Hydrolysis of gamma :epsilon Isopeptides by Cytosolic Transglutaminases and by Coagulation Factor XIIIa*

(Received for publication, October 3, 1996, and in revised form, February 4, 1997)

Kumarapuram N. Parameswaran , Xiang-Fei Cheng , Ellen C. Chen , Pauline T. Velasco , James H. Wilson and Laszlo Lorand Dagger

From the Department of Cell and Molecular Biology and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Nepsilon -(gamma -glutamyl)lysine cross-links, connecting various peptide chain segments, are frequently the major products in transglutaminase-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the gamma :epsilon linkage. Branched compounds were synthesized, in which the backbone on the gamma -side of the cross-bridge was labeled with a fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl) attached through an epsilon -aminocaproyl linker in the N-terminal position, and the other branch of the bridge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the Nalpha position. Hydrolysis of the cross-link could be followed in these internally quenched substrates by an increase in fluorescence. In addition to the thrombin and Ca2+-activated human coagulation Factor XIIIa, cytosolic transglutaminases from human red cells and from guinea pig liver were tested. All three enzymes were found to display good isopeptidase activities, with Km values of 10-4 to 10-5 M.

Inhibitors of transamidation were effective in blocking the hydrolysis by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of Nepsilon -(gamma -glutamyl)lysine isopeptides.


INTRODUCTION

Apart from obvious differences in substrate specificities for the alpha -carbonyl groups of endo-Lys and Arg residues by papain (EC 3.4.22.2) and for the gamma -carbonyl groups of certain endo-Gln residues by transglutaminases (EC 2.3.2.13), considerable kinetic and mechanistic similarities exist between these two families of enzymes. Both operate by acylation-deacylation pathways, with a Cys thiol in the catalytic center assisted by a His residue (1-6). However, because of the exceptional specificities of transglutaminases for amines mimicking the epsilon -amino groups of Lys side chains in proteins (7-9), this group of enzymes shows a unique ability for generating protein-to-protein Nepsilon -(gamma -glutamyl)lysine cross-links, a post-translational reaction of major biological significance. Transglutaminases are known to participate in various clotting phenomena (7, 10-16), in the assembly of extracellular matrices (17) and of intracellular polymeric structures in cells under Ca2+ stress (18-22), and in apoptosis (23).

While a great deal of attention has been paid to the amine transferase activities of transglutaminases (3, 4, 24), i.e. to the production of Nepsilon -(gamma -glutamyl)lysine bridges and the incorporation of small molecular weight amines into proteins, the isopeptide breaking potential of the enzymes has not yet been explored. Since lack of availability of appropriate substrates may have been a main reason, we embarked on synthesizing gamma -branched peptides with built-in features, which would facilitate the application of fluorescence methodologies for kinetic studies. Two cytosolic transglutaminases of different properties, isolated from human red blood cells (HTg)1 and from guinea pig liver (GTg) respectively, and a recombinant form of the human coagulation Factor XIIIa (rA2*) were employed as enzymes.


MATERIALS AND METHODS

Organic Synthesis

Reagents, solvents, and blocked amino acid derivatives were purchased from Aldrich, Sigma, and Bachem Bioscience Inc. TLC (thin-layer chromatography) was performed on Whatman K6F-Silica gel glass plates (0.25 mm) using the following solvent systems (v/v): (A) chloroform/methanol/glacial acetic acid (10:3:1); (B) chloroform/methanol/2-propanol (10:4:4); (C) n-butanol/glacial acetic acid/water (15:6:5); (D) 1-propanol/water (7:3); (E) propanol/water/concentrated ammonium hydroxide/ethanol (7:4:2:3). Plates were viewed under UV light (at 254 nm and 366 nm for detection of UV absorbing and fluorescent moieties, respectively) or were developed by ninhydrin (0.25% in 1-butanol for N-deblocked peptides) or by hypochlorite (10%) followed by starch/KI spray for N-blocked peptides (26). Melting points were determined with a Büchi apparatus and are uncorrected. After acid hydrolysis, amino acid analyses were kindly carried out by Dr. Thomas J. Lukas of the Department of Molecular Pharmacology and Biochemistry, Northwestern University Medical School, Chicago, IL. Elemental analyses were performed by G. D. Searle Laboratories, Skokie, IL.

Peptide Coupling

To a stirred and cooled (0 °C) 0.5-0.8 M solution of the pertinent Boc amino acid in dry DMF were added equimolar amounts of 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The mixture was stirred at 0 °C for 40 min and then added to a solution of the trifluoracetate salt of the peptide benzyl ester (obtained by acidolytic deblocking of the Boc-peptide benzyl ester described below) in dry DMF, which was pre-neutralized with N-methylmorpholine. The reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 18-36 h. The mixture was evaporated under reduced pressure to remove DMF, and the residue was stirred with 3% sodium bicarbonate for 15 min. The precipitated product was filtered off, or separated by centrifugation, washed with 5% NaHCO3, water, cold 0.5 N HCl, water and dried in vacuo in a P2O5 desiccator for ~18 h. When necessary, the product was reprecipitated from DMF-water. Alternatively, the product was extracted with ethyl acetate, and the organic extract was washed as above, dried over anhydrous Na2SO4, and the solvent evaporated to give the product.

Deblocking of the Boc Group

To 1.0 mmol of the Boc-peptide benzyl ester was added 2 ml of 50% trifluoroacetic acid in anhydrous dichloromethane. The solution was allowed to stand at room temperature for 1 h, and excess trifluoroacetic acid was removed by adding fresh dichloromethane to the mixture followed by evaporation under reduced pressure, then by addition of anhydrous ether to the residue. The precipitated trifluoroacetic acid salt of the peptide benzyl ester was filtered off, washed with anhydrous ether, and dried under vacuum in a desiccator for 2 h before proceeding with the coupling reaction.

