HYDROLYSIS OF GAMMA-EPSILON ISOPEPTIDES BY CYTOSOLIC TRANSGLUTAMINASES AND BY COAGULATION-FACTOR XIII(A)

N-epsilon (gamma-glutamyl)lysine cross-links, connecting various pepti de chain segments, are frequently the major products in transglutamina se-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the gamma:epsilon linkage. Branched comp ounds were synthesized, in which the backbone on the gamma-side of the cross bridge was labeled with a fluorophor (5-(dimethylamino)-1-napht halenesulfonyl or a-aminobenzoyl) attached through an epsilon-aminocap royl linker in the N-terminal position, and the other branch of the br idge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the N-alpha position, Hydrolysis of the cross-li nk could be followed in these internally quenched substrates by an inc rease in fluorescence, In addition to the thrombin and Ca2+-activated human coagulation Factor XIII(a), cytosolic transglutaminases from hum an 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 10(-5) M. Inhibitors of transamidation were effective in bl ocking the hydrolysis by the enzymes, indicating that expression of is opeptidase 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-epsilon-(ga mma-glutamyl)lysine isopeptides.