Mapping of antigenic sites to monoclonal antibodies on the primary structure of the F1-ATPase beta subunit from Escherichia coli: concealed amino-terminal region of the subunit in the F1.
To analyze relationships between the ternary and primary structures of the beta subunit of Escherichia coli F1 ATPase, we prepared two monoclonal antibodies beta 12 and beta 31 against the beta peptide. These antibodies bind to the beta subunit but do not bind to the F1 ATPase, resulting in no inhibition of the ATPase activities. Several different portions of the beta subunit peptide were prepared by constructing expression plasmids carrying the corresponding DNA segment of the beta subunit gene amplified by the polymerase chain reaction. Western blotting analysis using these peptides revealed that the antibodies bound to a peptide of 104 amino acid residues from the amino terminal end, which is outside the previously estimated catalytic domain between residues 140 and 350. These results indicated that the amino terminal portion of the maximal 104 residues is not exposed to the surface of the F1 ATPase. The binding spectrum of the antibodies to the subunit from various species including Vibrio alginolyticus and thermophilic bacterium PS3 indicated possible epitope sequences within the 104 residues. The ternary structure of the beta subunit, in terms of cleavage sites by endopeptidases, was analyzed using the antibodies. A 43-kDa peptide without binding ability to beta 12 and beta 31 appeared upon cleavage by lysyl endopeptidase. The results suggested that lysyl residues from around 70 to 100 from the amino terminus are exposed to the surface of the beta subunit.
Miki
J
J
Department of Biotechnology, Faculty of Engineering Sciences, Okayama University, Japan.
Matsuda
T
T
Kariya
H
H
Ohmori
H
H
Tsuchiya
T
T
Futai
M
M
Kanazawa
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D000939:Epitopes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D006180:Proton-Translocating ATPases