Deblocking of the Benzyl Ester Group

The blocked peptide benzyl ester was dissolved in a mixture of DMF/ethanol/water in the approximate volume ratio of 10:20:2 at 50 °C and hydrogenated in presence of 10% Pd/C catalyst for 3.5 h at 50 °C with stirring. After cooling to room temperature, water and 1 N NH4OH to pH 9 were added and the mixture filtered to remove the catalyst through a Celite filter pad. The filtrate was evaporated under reduced pressure, the residue was taken up in a minimum volume of absolute ethanol, and about 10 volumes of anhydrous ether added and cooled to 0 °C. The precipitated product was filtered off, washed with anhydrous ether, and dried in vacuo.

Compounds Used as First Substrates

Dns-Eaca-Gln-Gln-Ile-Val (I)

This was synthesized as described in Ref. 27.

Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (II)

This was synthesized by a multistep procedure as follows. First, Boc-Glu-alpha -benzyl ester was coupled to mono-Z-cadaverine (24), essentially according to the general procedure above and the product isolated from an ethyl acetate extract of the reaction mixture to give an 80% yield of Boc-Glu[gamma -(Cad-Z)]-alpha -benzyl ester, melting point (m.p.) 68-71 °C, RF 0.91 (B), 0.92 (C), 0.87 (D), and 0.89 (E). Catalytic hydrogenation of this intermediate followed by reaction with N-(benzyloxycarbonyloxy)succinimide in presence of NaHCO3 according to the general procedures gave Boc-Glu-[gamma -(Cad-Z)] in 75% yield, RF 0.34 (B), 0.80 (C), 0.66 (D), and 0.72 (E). This was coupled by the above general procedure with the trifluoracetate salt of Gln-Ile-Val-O-Bzl (28) to give an 83% yield of Boc-Glu[gamma -(Cad-Z)]-Gln-Ile-Val-O-Bzl, m.p. 229-231 °C, RF 0.91 (A), 0.89 (C), 0.82 (D), and 0.89 (E). This material was deblocked to remove the Boc group and coupled with equimolar amount of Dns-Eaca according to the general procedures to give the fluorescent peptide intermediate, Dns-Eaca-Glu[gamma -(Cad-Z)]-Gln-Ile-Val-O-Bzl, yield 81%, m.p. (dec.) 238-240 °C, RF 0.94 (A), 0.88 (C), 0.86 (D), and 0.91 (E). This was catalytically hydrogenated to give Dns-Eaca-Glu-(gamma -Cad)-Gln-Ile-Val, RF 0.26 (A), 0.31 (C), 0.55 (D), and 0.63 (E). This was then reacted in 50% aqueous ethanol with a 40% molar excess of 2,4-dinitrofluorobenzene and NaHCO3. The reaction mixture was stirred at 0 °C for 0.5 h and at room temperature for 14 h while protected from light. About 15 drops of concentrated Na2CO3 solution were added to the mixture to raise the pH to 9-9.5 (for decomposing excess 2,4-dinitrofluorobenzene), and ethanol was evaporated under reduced pressure. After adding 5 ml of water, the solution was centrifuged to remove a small amount of precipitate. The clear supernatant was separated, cooled (0 °C), and acidified to pH 2.5 with cold 2 N HCl with stirring. The yellow precipitate was collected by centrifugation, washed with cold water (3 × 3 ml), and finally washed with ether (5 × 5 ml). The precipitate was triturated with ethanol-ether (1:10) to remove the contaminating 2,4-dinitrophenol and dried in vacuo to give a 40% yield of the titled quenched fluorescent peptide, Dns-Eaca-Glu[gamma -(Cad-Dnp)]-Gln-Ile-Val, as a yellow solid, m.p. (dec.) 223-225 °C, RF 0.77 (A), 0.74 (C), 0.69 (D), and 0.83 (E). 1H NMR (Me2SO-d6) was in agreement with the structure. Amino acid analysis gave Glu 2.17 (2), Ile 0.92 (1), and Val 0.91 (1).
<UP>C<SUB>50</SUB>H<SUB>73</SUB>N<SUB>11</SUB>O<SUB>14</SUB>S</UP> · 1/2<UP>H<SUB>2</SUB>O</UP>
<UP>Calculated: </UP><UP>C 54.93% </UP><UP>H 6.82% </UP><UP>N 14.09%</UP>
<UP>Found: </UP><UP>C 55.20% </UP><UP>H 7.16% </UP><UP>N 13.68%</UP>

Dns-Eaca-Glu-[gamma -epsilon -(alpha -Dnp-Lys-NHCH3)]-Gln-Ile-Val (III)

This was synthesized essentially as the gamma -Cad-Dnp analog peptide. First, Z-Lys(epsilon -Boc) was coupled with methylamine hydrochloride in presence of N-methylmorpholine by the general method to give Z-Lys(epsilon -Boc)-NHCH3, yield 92%, m.p. 125-127 °C, RF 0.94 (B), 0.87 (C), 0.77 (D), and 0.85 (E). Removal of Boc group followed by coupling with Boc-Glu-alpha -O-Bzl gave a 94% yield of Boc-Glu-[gamma -epsilon -(alpha -Z-Lys-NHCH3)]-alpha -O-Bzl, m.p. 162-164 °C, RF 0.92 (B), 0.86 (C), 0.82 (D), and 0.87 (E). Further reactions similar to those described for peptide II gave Dns-Eaca-Glu-[gamma -epsilon (alpha -Z-Lys-NHCH3)]-Gln-Ile-Val-O-Bzl in about 60% yield, m.p. (dec.) 234-236 °C, RF 0.9 (B), 0.76 (C), 0.79 (D), and 0.89 (E). This was catalytically hydrogenated and reacted with 2,4-dinitrofluorobenzene by a similar procedure as above to give the titled quenched fluorescent peptide (III), yield 21%, yellow solid, m.p. (dec.) 216-218 °C, RF 0.15 (B), 0.73 (C), 0.68 (D), and 0.84 (E). Amino acid analysis gave Glu 2.0 (2), Val 0.79 (1), and Ile 0.81 (1).

