Fluorescence of NBD-Labelled Troponin-I as a Probe for the Kinetics of Thin Filament Activation in Skeletal Muscle Fibers
- 147 Downloads
Using fluorescence of NBD-labelled troponin I incorporated into skinned fibers of the rabbit psoas muscle by chasing native troponin by troponin with the NBD-labelled TnI subunit we attempted to study kinetics of thin filament activation at different Ca++-concentrations. Since fluorescence of NBD-labelled TnI is sensitive to both, changes in Ca++, and strong cross-bridge attachment, we were able to induce changes thin filament activation by rapidly dropping the fraction of strongly attached cross-bridges to very low levels, e.g. by switching from isometric to isotonic contraction conditions. At any [Ca++], the time course of the resulting changes in fluorescence of NBD-labelled TnI was found at least an order of magnitude faster than the time course of force redevelopment subsequent to the period of isotonic contraction. Modelling shows that with the kinetics of thin filament activation derived from these studies, common kinetic schemes of the actomyosin ATPase predict regulation to act via changes in cross-bridge cycling kinetics, as we had previously proposed.
KeywordsThin Filament Myosin Head Fluorescence Change Isotonic Contraction Actomyosin ATPase
Unable to display preview. Download preview PDF.
- 10.Kraft, T., Schnekenbühl, S., Messerli, M., Yu, L.C., Chalovich, J.M. & Brenner, B. J. Muscle Res. Cell Mo-til. 15, 202 (1994)Google Scholar
- 13.Brenner, B. & Chalovich, J.M. Biophys. J. 68, A142 (1995)Google Scholar
- 14.Chalovich, J.M. & Brenner, B. Biophys. J. 68, A365 (1995)Google Scholar
- 16.Brenner, B. in Molecular Mechanisms in Muscular Contraction (ed. J.M. Squire) 77–149 (Macmillan Press, London, 1990)Google Scholar
- 17.Miliar, N.C. & Homsher, E. J. Biol. Chem. 265, 20234–20240 (1990)Google Scholar