N⑀ -(␥-glutamyl)lysine cross-links, connecting various peptide chain segments, are frequently the major products in transglutaminase-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the ␥:⑀ linkage. Branched compounds were synthesized, in which the backbone on the ␥-side of the cross-bridge was labeled with a fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl) attached through an ⑀-aminocaproyl linker in the N-terminal position, and the other branch of the bridge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the N ␣ position. Hydrolysis of the cross-link could be followed in these internally quenched substrates by an increase in fluorescence. In addition to the thrombin and Ca 2؉-activated human coagulation Factor XIII a , cytosolic transglutaminases from human red cells and from guinea pig liver were tested. All three enzymes were found to display good isopeptidase activities, with K m values of 10 ؊4 to 10Inhibitors of transamidation were effective in blocking the hydrolysis by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of N ⑀ -(␥-glutamyl)lysine isopeptides.Apart from obvious differences in substrate specificities for the ␣-carbonyl groups of endo-Lys and Arg residues by papain (EC 3.4.22.2) and for the ␥-carbonyl groups of certain endo-Gln residues by transglutaminases (EC 2.3.2.13), considerable kinetic and mechanistic similarities exist between these two families of enzymes. Both operate by acylation-deacylation pathways, with a Cys thiol in the catalytic center assisted by a His residue (1-6). However, because of the exceptional specificities of transglutaminases for amines mimicking the ⑀-amino groups of Lys side chains in proteins (7-9), this group of enzymes shows a unique ability for generating protein-to-protein N ⑀ -(␥-glutamyl)lysine cross-links, a post-translational reaction of major biological significance. Transglutaminases are known to participate in various clotting phenomena (7, 10 -16), in the assembly of extracellular matrices (17) and of intracellular polymeric structures in cells under Ca 2ϩ stress (18 -22), and in apoptosis (23).While a great deal of attention has been paid to the amine transferase activities of transglutaminases (3, 4, 24), i.e. to the production of N ⑀ -(␥-glutamyl)lysine bridges and the incorporation of small molecular weight amines into proteins, the isopeptide breaking potential of the enzymes has not yet been explored. Since lack of availability of appropriate substrates may have been a main reason, we embarked on synthesizing ␥-branched peptides with built-in features, which would facilitate the application of fluorescence methodologies for kinetic studies. Two cytosolic transglutaminases of different properties, isolated from human red blood cells (HTg) 1 and fro...