Availability to monoclonal antibodies of antigenic sites of the α and β subunits in active, denatured or membrane-bound mitochondrial F1-ATPase

Abstract

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 β-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-β monoclonal antibodies bound more easily to the β-subunit than to active F1, and recognized equally active F1 and F1 integrated in membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated β-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-β monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the β-subunit. One anti-α 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 α-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the α-subunit well exposed at the surface of the inner face of the mitochondrial membrane.