Abstract
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.
Highlights
From the AM1⁄7ADP1⁄7Pi3 complex is closely related to the initiation of the power-stroke [4] and to the transition of the myosin head domain from states of weak and non-stereospecific actin binding to states of strong and stereospecific binding to actin [5,6,7]
Effect of Inorganic Phosphate on Actin Filament Gliding Velocity—First we examined the effect of inorganic phosphate on the velocity of actin filament gliding on a myosin-coated surface to reproduce previous data [25]
Actin Filaments Tend to Fragment in the Presence of Pi but Only at Low MgATP Concentrations— the inhibitory effect of inorganic phosphate on actin gliding velocity was fully reversible, to our surprise actin filaments in the motility assay tended to break in the presence of inorganic phosphate but only at the low MgATP concentrations (Fig. 2)
Summary
M, myosin; A, actin; BSA, bovine serum albumin; Cy3-EDA-ATP, 2Ј(3Ј)-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP; TRITC, tetramethylrhodamine isothiocyanate. M1⁄7Pi and AM1⁄7Pi are the states formed when Pi binds to nucleotide free myosin or actomyosin, respectively. Consistent with concepts in which actin filament gliding velocity is reduced by binding of Pi to the empty nucleotide binding pocket of found slowed down by inorganic phosphate at low MgATP myosin, increasing the resistive load against filament glidconcentrations [25]. Preliminary results have been prealso proposed competitive inhibition of ATP-binding to the sented in abstract form [30]. Nucleotide-free myosin head by inorganic phosphate, assuming that Pi may bind to the nucleotide free active site, forming an AM1⁄7Pi complex. These authors proposed that inorganic phosphate, before ADP release, may bind to a secondary site on the myosin head that is not the site
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