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J. Biol. Chem., Vol. 283, Issue 7, 3773-3781, February 15, 2008

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Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II^*

Mamta Amrute-Nayak, Massimo Antognozzi¹, Tim Scholz, Hiroaki Kojima, and Bernhard Brenner²

From the Department of Molecular and Cell Physiology, Hannover Medical School, Hannover D-30625, Germany and Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe 651-2492, Japan

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.

Received for publication, August 14, 2007 , and in revised form, November 20, 2007.

^* This study was funded by the Deutsche Forschungsgemeinschaft as part of the Priority Program SPP 1068 (Br849/21-3). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

¹ Present address: Dept. of Physics, University of Bristol, Bristol, UK.

² To whom correspondence should be addressed. Tel.: 49-511-532-6396; Fax: 49-511-532-4296; E-mail: brenner.bernhard{at}mh-hannover.de.

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