Gray, A., and R. Clothier. Control of Transglutaminase Activity in Human Keratinocytes by Using the Newly Developed Fluorescein Cadaverine Incorporation Technique. ATLA. 1999. 27: 410.

cysteine - 4371-52-2; cysteamine - 60-23-1; retinoic acid - 00302-79-4; phosphatidylcholine - 8002-43-5;

A novel technique for detecting transglutaminase activity and the production of cornified envelopes in keratinocytes has been devised, based on the transglutaminase catalysed incorporation of fluorescein-labelled cadaverine (FC) into isodipeptide cross-links Gray & Clothier. Toxicology In Vitro, in press). The addition of FC (20µm) to the incubation medium serves as an amine donor in place of protein lysine residues. Cells incorporating the label are visible with fluorescence microscopy and quantifiable by fluorimetry. The newly developed technique has been used to demonstrate how FC incorporation can be modified in keratinocyte cultures over time. Cornified envelope formation was significantly enhanced over 6 or 9 days as a result of increased cross-linking activity in keratinocytes cultured in Green's medium without serum, compared to cultures in keratinocyte growth medium, where no activity was observed. These conditions served as controls in later experiments. FC incorporation was inhibited using competitive substrates providing the essential amine group during the formation of isodipeptide cross-links. Transglutaminase activity was inhibited non-competitively by a range of well-known inhibitors. Cysteine was inhibitory at a dose close to that which proved to be cytotoxic. Cysteamine, the decarboxylation product of cysteine, inhibited activity at a tenth of the concentration, without cytotoxicity. Transglutaminase activity was also inhibited by 1 X 10^-5M retinoic acid. The lipid moiety phosphotidylcholine protects the cell against cytolysis. Inhibition by retinoic acid was greater when cultured with 50µg/ml phosphotidylcholine. The technique is simple and less time consuming than currently available techniques, and allows transglutaminase activity and terminal differentiation to be assessed in living epithelia.