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 membrane bound segments of the gastric H+/K+-ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane-anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational 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 presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 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 vanadate begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformation. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7-M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.
Baeyens
N
N
Structure and Function of Biological Membranes, Free University of Brussels, Boulevard du Triomphe, B1050 Brussels, Belgium.
Wattiez
R
R
Raussens
V
V
Ruysschaert
J M
JM
Goormaghtigh
E
E
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation; D013270:Stomach / Q000201:enzymology*; D013552:Swine; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D014638:Vanadates; D000255:Adenosine Triphosphate; D014357:Trypsin; D017506:H(+)-K(+)-Exchanging ATPase