Purified Guinea Pig Liver Transglutaminase Research Articles

Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca 2+-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation ( r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose–response curves over a wide range of hrTGase2 concentrations (2.5–40 μU/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 μg of guinea pig liver extracts.

Kinematic changes following orthopedic surgery in persons status post traumatic brain injury

Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation (r2=0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9±1.5% (n=8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N′,N′-bis (γ-glutamyl) polyamine bridges is measured. N′,N′-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N′,N′-bis (γ-glutamyl) polyamine linkages.

A Transglutaminase-Related Antigen Associates with Keratin Filaments in Some Mouse Epidermal Cells

A mouse monoclonal IgG, G82, directed against guinea pig liver transglutaminase recognizes a transglutaminase-related antigen that is associated with the keratin intermediate filament network in some primary mouse keratinocytes. The association can be seen at the resolution of individual keratin tonofibrils following fixation and staining for double-label indirect immunofluorescence. Western blots indicate that G82 reacts with two proteins of 95 kDa and 280 kDa, respectively, in extracts of these cells. The 95-kDa band is also recognized by a polyclonal antibody against purified guinea pig liver transglutaminase, and the 280-kDa protein seems to correspond to a similar protein that was shown to be recognized by G92.1.2 in the intermediate filament fraction of primary mouse fibroblasts. The transglutaminase-related antigen was shown by confocal microscopy to co-localize only with nonbasal cell specific keratin intermediate filaments.

Assays for the measurement of tissue transglutaminase (type II) mediated protein crosslinking via ϵ-( γ-glutamyl) lysine and N′,N′-bis ( γ-glutamyl) polyamine linkages using biotin labelled casein

Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation ( r 2=0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9±1.5% ( n=8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N′,N′-bis ( γ-glutamyl) polyamine bridges is measured. N′,N′-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N′,N′-bis ( γ-glutamyl) polyamine linkages.

Neurokinin receptors could be differentiated by their capacity to respond to the transglutaminase-synthesized gamma-(glutamyl5)spermine derivative of substance P.

Four different gamma-(glutamyl5)amine derivatives of substance P (SP) were synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The 1,3-diaminopropane, spermidine, spermine (Spm), and monodansylcadaverine adducts of the neuropeptide were purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The gamma-(glutamyl5)Spm derivative of SP (Spm-SP) was found to be able, like the parent neuropeptide, to provoke rabbit aorta relaxation, to decrease rat arterial blood pressure, and to inhibit collagen-induced platelet aggregation. Unlike SP, only a weak inflammatory response was observed when Spm-SP was injected in the rat hind limb. All these effects were found to be prevented by N omega-nitro-L-arginine methyl ester, a well-known nitric oxide synthesis inhibitor. In contrast, Spm-SP was completely ineffective in contracting guinea pig ileal segments, thus confirming our preliminary observations indicating that Spm-SP does not evoke SP-like spasmogenic effects on isolated smooth muscle preparations. The specificity of the effects due to the selective introduction of a Spm moiety at the glutamine5 level was demonstrated by the SP agonist pharmacological profile of the other gamma-(glutamyl5)amine derivatives tested. These results suggest that neurokinin receptors could be differentiated by their capacity to respond to Spm-SP.

Calmodulin regulates nucleotide hydrolysis activity of tissue transglutaminase.

The interaction of calmodulin with purified guinea pig liver transglutaminase was studied. The nucleotide (ATP and GTP) hydrolysis activity of this tissue transglutaminase was transiently increased and then gradually decreased depending on calmodulin concentration. The peak activation was obtained in the presence of a stoichiometric amount of calmodulin. The effect of calmodulin on the classical transglutaminase activity was minimal. Fluorescence spectroscopy demonstrated that the enzyme produced a significant blue shift in the emission peak of dansylated calmodulin. Interestingly, Ca2+ was not required for the interaction between the two proteins. The results described here give an additional regulatory role to calmodulin.

A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate

Transglutaminases belong to an important family of enzymes involved in hemostasis, skin formation, and wound healing. We describe a technique for the measurement of transglutaminase activity using polystyrene microtiter plates coated with N,N′-dimethylcasein. The substrate 5-(biotinamido)pentylamine is convalently incorporated into N,N′-dimethylcasein by transglutaminase in a calcium-dependent reaction. The biotinylated product is detected by streptavidin-alkaline phosphatase and quantitated by measuring the absorbance at 405 nm following the addition of p-nitrophenyl phosphate. The assay is sensitive, specific, and linear at plasma factor XIIIa concentrations between 0.08 and 1.25 μg/ml and at purified guinea pig liver transglutaminase concentrations between 0.05 and 0.8 μg/ml. The intra-assay coefficient of variation is less than 8%. The solid-phase assay was used to quantitate the transglutaminase activity in Escherichia coli extracts expressing recombinant factor XIII A-chains and to analyze factor XIIIa inhibitors. This method will facilitate the analysis of structure-function relationships of the transglutaminases using recombinant DNA methods. Furthermore, screening of natural and synthetic factor XIIIa inhibitors will be expedited by this solid-phase microtiter plate assay.

Substance P inactivation by transglutaminase in vitro

Gamma(glutamyl 5)spermine derivative of substance P (Spm-SP) was synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca 2+. The spermine adduct of the neuropeptide was purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The biological activities of Spm-SP were tested by assaying, in comparison with substance P, its ability to induce both the contractions of smooth muscle in vitro and the edema formation in vivo. Spm-SP was shown not to elicit contractile responses in the isolated rat stomach strip and duodenum and not to antagonize the spasmogenic effect evoked by the native neuropeptide. Furthermore, Spm-SP was unable, when administered into rats by plantar injection, either to provoke an acute inflammatory response in the hind limb or to antagonize the edema formation induced by a concurrent administration of substance P. These results indicate that the introduction of a large size hydrophilic moiety at the glutamine 5 level negatively affects the ability of the neuropeptide to bind to its receptor(s), thus supporting the view that the hydrophobic middle portion of substance P plays a key role in receptor recognition.

Colorimetric assay for cellular transglutaminase

A colorimetric assay for cellular transglutaminase using 5-(biotinamido)pentylamine and polyvinylidine difluoride membranes for crude cellular preparations and purified enzyme has been developed. The biotinpentylamine substrate was incorporated into N,N-dimethylcasein by transglutaminase, the biotinylated products were adsorbed onto the membrane disks and conjugated with streptavidin-β-galactosidase, and the absorbance resulting from the formation of p-nitrophenol from hydrolysis of p-nitrophenyl-β- d-galactopyranoside was measured at 405 nm. The validity of the assay was established by showing a good correlation, γ = 0.922, between the colorimetric procedure and the commonly used radiometric filter paper method for the enzyme. The procedure offers a rapid, sensitive, and nonisotopic method for the estimation of cellular transglutaminase activity in as low as 20 ng of purified guinea pig liver transglutaminase and 10 μg of crude fibroblast cytosol protein.

GTP hydrolysis by guinea pig liver transglutaminase

Homogeneous guinea pig liver transglutaminase was purified from a commercially available enzyme preparation by affinity chromatography on GTP-agarose. The purified transglutaminase exhibited a single band of apparent Mr=80,000 on sodium dodecyl sulfate polyacrylamide gel and Western blotting and had enzyme activity of both transglutaminase and GTPase. The guinea pig liver transglutaminase has an apparent Km value of 4.4 μM for GTPase activity. GTPase activity was inhibited by guanine nucleotides in order GTP-γ-S > GDP, but not by GMP. These results demonstrate that purified guinea pig liver transglutaminase catalyzes GTP hydrolysis.

Substance P as a transglutaminase substrate: Identification of the reaction products by fast atom bombardment mass spectrometry

Substance P was found to be an effective acyl donor substrate of transglutaminase in vitro, the reaction products having been examined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fast atom bombardment mass spectrometry. Electrophoretic experiments showed that Substance P incorporated 14C-labeled polyamines when incubated with purified guinea pig liver transglutaminase and Ca 2+. Extensive use of fast atom bombardment mass spectrometry allowed to establish that: i) a 1:1 adduct Substance P-spermine is formed; ii) only a single glutamine residue out of two, i.e. Gln-5, acts as acyl donor; iii) the single lysine residue of the neuropeptide is unable to act as acyl acceptor. A direct analytical methodology to detect transglutaminase reaction products is described.

