An in vitro motility assay approach was used to understand the mechanisms of the functional differences between myosin isoforms, studying the effect of MgATP and MgADP on actin sliding velocity (Vf) on pure slow and fast rat skeletal myosin at different temperature. Vf depended on [MgATP] according the Michaelis Menten kinetic with apparent constants (Km) of 54.2, 64.4 and 200muM for fast and of 18.6, 36.5 and 45.5muM for slow isoform at 20 degrees C, 25 degrees C and 35 degrees C respectively. The presence of 2mM MgADP decreased Vf and yielded inhibition constants (Ki) of 377, 463 and 533muM for fast isoform at 20 degrees C, 25 degrees C and 35 degrees C respectively and of 120 muM and 355muM for slow isoform at 25 degrees C and 35 degrees C. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover the higher sensitivity of fast myosin to a drop in [MgATP] is consistent with the higher fatigability of fast then slow fibres. From the Michaelis Menten relationship in 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, 6.5 and 6.6 106M-1sec-1 for fast and of 3.3, 2.9 and 6.7 106M-1sec-1 for slow isoform and values of k-ADP of 263; 420 and 1320sec-1for fast and of 62; 107 and 306sec-1 for slow isoform at 20 degrees C; 25 degrees C and 35 degrees C respectively. The results suggest that k-ADP could be the major determinant of functional differences between fast and slow myosin isoforms at physiological temperature.

Actomyosin kinetic of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach

CANEPARI, MONICA;MAFFEI, MANUELA;BOTTINELLI, ROBERTO
2012

Abstract

An in vitro motility assay approach was used to understand the mechanisms of the functional differences between myosin isoforms, studying the effect of MgATP and MgADP on actin sliding velocity (Vf) on pure slow and fast rat skeletal myosin at different temperature. Vf depended on [MgATP] according the Michaelis Menten kinetic with apparent constants (Km) of 54.2, 64.4 and 200muM for fast and of 18.6, 36.5 and 45.5muM for slow isoform at 20 degrees C, 25 degrees C and 35 degrees C respectively. The presence of 2mM MgADP decreased Vf and yielded inhibition constants (Ki) of 377, 463 and 533muM for fast isoform at 20 degrees C, 25 degrees C and 35 degrees C respectively and of 120 muM and 355muM for slow isoform at 25 degrees C and 35 degrees C. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover the higher sensitivity of fast myosin to a drop in [MgATP] is consistent with the higher fatigability of fast then slow fibres. From the Michaelis Menten relationship in 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, 6.5 and 6.6 106M-1sec-1 for fast and of 3.3, 2.9 and 6.7 106M-1sec-1 for slow isoform and values of k-ADP of 263; 420 and 1320sec-1for fast and of 62; 107 and 306sec-1 for slow isoform at 20 degrees C; 25 degrees C and 35 degrees C respectively. The results suggest that k-ADP could be the major determinant of functional differences between fast and slow myosin isoforms at physiological temperature.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/444693
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