title: Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity.
abstract: Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca2+-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 microU/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 4ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25mug of guinea pig liver extracts.
authors.affiliation: CovalAb S.A.R.L, Research Department, 11 Avenue Albert Einstein, 69100 Villeurbanne, Lyon, France.
authors.forename: Mileidys
authors.initials: M
authors.lastname: Perez Alea
authors.forename: Miyako
authors.initials: M
authors.lastname: Kitamura
authors.forename: Guillaume
authors.initials: G
authors.lastname: Martin
authors.forename: Vincent
authors.initials: V
authors.lastname: Thomas
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Hitomi
authors.forename: Saïd
authors.initials: S
authors.lastname: El Alaoui