The binding of monoclonal and polyclonal antibodies to the Ca 2+-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules

Abstract

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca 2+-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATP-ase-ATPase interactions and Ca 2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca 2+-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca 2+-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E 1 and E 2V conformations. Therefore these regions of the Ca 2+-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca 2+-ATPase only in the E 1 conformation stabilized by 0.5 mM Ca 2+ but not in the E 2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca 2+-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca 2+-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A 1 tryptic fragment of the Ca 2+-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca 2+-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca 2+-ATPase into the A 1, A 2 and B fragments did not promote the reaction of anti-A 1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C 12E 8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca 2+ transport, with significant inhibition of ATPase activity; this may suggest a role for ATPase oligomers in the regulation of Ca 2+ transport. The other antibodies that interact with the native Ca 2+-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca 2+ transport.