Two antigenic sites of tissue transglutaminase.

The immunization of rabbits with purified guinea pig liver transglutaminase resulted in the appearance of two antibody populations against the enzyme: one which reacted only with the Ca2+-enzyme complex and another which reacted with the intact as well as the Ca2+-enzyme. The Ca2+-induced confomrational change of the enzyme molecule exposes a new antigenic determinant which initiates the production of a specific antibody population. When the glutamine substrate of the enzyme was a dipeptide, the result of the interaction of the Ca2+-enzyme and its isolated specific antibody was an apparent activation of the catalytic activity. However, when protein substrates were used, an inhibition was observed. The characterization of the mechanism of the activation and the inhibition has led to the conclusion that the consequence of the interaction of Ca 2+-enzyme and its specific antibody is not only a limited steric hindrance of the active center but, besides that, a stabilization of the otherwise labile Ca2+-enzyme. The other antibody population reacts with both forms of the enzyme and its inhibitory effect, which has been observed in each assay, could be due to a prevention of the Ca2+-induced formation of the active enzyme.

Catalytic formation of ϵ-(γ-glutamyl)lysine in guinea pig liver transglutaminase

Incorporation of putrescine into purified guinea pig liver transglutaminase was observed when the enzyme was incubated under catalytic conditions in the absence of the exogenous acceptor substrate casein. Furthermore, in the absence of exogenous substrates, putrescine and casein, the enzyme monomer of 85–90,000 daltons was converted to polymeric material. ϵ(γ-Glutamyl)lysine isopeptide was isolated from polymeric transglutaminase samples following proteolytic digestion. The results were consistent with the formation of an intermolecular isopeptide, ϵ(γ-glutamyl)lysine, between two or more enzyme monomers apparently by an autocatalytic reaction.

Mechanism of Action of Guinea Pig Liver Transglutaminase: IV. THE TRIMETHYLACYL ENZYME

Abstract Evidence is presented that purified guinea pig liver transglutaminase catalyzes the hydrolysis of p-nitrophenyl trimethylacetate. As in the case of the other transglutaminase-catalyzed reactions, calcium ion is required for this esterolysis. This reaction involves the formation of a trimethylacetyl enzyme complex which has been shown by two procedures, burst in p-nitrophenol liberation and incorporation of 14C-trimethylacetyl, to have one trimethylacetyl group per molecule of protein. A trimethylacetyl enzyme complex containing 0.9 eq of trimethylacetyl has been isolated. Incorporation of trimethylacetyl is reflected in a concomitant loss in activity toward p-nitrophenyl acetate and for hydroxylamine incorporation into carbobenzoxy-l-glutaminylglycine. Studies on the inhibition of transglutaminase activity by p-nitrophenyl trimethylacetate and formation of 14C-trimethylacetyl enzyme, both as a function of calcium ion concentration, indicate that the same divalent cation-enzyme complexes are involved in p-nitrophenyl acetate hydrolysis and in p-nitrophenyl trimethylacetate hydrolysis. Thus the apparent dissociation constants for the calcium-transglutaminase complex, as determined by inhibition and by trimethylacetyl enzyme formation at pH 7.0, are 0.37 ± 0.2 x 10-3 m and 0.54 ± 0.2 x 10-3 m, respectively. These values are comparable to the reported activator constant of calcium, 0.4 ± 0.2 x 10-3 m at pH 7.0, determined from the kinetics of p-nitrophenyl acetate hydrolysis. A single radioactive peptide has been separated from a proteolytic digest of 14C-trimethylacetyl transglutaminase. Studies on the isolated peptide indicate that the trimethylacetyl group is bound in thioester linkage to a cysteine —SH group and to the same —SH group in the enzyme with which iodoacetamide reacts to form the inactive carbamidomethyl enzyme. These results are presented in support of a previously suggested cation-controlled mechanism of enzyme action involving the intermediate formation of an acyl enzyme through thioester linkage.