Structural aspects of the gastric H,K-ATPase.
The gastric H,K-ATPase is an alpha, beta heterodimer. The large catalytic subunit is composed, in the case of the hog enzyme, of 1033 amino acids, whereas the beta subunit is composed of about 291 amino acids and is heavily glycosylated. The membrane topology of the alpha subunit is difficult to predict using hydropathy analysis. Tryptic hydrolysis of intact, inside out vesicles followed by cysteine labelling with fluorescein-5-maleimide provided experimental evidence for an 8 membrane spanning model for the alpha subunit, between residues 104 and 162 (M1/M2), 291 and 358 (M3/M4), 776 and 835 (M5/M6), and 853 and 946 (M7/M8). No evidence was found for a pair of segments (M9/M10) towards the C terminal end of the molecule, contrary to predictions for the Na,K- and Ca-ATPases. Iodination of intact vesicles followed by carboxypeptidase Y cleavage of the C terminal tyrosines showed that the C terminal end of the alpha subunit was cytoplasmic. The epitope for antibody 146 was extracytoplasmic and located between residues 871 to 874 between M7/M8. The binding site of the K competitive imidazo-pyridine, SCH28080, was to the extracytoplasmic loop between M1 and M2, whereas the binding of the covalent SH reagent generated from acid activation of omeprazole in acid transporting vesicles was to 2 cysteines at positions 813 (or 822) and 892 predicted to be in the extracytoplasmic loops connecting M5/M6 and M7/M8, respectively. The beta subunit was only hydrolysed in broken vesicles. A fragment beginning at position 236 was liberated under these conditions only in the presence of reducing agents, showing that cysteine 210 and 263 were disulfide linked.(ABSTRACT TRUNCATED AT 250 WORDS)
Sachs
G
G
UCLA.
Besancon
M
M
Shin
J M
JM
Mercier
F
F
Munson
K
K
Hersey
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D000939:Epitopes; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation
D000939:Epitopes; D006859:Hydrogen; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium