Canepari, Monica and Maffei, Manuela and Longa, Emanuela and Geeves, Michael A. and Bottinelli, Roberto (2012) Actomyosin kinetics of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach. Experimental Physiology, 97 (7). pp. 873-881. ISSN 0958-0670. (The full text of this publication is not available from this repository)
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An in vitro motility assay approach was used to investigate the mechanisms of the functional differences between myosin isoforms, by studying the effect of MgATP and MgADP on actin sliding velocity (Vf) of pure slow and fast rat skeletal myosin at different temperatures. The value of Vf depended on [MgATP] according to Michaelis–Menten kinetics, with an apparent constant (Km) of 54.2, 64.4 and 200 μM for the fast isoform and 18.6, 36.5 and 45.5 μM for the slow isoform at 20, 25 and 35°C, respectively. The presence of 2 mM MgADP decreased Vf and yielded an inhibition constant (Ki) of 377, 463 and 533 μM for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 μM for the slow isoform at 25 and 35°C, respectively. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover, the higher sensitivity of the fast myosin isoform to a drop in [MgATP] is consistent with the higher fatigability of fast fibres than slow fibres. From the Michaelis–Menten relation in the absence of MgADP, we calculated the rate of actomyosin dissociation by MgATP (k+ATP) and the rate of MgADP release (k-ADP). We found values of k+ATP of 4.8 × 106, 6.5 × 106 and 6.6 × 106 M−1 s−1 for the fast isoform and 3.3 × 106, 2.9 × 106 and 6.7 × 106 M−1 s−1 for the slow isoform and values of k-ADP of 263, 420 and 1320 s−1 for the fast isoform and 62, 107 and 306 s−1 for the slow isoform at 20, 25 and 35°C, respectively. The results suggest that k-ADP could be the major determinant of functional differences between the fast and slow myosin isoforms at physiological temperatures.
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||Sue Davies|
|Date Deposited:||17 Jul 2012 09:25|
|Last Modified:||23 Apr 2014 15:27|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/29889 (The current URI for this page, for reference purposes)|