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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 (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 …