Transient kinetics and time-resolved X-ray diffraction studies in isolated single muscle fibres.

The timing of events associated with the contraction and relaxation of the force cycle is described in isolated single arthropod muscle fibers using the fluorescently labelled derivatives of the Ca2+ binding sub-unit of troponin TnC. The kinetics of the subtracted fluorescence (490-410 nm) response from injected TnCDANZ, labelled at the Ca2+ specific sites, shows a rapid rise which is some 90% complete at 50% force consistent with rapid Ca2+ binding to this sub-unit. Subsequently the TnCDANZ fluorescence decays 2x more slowly, at 12 degrees C, than force consistent with a slower release of this bound Ca2+. In fibers injected with both aequorin and TnCDANZ, the aequorin kinetics are essentially unaltered compared to control fibers in the presence of 10-100 microM TnCDANZ. The peak of the aequorin response occurs some 150-170 msec in front of the TnCDANZ peak and the T 1/2 for light decay is faster than either force or TnCDANZ decay, but there is a 'tail' to the aequorin light response (elevated free Ca2+) well into the relaxation phase, seen both in cannulated and intact muscle fibers. The kinetics of the fluorescence of TnCIAANS, labelled of the Ca2+-Mg2+ sites, shows a slow decrease (T 1/2 1.8 sec) and subsequent increase (T 1/2 2.5 sec) in fluorescence consistent with a slow loading and unloading of these sites with Ca2+ during a tetanus. Time resolved X-ray diffraction from intact muscle fibers indicate that forces of up to 600 kN/m2 can be developed at sarcomere lengths of 8-10 micron. Force shows a marked sarcomere dependency while the aequorin response is relatively insensitive. At these high forces, there is a marked change in intensity of the first actin layer line (A2 at 38 nm), consistent with S1 (cross-bridge) attachment, which has a T 1/2 for rise of 125-150 msec.