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0; Graphpad Software Inc., La Jolla, CA, USA), which provided the values of Km and Vmax expressed as means �� SEM. The statistical significance of the differences was assessed by two-way ANOVA for repeated measures followed by the Student�CNeuman�CKeuls post hoc test. A probability of less than 5% (P < 0.05) was considered to be significant. The temperature dependence of the actin filament sliding velocity was analysed with Arrhenius plots. From the slopes of the linear regression lines, the kinetic energy that must be provided Selleckchem Anti-cancer Compound Library to start the reaction or activation energy (Ea) and the temperature coefficient (Q10) were calculated as described by Rossi et al. (2005). Actin sliding velocity was studied on pure slow (type 1) and pure fast (type 2B) myosin isoforms at MgATP concentrations of 0.01, 0.02, 0.1, 0.5, 1.0 and 2.0 mm (Fig. 2). Experiments were performed at 20, 25 and 35��C, in the absence and in the presence of 2 mm MgADP (Fig. 2). As expected, Vf was higher for the fast than for the slow isoform and increased with temperature (Fig. 2). The Vf increased with MgATP concentration, both in the presence and in the absence of MgADP. Velocities were lower in the presence of 2 mm MgADP at all MgATP concentrations (Fig. 2). Assuming that the relation between Vf and MgATP concentration followed the behaviour of a simple enzymatic reaction with MgATP as a substrate, we fitted the data with Sitaxentan a Michaelis�CMenten relation [eqn (1)] and calculated the following Alectinib in vivo parameters: (a) the Michaelis constant (Km), which is the [MgATP] at which velocity is half-maximum; (b) Vmax, which is the maximal velocity at infinite MgATP concentration; and (c) the ratio Vmax/Km, which characterizes myosin enzyme kinetics at subsaturating MgATP concentrations. The values of Km were lower for the slow than for the fast isoform at all temperatures, indicating that the velocity saturates at a lower MgATP concentration in the slow than in the fast isoform (Table 1). The Km increased with temperature in both isoforms. In the temperature range 25�C35��C, the increase was much more evident for the fast (3.1-fold change) than for the slow isoform (1.25-fold change), indicating that MgATP binding to the slow isoform was little affected by temperature. The opposite was observed in the range 20�C25��C, i.e. Km of the fast isoform had a lower temperature sensitivity (1.42-fold change) than that of the slow isoform (3.8-fold change). Values of Vmax were three- to fourfold higher for the fast than for the slow isoform at all temperatures (Table 1), consistent with Vf values. In the range 25�C35��C, Q10 values for Vmax were 2.86 and 3.14 and activation energies were 77.73 and 76.58 kJ mol?1 for the slow and fast isoform, respectively, indicating similar temperature sensitivity of the two isoforms, consistent with what was previously observed (Rossi et al. 2005).
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