The molecular mechanisms of contraction is probed in a skinned fiber system, in which the lattice spacing of the thick and thin filaments is osmotically altered. The length of an active muscle fiber is changed sinusoidally or in a stepwise fashion, and a concomitant tension time course is analyzed in terms of exponential rate constants. Two hypotheses predicting the effect of the lattice spacing on the crossbridge kinetics are tested. These include: (i) a reduction of the rate constant of actomyosin dissociation at a low level of compression, and (ii) a reduction of the rate constant of the power stroke at a high level of compression. Both the rate constants of the MgATP binding to actomyosin and of the dissociation which ensues the binding can be obtained by studying the MgATP dependence of the exponential rate constants. Th rate constant(s) which involves the power stroke reaction(s) can be obtained by studying the Ca effect. The equilibrium constant between the detached stae and the pre-tension state is estimated, based on the ionic strength effect on tension, stiffness, and the rate constants. We will then change the sarcomere length, and study the rate constants to determine whether the results can be predicted based on the change in the lattice spacing. The sarcomere length study will also be carried out on intact preparations where the quality of the data is better, although the solution parameters cannot be controlled as in skinned fibers. In other series of experiments, the lattice spacing is measured by low angle equitorial X-ray diffraction studies, and their results are correlated with the crossbridge kinetics. In skinned fibers, the ATPase rate is measured to examine if the rate limiting step is influenced by the lattice spacing change. Nonlinear profiles of the exponential rate constants are measured primarily in the step analysis method. All of these results are used to construct a crossbridge model, which will account for the Ca and MgATP effects on the exponential rate constants, as well as the effect of the lattice spacing and the ionic strength. The knowledge acquire and the technique developed during the course of this research can ultimately be applied to the study of muscle dysfunction.
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