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

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 MichaelisMenten kinetics, with an apparent constant (Km) of 54.2, 64.4 and 200 mu m for the fast isoform and 18.6, 36.5 and 45.5 mu m for the slow isoform at 20, 25 and 35 degrees C, respectively. The presence of 2 mm MgADP decreased Vf and yielded an inhibition constant (Ki) of 377, 463 and 533 mu m for the fast isoform at 20, 25 and 35 degrees C, respectively, and 120 and 355 mu m for the slow isoform at 25 and 35 degrees 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 MichaelisMenten 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 x 10(6), 6.5 x 10(6) and 6.6 x 10(6)m-1 s-1 for the fast isoform and 3.3 x 106, 2.9 x 10(6) and 6.7 x 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 degrees 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.