首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity
Authors:Mileidys Perez Alea  Guillaume Martin  Kiyotaka Hitomi
Institution:a CovalAb S.A.R.L, Research Department, 11 Avenue Albert Einstein, 69100, Villeurbanne, Lyon, France
b Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, 464-8601, Japan
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 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.
Keywords:TG2  Tissue transglutaminase  Colorimetric assay  Solid-phase assay  Spermine  TG2-specific assay
本文献已被 ScienceDirect 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号