When transglutaminase is heated at temperatures above 40 degrees C, it loses its activity according to a two-step mechanism [Nury, Meunier & Mouranche (1989) Eur. J. Biochem. 180, 161-166]: N----X(TD)----D However, the nature of the molecular events responsible for the irreversible denaturation is still unknown. Investigation of the effects of dithiothreitol and 5,5'-dithiobis-2-nitrobenzoate on the kinetics of inactivation, titrations of ammonia released by deamidation and of thiol groups on the native and denatured enzymes and SDS/PAGE rule out the involvement of covalent processes during the denaturation of transglutaminase at 55 degrees C and pH 7. Of the two possible kinds of non-covalent events, i.e. unfolding of the polypeptide chain and aggregation of enzyme molecules, we show that both occur, though only the former process is responsible for the denaturation. The latter process, aggregation, follows the unfolding of the molecules.
By incubating native (N) transglutaminase from guinea-pig liver at various temperatures and assaying it at 2572, two steps in the irreversible deactivation process to the denatured form (D) have been found. The fitting of the data to the equations of two possible models (the two-steps model and the two-isoenzymes model) is only compatible with the first one (N ---* X + D). It is shown that the structure of the active intermediate, X, depends on the deactivation temperature and on the thermal history of the enzyme. This may mean that transglutaminase exists in a large number of microstates. Surprisingly, the activation energy of deactivation is lower than that of activity (36.6 & 3.4 against 47.2 2.2 kJ . mol-I). By deactivating transglutaminase at a constant temperature (55°C) and assaying it at variable temperatures, the activation energy of the intermediate, (X55), has been determined to be 40.2 _+ 5 kJ . mol-l, of the same order of magnitude as the native form.Among several agents assayed, only Ca2+ had a positive effect on the thermal stability of this enzyme. At 40°C, transglutaminase was quite stable in the presence of Ca2+ (in its absence, the half-life was 65 min) and at 45 "C, its thermostability had been considerably increased, the half-life being raised from 47 min to 275 min.Transglutaminase is a Ca2 +-dependent enzyme responsible for post-translational modifications of proteins. It allows the cross-linking of proteins like fibrin, collagen and uteroglobulin [l -51. It is a tool for in vitro modifications ofproteins and especially for making gels [6 -81. Although the kinetics of the reversible thermal deactivation of proteins (and especially enzymes) has been extensively studied [9], the kinetics of the irreversible process has been the object of only a few papers [lo -121. To increase our knowledge in this basic area, we have carried out such a study on the transglutaminase from guinea-pig liver. This basic study may also be of some interest on potential uses of this enzyme. Knowledge of the kinetics of its irreversible deactivation may be essential for the development of rational approaches to enzyme stabilization, e. g. enhancement of enzyme thermostability by such agents as CaZ + . Therefore, the aim of this paper was to study the effect of temperature on the activity and on the stability of transglutaminase from guinea-pig liver and the role of Ca2+ in the enhancement of its thermostability. MATERIALS AND METHODS Purification of ' transglutaminaseEnzyme was extracted and purified from guinea-pig liver according to the method of Brookhart et al. [13], slightlyCorrespondence to J
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