Structural modifications in the membrane-bound regions of the gastric H+/K+-ATPase upon ligand binding

Extensive trypsin proteolysis was used to examine the accessibility of memb rane bound segments of the gastric H+/K+-ATPase under different experimenta l conditions known to induce either the E1 or the E2 conformation. Membrane -anchored peptides were isolated after trypsinolysis and identified by sequ encing. We show that several membrane bound segments are involved in the co nformational change. In the N-terminal region, a M1-M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the p resence of K+ or in the presence of vanadate (12 kDa. and 15 kDa). In the M 3 and M4 region, no difference was observed for the peptide obtained in E1 or E2-K conformations, but the peptide generated in the presence of vanadat e begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop r egion: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformat ion. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 3 9 amino acids shorter than the E2 peptide. In the M7-M8 region, the same pe ptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.