PMID: 8611158Feb 1, 1996Paper

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

The Biochemical Journal
P A Smethurst, M Griffin

Abstract

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.

Citations

Dec 13, 2005·Placenta·Nicola J RobinsonJohn D Aplin
Feb 26, 2011·International Journal of Alzheimer's Disease·Antonio MartinVittorio Gentile
Mar 9, 2005·Immunology Letters·Ewa G GendekHanna Gendek-Kubiak
Aug 14, 2001·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·D J HeathM Griffin
Mar 25, 2017·American Journal of Hypertension·Chen LiuYang Xia
Feb 19, 2004·The Journal of Biological Chemistry·Morgan DupuisSakina Mhaouty-Kodja
Jul 20, 1999·European Journal of Biochemistry·R CasadioC M Bergamini
Apr 16, 2004·The Journal of Biological Chemistry·Bijan AhvaziPeter M Steinert
Nov 20, 2004·Circulation Research·Erik N T P BakkerEd Vanbavel
Jun 22, 2007·Amino Acids·P Kotsakis, M Griffin
Apr 23, 2005·Oncogene·Michael T SturnioloRichard L Eckert
Dec 25, 2009·Amino Acids·Carlo M BergaminiRita Casadio
Nov 16, 2007·Journal of the American Society of Nephrology : JASN·Tim S JohnsonMartin Griffin
Feb 4, 2003·Nature Reviews. Molecular Cell Biology·Laszlo Lorand, Robert M Graham
Aug 8, 2006·Oncogene·H PelicanoP Huang
Aug 8, 2006·Oncogene·C Brenner, S Grimm
Mar 17, 2006·Journal of Neurochemistry·Janusz TucholskiGail V W Johnson
Aug 13, 2008·Amino Acids·Carlo CervellatiAlberto Spisni
Aug 2, 2011·Amino Acids·Daniela CaccamoRiccardo Ientile
Aug 6, 2011·Amino Acids·Zhuo Wang, Martin Griffin
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Sep 22, 2001·Journal of Biomolecular Structure & Dynamics·A AmbrusL Fésüs
Dec 12, 2012·Breast Cancer Research and Treatment·Kevin D Brown
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Jul 31, 2010·Expert Opinion on Therapeutic Targets·Daniela CaccamoRiccardo Ientile

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