Abz-Eaca-Glu-[gamma -(Cad-Dnp]-Gln-Ile-Val (IV)

Starting with 2-aminobenzoic acid (anthranilic acid), Boc-Abz was prepared according to the literature procedure (29), m.p. 152-154 °C, m.p. (literature) 149-150 °C, RF 0.79 (B), 0.93 (C), 0.76 (D), and 0.77 (E). Coupling of this with epsilon -aminocaproic acid by the general procedure gave a 74% yield of Boc-Abz-Eaca, RF 0.79 (B), 0.79 (C), 0.74 (D), and 0.66 (E). Further reactions similar to those carried out for the synthesis of II above gave a 32% yield of Boc-Abz-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val, yellow solid, m.p. (dec.) 225-227 °C, RF 0.72 (C), 0.68 (D), and 0.8 (E). This was finally deblocked to remove the Boc group according to the general procedure above to give a 66% yield of the trifluoroacetate salt of the titled compound, IV, as a yellow solid m.p. (dec.) 230-232 °C, RF 0.64 (C), 0.56 (D), and 0.74 (E). Amino acid analysis gave Glu 2.37 (2), Ile 0.86 (1), and Val 0.76 (1).

Boc-Glu[gamma -(Cad-Dns)]-Gln-Ile-Val (V)

Coupling of dansylcadaverine (Cad-Dns) with Boc-Glu-alpha -O-Bzl followed by hydrogenation according to the published procedure (30) gave Boc-Glu[gamma -(Cad-Dns)], which was subsequently coupled by the general procedure with the trifluoroacetate salt of Gln-Ile-Val-O-Bzl (28) to give a 76% yield of Boc-Glu[gamma -(Cad-Dns)]-Gln-Ile-Val-O-Bzl, m.p. 210-212 °C, RF 0.94 (A), 0.94 (B), 0.91 (C), 0.86 (D), and 0.91 (E). Catalytic hydrogenation of this material gave the titled fluorescent peptide, V, yield 84%, m.p. (dec.) 212-214 °C, RF 0.8 (A), 0.3 (B), 0.84 (C), 0.74 (D), and 0.88 (E). Amino acid analysis gave Glu 2.1 (2), Ile 0.88 (1), and Val 0.98 (1).

pGlu-Ala-Glu[gamma -(Cad-Dns)]-Gln-Ile-Val (VI)

The above peptide intermediate was deblocked to remove Boc group and coupled with pGlu-Ala according to the general procedures to give pGlu-Ala-Glu[gamma -(Cad-Dns)]-Gln-Ile-Val-O-Bzl, yield 81%, m.p. (dec.) 276-278 °C, RF 0.1 (B), 0.64 (C), 0.68 (D), and 0.79 (E). Finally, hydrogenation, followed by recrystallization, gave a 20% yield of the titled peptide, VI, m.p. (dec.) 259-261 °C, RF 0.18 (A), 0.1 (B), 0.62 (C), 0.65 (D), and 0.73 (E). Amino acid analysis gave Glu 3.23 (3), Ala 1.1 (1), Ile 0.83 (1), and Val 0.83 (1).

Boc-Glu-[gamma -epsilon -(alpha -Dns-Lys-NHCH3)]-Gln-Ile-Val (VII)

First, Dns-Lys-NHCH3 (XIII) described below was coupled with Boc-Glu-O-Bzl and the product hydrogenated to give an 80% yield of Boc-Glu-[gamma -epsilon -(alpha -Dns-Lys-NHCH3)], as an amorphous solid, RF 0.29 (B), 0.73 (c), 0.66 (D), and 0.78 (E). This was coupled by the general procedure with the trifluoroacetate salt of Gln-Ile-Val-O-Bzl (see Ref. 28) to give a 60% yield of Boc-Glu-[gamma -epsilon -(alpha -Dns-Lys-NHCH3)]-Gln-Ile-Val-O-Bzl, m.p. (dec.) 206-208 °C, RF 0.93 (B), 0.81 (C), 0.82 (D), and 0.89 (E). Hydrogenation of this material gave the titled fluorescent peptide, VII, yield 89%, m.p. (dec.) 210-212 °C, RF 0.18 (B), 0.74 (C), 0.68 (D), and 0.79 (E). Amino acid analysis gave Glu 2.24 (2), Ile 0.87 (1), and Val 0.88 (1).

Boc-Glu-[gamma -epsilon -(alpha -Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu (VIII)

First, Boc-Gly-Pro was coupled to Leu-O-Bzl by the general procedure to give a 80% yield of Boc-Gly-Pro-Leu-O-Bzl, m.p. 118-120 °C, RF 0.93 (B), 0.9 (C), 0.82 (D), and 0.88 (E). This peptide was deblocked to remove Boc group and coupled to Boc-Gln-Ile-Val (31) to give a 54% yield of Boc-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl, m.p. 226-228 °C, RF 0.93 (B), 0.8 (C), 0.77 (D), and 0.87 (E). This was subsequently deblocked to remove Boc group and coupled to Boc-Glu-[gamma -epsilon -(alpha -Dns-Lys-NHCH3)] as described under compound VII above to give a 71% yield of Boc-Glu-[gamma -epsilon -(alpha -Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl, m.p. (dec.) 209-212 °C, RF 0.9 (B), 0.79 (C), 0.77 (D), and 0.91 (E). Hydrogenation of this material gave the titled fluorescent peptide, VIII, yield 90%, m.p. (dec.) 216-218 °C, RF 0.23 (B), 0.66 (C), 0.68 (D), and 0.84 (E). Amino acid analysis gave Glu 2.25 (2), Gly 0.94 (1), Leu 1.01 (1), Pro 1.09 (1), Ile 0.79 (1), and Val 0.76 (1).

Dns-Eaca-Ala-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (IX)

This was synthesized by a procedure similar to that for peptide II above. First, Dns-Eaca was coupled with alanine benzyl ester and the product hydrogenated to give a 95% yield of Dns-Eaca-Ala, m.p. 82-85 °C, RF 0.36 (B), 0.78 (C), 0.65 (D), and 0.74 (E). This was then coupled to Glu[gamma -(Cad-Z)]-Gln-Ile-Val-O-Bzl to give a 80% yield of Dns-Eaca-Ala-Glu-[gamma -(Cad-Z)]-Gln-Ile-Val-O-Bzl, m.p. (dec.) 258-261 °C, RF 0.89 (B), 0.83 (C), 0.79 (D), and 0.88 (E). Amino acid analysis gave Ala 1.18 (1), Glu 2.3 (2), Ile 1.0 (1), and Val 0.86 (1). This was hydrogenated and reacted with 2,4-dinitrofluorobenzene by a procedure similar to that above to give the titled quenched fluorescent peptide, IX, yield 11%, yellow solid, m.p. (dec.) 235-237 °C, RF 0.3 (B), 0.68 (C), 0.62 (D), and 0.86 (E). Amino acid analysis gave Ala 1.03 (1), Glu 2.39 (2), Ile 1 (1), and Val 0.9 (1).

Dns-Eaca-Pro-Ala-Gln-Gln-Ile-Val (X)

Boc-Ala-Gln-Gln-Ile-Val-O-Bzl (28) was deblocked with trifluoroacetic acid to remove the Boc group and coupled to Boc-Pro to give Boc-Pro-Ala-Gln-Gln-Ile-Val-O-Bzl, yield 91%, m.p. (dec.) 270-272 °C, RF 0.78 (A), 0.77 (C), 0.73 (D), and 0.8 (E). This was deblocked with trifluoroacetic acid and coupled to Dns-Eaca to give Dns-Eaca-Pro-Ala-Gln-Gln-Ile-Val-O-Bzl, yield 88%, m.p. (dec.) 270-272 °C, RF 0.81 (A), 0.55 (B), 0.63 (C), 0.57 (D), and 0.82 (E). Catalytic hydrogenation of this material gave the titled fluorescent peptide, X, yield 77%, m.p. (dec.) 248-250 °C, RF 0.44 (A), 0.08 (B), 0.55 (C), 0.55 (D), and 0.72 (E). Amino acid analysis gave Glu 2.03 (2), Pro 1.29 (1), Ala 1.28 (1), Ile 0.82 (1), and Val 0.92 (1).

Amine Compounds Used as Second Substrates for Studying the Enzyme-catalyzed Formation of Isopeptides

Dnp-Cadaverine (XI)

This was prepared according to the published procedure (24).

alpha -Dnp-Lys-NHCH3 (XII)

The Z-Lys(epsilon -Boc)-NHCH3 intermediate was catalytically hydrogenated and reacted with 2,4-dinitrofluorobenzene to give Dnp-Lys(epsilon -Boc)-NHCH3, yield 79%, a yellow solid, m.p. 149-151 °C, RF 0.96 (B), 0.88 (C), 0.80 (D), and 0.86 (E). This was deblocked with trifluoroacetic acid according to the general procedure to give an 80% yield of the initially hygroscopic trifluoroacetate salt, which was converted to the hydrochloride derivative by adding 1N HCl in 2-propanol followed by anhydrous ether to give the HCl salt of Dnp-Lys-NHCH3, yellow solid, m.p. 135-137 °C, RF 0.4 (B), 0.49 (C), 0.57 (D), and 0.59 (E).

alpha -Dns-Lys-NHCH3 (XIII)

This was prepared similarly to the Dnp analog, with dansyl chloride replacing the reagent in the preparation to give the HCl salt of Dns-Lys-NHCH3, yield 76%, m.p. 194-196 °C, RF 0.08 (B), 0.38 (C), and 0.58 (E).

Dbc-Cadaverine (XIV)

First, 4-(4-dimethylaminophenylazo)benzoic acid sodium salt was coupled to Boc-cadaverine hydrochloride (32) in presence of two equivalents of N-methylmorpholine by a slightly modified peptide coupling method. A 2-fold molar excess of 1-hydroxybenzotriazole was used in the coupling, and the reaction was carried out at room temperature for 24 h and the product isolated from an ethyl acetate extract of the reaction mixture to give dabcylcadaverine-Boc, a bright red solid, yield 71%, m.p. 159-161 °C, RF 0.97 (A), 0.94 (C), 0.87 (D), and 0.93 (E). 1H NMR (Me2SO-d6) was in agreement with the structure. This was then deblocked with 50% trifluoroacetic acid in anhydrous dichloromethane as described above to give a 77% yield of dabcylcadaverine trifluoroacetate as a red solid, m.p. 177-179 °C, RF 0.33 (A), 0.55 (C), 0.21 (D), and 0.65 (E). The absorption spectrum showed maxima at 470 nm (epsilon  = 23, 610; pH 7.5) and 502 (epsilon  = 45, 860; pH 1.0).

Reference Compound: Boc-Glu-Gln-Ile-Val-Gly-Pro-Leu (XV)

The intermediate in the synthesis of VIII above, Boc-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl was deblocked by removing the the Boc group and was then coupled to Boc-Glu-gamma -O-Bzl to give a 76% yield of Boc-Glu-(gamma -O-Bzl)-Gln-Ile-Val-Gly-Pro-Leu-O-Bzl, m.p. 237-239 °C, RF 0.94 (B), 0.87 (C), 0.80 (D), and 0.86 (E). This was hydrogenated according to the general procedure to give a 76% overall yield of Boc-Glu-Gln-Ile-Val-Gly-Pro-Leu (XV), m.p. (dec.) 249-252 °C , RF 0.06 (B), 0.65 (C), 0.64 (D), and 0.66 (E). Amino acid analysis gave Glu 2.26 (2), Gly 1.04 (1), Pro 1.14 (1), Val 0.8 (1), Ile 0.83 (1), and Leu 1.24 (1).

Enzymatic Studies

Guinea pig liver transglutaminase (GTg; Refs. 33 and 34) and human red blood cell transglutaminase (HTg; Refs. 35 and 36) were purified as described previously and stored at -80 °C. Protein concentrations for GTg were measured by absorbance at 280 nm (epsilon 1 cm1% = 15.8). Protein concentrations for HTg were determined with the BCA protein assay (Pierce), utilizing bicinchoninic acid with bovine serum albumin for standard. Calculations were based on molecular weights of 76,600 for GTg (37) and 80,000 for HTg (35).

Transglutaminase activity was measured in a CytoFluor model 2300 (Millipore, Bedford, MA), upgraded to a model 2350, by monitoring the rate of increase in fluorescence during the transglutaminase-catalyzed incorporation of dansylcadaverine (38) into N,N-dimethylcasein (excitation filter = 360 ± 40 nm; emission filter = 490 ± 40; sensitivity 6). Incubations were carried out at 37 °C in a Millipore 96-well low fluorescence CytoPlate in 125-µl reaction mixtures which comprised 50 mM Tris-HCl, pH 7.5, 0.1 mM dansylcadaverine, 2 mg/ml N,N-dimethylcasein, 1 mM dithiothreitol, 1 mM CaCl2, and 0-0.124 µM GTg or HTg.

Recombinant human factor XIII A subunit (rA2; Ref. 39), a gift from Dr. Paul D. Bishop (Zymogenetics, Seattle, WA), was converted to rA2' by incubating 4-8 µM rA2 with 32-64 NIH units/ml human alpha -thrombin (a gift from Dr. J. W. Fenton III, New York State Department of Health, Albany, NY) in 50 mM N-methylmorpholine, pH 7.5, for 30 min at room temperature. Thrombin activity was then quenched by the addition of a 4-fold excess of hirudin (128-256 units/ml; Sigma). Protein concentration for rA2 was determined using absorbance at 280 nm (epsilon 1 cm1% = 14.9).

Thin Layer and High Performance Liquid Chromatography (TLC and HPLC)

Mixtures of 100 µl comprising 50 mM N-methylmorpholine-HCl, pH 7.5, ionic strength 0.1 (adjusted with NaCl), 100 µM Boc-Glu[gamma -epsilon -(alpha -Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu (VIII) or alpha -Dns-Lys-NHCH3 (XIII), 0.65 µM GTg, 1 mM DTT, and either 1 mM CaCl2 or 1 mM EDTA were incubated at 37 °C for 120 min, when 2 µl of 100 mM EDTA were added to stop the reaction; 25-µl samples were spotted (2 × 10 mm) on a Polygram TLC plate (0.1-mm Polyamide-6, 20 × 20 cm; Macherey & Nagel, Alltech Associates, Deerfield, IL), and separation was accomplished in an ascending mode in aqueous 1% pyridine, pH 5.4, for 60 min (40). The dried plate was photographed under UV light (366 nm).

HPLC separations were also performed on the same mixtures. Approximately 50 µl of sample was mixed with 60 µl of 0.6 M perchloric acid and centrifuged (2 min, 15,600 × g), and 100 µl was injected onto an Ultrasphere C8 column (Beckman, Fullerton, CA) using gradients formed with H2O (containing 0.1% trifluoroacetic acid) and MeCN (containing 0.1% trifluoroacetic acid): from injection to 20 min, linear increase of MeCN to 20%; 20 to 22 min, isocratic 20% MeCN; 22 to 32 min, linear increase of MeCN to 30%; at 32 min, MeCN was eliminated (0.1 min) and the column was then re-equilibrated with 0.1% trifluoroacetic acid in H2O for 15 min. Peaks were detected by absorbance at 220 nm and by fluorescence (lambda exc = 338 nm; lambda em = 500 nm), recorded on a Hewlett-Packard 3390A integrator and collected with a Foxy 200 fraction collector (ISCO, Lincoln, NE) set for detection of slope. Collected fractions were concentrated on a Savant (Farmingdale, NY) Speed-Vac concentrator.

Monitoring Transglutaminase-catalyzed Isopeptide Formation or the Breaking of Isopeptide Bonds by Changes in Fluorescence

Conditions for individual experiments are specified in the figure legends. Fluorescence measurements in the CytoFluor instrument were carried out with an excitation filter = 360 ± 40 nm and an emission filter = 590 ± 35 nm at sensitivities of 7 or 8. Incubations were set up in a CytoPlate at 37 °C in 150-µl reaction mixtures, which, in addition to the specified substrates and inhibitors, comprised 50 mM buffer (either Tris-HCl, N-methylmorpholine-HCl or sodium acetate:acetic acid), 1 mM DTT, 1 mM CaCl2, and one of the transglutaminases or rA2'. Ionic strength was adjusted with NaCl as specified. For monitoring isopeptide formation, fluorescence readings were expressed as a percentage of the initial fluorescence measured in the absence of enzyme. Fluorescence readings for the breaking of the isopeptide bonds were corrected for the initial background fluorescence in the absence of enzyme.

Inhibition of the rA2*-catalyzed hydrolysis of Abz-Eaca-Glu-[gamma -(Cad-Dnp]-Gln-Ile-Val (IV) by 1,3,4,5-tetramethyl-2[(2-oxopropyl)thio]imidazolium chloride (L-682,777 (41); prepared in Me2SO and stored at -20 °C; kindly provided by Dr. Andrew M. Stern of Merck Research Laboratories, West Point, PA) was measured on a SLM (Urbana, IL) model 8000C spectrofluorometer (excitation wavelength = 320 nm, emission wavelength = 410 nm, high voltage = 400 V, gain = 100) at 37 °C. GTP and GTPgamma S (Sigma) were used as equimolar mixtures with MgCl2.


