title: Actomyosin kinetics of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach.
abstract: 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 (V(f)) of pure slow and fast rat skeletal myosin at different temperatures. The value of V(f) depended on [MgATP] according to Michaelis-Menten kinetics, with an apparent constant (K(m)) 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 V(f) and yielded an inhibition constant (K(i)) 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 K(m) and K(i) suggested that slow and fast isoforms differ in the kinetics limiting V(f). 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 × 10(6), 6.5 × 10(6) and 6.6 × 10(6) M(-1) s(-1) for the fast isoform and 3.3 × 10(6), 2.9 × 10(6) and 6.7 × 10(6) 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.
authors.affiliation: Department of Molecular Medicine and Interuniversity Institute of Myology, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy. canepari@unipv.it
authors.forename: Monica
authors.initials: M
authors.lastname: Canepari
authors.forename: Manuela
authors.initials: M
authors.lastname: Maffei
authors.forename: Emanuela
authors.initials: E
authors.lastname: Longa
authors.forename: Michael
authors.initials: M
authors.lastname: Geeves
authors.forename: Roberto
authors.initials: R
authors.lastname: Bottinelli
mesh_terms: D008841:Actin Cytoskeleton; D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D007700:Kinetics; D018482:Muscle, Skeletal / Q000502:physiology; D009218:Myosins / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000199:Actins; D020033:Protein Isoforms; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins