Location of the cytoplasmic epitope for a K(+)-competitive antibody of the (H+,K+)-ATPase.
The monoclonal antibody (mAb) 95-111 binds the alpha subunit of (H+,K+)-ATPase and inhibits the K(+)-ATPase activity. To map the epitope, all of the partial sequences of the alpha subunit were expressed in Escherichia coli HB101 using rabbit alpha subunit cDNA restriction fragments ligated into PuEx vector. Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting. The antibody site was mapped between Cys529 and Glu561. This is close to the Lys517 that binds fluorescein isothiocyanate (FITC) and is considered to be between M4 and M5 close to the ATP binding domain. However, the mAb inhibition of ATPase is not ATP-competitive but is K(+)-competitive with a KI of 2 x 10(-9) M. The mAb also inhibits K+ quench of FITC fluorescence competitively with a KI of 8 x 10(-9) M. The K+ activation of ATPase activity and quench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme from the extracytoplasmic surface. The mAb epitope is cytoplasmic since the K(+)-ATPase activity of ion-tight gastric vesicles is inhibited. The 125I-mAb 95-111 binds to a single class of sites with an apparent KD of 2.3 +/- 0.8 x 10(-9) M and K+ does not displace bound mAb. Hence, antibody binding to a cytoplasmic Cys529-Glu561 epitope allosterically competes with K(+)-dependent reactions at extracytoplasmic sites.
Bayle
D
D
Institut National de la Santé et de la Recherche Médicale Unité 10, Hôpital Bichat, Paris, France.
Robert
J C
JC
Bamberg
K
K
Benkouka
F
F
Cheret
A M
AM
Lewin
M J
MJ
Sachs
G
G
Soumarmon
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003593:Cytoplasm; D000939:Epitopes; D005753:Gastric Mucosa / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium