanosine-5'-0-(3 thiotriphosphate); HPLC, high pressure liquid cbro- matography.

using bovine serum albumin as a standard. TO verify the inhibition of GTP binding to transglutaminase by

calcium ions, GTP-agarose columns were equilibrated with the Tris- glycerol buffer to which 10 mM CaC1' had been added. Sixty micrograms of guinea pig liver transglutaminase were applied to the columns and washed through with buffer containing calcium chloride. Fractions were collected and assayed for enzyme activity and protein content as described above. Results were expressed as the average of duplicate experiments.

SDS-PAGE and Western Blotting Protein samples were solubilized in a buffer containing 1% sodium

dodecyl sulfate, 1.5 M urea, 2.5 mM EDTA, 30 mM Tris-HC1 (pH 7.5), 5% 2-mercaptoethanol, and 0.001% bromphenol blue and boiled for 5 min. The proteins were separated by 8% polyacrylamide gel electrophoresis as described by Laemmli (1970). Following electrophoresis, the proteins were transferred to a nitrocellulose filter (Western blotting) as described by Towbin et al. (1979). The gel was stained with Coomassie Brilliant Blue to demonstrate the absence of detectable proteins remaining adherent to the polyacrylamide. The nitrocellulose filter was incubated in 10 mM Tris-HC1, 150 mM NaCl, and 3% bovine serum albumin (Tris-bovine serum albumin, pH 7.4) with gentle shaking at 4 "C overnight to block residual binding sites. The filter was then exposed to a 1:lOOO dilution of monoclonal antibody (CUB 7401) to guinea pig transglutaminase in Tris-bovine serum albumin, incubated with gentle shaking for 8-12 h at 4 "C, washed with buffer, and incubated with a 1:2000 dilution of goat anti-mouse horseradish peroxidase conjugate for 4-6 h at 4 "C. Following an additional wash in buffer, the nitrocellulose was developed in a color development reagent from Bio-Rad.

Photoaffinity Labeling of Guinea Pig Liver Transglutaminase

Photoaffinity labeling of purified guinea pig liver transglutaminase was carried out as described by Kohnken and McConnell (1985) and by Takemoto et al., (1981) using 8-AGTP. In this procedure, 330 pmol of transglutaminase was mixed with 265 pmol of 8-AGTP (5 pCi) in the presence or absence of either CaC1, or unlabeled GTP to a final volume of 50 pl in 100 mM Tris-HC1 (pH 8.5). The reagents were mixed under a dim red safelight on ice in 1.5-ml conical poly- propylene centrifuge tubes. The samples were irradiated at a distance of 1 cm for 10 min using long wavelength UV light (Ultra-Violet Products, model UVSL-25). A 5-10 pl portion of each reaction mixture was removed and added to 100 p1 of bovine serum albumin (3 mg/mL). To this, 100 p1 of 50% trichloroacetic acid was added to precipitate the proteins. The precipitate was collected on Whatman GF/C glass filters, washed three times in each of 5% trichloroacetic acid, methanol, and ether, and then air-dried. Radioactivity of the proteins was determined by liquid scintillation. The remaining portion of each sample was analyzed by SDS-PAGE, Western blotting, and exposure to monoclonal antibody to guinea pig liver transglutaminase as described. After visualization and photography of the antigen bands, autoradiography was performed. The radiolabeled bands were compared with the antigen profile and quantitated with a scanning densitometer (Hoefer Scientific).

Effect of GTP on Trypsin Proteolysis of Guinea Pig Liver Transglutaminase

Purified guinea pig liver transglutaminase (2 pg) was incubated at 37 "C for 1 h with 2-200 ng of trypsin in 50 pl of 100 mM Tris-HC1 (pH 8.5). Incubations were carried out in the presence of 1 mM EDTA and various combinations of CaClz and GTP. Proteolysis was stopped by the addition of SDS-PAGE buffer and the samples were analyzed by SDS-PAGE and Western blotting as described. Both radioactive and nonradioactive reference proteins were electrophoresed and the molecular weights of the transglutaminase fragments calculated from a curve constructed from the relative mobilities of the molecular weight standards.

Trypsin (0.3 pg/ml) was incubated at 22 "C for 30,60, and 120 min in 50 mM Tris-HC1 (pH 8.5) containing 1 mM benzoylarginine-p- nitroanilide. The reaction was stopped by the addition of 5% acetic acid. Absorbance was recorded at 405 nM using a LKB spectropho- tomer. Assays were also performed in the presence of either 1 mM GTP alone or 1 mM GTP with 1 mM CaClZ.

GTP Binds to Guinea Pig Liver Transglutaminuse 1903

Ionized Calcium Determinations Ionized calcium measurements of calcium chloride solutions in

presence of 1 mM GTP were conducted using a NOVA Ca2+ selective electrode (NOVA Biomedical, Waltham, MA).

RESULTS

Effect of Guanine Nucleotide Derivatives on Purified Guinea Pig Liver Transglutaminase Activity-GTP, GTP-yS, GMP, cyclic GMP, GDP, and polyguanylic acid were each tested at a concentration of 1 mM to determine their relative affects on guinea pig liver transglutaminase activity. GTP, GTP-y-S, and GDP were found to exhibit concentration-dependent inhibition of enzyme activity, with 50% inhibition occurring at concentrations of 90,30, and 400 pM, respectively (Fig. 1). Higher GTP, GTP-y-S, and GDP levels were found to completely inhibit transglutaminase activity. Both the sodium and lithium salts of GTP inhibited two different preparations of purified guinea pig and rat liver transglutaminases as well as the ABAE cell transglutaminase. The other compounds tested had no affect on enzyme activity.

Specificity of GTP Inhibition-To determine the specificity of GTP inhibition for tissue transglutaminase, the activity of purified platelet Factor X U , was assayed. Amine (putrescine, methylamine) incorporation into casein and fibrinogen was not inhibited by the presence of 1 mM GTP.

Kinetics of GTP Inhibition-Dixon plot analysis demonstrated that GTP was a noncompetitive inhibitor of purified guinea pig liver transglutaminase activity in the presence of 1 mM CaC1, (K; = 90 pM). The inhibition of rat liver transglutaminase (Ki = 150 p ~ ) and ABAE cell transglutaminase (K, = 280 p ~ ) was also noncompetitive. The GTP concentration required to achieve 50% inhibition of transglutaminase activity in ABAE cells was reduced by 2 to %fold in the presence of 10 mM sodium fluoride or 10 mM p-nitrophenylphosphate. The addition of 10 mM NaCl had no affect.

Effect of Calcium and Magnesium Ions on GTP-mediated Inhibition-Complete inhibition of guinea pig liver transglutaminase occurred with 1 mM GTP in the presence of 1 mM CaC12, and the level of inhibition was reduced by 50% when 5 mM CaCI, was present. Similar effects of calcium chloride on GTP inhibition were observed using rat liver and ABAE cell transglutaminases. The inhibition of enzyme activity by 1 mM GTP in presence of 1 mM CaC12 was not reduced by the addition of 5 mM MgC1,.

GTP-Affinity Chromatography-Purified guinea pig liver transglutaminase bound to GTP-agarose but did not bind to CTP-agarose (Fig. 2). A CTP-agarose affinity chromatogra-

phy column was used as a control since 0.8 mM CTP had no inhibitory effect on enzyme activity (data not shown). The protein recovery from the CTP-agarose column was 82% and contained 96% of the transglutaminase activity applied to the column. In contrast, protein recovery from the GTP-agarose column was 38% and represented only 22% of the total enzyme activity applied. In four separate experiments, we found that 7585% of the transglutaminase was bound to the GTP-agarose columns.

