THE USE OF FLUORESCEINCADAVERINE FOR DETECTING AMINE ACCEPTOR PROTEINSUBSTRATES ACCESSIBLE TO ACTIVE TRANSGLUTAMINASE IN LIVING CELLS

The use of Fluoresceincadaverine as a primary amine donor for detectin g the endogenous substrates for active transglutaminase in living cell s was studied. Fluoresceincadaverine was found to be suitable for labe lling cells in culture as it did not induce cytotoxicity when used at 0.5 mM in culture media and diffused throughout the tell. After approp riate fixation using methanol, Fluoresceincadaverine-labelled cells we re observed by direct fluorescence microscopy, allowing visualization of the substrates for active transglutaminase. Simultaneous detection of transglutaminase and of Fluoresceincadaverine incorporated into pro teins strongly suggested that cytosolic transglutaminase was inactive in these living cells. However, transglutaminase co-distributed with F luoresceincadaverine-labelled structures, which resembled a lattice. F luoresceincadaverine-labelled proteins detected by Western blotting us ing an anti-Fluorescein antibody showed that, in Living cells, the maj or transglutaminase substrate migrated at an apparent molecular weight of 220 kDa, as does fibronectin. Fibronectin was found to co-distribu te with Fluoresceincadaverine-labelled lattice. This confirmed that th ese lattice structures were extracellular and, therefore, that transgl utaminase is in an active form in this compartment. This opportunity t o perform morphological and biochemical analyses in the search for tra nsglutaminase substrates in living cells should help in determining th e specific function of transglutaminases in a particular cell type as well as in universal cellular events, such as apoptosis or cell growth .