Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides

摘要: Electropermeabilized human endothelial cells (ECV-304) were used to study the regulation of tissue transglutaminase (tTGase) activity in intracellular environment. An ELSA (enzyme-linked sorbent assay) plate assay was developed for tTGase activity, using incorporation a biotinylated primary amine, 5-?[(N-biotinoylamino)hexanoyl]amino?pentylamine(biotin-x-cadaveri ne; BTC), into endogenous protein substrates tTGase. This process inhibited by competitive inhibitors tTGase, cystamine and monodansylcadaverine, dose-dependent manner. Over 30 min period its did not leak out cell, no BTC occurred unpermeabilized cells, indicating reaction be intracellular. In presence 10 nM or muM CA2+, when nucleotides ATP GTP added at concentrations mimicking cytosolic levels, decreased virtually zero. Only 100 Ca2+, low absent observed. Under these conditions variety proteins labelled enzyme, with major labelling found molecular mass around 51 kDa analysed SDS/PAGE/Western blotting.