The effect of calcium ions on guinea pig liver transglutaminase binding to GTP-agarose columns was studied by load- ing and eluting the enzyme in buffer containing 10 mM CaCL In these experiments, 60% of the total protein containing 75% of the total transglutaminase activity was eluted from the columns.

Photoaffinity Labeling-The photoaffinity label, 8-AGTP, was covalently bound to guinea pig liver transglutaminase at a ratio of 27-30 pmol 8-AGTP/330 pmol of enzyme in the presence of either 1 mM EDTA or 1 mM CaC12. GTP compet- itively inhibited photoaffinity labeling of the transglutaminase (Fig. 3) with 50% inhibition occurring at 2 WM GTP. CaC12 concentrations greater than 2 mM completely inhibited 8- AGTP labeling (Fig. 4). In presence of 0.1 mM GTP, 8-AGTP labeling of guinea pig liver transglutaminase was inhibited by 66%. CaCl, (1 mM) completely reversed GTP (0.1 mM) inhibition of photoaffinity labeling.

Effects of GTP and Calcium on Trypsin Proteolysis of Guinea Pig Liver Transglutaminuse-GTP concentrations of 50 nM and higher completely inhibited trypsin proteolysis of purified transglutaminase (Fig. 5). In the absence of calcium, trypsin was found to degrade transglutaminase into fragments that were nonreactive with anti-guinea pig liver transglutaminase monoclonal antibody (Fig. 6). However, in presence of 10 mM CaCL, fragments of 44 and 78 kDa were formed (Fig. 6). In the presence of 0.1 mM GTP, only a minor 55-kDa proteolytic fragment was produced. The presence of 10 mM CaClz completely reversed the proteolytic inhibition by 0.1 mM GTP. Proteolysis in the presence of either 10 mM CaClz or 10 mM CaC& and 0.1 mM GTP were similar (Fig. 6). In the presence of 500 nM GTP, CaC1, concentrations greater than 1 mM promoted proteolysis of guinea pig liver transglutaminase (Fig. 7). Under these conditions, the enzyme was degraded into 70-, 75 , and 78-kDa fragments when 5 mM CaC12 was present (Fig. 7, lane 6). Control experiments demonstrated that the GTP and CaCl, concentrations used in these experiments did not affect trypsin cleavage of the chromogenic substrate, benzoyl-D,t-arginine-p-nitroanilide.

.. c 20 -

C I

0 20 40 60 80 100 GTP-IS ( r M l

FIG. 1. Effect of GTP, GDP, and GTP-7-S on guinea pig liver transglutaminase activity. Increasing concentrations of GTP (A) , GDP ( B ) and GTP-7-S (C) were incubated with guinea pig liver transglutaminase (3 fig) in a total volume of 0.1 ml 50 mM Tris-HC1 (pH 8.5), 20% (v/v) glycerol, and 1 mM CaC1,. TG activity was assayed as described under the “Materials and Methods” section.

.

1904 GTP Binds to Guinea Pig Liver Transglutaminase

I 4 ,

0 2 4 6 8 1 0 1 2 Fraction Number

FIG. 2. Affinity chromatography of guinea pig liver transglutaminase. Sixty micrograms of the enzyme was applied to either a 1-ml column of GTP-agarose (broken lines) or a 1-ml column of CTP-agarose (solid lines). The columns were equilibrated and eluted with 0.1 M Tris-HC1 (pH 8.5) containing 10% glycerol. Fractions (250 pl) were collected and assayed for transglutaminase activity. Trans- glutaminase activity is expressed as nanomoles of putrescine incorporated into casein in 1 h at 37 "C.

a\ OO 0.1 1.0 1 0 0 1000

GTP (pM) FIG. 3. Effect of GTP on covalent incorporation of the pho-

toaffinity label, 8-azidoguanosine 5'-[-pS2P]triphosphate into purified guinea pig liver transglutaminase. Photoaffinity labeling was carried out in presence of increasing concentrations of unlabeled GTP and the amount of label incorporated into transglutaminase was quantitated by measuring the radioactivity in the trichloroacetic acid-precipitated protein, as described under the "Materials and Methods" section.

