CA2+-DEPENDENCE OF STRUCTURAL-CHANGES IN TROPONIN-C IN DEMEMBRANATED FIBERS OF RABBIT PSOAS MUSCLE

The Ca2+-dependence of structural changes in troponin-C (TnC) has been detected by monitoring the fluorescence from TnC labeled at Methionine-25, in the NH2-terminal domain, with danzylaziridine (TnC-DANZ) and then exchanged for endogenous TnC in glycerinated single fibers. The fluorescence-pCa relation obtained from fibers stretched to a sarcomere length > 4.0-mu-m evidenced two transitions: a small one, attributable to the binding of Ca2+ to the high affinity, Ca 2+-Mg2+-binding sites of TnC; and a large one, attributable to the binding of Ca2+ to the low affinity, Ca2+-specific binding sites of TnC. In the fluorescence-pCa relation determined with fibers set to a sarcomere length of 2.4-mu-m, hence obtained in the presence of cycling cross-bridges, the large transition had the same Ca2+-dependence as did the development of tension. These results indicate that the NH2-terminal globular domain of TnC is modified by the binding of Ca2+ to Sites located in both globular domains and that the structural changes in TnC resulting from the binding of Ca2+ to the low-affinity sites, but not to the high-affinity sites, are directly associated with the triggering of contraction.