The contractile mechanism of vertebrate striated muscle is activated by the binding of calcium to troponin.  Studies relating force to free calcium concentration show that the activation mechanism has the properties of a cooperative system.  One manifestation of cooperativity is the enhanced calcium-troponin affinity evident when myosin is linked to actin, as in rigor.  Work in this laboratory indicates that in glycerinated muscle fibers in rigor the number of calcium-binding sites and the degree of interaction between sites is controlled by the number of rigor complexes formed between actin and myosin filaments.  Certain dynamic properties of living muscle, such as the effect of force and length on activation, could be accounted for if it can be shown that force-generating complexes formed in the intact myofilament lattice also alter calcium-troponin affinity.  The aim of this investigation will be to determine how the binding of calcium to glycerinated fibers is influenced by mechanical variables such as cross-bridge attachment, force, and length.  A double isotope technique and EGTA buffers will be used to measure the binding of calcium to glycerinated fibers.  Measurements will be made in the presence of ATP and ATP analogs which "set" the contractile system into various modes of cross-bridge orientation and attachment that are believed to represent stages of the normal cross-bridge cycle.  Calcium binding will be characterized quantitatively under conditions in which force and length are varied.  From these studies it should be possible to draw some conclusions about how calcium mobilization and binding are controlled by mechanical conditions during the contraction cycle of living muscle.