Journal article
Correlation between cross-bridge kinetics obtained from Trp fluorescence of myofibril suspensions and mechanical studies of single muscle fibers in rabbit psoas
Journal of muscle research and cell motility, Vol.32(4), pp.315-326
12/2011
DOI: 10.1007/s10974-011-9264-7
PMID: 22006015
Abstract
Our goal is to correlate kinetic constants obtained from fluorescence studies of myofibril suspension with those from mechanical studies of skinned muscle fibers from rabbit psoas. In myofibril studies, the stopped-flow technique with tryptophan fluorescence was used; in muscle fiber studies, tension transients with small amplitude sinusoidal length perturbations were used. All experiments were performed using the equivalent solution conditions (200 mM ionic strength, pH 7.00) at 10°C. The concentration of MgATP was varied to characterize kinetic constants of the ATP binding step 1 (K
1: dissociation constant), the binding induced cross-bridge detachment step 2 (k
2, k
−2: rate constants), and the ATP cleavage step 3 (k
3, k
−3). In myofibrils we found that K
1 = 0.52 ± 0.08 mM (±95% confidence limits), k
2 = 242 ± 24 s−1, and k
−2 ≈ 0; in muscle fibers, K
1 = 0.46 ± 0.06 mM, k
2 = 286 ± 32 s−1, and k
−2 = 57 ± 21 s−1. From these results, we conclude that myofibrils and muscle fibers exhibit nearly equal ATP binding step, and nearly equal ATP binding induced cross-bridge detachment step. Consequently, there is a good correlation between process C (phase 2 of step analysis) and the cross-bridge detachment step. The reverse detachment step is finite in fibers, but almost absent in myofibrils. We further studied partially cross-linked myofibrils and found little change in steps 2 and 3, indicating that cross-linking does not affect these steps. However, we found that K
1 is 2.5× of native myofibrils, indicating that MgATP binding is weakened by the presence of the extra load. We further studied the phosphate (Pi) effect in myofibrils, and found that Pi is a competitive inhibitor of MgATP, with the inhibitory dissociation constant of ~9 mM. Similar results were also deduced from fiber studies. To characterize the ATP cleavage step in myofibrils, we measured the slow rate constant in fluorescence, and found that k
3 + k
−3 = 16 ± 1 s−1.
Details
- Title: Subtitle
- Correlation between cross-bridge kinetics obtained from Trp fluorescence of myofibril suspensions and mechanical studies of single muscle fibers in rabbit psoas
- Creators
- Robin Candau - UMR 866 Institut National de la Recherche Agronomique, Faculty of Sport Sciences University of Montpellier 1 34000 Montpellier FranceMasataka Kawai - Department of Anatomy and Cell Biology, College of Medicine University of Iowa Iowa City IA 52242 USA
- Resource Type
- Journal article
- Publication Details
- Journal of muscle research and cell motility, Vol.32(4), pp.315-326
- Publisher
- Springer Netherlands; Dordrecht
- DOI
- 10.1007/s10974-011-9264-7
- PMID
- 22006015
- ISSN
- 0142-4319
- eISSN
- 1573-2657
- Language
- English
- Date published
- 12/2011
- Academic Unit
- Anatomy and Cell Biology; Internal Medicine
- Record Identifier
- 9984025668502771
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