Molecular mechanisms of the irreversible thermal denaturation of guinea-pig liver transglutaminase

Abstract

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.

Kinetic stability of membrane proteins

“…or non-covalent changes leading to aggregation or the formation of kinetically trapped conformations (Ahern and Klibanov 1985;Nury and Meunier 1990;Yan et al 2004).…”

The Importance of Sample Handling in Neuropeptidomics

“…It is the heat generated from the microwaves that inactivates the proteins responsible for the degradation [14]. Proteins exposed to increasing temperature lose their enzymatic activity [15]. Exposure of most proteins to high temperatures results in irreversible denaturation [16].…”

Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase

“…To understand the relevance of degradative effects in the biology of transglutaminase, we decided to study the effects of regulatory ligands on the in vitro thermal stability of tissue transglutaminase, complementing previous investigations on the sensitivity to proteinases (Casadio et al 1999) and to chemical denaturants (Cervellati et al 2009). The issue of thermal stability of tissue transglutaminase has been marginally dealt with previously, mostly in relation to exposition to shifts in pH (Lichti et al 1985;Nury et al 1989;Nury and Meunier 1990;Bergamini et al 1999) but not in relation to effects of physiologically relevant ligands. We now discuss the new results we have obtained in relation to transglutaminase stability and unfolding by combined approaches aimed to correlate inactivation and structural perturbations of the protein.…”