Localization on the mitochondrial F1 ATPase alpha subunit of an epitope masked in the membrane-bound enzyme using a monoclonal antibody and synthetic peptides.
The epitope of the monoclonal antibody 20D6 was localized by N-terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1 alpha subunit of the mitochondrial ATPase/ATP synthase. Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven-amino-acid sequence recognized by 20D6 as 84EGDIVKR90. The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the alpha subunit sequence of Escherichia coli or chloroplast ATPase, respectively. This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope. Immunochemical analysis of synthetic peptides by polyclonal anti-F1 antisera showed that this region is highly immunodominant. In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the alpha subunit and not buried between F1 subunits. The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane. This suggests that it is located at the interface between F1 and the membrane.
Moradi-Améli
M
M
Laboratoire de Biologie et Technologie des Membranes du CNRS, Villeurbanne, France.
Clerc
F F
FF
Cieur
F
F
Seiberras
G
G
Godinot
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D007106:Immune Sera; D007425:Intracellular Membranes / Q000201:enzymology*; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000276:immunology* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007106:Immune Sera; D046911:Macromolecular Substances; D010455:Peptides; D006180:Proton-Translocating ATPases