A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells.
Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis andinflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation between spots was 2.6% (n=3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n=3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.
Park
Jin-Young
JY
Department of Molecular and Cellular Biochemistry and Vascular System Research Center, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Jung
Se-Hui
SH
Jung
Jae-Wan
JW
Kwon
Mi-Hye
MH
Yoo
Je-Ok
JO
Kim
Young-Myeong
YM
Ha
Kwon-Soo
KS
D002103:Cadaverine / Q000494:pharmacology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D003538:Cystamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000201:enzymology* / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008387:Marine Toxins / Q000494:pharmacology; D035964:Oxocins / Q000494:pharmacology; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D000095702:Polyether Toxins
D002103:Cadaverine; D016895:Culture Media, Serum-Free; D003538:Cystamine; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D035964:Oxocins; D011503:Transglutaminases; C020394:maitotoxin; D000095702:Polyether Toxins