Availability to monoclonal antibodies of antigenic sites of the alpha and beta subunits in active, denatured or membrane-bound mitochondrial F1-ATPase.
The binding of five monoclonal antibodies to mitochondrial F1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F1 integrated in the mitochondrial membrane, for purified beta-subunit and for purified F1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-beta monoclonal antibodies bound more easily to the beta-subunit than to active F1, and recognized equally active F1 and F1 integrated in the membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated beta-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-beta monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the beta-subunit. One anti-alpha monoclonal antibody (20 D6) bound poorly to F1 integrated in the membrane, while the other (7 B3) had a higher affinity for F1 integrated in the membrane than for soluble F1. Therefore, 20 D6 recognizes an epitope of the alpha-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the alpha-subunit well exposed at the surface of the inner face of the mitochondrial membrane.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D011489:Protein Denaturation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013552:Swine
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases