Detection Of Tissue Transglutaminase Activation By Cell Resistance To Detergents

Extensive protein crosslinking takes place during apoptosis. The ubiquitous transglutami-nase TGase 2 (also called "tissue transglutaminase"; tTGase) was identified as the enzyme responsible for this reaction (Fesus et al., 1987; Melino and Piacentini, 1998). It is presumed that activation of TGase 2 during apoptosis prevents release of soluble and immunogenic proteins from dying cells because protein crosslinking makes these proteins less soluble, and thereby decreases a possibility of induction of autoimmune reaction. Furthermore, protein packaging into apoptotic bodies may be facilitated when proteins remain in solid state rather than in solution. The additional role of TGase 2 as one of the "executor enzymes" during apoptosis is still being debated.

This protocol is a simple and rapid approach to identify apoptotic cells with activated TGase 2. The method is based on the propensity of crosslinked protein to withstand treatment with detergents. The authors have noticed that when live, nonapoptotic cells are subjected to treatment with solutions of nonionic detergents, lysis of their plasma membrane and release of the content of cytoplasm is complete, resulting in preparation of isolated nuclei. In contrast, apoptotic cells resist the detergent treatment; their cyto-plasmic protein remains insoluble, attached to the nucleus in the form of a shell-like cover (Grabarek et al., 2002). It is possible, therefore, by flow or laser scanning cytometry to distinguish apoptotic cells from the nuclei isolated from nonapoptotic cells, by means of the abundance of protein in the former. In addition, bivariate gating analysis of cellular DNA and protein content makes it possible to reveal the cell cycle distribution separately for the population of cells with crosslinked protein (activated TGase 2) and for the population of cells that did not show protein crosslinking (Grabarek et al., 2002).

Alternate Protocol 2 combines the detection of TGase 2 activity by binding of fluoresce-inated cadaverine (F-CDV) with analysis of the cell cycle.

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