title: Transient kinetics and time-resolved X-ray diffraction studies in isolated single muscle fibres.
abstract: 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.
authors.affiliation: Department of Physiology, Oxford, U.K.
authors.forename: P J
authors.initials: PJ
authors.lastname: Griffiths
authors.forename: J D
authors.initials: JD
authors.lastname: Potter
authors.forename: Y
authors.initials: Y
authors.lastname: Maéda
authors.forename: C C
authors.initials: CC
authors.lastname: Ashley
mesh_terms: D000199:Actins / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials; D009119:Muscle Contraction; D009132:Muscles / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism; D019209:Troponin C; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D000199:Actins; D014336:Troponin; D019209:Troponin C; D002118:Calcium