Factors contributing to troponin exchange in myofibrils and in solution

The troponin complex in a muscle fiber can be replaced with exogenous tropo nin by using a gentle exchange procedure in which the actin-tropomyosin com plex is never devoid of a full complement of troponin (Brenner et al. (1999 ) Biophys J 77: 2677-2691). The mechanism of this exchange process and the factors that influence this exchange are poorly understood. In this study, the exchange process has now been examined in myofibrils and in solution. I n myofibrils under rigor conditions, troponin exchange occurred preferentia lly in the region of overlap between actin and myosin when the free Ca2+ co ncentration was low. At higher concentrations of Ca2+, the exchange occurre d uniformly along the actin. Ca2+ also accelerated troponin exchange in sol ution but the effect of S1 could not be confirmed in solution experiments. The rate of exchange in solution was insensitive to moderate changes in pH or ionic strength. Increasing the temperature resulted in a two-fold increa se in rate with each 10 degreesC increase in temperature. A sequential two step model of troponin binding to actin-tropomyosin could simulate the obse rved association and dissociation transients. In the absence of Ca2+ or rig or S1, the following rate constants could describe the binding process: k(1 ) = 7.12 muM(-1)s(-1), k(-1) = 0.65 s(-1), k(2) = 0.07 s(-1), k(-2) = 0.001 4 s(-1). The slow rate of detachment of troponin from actin (k(-2)) limits the rate of exchange in solution and most likely contributes to the slow ra te of exchange in fibers.