DISCUSSION

Although the role of calcium ions in the regulation of transglutaminase activity is well established, many of the intracellular cofactors regulating transglutaminase activity remain poorly understood (Folk, 1980). A growing body of evidence implicates nucleotides as a group of factors influenc- ing transglutaminase activity. In our examination of guanine nucleotides and their effects on guinea pig liver transglutaminase, the prototype for intracellular tissue transglutaminase, we found that GTP, GTP-y-S, and GDP significantly inhibited the activity of purified enzyme. These nucleotides also inhibited the activity of transglutaminases isolated from ABAE cells and rat liver, demonstrating species nonspecific- ity.

GTP bound approximately 62% of the guinea pig liver transglutaminase applied to a GTP-agarose column and reduced total enzyme activity by 78%. CTP-agarose columns bound very little of the enzyme and the presence of CaCl, was found to reduce binding of transglutaminase to GTP-agarose

I 2 3 4 5 6 7 FIG. 4. Effect of increasing CaClz concentrations on the

incorporation of the photoaffinity label 8-AGTP into guinea pig liver transglutaminase. Reaction conditions were as described in the text. Lunes 1-7 represent incubations in presence of 0, 0.1, 1.0, 2.0,4.0,6.0, and 10.0 mM CaC12. Quantitation of the radioactive bands on the x-ray film using a scanning densitometer revealed incorporation of the photoaffinity label was inhibited bv more than 90% at 4

-94

1 2 3 4 5 6 7 8 9

-55

IO FIG. 5. Effect of GTP concentration on proteolysis of guinea

pig liver transglutaminase. Guinea pig transglutaminase (2 pg) was incubated with trypsin (0.2 pg) and various GTP concentrations. Lunes 1-10 (from kft to right) represent incubations in presence of 0, 1, 5, 10, 25, 50, 100, 500, and 1000 nM GTP. The samples were then analyzed by SDS-PAGE and Western blotting as described in the text.

columns. It is likely that GTP, when it is bound to agarose, might exist in a conformational state unlike soluble GTP. Such conformational changes are supported by the finding that some transglutaminase could pass through the affinity columns despite a 1500-fold excess of GTP bound to agarose. The selective GTP-agarose binding of transglutaminase may provide a powerful step in purification of this enzyme, similar to the purification scheme utilizing carbobenzoxy-L-phenyl- alanine-Sepharose 4B columns developed by Brookhart, et al. (1983).

Photoaffinity labeling and trypsin proteolysis experiments provided additional evidence that GTP bound to guinea pig liver transglutaminase and that both GTP and CaCl, acted to modulate enzyme structure. Covalent bonding of 8-AGTP to the enzyme was optimal in the absence of GTP and calcium ions, and inhibited by 2 ~ L M GTP and calcium ions in excess of 2 mM. The binding of GTP to transglutaminase protected the enzyme from trypsin proteolysis and the presence of calcium ions was found to reverse this effect. In addition, the

GTP Binds to Guinea Pig Liver Tramglutaminase 1905

-94

-10

-55

4 4

1 2 3 4 FIG. 6. Effect of GTP and CaCla on trypsin proteolysis of

guinea pig liver transglutaminase. Guinea pig liver transglutaminase (2 pg) was incubated with trypsin (0.2 pg) in presence of 1 mM EDTA ( l a n e I ) , 10 mM CaC12 ( l a n e 2), 0.1 mM GTP ( l a n e 3), and 0.1 mM GTP and 10 mM CaC12 ( l a n e 4). After incubation for 1 h at 37 "C, the samples were analyzed by SDS-PAGE and Western blotting as described in the text.

proteolytic fragments obtained in varying concentrations of GTP, CaC12, and EDTA were quite dissimilar, arguing in favor of varying conformational changes in transglutaminase which are dependent upon the relative concentrations of these cofactors. It seems unlikely that such variations could be due to chelation of calcium ions by GTP because the molar ratio of CaC12/GTP was greater than 20001.

GTP concentrations of 0.1-2 PM were sufficient to block trypsin proteolysis and photolabeling of transglutaminase, yet the level required for inhibition of enzyme activity was much higher, 90 PM. Distinct domains within the enzyme must be perturbed to varying degrees by GTP binding, with the photolabeling and trypsin-sensitive residues severely affected and the catalytic center more resistant. This characteristic of the enzyme appears applicable to CaC12-induced changes in structure as well. Both GTP and CaC12 bind to the enzyme but induce different conformational states which result in altered patterns of proteolysis and photolabeling, as well as variations in the level of enzyme activity.

The nonhydrolyzable GTP analog, GTP-7-S, was found to function as a potent inhibitor of transglutaminase activity, even more so than GTP. The increased level of inactivity produced by GTP-7-S may well relate to changes in Van der Wall's ratios, acidity, charge distributions, and hydrogen- bonding properties that occur when the oxygen in GTP is replaced by sulfur in GTP-7-S (Frey and Sammons, 1985). Alternatively, the higher GTP concentrations required to inhibit transglutaminase in extracts of ABAE cells could be

-94

".>-78 -7 5 -7 0

-44

1 2 3 4 5 6 7

FIG. 7. Effect of increasing CaCll concentrations on trypsin proteolysis of guinea pig liver transglutaminase in presence of 500 nM GTP. Guinea pig liver transglutaminase (2 pg) was incubated with trypsin (0.2 pg) in presence of 500 nM GTP and increasing CaCI2 concentrations. Lanes 1-7 (from left to right) represent incubations carried out in presence of 0, 1 pM, 10 pM, 100 pM, 1 mM, 5 mM, and 10 mM CaCl,; samples were analyzed by SDS- PAGE and Western blotting as described in the text.

due to the presence of endogenous phosphatases or GTPases, degrading the GTP. Phosphatase inhibitors (sodium fluoride andp-nitrophenylphosphate) reduced by 2 to %fold the GTP concentration required to inhibit ABAE cell transglutaminase. GTP-mediated inhibition was not the result of nonspecific effects due to either the presence of polyphosphate residues, the chelation of calcium ions, or the reaction of GTP with primary amines or proteins, since neither factor XII1.- mediated amine incorporation in presence of GTP nor transglutaminase activity in the presence of polyguanylic acid were affected. Presence of M e ions also did not mitigate GTP inhibition of guinea pig transglutaminase. Furthermore, ionized calcium measurements demonstrated no reduction in calcium ions in presence of 1 mM GTP.

Calcium ions were found to partially reverse the GTP inhibition of transglutaminases from all sources studied. We found that the GTP concentration required to inhibit transglutaminase activity in the presence of 0.1 mM CaC12 was reduced nearly 100-fold.' We believe that calcium ions induce a conformational change in transglutaminase, resulting in an alteration of GTP binding site(s). A similar conclusion was reached by Folk et al. (1967) in explaining the partial protection conferred by calcium ions against inactivation of guinea pig liver transglutaminase by the sulfhydryl agent, 5,5'-di- thiobis-(2-nitrobenzoic acid). The balance between local GTP, Ca2+, and other nucleotide concentrations may function to synchronously regulate intracellular transglutaminase activity. It appears likely that the nucleotide triphosphate- binding site may be occupied by several compounds, since ATP and CTP (>1 mM) were found to partially inhibit the activity of guinea pig liver transglutaminase in preliminary studies.

Acknowledgments-The critical comments of Dr. K. V. Rajagopa- lan are gratefully acknowledged.

K. E. Achyuthan and C. S. Greenberg, unpublished observations.

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identification of a guanosine triphosphate-binding site on guinea pig

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