Inorganic phosphate inhibits movement by forming a strong-binding actomyosin-phosphate intermediate, reducing the number of Cy3-signals per field of view by nearly 50% at 1-2 mM.
Inorganic phosphate inhibits movement by forming a strong-binding actomyosin-phosphate intermediate rather than binding to an actomyosin-ADP intermediate.
In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2'(3')-O-[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1-2 mM. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513-3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.
Amrute‐Nayak et al. (Fri,) reported a other. Inorganic phosphate was evaluated on Inhibition of filament gliding and dwell times of Cy3-EDA-ATP. Inorganic phosphate inhibits movement by forming a strong-binding actomyosin-phosphate intermediate, reducing the number of Cy3-signals per field of view by nearly 50% at 1-2 mM.