A monoclonal antibody that recognizes different conformational states of skeletal muscle troponin C and other calcium binding proteins

Summary

Skeletal muscle troponin C contains four Ca²⁺-binding sites, two with a high affinity for Ca²⁺ that also bind Mg²⁺ competitively (Ca²⁺/Mg²⁺ sites) and two sites of lower affinity that are specific for Ca²⁺. We have characterized a monoclonal antibody (B₉D₉) that was produced against rabbit skeletal troponin C. The binding of this antibody to rabbit skeletal troponin C is sensitive to the binding of Ca²⁺. Increasing the Ca²⁺ concentration produces a decrease in the amount of antibody bound with a pK of approximately 6.9 which correlates with Ca²⁺ binding to the Ca²⁺/Mg²⁺ sites. Magnesium binding to rabbit skeletal troponin C had no effect on antibody binding. Thus the conformation of rabbit skeletal troponin C brought about by Ca²⁺ binding to these sites affects the antibody binding to its epitope. This epitope was unavailable for antibody binding in whole troponin. The antibody-binding site was localized in cyanogen bromide fragment CB9 of rabbit skeletal troponin C (residues 84–135). This antibody was also shown to cross-react with bovine cardiac troponin C., barnacle (Balanus nubilus) troponin C, bovine testis calmodulin and carp parvalbumin. In addition, the effect of Ca²⁺ on antibody binding seen with rabbit skeletal troponin C was also seen with bovine cardiac troponin C, and calmodulin. Thus these proteins appear to share a similar epitope and undergo similar structural changes. Wang and colleagues (1987) have presented evidence that rabbit skeletal troponin C at low pH has an elongated structure similar to that seen in the crystal structure and that at neutral pH its structure is more compact. We have found that in the absence of Ca²⁺ this antibody binds best to rabbit skeletal troponin C at low pH and its binding is reduced with increasingly alkaline pH. It is possible that the structural alterations brought about by changes in pH may also be responsible for the reduction in antibody binding. Since pH and Ca²⁺ have the same effect on antibody binding, this may mean that Ca²⁺ binding to the Ca²⁺/Mg²⁺ sites may also make rabbit skeletal troponin C more compact.