par Gasset, María;Laynez, J;Menéndez, M;Raussens, Vincent 
;Goormaghtigh, Erik
Référence The Journal of biological chemistry, 272, 3, page (1608-1614)
Publication Publié, 1997-01
;Goormaghtigh, Erik Référence The Journal of biological chemistry, 272, 3, page (1608-1614)
Publication Publié, 1997-01
Article révisé par les pairs
| Résumé : | Differential scanning calorimetry has been used to characterize the thermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacity function of (H+,K+)-ATPase in highly oriented gastric vesicles displays two peaks at 53.9 degrees C (Tm1) and 61.8 degrees C (Tm2). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state processes. They can be assigned to two cooperative domains located in the cytoplasmic loops of the alpha-subunit, according to the disappearance of the endothermic signal upon removal of these regions by proteinase K digestion. Analysis of the thermal-induced unfolding of the enzyme trapped in different catalytic cycle intermediates has allowed us to get insight into the E1-E2 conformational change. In the E1 forms both transitions are always observed. As Tm1 is shifted to Tm2 by vanadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions. Stabilization of the E2 forms results in the disappearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in Tm2 and DeltaH1. The catalytic domain melts following a process in which intermolecular interactions either in the native or in the unfolded state might be involved. Interestingly, the E2-vanadate-K+ form displays intermediate properties between the E1 and E2 conformational families. |
