Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides.
Electropermeabilized human endothelial cells (ECV-304) were used to study the regulation of tissue transglutaminase (tTGase) activity in the intracellular environment. An ELSA (enzyme-linked sorbent assay) plate assay was developed for intracellular tTGase activity, using the incorporation of a biotinylated primary amine, 5-{[(N-biotinoylamino)hexanoyl]amino}pentylamine(biotin-x-cadaveri ne; BTC), into endogenous protein substrates of tTGase. This incorporation process was inhibited by competitive inhibitors of tTGase, cystamine and monodansylcadaverine, in a dose-dependent manner. Over a 30 min period tTGase and its protein substrates did not leak out of the cell, and no incorporation of BTC occurred in unpermeabilized cells, indicating the reaction to be intracellular. In the presence of 10 nM or 10 muM CA2+, when nucleotides ATP and GTP were added at concentrations mimicking cytosolic levels, tTGase activity was decreased virtually to zero. Only at 100 muM Ca2+, when nucleotides were low or absent was tTGase activity observed. Under these conditions a variety of proteins was labelled by the enzyme, with the major labelling found in a protein of molecular mass around 51 kDa when analysed by SDS/PAGE/Western blotting.
Smethurst
P A
PA
Department of Life Sciences, Faculty of Science and Mathematics, Nottingham Trent University, U.K.
Griffin
M
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000588:Amines / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004560:Electricity; D004730:Endothelium, Vascular / Q000201:enzymology; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology*; D010539:Permeability; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry; D011503:Transglutaminases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000588:Amines; D009711:Nucleotides; D011506:Proteins; C076022:5-(biotinamido)pentylamine; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D001710:Biotin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011503:Transglutaminases; D002118:Calcium