Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H 1 /K 1 -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 1 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. . It contains the phosphorylation site (Asp385) and Lys517, which is specifically labeled by fluorescein isothiocyanate (FITC). The models based on hydrophobicity profiles and different experimental results predict 8-10 membrane spanning segments for the a subunit, even though segments M9 and M10 have never been isolated after proteolysis. Sequence analyses support this view [1,2]. The b subunit consists of 225 aminoacid residues with 40 amino-acid residues on the cytoplasmic side and a single predicted transmembrane segment of 27 amino-acid residues. The gastric H 1 /K 1 -ATPase is a member of the P-ATPase family and shares many features, including sequence homologies and enzymatic mechanism with other members such as the Ca 21 -ATPase [3], and especially the Na. Two major conformations, E1 and E2, have been identified. The E1 conformation is characterized by a high affinity for ATP and by exposure of the proton binding site on the cytoplasmic side. The E2 conformation is characterized by a low affinity for ATP and by exposure of the potassium binding site on extracytoplasmic side [4]. Different qualitative methods have been used to characterize the E1 -E2 conformational change. During limited tryptic digestion, the E1 conformation produces two major peptides from the a subunit, 67 and 35 kDa in size, respectively, while the E2 conformation produces two other peptides from the a subunit, 56 and 42 kDa, respectively [5]. Fluorescence measurement of the FITC-labeled a subunit also demonstrates the conformational change between E1 and E2 [6,7]. As far as the K 1 binding site is concerned, different results using the competitive inhibitor 8-[(4-azidophenyl)methoxy]-1-trithiomethyl-2,3-dimethylimidazo-(1,2-a)pyrimidium iodide (mDazip, a photoactivable derived from Schering 28080) suggest a binding site on the lum...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.