C-terminal topology of gastric H+,K(+)-ATPase.
An antibody was prepared against a peptide corresponding to residues 1024-1034 (the putative C-terminus) of the alpha-subunit of hog gastric H+,K(+)-ATPase. The antibody bound to a 95 kDa band of H+,K(+)-ATPase that was solubilized in SDS, but not to that of Na+,K(+)-ATPase. It also bound to products of tryptic digestion that included C-terminal fragments of the H+,K(+)-ATPase alpha-subunit. The same amount of the antibody bound to both intact (tight) and lyophilized (leaky) inside-out gastric vesicles, indicating that its epitope is present on the cytosolic side of the vesicles. This finding was further confirmed by using fluorescence-immunolocalization techniques and streptolysin-O to permeabilize newt oxyntic cells. Stimulation of isolated newt oxyntic cells with dibutyryl cyclic AMP induces fusion of tubulovesicles with the apical membrane, so that the luminal domains of the H+,K(+)-ATPase alpha-subunit directly face the cell-suspension medium. The antibody did not bind to the stimulated intact cell, but bound to cells permeabilized with streptolysin-O, indicating that it binds from the cytoplasmic side to the C-terminus of the H+,K(+)-ATPase alpha-subunit in apical and tubulovesicular membrane, and also that the H+,K(+)-ATPase alpha-subunit has an even number of transmembrane domains.
Asano
S
S
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Arakawa
S
S
Hirasawa
M
M
Sakai
H
H
Ohta
M
M
Ohta
K
K
Takeguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000276:immunology; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010295:Parietal Cells, Gastric / Q000201:enzymology*; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D012455:Salamandridae; D013552:Swine
D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium