Enzimology
 

Tissue transglutaminase or type II transglutaminase (tTG) is a Ca2+ dependent enzyme catalyzing an acyl transfer reaction in which new g amide bonds are formed between a g-carboxamide group of peptide-bound glutamine residues and various primary amines.

Binding of Ca2+ ions to tTG and exposure of the active site cysteine are essential for enzymatic activity. The active site cysteine reacts with the g-carboxamide of the glutamine forming a g-glutamyl thioester and releasing ammonia. The transient acyl-enzyme intermediate then reacts with any nucleophilic primary amine, yielding either an isopeptide bond or a (g-glutamyl) polyamine bond.

Crosslinking either through a polyamine linker or through the formation of the Ne(glutamyl)-lysine bridge may be not the only reaction catalyzed by transglutaminases. Their catalytic mechanism, in fact, is very similar to papain's and can function also in a hydrolytic mode (1). The hydrolysis of a single Gln to a Glu residue in a protein, though representing a charge of just a few kilocalories in the thermodynamic behaviour of the side chain, can have an enormous effect on the equilibria governing the conformation, the oligomeric association-dissociation and the interactions of the macromolecule with other proteins.

Reactions catalyzed by transglutaminases are here reported (2). The backbones or peptide substrates carrying TG-reactive Gln (i.e. acceptor) and Lys (i.e. donor) side chains, are illustrated by rectangles and ovals, respectively. Reactions I and V reflect the hydrolytic nature of this group of enzymes, while transamidating possibilities are shown in reactions II-IV.

TG has a broad specificity for primary amine acceptors (peptide bound lysines or polyamines), but in contrast relatively few proteins contain glutamine residues that form acyl-enzyme intermediates.

Tissue distribution and regulation of expression

Tissue transglutaminase is a 75 kDa monomeric globular protein expressed in the majority of cells and tissues. Its subcellular localization is in the cytosolic fraction and no interactions with other specific subcellular compartments are described. It is translated as a fully active enzyme and there is no evidence for a proteolytic activation (3).

Cells such as endothelial cells, vascular smooth muscle cells, platelets, and epithelial cells of the lens express the enzyme constitutively and accumulate high levels of active enzyme (4). In other cells, such as neurons and skeletal muscle cells, the tTG constitutive expression is very low and not easily detectable.

The promoter of the tTG gene is constitutively active, due to the presence of a CAAT box as well as of four SP1 sites. Two SP1 sites are located directly upstream of the TATA box elements and two are in the 5’UTR (5). The presence of a constitutively active promoter suggests that the tTG gene expression is regulated by negative or tissue specific elements; nevertheless no cis-regulatory regions responsible for cell and tissue specific expression have been identified so far. Alternatively, posttranslational modifications of the protein structure could be responsible for the regulation of the catalytic activity of the enzyme in response to specific stimuli.

Since tTG is expressed in the vast majority of cells and tissues, it has been also implicated in a wide variety of functions resulting in intra and/or extracellular structural alterations. These include modeling of the extracellular matrix (6), stimulus-secretion coupling (7), receptor mediated endocytosis (8), cell differentiation (9), tumor growth (10) and programmed cell death (11).

During apoptosis tTG expression is increased and its catalytic function is activated (11). The fast increase in the expression of the enzyme in cells undergoing programmed cell death may be due to a specific induction of the enzyme or rather to the loss of factors that normally suppress its expression. Cell death may serve to unmask the activity of the constitutive core-promoter of the tTG gene and in turn this may lead to the accumulation of the enzyme that occurs in many dying cells.

Immunohistological studies together with the isolation of highly polymerized protein products from apoptotic cells have established tissue transglutaminase as an useful biochemical marker of programmed cell death. Its precise role in the death process, though, is still under discussion (12-14). The enzyme could participate to the death program as part of the killing mechanism or alternatively it could modulate the course of apoptosis by temporary stabilization of the dying cell.
 

The detection of the tTG protein can be pursued by different strategies: i) evaluation of the catalytic activity of the enzyme, ii) identification of the protein by immunochemical methods, and iii) evaluation of the products of the crosslinking reaction.
 

The measurement of tTG activity is based on the incorporation of a labeled amine into N,N’-dimethylcasein. The use of dimethylcasein, where the lysine side chains are blocked by methylation, instead of casein as the substrate protein prevents the intrachain reaction between glutamyl and lysine residues and favours the binding with amino-groups of polyamines.

Three methods, differing in the usage of the labeled amine substrate and in the requirement for different pieces of equipment, are here described.
 

• The first is based on the incorporation of a radioactive polyamine (e.g. [1,4(n)-3H]putrescine dihydrochloride) into the N,N’-dimethylcasein substrate.
• The second is based on the incorporation of a fluorescent polyamine (e.g. monodansyl-cadaverine) into the N,N’-dimethylcasein substrate.
• The third is based on the incorporation of a biotin labeled polyamine [e.g. N-(5’-aminopentyl)biotinamide] into the N,N’-dimethylcasein substrate.
 

This assay is based on the incorporation of a radiolabeled polyamine (either [H3]putrescine or [C14]putrescine) into N,N'-dimethylcasein. The proteins are subsequently precipitated by the addition of TCA, washed and counted in a liquid scintillation spectrometer (15).

Materials

• Prepare two reaction mixture 10x, one (A) with and one (B) without casein
      A)    Tris HCl 1 M pH 8.3
              CaCl2 50 mM
              NaCl 1.5 M
              N,N’-dimethylcasein 2 mg/ml

      B)    Tris HCl 1 M pH 8.3
              CaCl2 50 mM
              NaCl 1.5 M

• DTT 100 mM
• [1,4(n)-3H]putrescine dihydrochloride 100 mCi/ml
• Purified tTG from Guinea pig liver 1-5 mg/sample (Sigma Chemical Co.)
• Crude extracts 50-200 mg/sample
• Test samples should be prepared in Tris HCl 0.1 M pH 8.3, supplemented with EDTA 1 mM and protease inhibitors (e.g. Leupeptin 0.1 mM, PMSF 1 mM)
 

Special equipment

• Liquid scintillation spectrometer
 

Methodology

• In order to measure the incorporation of putrescine both in the presence and in the absence of substrate (N,N’-dimethylcasein), set up two samples adding, respectively, 0.03 ml of sln.A and 0.03 ml of sln.B in Eppendorf microfuge tubes.
• Add 15 ml of DTT 100 mM
• Add crude extracts (50-100 mg) or purified tTG (0.05-1 mg) in Tris HCl 0.1 M pH 8.3 supplemented with protease inhibitors.
• Add H2O to 0.3 ml final volume
• Add [3H]putrescine 0.2-1 mCi/sample
 

Each sample, therefore, must have a final volume of 0.3 ml and the following composition:

Tris HCl 0.1 M pH 8.3
CaCl2 5 mM
NaCl 0.15 M
DTT 5 mM Note: DTT must be added just before the incubation.
N,N’-Dimethylcasein 0.2 mg/ml
[3H]putrescine 0.2-1 mCi/sample
Crude extracts (50-100 mg) or purified tTG (0.05-1 mg)
 

• Incubate 90 min at 37°C
• Stop the reaction by addition of 100 ml of TCA 50% and 10 ml of BSA 1 mg/ml to each sample.
• Let sit overnight at 4°C
• Collect precipitates by a 10 min centrifugation at 10000 rpm in a microfuge (e.g. Haereus Biofuge), wash the pellet once with 200 ml of TCA 10%, once with ethyl alcohol, once with a 50% ethyl alcohol 50% ethyl ether mix and finally with 100% ethyl ether. Air dry the pellet and dissolve it in 1% SDS vortexing at maximum speed and boiling. Mix the sample with 5 ml of scintillation fluid (e.g. Insta Gel, Packard Instrument Co.) and analyse it in a liquid scintillation spectrometer (e.g. Tri Carb, Packard Instrument Co.).
 
 

tTG activity can be measured in cell extracts or alternatively in purified proteins. tTG from guinea pig liver, commercially available from Sigma, is a very useful positive control for this test. This enzyme has to be equilibrated in Tris 0.1 M pH 8.3 and kept on ice until the beginninng of the reaction. Good values are obtained with 0.05-1 mg of the commercial enzyme.