RESULTS AND DISCUSSION

Three representative preparations from the family of transglutaminases, exhibiting different kinetic and physical properties, were employed in this study. The cytosolic transglutaminases were purified from human red cells and from guinea pig liver, whereas the activated form of human fibrin stabilizing factor (Factor XIIIa = A2*) was generated by treatment with thrombin and Ca2+ from the recombinant placental rA2 zymogen. The findings presented in Fig. 1 serve as evidence that transglutaminases can be effective in catalyzing the cleavage of the gamma :epsilon isopeptide bond. In panel A (lane 2), thin layer chromatographic separation was used to demonstrate the production of fluorescent alpha -Dns-Lys-NHCH3, marked as P1, during the liver transglutaminase-catalyzed hydrolysis of the branched peptide substrate: Boc-Glu-[gamma -epsilon (alpha -Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu (VIII). No release of P1, identified by the mobility (RF ~ 0.6) of the reference compound XIII in lane 3, occurred with the enzyme when Ca2+ was replaced by EDTA, as in lane 1. Analysis by HPLC, presented in panel B, confirmed the formation of alpha -Dns-Lys-NHCH3 as the fluorescent product P1 (graph 2) eluting at the position (~14.5 min) of the reference compound XIII (graph 3), while the amount of the starting fluorescent substrate S (i.e. compound VIII, eluting at ~36.5 min) diminished by about 60% (compare graphs 1 and 2). Monitoring by absorbance at 220 nm revealed the production of a non-fluorescent peak, representing the second product of hydrolysis in the Ca2+-containing enzymatic mixture, with an elution time (~32.5 min) corresponding to that of the Boc-Glu-Gln-Ile-Val-Gly-Pro-Leu reference (XV; data not shown). These findings established the catalytic potential of transglutaminases for hydrolyzing the isopeptide bond. However, it became obvious that a more penetrating evaluation of branched substrates would require a different analytical approach, and, with this in mind, fluorescence quenching procedures were explored first for the enzyme-catalyzed conventional reaction of a Gln residue-containing acceptor with a primary amine as the donor substrate.


Fig. 1. GTg-catalyzed hydrolysis of the gamma :epsilon isopeptide bond in the branched substrate: Boc-Glu-[gamma -epsilon -(alpha -Dns-Lys-NHCH3)]-Gln-Ile-Val-Gly-Pro-Leu. The substrate (marked as S, i.e. compound VIII, 100 µM) was incubated (37 °C) with GTg (0.65 µM) in 50 mM N-methylmorpholine-HCl (pH 7.5, µ ~ 0.1 adjusted with NaCl) and 1 mM DTT either in the presence of 1 mM CaCl2 (lane 2 in panel A and graph 2 in panel B) or of 1 mM EDTA (lane 1 and graph 1). After a reaction time of 2 h, separation of product (marked as P1) was accomplished by Polyamide TLC (viewed under UV, panel A) and by HPLC on a C8 column (monitored by dansyl fluorescence, panel B), as described under "Materials and Methods." Identity of the fluorescent P1 product as alpha -Dns-Lys-NHCH3 was established in relation to compound XIII as reference (lane 3 and graph 3). Both P1 and XIII exhibited the same RF ~ 0.6 on polyamide (origin of application marked as O), and eluted at the same position from the C8 column (~14.5 min). Comparison of the substrate peaks (S) in graphs 1 and 2 showed that, under the conditions of this experiment, the hydrolytic reaction progressed to ~60% of completion.
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Isopeptide Formation Monitored by the Quenching of Fluorescence

Acceptor substrates were synthesized with a fluorescent N-terminal blocking group (Dns), whereas the donor substrates (cadaverine or Lys-NHCH3) contained some quenching moiety (Dnp or Dbc) in the N-terminal position. Affinities of the new substrates for the enzymes were sufficiently favorable so that they could be employed at low enough concentrations for minimizing bimolecular quenching in the starting mixtures. However, as the coupling reaction for forming the gamma :epsilon product progressed with the addition of enzymes, a significant drop in fluorescence ensued. This was attributed to the intramolecular quenching effect exerted by the Dnp or Dbc group on the Dns fluorophore concomitant with forming the isopeptide linkage. This approach, tested for a variety of substrate pairs, proved to be highly sensitive for measuring the rate of isopeptide formation. Catalysis by the human red cell and the guinea pig liver enzyme was explored either with Dnp-cadaverine (XI), Dbc-cadaverine (XIV), or alpha -Dnp-Lys-NHCH3 (XII) as donor, in conjunction with Dns-Eaca-Gln-Gln-Ile-Val (I) as the acceptor substrate. Figs. 2 and 3 illustrate the findings for the tissue type of transglutaminases. Fig. 2 pertains to the reaction of the Dns-labeled acceptor (I) with Dnp-Lys-NHCH3 (XII) as promoted by guinea pig liver transglutaminase, whereas Fig. 3 presents the data for the human red cell enzyme-catalyzed reaction between I and Dbc-cadaverine (XIV). Dns-Eaca-Pro-Ala-Gln-Gln-Ile-Val (X) could be used as first substrate either with Dbc-cadaverine (XIV) or with Dnp-cadaverine (XI) as second substrate for following the coupling reactions catalyzed by rA2* (data not shown). These results guided our synthetic work for designing gamma -branched peptides for examining the isopeptide breaking potentials of transglutaminases.


Fig. 2. Quenching of fluorescence in the reaction of Dns-Eaca-Gln-Gln-Ile-Val with alpha -Dnp-Lys-NHCH3, catalyzed by GTg. Dns-Eaca-Gln-Gln-Ile-Val (compound I, 0.2 mM) and Dnp-Lys-NHCH3 (compound XII, 0.5 mM) were incubated at 37 °C with 0.05 µM (bullet ), 0.10 µM (down-triangle), 0.21 µM (black-down-triangle ), or no (open circle ) GTg in 1 mM CaCl2, 1 mM DTT, and 50 mM Tris-HCl, pH 7.5. Fluorescence, measured on the CytoFluor (sensitivity 8), is expressed as a percentage of the initial fluorescence reading in the absence of GTg.
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Fig. 3. Decrease of fluorescence in the HTg-catalyzed reaction between Dns-Eaca-Gln-Gln-Ile-Val and dabcylcadaverine. Dns-Eaca-Gln-Gln-Ile-Val (compound I, 0.1 mM) and dabcylcadaverine (Dbc-Cad, compound XIV, 25-100 µM) were incubated at 37 °C with HTg (0.55 µM) in 1 mM CaCl2, 1 mM DTT, and 50 mM methylmorpholine-HCl, pH 7.5. Fluorescence, measured on the CytoFluor (sensitivity 7), is expressed as a percentage of the initial fluorescence reading in the absence of HTg.
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Transglutaminase-catalyzed Breaking of the Isopeptide Bond

We synthesized a number of intramolecularly quenched compounds containing an isopeptide linkage (described under "Materials and Methods") and followed the actions of the enzymes by the increase in fluorescence resulting from the release of the quenching moiety (Dnp) attached to the leaving amine group. On the gamma  side of the backbone the substrates carried either a Dns or an Abz fluorophor in an N-terminal position.

