Table of ContentsIn this study we used an “in vitro motility assay” (IVMA) approach to investigate the effect of the variation of [MgATP] in determining the number and the sliding velocity (Vf) of actin filaments moved by fast skeletal myosin. Vf was studied on type 2B HMM from rats at 25C°, 100mM ionic strength and at varius [MgATP], [MgADP] and [Pi]. We designed a new experimental set-up to perform experiments at lower ionic strength and in buffers that had no interference with the ionic environment, in order to mimic physiological condition. This set-up allowed a complete and chemical speciation of the solutions opening the possibility to perform an accurate thermodynamic study. Therefore, along with kinetic measurements also quantitative thermodynamics measurements were carried obtaining the ∈″G of MgATP hydrolysistaking into account pH and [Mg2+]. We correlate the thermodynamics property of the system to Vf and to the number of sliding actin filaments which were assessed by a purpose-designed software. Preliminary results indicate: a) no straightly correlation between values of ∈″GATPand the velocity of actin filaments, b) an increase in thenumberof sliding actin filaments at low [MgATP] and no changes when the ratio [MgATP] / [HMM] was kept constant c) a decrease in the velocity of actin filaments at [Pi]=30mM. More investigations are required to confirm the unexpected results that indicate a complex role of MgATP and its metabolites in the modulation of actomyosin interaction.