Although tTG is described as an ubiquitous enzyme, its activity is detectable in endothelial and smooth muscle cells, in arteries, in veins and in capillaries but it might be difficult to measure it in other cell types and tissues. In the latter case, tTG activity may result undetectable either because the enzyme concentration is below treshold or because post-translational modifications or intracellular inhibitors could affect its catalytic activity.

This method is very efficient when guinea pig liver tTG is used. On the contrary, very low values can be obtained with cell extracts. In such a case, it is necessary to set up the assay in triplicate in order to get reliable results.

The protocol here described includes several washes with toxic and dangerous reagents, such as TCA and ethyl ether. This procedure is tedious and time-consuming, but it yields more precise results than simply washing the precipitates on glass fiber filters.

When the tTG activity is determined in crude extracts, the proteins present in the sample can compete with [3H]putrescine. A two-threefold increase in the amount of [3H]putrescine per sample can solve the problem.
 

The fluorescent molecule monodansyl cadaverine, i.e. N-(5’-aminopentyl)-5-dimethylamino-1-naphtalensulfonamide (Serva Fine Biochemicals) can be used as the amine donor in the tTG activity detection assay (16).

The protocol is identical to the one described in paragraph 1), except that the washed pellet is dissolved in urea 4 M, 2-mercaptoethanol 0.5 mM and is subjected to SDS-PAGE. The acrylamide gels are then fixed in 25% ethyl alcohol, 10% acetic acid and photographed on a 300 nm UV screen using a Polaroid camera. Exposure time is 14 min when positive/negative 665 instant pack films, 80 ASA, are used (17).
 

• Prepare two reaction mixture 10x, one (A) with and one (B) without casein

A) Tris HCl 1 M pH 8.3
     CaCl2 50 mM
     NaCl 1.5 M
     N,N’-dimethylcasein 2 mg/ml

B) Tris HCl 1 M pH 8.3
     CaCl2 50 mM
     NaCl 1.5 M

• DTT 100 mM
• Monodansylcadaverine 39 mM. From a 50 mM stock solution in methyl alcohol (stored at -20° C) take 7.5 ml and dilute in 300 ml Tris HCl 0.1 M pH 8.3
• Purified tTG from Guinea pig liver 1-5 mg/sample (Sigma)
• Crude extracts 50-200 mg/sample
• Test samples should be prepared in Tris HCl 0.1 M pH 8.3, supplemented with EDTA 1 mM and protease inhibitors (e.g. Leupeptin 0.1 mM, PMSF 1 mM)

Special equipment

• 300 nm UV screen
• Polaroid camera with positive/negative 665 instant pack film, 80 ASA

• In order to measure the incorporation of dansylcadaverine both in the presence and in the absence of substrate (N,N’-dimethylcasein), set up two samples adding, respectively, 0.03 ml of sln.A and 0.03 ml of sln.B in Eppendorf microfuge tubes.
• Add 15 ml of DTT 100 mM
• Add crude extracts (50-100 mg) or purified tTG (0.05-1 mg) in Tris HCl 0.1 M pH 8.3 supplemented with protease inhibitors.
• Add 30 ml Monodansylcadaverine 39 mM
• Add H2O to 0.3 ml final volume

Each sample, therefore, must have a final volume of 0.3ml and the following composition:
 

Tris HCl 0.1 M pH 8.3
CaCl2 5 mM
NaCl 0.15 M
DTT 5 mM (Note: DTT must be added just before the incubation)
N,N’-Dimethylcasein 0.2 mg/ml
Monodansylcadaverine 3.9 mM
Crude extracts (50-100 mg) or purified tTG (0.05-1 mg)
 
• Incubate 90 min at 37°C
• Stop the reaction by addition of 100 ml of TCA 50% and 10 ml of BSA 1 mg/ml to each sample.
• Let sit overnight at 4°C
• Collect precipitates by a 10 min centrifugation at 10000 rpm in a microfuge (e.g. Haereus Biofuge)
• Wash the pellet once weled polyamine (e.g. N-(5’-aminopentyl)biotinamide) into N,N’-dimethylcasein.

This method is an ELISA based on the incorporation of a biotin labeled amine into the N,N'-dimethylcasein substrate bound to an immunoplate. The tTG activity is then revealed by addition of streptavidine conjugated with alkaline phosphatase (AP) or horseradish peroxidase (HRPO) (18).
 
 

• N,N’-dimethylcasein 10-20 mg/ml in PBS or in NaHCO3 50 mM for coating the immunoplates
• 2% (w/v) powdered skimmed milk in PBS
• Reaction mixture 10x

• DTT 100 mM
• N-(5’-aminopentyl)biotinamide 5 mM (Molecular Probes Inc.). It can be stored at -70°C for up to 2 weeks.
• Purified tTG from Guinea pig liver 1-5 mg/sample (Sigma)
• Crude extracts 50-200 mg/sample. Total volume maximum 0.2 ml/sample
• Test samples should be prepared in Tris HCl 0.1 M pH8.3, supplemented with EDTA 1 mM and protease inhibitors (e.g. Leupeptin 0.1 mM, PMSF 1 mM).
• Streptavidin AP- or HRPO-conjugated 200 mg/ml
• Phosphatase substrate (Sigma 104 Phosphatase Substrate Tablets, Sigma) or peroxidase substrate (BM Blue POD Substrate, Boehringer)
• TBST solution

Equipment

• Flat bottom, high binding 96 wells immunoassay microtiter plates (Nunc Maxisorp)
• ELISA reader provided with the appropriate filters (wavelenght: 405 nm for Substrate 104; 495 nm for POD solution)

• Coat the immunoplates by adding 200 ml of N,N’-dimethylcasein (10-20 mg/ml in PBS or NaHCO3 50 mM) to each well
• Incubate at 4°C overnight or at 37°C for 1 hour
• Wash with PBS and add 200 ml/well of 2% milk in PBS to block free protein-binding sites. Incubate for 1 hour at 37°C
• Wash three times with PBS. The washed plates can be stored at -20 for up to 4 months
• Set up the samples adding to the crude extracts (50-100 mg/well) or to the purified tTG (1-10 mg/well) the appropriate amount of reaction mixture 10x and of DTT 100 mM for a final reaction volume of 200 ml
• Add N-(5’-aminopentyl)biotinamide, 0.5 mM final concentration
• Adjust the final volume (200ml) with H20

Tris HCl 0.1 M pH 8.5
CaCl2 5 mM
NaCl 0.15 M
DTT 5 mM (Note: DTT must be added just before the incubation)
0.5 mM biotin labeled amine.
Crude extracts (50-200 mg) or purified tTG (1-10 mg)

• Prepare negative controls replacing cell extracts with extraction buffer
• Incubate the plate at 37°C for 1 hour
• Wash the plate several times with TBST solution
• Dilute streptavidin 1-2 mg/ml in TBST supplemented with 2% milk. Add 200 ml per well and incubate for 1 hour at room temperature
• Wash the plate several times (at least five) with TBST solution
• Develop the reaction by adding 200 ml of the appropriate substrate (Substrate 104 for AP-labeled and POD Substrate for HRPO-labeled streptavidin)
• Read the plate at 405 nm wavelenght when the yellow substrate 104 is used, and at 495 nm when the blue POD is used
• Determine tTG activity by comparison of the absorbance values per min at the addition of substrate and after 30 min. Subtract the negative controls' values. Since the differences are in the order of magnitude of 0.1 OD, each measurement must be performed in triplicate

Detection and localization of tTG protein

Detection and localization of the tTG protein can be pursued directly via biochemical and immunochemical methods (enzymatic assays and western blot analysis) or indirectly using molecular biology techniques (mRNA detection by in situ hybridization).

Hereinafter are listed few studies employing antibodies against tTG. Monoclonal antibodies have been used by P.J. Birckbichler et al. (19, 20), by A. Monsonego et al. (21) and by A.V. Trejo-Skalli et al. (22). Polyclonal antibodies have been used by G. Melino et al. (12), by L. Fesus et al. (11) and by Z. Chowdhury et al. (4). Some of them are suitable for immunohystochemistry and immunocytochemistry, others for western blot analysis.

These reagents are mainly used to detect by immunohystochemistry the increased levels of tTG described in apoptosis.

On the other hand, it has been suggested that tTG activity could be regulated by post-translational modifications of the enzyme occuring during the cell death process (11).

The identification of such a modification could represent a convenient marker of the progress through the different steps of apoptosis, as much as is the cleavage of PARP (poly(ADP-ribose)polymerase). The digestion of PARP by caspase 3 (apopain-CPP32) is in fact used as a marker of the early stages of the apoptotic process (23).

We have recently found that tTG is cleaved in cells undergoing the late stages of apoptosis (manuscript in preparation). The availability of a reagent (e.g. scFv (24)) detecting also the cleaved product allows to monitor the progress of apoptosis by western blot analysis.

 
 

• scFv specific for Guinea pig liver tTG, selected from a phage display library.
• Anti myc mAb (clone 9E10, from ATCC) specific for the scFv tag sequence.
• Anti mouse IgG1 HRPO-labeled (Southern Biotechnology Associates, Inc.)
• Lysing solution for cell extracts

TrisHCl 0.1 M pH 8.3
Triton-X-100 1%
EDTA 1 mM
protease inhibitors (Leupeptin 0.1 mM, PMSF 1 mM).
• Micro BCA Protein Assay Reagent (Pierce), for determination of protein concentration
• TBST solution
TrisHCl 10 mM pH 8.0N
NaCl 150 mM
Tween 20 0.001% (v/v)

• Resuspend a 107 cells pellet in 100 ml of lysing solution. Let sit 30 min on ice, then spin at 10000 rpm for 10 min at 4°C in an Eppendorf microfuge
• Normalize the extracts for protein concentration
• Load a 10% SDS-PAGE gel with 20 mg/lane of crude extracts and with 1-5 mg/lane of guinea pig liver tTG as control
• Process the gel by standard western blot technique
• Saturate the nitrocellulose filter by soaking in TrisHCl 0.1 M pH 8.5 supplemented with 4% powdered skimmed milk, for at least 1hour at room temperature
• Rinse the nitrocellulose filter in TBST
• Incubate with scFv anti-tTG plus anti-myc mAb (10mg/ml final dilution) for 1 hour at room temperature
• Wash with TBST. Four washes 10 min each at room temperature
• Incubate with HRPO-conjugated goat anti-mouse IgG1 antibody, 1 mg/ml in TBST 2% milk, for 1 hour at room temperature
• Wash with TBST as above
• Develop the assay by ECL (Enhanced Chemioluminescence) western blotting detection reagents (Amersham)

This method has been developed in the attempt to identify a molecular marker suitable for the detection of cells irreversibly committed to apoptosis.

A scFv selected for reactivity with guinea pig liver tTG and specific for the carboxyl terminal portion of human tTG, detects in western blot analysis a single 75 kDa band in cytoplasmic extracts from human thymocytes as well as from a large panel of human cell lines. In crude extracts from human thymocytes undergoing cell death following dexamethasone treatment, a new 48 kDa band is detected. A similar band is also observed in extracts from human tumor cell lines that undergo DNA fragmentation in the presence of Cycloheximide (CHX) or Actinomycin D (ActD). These findings suggest a correlation between the apoptotic process and the appearance of the 48kDa band (manuscript in preparation).

The tTG cleavage yielding the 48 kDa fragment occurs in the late phases of apoptosis, simultaneously to DNA fragmentation and about 8 hours later than PARP cleavage.

Measurement of e(g -glutamyl)lysine cross-link and isopeptide

e(g-glutamyl)lysine cross-links are the footprints of past tTG activity. The presence of isopeptide bonds in tissues and in apoptotic bodies can be successfully quantitated. Since the stability of the side chain isopeptide bridge is not different from that of the peptide backbone, there are no chemical methods of obtaining differential hydrolysis. Digestion of the pellet with proteolytic enzymes is therefore used to brake down the backbone to the level of single aminoacid without hydrolysis of the e-g isopeptide. The enzymatic digestion is carried out with the sequential addition of pronase, carboxypeptidase A, B and Y, and leucine aminopeptidase. The determination of e(g-glutamyl)lysine peptide is then carried out by an amino acid analyzer using ninhydrin as detecting reagent.

Reading out the results requires the comparison between the profile of the test sample and the profiles of a negative and a positive control. The positive control is represented by a known amount of e(g-glutamyl)lysine peptide. The negative control is represented by an aliquot of the test sample where g-glutamyl cyclo-transferase has been added. This enzyme degrades the isodipeptide by cleaving the e(g-glutamyl)lysine bond and catalysing its conversion to free L-lysine and 5-oxo-1-proline.
 

Materials

• Synthetic e(g-glutamyl)lysine isopeptide (SERVA)
• g-glutamyl cyclo-transferase
• 0.1 M Ammonium bicarbonate
• Pronase, carboxypeptidase A, B and Y, and leucine aminopeptidase

Special equipment

• Lyophilizer or SpeedVac (Savant)
• Amino acid analyzer

Methodology

• Solubilize cells in TrisHCl 50 mM pH 7.0, SDS 2%, 2-mercaptoethanol 0.1 M with a 2 hours incubation at room temperature
• Heat the sample at 100°C for 10 min, pass it through a 0.1 mm mesh and centrifuge at 1500xg
• Collect supernate (0.2-0.5 mg protein/sample maximum) and mix it with trichloroacetic acid, final concentration 30% (w/v). Let sit overnight at 4°C
• Collect the precipitated proteins by centrifugation and wash the pellet in succession with 1 ml of 5% trichloroacetic acid (3 times), 1 ml of 50% ethyl alcohol/50% aceton mix (3 times) and with 1 ml of aceton (twice)
• Air dry the pellet, then resuspend it in 0.5 ml of 0.1 M ammonium bicarbonate pH 8.0
• Carry out the enzymatic digestion in NH4HCO3 0.1 M by sequential addition of the proteolytic enzymes directly to the reaction mixture at 37°C in the presence of 0.02% sodium azide (25): pronase 5 mg/ml for 18 hours; a second pronase addition (5 mg/ml) for 18 more hours; leucine aminopeptidase 1 mg/ml for 65 hours; carboxypeptidase A 0.4 mg/ml, carboxypeptidase B 0.4 mg/ml and carboxypeptidase Y 0.4 mg/ml for 24 hours. Before each new enzyme addition the sample is boiled for 5 min to inactivate the previously added protease.
• Add a volume of trichloroacetic acid equal to that of the final and remove the precipitate by centrifugation.
• Extract the supernatant three times with 1.5 ml of ethyl ether and concentrate the acqueous layer to a volume suitable for the analysis.
• Split the protein free fraction of the digest in three aliquots.
• Add 10 pmol of e(g-glutamyl)lysine to the first aliquot, 0.1 U/ml g-glutamyl cyclo-transferase to the second aliquot and nothing to the third aliquot. The first two aliquots represent the positive and the negative control, respectively, the third aliquot represents the test sample. Incubate for 90 min at 37°C
• Analyse each aliquot by ion-exchange chromatography on a amino acid analyzer with litium citrate buffer and ninhydrin as detecting reagent (26).
• Elute (0.2 ml/min) according to the following program: buffer A (pH 2.90) is pumped for 30 min at 40°C, buffer B (pH 3.04) for 22 min at 40°C, buffer C (pH 2.95) for 43 min at 40°C, buffer D (pH 3.34) for 25 min at 40°C, buffer D (pH 3.34) for 5 min during which column temperature is raised to 65°C and buffer E (pH 4.24) for 60 min at 65°C. Ninhydrin flow rate is 0.1 ml/min with the reaction chamber at 115°C.
• At the completion of runs, wash the column for 30 min with 0.3 M lithium hydroxide containing EDTA 5 mM at a temperature of 65°C.
• Reequilibrated then the column in buffer A with the column temperature returning to 40°C.
• The amount of e(g-glutamyl)lysine isopeptide in a sample is calculated by the comparison of the profiles obtained from each of the three aliquots. A control containing enzymes but not cellular extracts should be treated in parallel for each determination.

A different chromatographic technique can also be used to identify e(g-glutamyl)lysine isopeptide. In this case the protein-free sample derived from enzymatic digestion is derivatized with o-phtalaldehyde and then loaded on a HPLC C18 reverse-phase column (26). Also with this technique the quantification of the e(g-glutamyl)lysine isopeptide results from the comparison between the profile of the test sample and the profiles of a negative and a positive control, prepared as described above.

References

 

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