Figs. 4 and 5 show the progression curves for the hydrolysis of the Dns-Eaca-Glu-[gamma -epsilon (alpha -Dnp-Lys-NHCH3)]-Gln-Ile-Val (III) (10-4 M) substrate with the guinea pig liver and of compound II with the human red blood cell enzyme, respectively. Approximately twice as much GTg (0.43 µM) was used for demonstrating the hydrolytic cleavage of compound III (0.1 mM) in Fig. 4 than the concentration of the enzyme (0.21 µM) for generating the highest rate of cross-bridge formation in the coupling reaction between compounds (I; 0.24 mM) and XII (0.5 mM), depicted by solid triangles (black-down-triangle ) in Fig. 2. Because of inherent ambiguities in comparing rate constants for the reaction of a single substrate (as in the experiment in Fig. 4) with those for a two-substrate reaction (as in Fig. 2), we did not attempt to draw kinetic comparisons between these different enzymatic processes.


Fig. 4. Increase of fluorescence accompanying the reaction of Dns-Eaca-Glu-[gamma -epsilon -(alpha -Dnp-Lys-NHCH3)]-Gln-Ile-Val with GTg. Dns-Eaca-Glu-[gamma -epsilon -(alpha -Dnp-Lys-NHCH3)]-Gln-Ile-Val (compound III, 100 µM) was incubated at 37 °C with GTg (0.43 µM) in 1 mM CaCl2, 1 mM DTT, and 50 mM methylmorpholine-HCl, pH 7.5. Fluorescence, measured on the CytoFluor (sensitivity 8), is expressed as the increase in fluorescence compared with the initial fluorescence reading without enzyme.
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Fig. 5. Reaction of HTg with Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val. HTg (0.55 µM) was incubated at 37 °C with 2 µM (bullet ), 3 µM (down-triangle), 5 µM (black-down-triangle ), 8 µM (square ), or 10 µM (black-square) Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (compound II) in 1 mM CaCl2, 1 mM DTT, 50 mM methylmorpholine-HCl, pH 7.5; for each experiment ionic strength was adjusted to 0.1 with NaCl. Fluorescence, measured on the CytoFluor (sensitivity 8), is expressed as the increase in fluorescence compared with the initial fluorescence reading in the absence of HTg.
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Some of the gamma -branched peptides also satisfied the more restrictive specificity requirement of human Factor XIIIa. Fig. 6 presents our data by for the hydrolysis of Dns-Eaca-Ala-Glu[gamma -(Cad-Dnp)]-Gln-Ile-Val (IX) by rA2* and Fig. 7 for the reaction of the same enzyme with Abz-Eaca-Glu[gamma -(Cad-Dnp)]-Gln-Ile-Val (IV). Because of the limitations of the CytoFluor plate reader, fluorescence of the Abz-blocked compound (lambda exc = 320 nm and lambda em = 410 nm), could only be monitored in the spectrophotofluorimeter. Fig. 7 shows that the hydrolytic activity of rA2* on the gamma -branched peptide could be abolished by an active site-directed inhibitor of Factor XIIIa: 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (41). At the same concentration (10-4 M), this compound also inhibited totally the hydrolysis of Dns-Eaca-Glu[gamma -(Cad-Dnp)]-Gln-Ile-Val (II) by the human red cell transglutaminase (5.5 × 10-7 M) measured in the CytoFluor under similar conditions (data not shown).


Fig. 6. The rA2*-catalyzed reaction of the Dns-Eaca-Ala-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val substrate. Dns-Eaca-Ala-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (compound IX, 40 µM) was incubated at 37 °C with 0.5 µM (bullet ), 1 µM (down-triangle), 2 µM (black-down-triangle ), or 4 µM (square ) thrombin-treated rA2 in 1 mM CaCl2, 1 mM DTT, and 50 mM methylmorpholine-HCl, pH 7.0. Fluorescence, measured on the CytoFluor (sensitivity 8), is expressed as the increase in fluorescence compared with the initial fluorescence reading without rA2 added.
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Fig. 7. Inhibition of the rA2*-catalyzed reaction of Abz-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val by 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride. Abz-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (compound IV, 60 µM) was incubated at 37 °C with thrombin-treated rA2 (8 µM) in 1 mM CaCl2, 1 mM DTT, and 50 mM methylmorpholine-HCl, pH 6.5, in the absence (solid line) or presence (broken line) of 0.1 mM 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride. Continuous fluorescence measurements were taken on a SLM spectrofluorometer (excitation wavelength = 320 nm, emission wavelength = 410 nm, high voltage = 400 V, gain = 100). The enzymatic reaction was initiated by the addition of rA2', which would be immediately converted to rA2*, at 300 s.
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Hydrolysis of Dns-Eaca-Glu[gamma -(Cad-Dnp)]-Gln-Ile-Val (II) by the human red cell transglutaminase proceeded fastest in the pH 6.5-7.5 range (ionic strength ~ 0.1), with reaction rates falling off on either side of this pH range (Fig. 8). For the guinea pig liver enzyme, the apparent optimum for the hydrolysis of the same substrate was in the pH 5.5-7 range (data not shown).


