STRUCTURAL DOMAIN ORGANIZATION OF GASTRIC H-ATPASE AND ITS REARRANGEMENT DURING THE CATALYTIC CYCLE(,K+)

Differential scanning calorimetry has been used to characterized the t hermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacit y function of (H+,K+)-ATPase in highly oriented gastric vesicles displ ays two peaks at 53.9 degrees C (T-m1) and 61.8 degrees C (T-m2). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state proce sses. They can be as signed 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 g et insight into the E(1)-E(2) conformational change. In the E(1) forms both transitions are always observed. As T-m1 is shifted to T-m2 by v anadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdo main interactions. Stabilization of the E(2) forms results in the disa ppearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in T-m2 and Delta H-1. The catalytic domain melts following a process in which intermolecula r interactions either in the native or in the unfolded state might be involved. Interestingly, the E(2)-vanadate-K+ form displays intermedia te properties between the E(1) and E(2) conformational families.