Enhancement of Escherichia coli H(+)-ATPase caused by binding of monoclonal antibodies is attributed to structural changes of Leu-456 and Ser-440 in the alpha subunit.
Five monoclonal antibodies against the alpha subunit of F1-ATPase from Escherichia coli alpha 104, alpha 105, alpha 107, alpha 109, and alpha 110 were prepared. The monoclonal antibodies alpha 104 and alpha 110 enhanced the F1-ATPase activity maximally to 1.6- and 1.7-fold that of the wild-type, respectively, while alpha 105 did not. Both antibodies bound to a peptide corresponding to the region between residues 354 and 513. Mutations in this region which caused reduced binding of the alpha subunit to the antibodies were identified at residues Ser-440, Leu-456, Leu-471, Leu-482, Met-483, and Ser-506 for alpha 104 and residues Ser-440, Leu-456, Leu-471, Asp-476, Leu-482, Met-483, and Ser-506 for alpha 110. These residues are possibly involved in the epitopes for the antibodies and are located close together on the surface of the alpha subunit. Among the mutations, Leu-456 to Pro and Ser-440 to Pro mutations caused increase of the F1-ATPase activity up to 1.9 and 1.2 times that of the wild-type, respectively, while Leu-471 to Pro mutation caused a defect in assembly of the F1-ATPase on the membrane. The other mutations caused no significant change in ATPase activity. These results suggested that Ser-440 and Leu-456 have an important role in regulating catalysis by the F1-ATPase, but that the neighboring residue Leu-471 has an important role in assembly of the F1-ATPase complex. It was also suggested that binding of the monoclonal antibodies alpha 104 and alpha 110 to residues Ser-440 and Leu-456 caused local conformational changes, leading to enhancing effects on F1-ATPase activity similar to the Ser-440 to Pro and Leu-456 to Pro mutations.
Kanazawa
H
H
Department of Biotechnology, Faculty of Engineering Sciences, University of Okayama, Japan.
Yabuki
M
M
Miki
J
J
Fudemoto
T
T
Ikeda
H
H
Noumi
T
T
Shin
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007930:Leucine; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D012694:Serine; D006180:Proton-Translocating ATPases; D007930:Leucine