Fig. 8. The pH profile of the reaction of Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val with HTg. Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (compound II, 10 µM) was incubated at 37 °C with HTg (0.55 µM) in 1 mM CaCl2, 1 mM DTT at various pH values set either with 50 mM sodium acetate/acetic acid (solid symbols) or with 50 mM methylmorpholine-HCl (open symbols); ionic strengths were adjusted to 0.1 with NaCl. Fluorescence, measured on the CytoFluor (sensitivity 8), is expressed as the increase in fluorescence compared with the initial fluorescence readings of the enzyme-free solutions.
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Tissue transglutaminases are negatively regulated by GTP for incorporating amines into protein substrates (42-44). We tested the effects of equimolar GTP·Mg2+ complexes on the isopeptidase activities of the enzymes. As illustrated in Fig. 9, GTP was found to exert a very strong inhibitory effect on the hydrolysis of Dns-Eaca-Glu[gamma -(Cad-Dnp)]-Gln-Ile-Val (II) by the human red cell enzyme (I50 ~ 5-10 × 10-6 M GTP). However, even the ~90% inhibition caused by 2 × 10-5 M GTP·Mg2+ at low Ca2+ (10-3 M), could be overcome substantially at higher concentrations of Ca2+ (Fig. 10). This finding supports the concept (43) that the binding of GTP causes a reduction in the Ca2+ sensitivity of transglutaminase. The inhibitory effect of GTPgamma S was indistinguishable from that of GTP (data not shown).


Fig. 9. GTP·Mg2+ inhibits the reaction of Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val with HTg. Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (compound II, 10 µM) was incubated at 37 °C with HTg (0.55 µM) in 1 mM CaCl2, 1 mM DTT, 50 mM methylmorpholine, pH 6.5, and 1 µM (down-triangle), 2 µM (black-down-triangle ), 5 µM (square ), 10 µM (black-square), 20 µM (triangle ), or no (open circle ) GTP. Fluorescence, measured on the CytoFluor (sensitivity 8), is expressed as the increase in fluorescence compared with the initial fluorescence reading without HTg.
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Fig. 10. Excess Ca2+ can override the inhibitory effect of GTP·Mg2+ in the reaction of Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val with HTg. Dns-Eaca-Glu-[gamma -(Cad-Dnp)]-Gln-Ile-Val (compound II, 10 µM) was incubated at 37 °C with HTg (0.55 µM) and GTP·Mg2+ (20 µM) in 50 mM methylmorpholine-HCl of pH 6.5, 1 mM DTT, and 1 mM (bullet ), 2 mM (down-triangle), 4 mM (black-down-triangle ), or 6 mM (square ) CaCl2. Fluorescence, measured on the CytoFluor (sensitivity 8), is expressed as the increase in fluorescence compared with the initial fluorescence reading with HTg. The broken line (open circle ) represents the reaction at 1 mM CaCl2 in the absence of GTP.
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Transglutaminases are known to catalyze the hydrolysis of protein or peptide-bound Gln to Glu residues (45), and also to hydrolyze nitrophenyl and thiocholine esters (3, 4, 24). The hydrolytic nature of transglutaminases is brought even more to the forefront by the examples provided in this paper for the breaking of isopeptide linkages, with high affinities for the gamma -branched substrates (e.g. Km ~ 10-5 M for substrate II by the human red cell enzyme; Fig. 5). Inhibition by the active-site directed blocking agent: 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]-imidazolium chloride (Fig. 7) and also by GTP or GTPgamma S (Figs. 9 and 10), which modulate the Ca2+ sensitivities of cytosolic transglutaminases, show that the same functional domains are involved in the expression of isopeptidase activities as in the well studied transamidating reactions promoted by these enzymes.

Claims have been made (46, 47) and refuted (48) for having isolated an isopeptidase, called destabilase, from leech saliva with specificity for hydrolyzing the Nepsilon -(gamma -glutamyl)lysine bonds between the gamma -chains of solubilized fibrin. The primary structure of destabilase, derived from the cDNA clone, does not share significant homology with transglutaminase (49). Based on the findings described in the present paper, it may be suggested that if a select group of enzymes exists with properties that would uniquely define them as isopeptidases, they would probably also display transamidating activities, the characteristic attributes of transglutaminase. One of the enzymes, Factor XIIIa, which was shown in our experiments to exhibit isopeptidase activity (Figs. 6 and 7), has been reported to actually hydrolyze the cross-link formed between alpha 2-plasmin inhibitor and fibrinogen, and to a lesser extent also the cross-link between the inhibitor and fibrin (50, 51). Altogether, our findings with the cytosolic enzymes suggest that transglutaminases may play a more dynamic role in cell biology than hitherto envisaged, not only by catalyzing the formation but also the breaking of Nepsilon -(gamma -glutamyl)lysine bonds.


FOOTNOTES

*   This work was supported by Grants HL-16346, HL-02212 and EY-03942 from the National Institutes of Health. This work was presented as a poster at the ASBMB/ASIP/AAI Joint Meeting, New Orleans, LA, June, 1996 (25).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.
Dagger    To whom correspondence should be addressed. Tel.: 312-503-0591; Fax: 312-503-0590.
1   The abbreviations used are: HTg, human red blood cell transglutaminase; GTg, guinea pig liver transglutaminase; Boc, tert-butyloxycarbonyl; Z, benzyloxycarbonyl; Dns or dansyl, 5-(dimethylamino)-1-naphthalenesulfonyl; Cad, cadaverine (or 1,5-diaminopentane); Dns-Cad or Cad-Dns, dansylcadaverine (or N-(5-aminopentyl)-5-(dimethylamino)-1-naphthalenesulfonamide); Eaca, epsilon -aminocaproyl; O-Bzl, benzyl ester; Dnp, 2,4-dinitrophenyl; Dnp-Cad or Cad-Dnp, dinitrophenylcadaverine; Abz, 2-aminobenzoyl; DMF, N,N-dimethylformamide; pGlu, pyroglutamyl; Dbc or dabcyl, 4-[4-(dimethylamino)phenylazo]benzoyl; rA2, the recombinant human Factor XIII subunit A dimer; DTT, dithiothreitol; MeCN, acetonitrile; HPLC, high performance liquid chromatography; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; dec., with decomposition.

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