Chemomechanical coupling in the gastric H,K ATPase.
The gastric H,K ATPase is investigated in terms of its secondary structure by analysis of the binding sites of the extracytoplasmic inhibitors and by tryptic cleavage of intact, inside-out gastric vesicles. The inhibitors affect phosphorylation and other partial reactions of the ATPase that depend on cytoplasmic conformational changes. The K competitive imidazopyridine, SCH28080 binds to the first pair of transmembrane segments, M1/M2, probably at phe124 and asp136. Omeprazole which generates a cationic sulfenamide in acid spaces binds to either cys813 or cys822 at one site and cys892 at the other. These cysteines are located at the membrane spanning pairs, M5/M6 and at M7/M8. Tryptic cleavage of intact inside out vesicles followed by labelling with fluorescein-5-maleimide provides direct evidence for 8 membrane spanning segments between positions 104/162 (M1/M2), 291/358(M3/M4), 776/835 (M5/M6),853/946 (M7/M8). Evidence is lacking so far for M9/M10, postulated on the basis of hydrophobicity for the Ca ATPase. Conformational studies suggest that there is interaction between the cytoplasmic loop between M4 and M5 (ATP domain) and the extracytoplasmic domain of the enzyme at the inhibitor binding sites.
Besancon
M
M
Emory University, Atlanta, Georgia.
Shin
J M
JM
Mercier
F
F
Munson
K
K
Rabon
E
E
Hersey
S
S
Sachs
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001693:Biological Transport, Active; D015153:Blotting, Western; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007093:Imidazoles / Q000378:metabolism; D009853:Omeprazole; D010455:Peptides / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D013270:Stomach / Q000201:enzymology*; D013552:Swine; D014357:Trypsin
D000345:Affinity Labels; D007093:Imidazoles; D010455:Peptides; C035235:Sch 28080; D000255:Adenosine Triphosphate; D014357:Trypsin; D017506:H(+)-K(+)-Exchanging ATPase; D009853:Omeprazole