title: Quantitative and rapid analysis of transglutaminase activity using protein arrays in mammalian cells.
abstract: We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutami-nase activity in mammalian cells. Transglutaminases are a family of Ca2+-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N'-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the [3H]putrescine-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transgluta-minase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases.
authors.affiliation: Department of Molecular and Cellular Biochemistry and Vascular System Research Center, Kangwon National University School of Medicine, Chuncheon 200-701, Korea.
authors.forename: Mi-Hye
authors.initials: MH
authors.lastname: Kwon
authors.forename: Jae-Wan
authors.initials: JW
authors.lastname: Jung
authors.forename: Se-Hui
authors.initials: SH
authors.lastname: Jung
authors.forename: Jin-Young
authors.initials: JY
authors.lastname: Park
authors.forename: Young-Myeong
authors.initials: YM
authors.lastname: Kim
authors.forename: Kwon-Soo
authors.initials: KS
authors.lastname: Ha