A Structural Role for the Ca 2 ' - Mg 2 + Sites on Troponin Regulation of Muscle Contraction PREPARATION AND PROPERTIES OF TROPONIN C DEPLETED

Troponin C (TnC)-depleted myofibrils from rabbit skeletal muscle were prepared by consecutive washes of whole myofibrils with EDTA in solutions of low ionic strength, a method which has recently been described for the preparation of TnC (Cox, J. A., Compte, M., and Stein, E. A. (1981) Biochem. J. 195,205-211). Myofibrils treated in this manner exhibit a calcium independent loss of myofibrillar ATPase activity which is fully reversible with addition of TnC. Extractions of whole myofibrils, with metal chelators under conditions in which the Ca*+-specific or Ca2+-Ma;2+ sites of TnC have different metal occupancies, demonstrate that the removal of metal ions from the Ca2+-M&+ sites is responsible for the dissociation of TnC from myofibrils and that binding of either Caz+ or M 8 + to the Ca2+-M&+ sites prevents this dissociation. The extent of TnC dissociation from myofibrils with EDTA can be modulated by raising the KC1 concentration. At higher KC1 concentrations, a smaller percentage of TnC is extracted by EDTA. The TnC interaction with the Tn complex within myofilaments must be thus mediated by at least two mechanisms. One type of interaction is best demonstrated by a KC1 modulated interaction in the absence of divalent metal ions. Another type of interaction, which can be best detected at lower KC1 concentrations, involves Ca2+ and/or M 8 + binding to the Ca2+M&+ sites. Although both of these mechanisms probably function in the intact muscle cell, the effect of Ca2+ and Mg' on the TnC and TnI interaction is most important since it is this interaction which is thought to be one of the primary steps in the regulatory event of the contraction cycle. Since previous studies (Robertson, s. P., Johnson, J. D., and Potter, J. D. (1981) Biophys. J. 34, 559-569) have shown that the Ca2+-M&+ sites always contain either Ca2+ or M$+ in vivo, we conclude that the Ca2+-Mg+ sites of TnC play a structural role in maintaining the integrity of the troponin molecule.