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J. Biol. Chem., Vol. 283, Issue 2, 766-773, January 11, 2008

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Kinetics of ADP Dissociation from the Trail and Lead Heads of Actomyosin V following the Power Stroke^*

Eva Forgacs¹, Suzanne Cartwright, Takeshi Sakamoto², James R. Sellers, John E. T. Corrie^¶3, Martin R. Webb^¶3, and Howard D. White⁴

From the Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, ^¶Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom, and the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035–13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.

Received for publication, May 25, 2007 , and in revised form, October 17, 2007.

^* This work was supported by National Institutes of Health Grant EB00209 and a grant from the Carman Foundation. 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.

¹ Supported by an American Heart Association postdoctoral fellowship.

² Supported by a Japanese Society for the Promotion of Science Fellowship.

³ Supported by the United Kingdom Medical Research Council.

⁴ To whom correspondence should be addressed. Tel.: 757-446-5652; Fax: 757-624-2270; E-mail: whitehd{at}evms.edu.

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