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J. Biol. Chem., Vol. 283, Issue 13, 8527-8537, March 28, 2008

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Human Myosin Vc Is a Low Duty Ratio, Nonprocessive Molecular Motor^*

Yasuharu Takagi, Yi Yang, Ikuko Fujiwara, Damon Jacobs^¶, Richard E. Cheney^¶, James R. Sellers, Supported by funds from the intramural NHLBI program of the National Institutes of Health¹, and Mihály Kovács, Supported by National Institutes of Health Research Grants D43 TW006230 and 1 R01 TW007241-01 funded by the Fogarty International Center and the NHLBI, National Institutes of Health, an EMBO-HHMI Startup Grant, and the Bolyai Fellowship of the Hungarian Academy of Sciences^||2

From the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8015, Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8017, ^¶Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7545, and the ^||Department of Biochemistry, Eötvös University, H-1117 Budapest, Pázmány P. Sétány 1/C, Hungary

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V_max of 1.8 ± 0.3 s^-1 and a K_ATPase of 43 ± 11 µM. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (1.5 s^-1), rather than the ADP release step (12.0–16.0 s^-1). Nevertheless, the ADP affinity of actomyosin Vc (K_d = 0.25 ± 0.02 µM) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.

Received for publication, November 7, 2007 , and in revised form, January 2, 2008.

^* 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 To whom correspondence may be addressed: Laboratory of Molecular Physiology, NHLBI, Bldg. 50, Rm. 3523, National Institutes of Health, Bethesda, MD 20892-8015. Tel.: 301-496-6887; Fax: 301-402-542; E-mail: sellersj{at}mail.nih.gov. ² To whom correspondence may be addressed: Dept. of Biochemistry, Eötvös University, H-1117 Budapest, Pázmány P. Sétány 1/C, Hungary. Tel.: 36-1-209-0555/8401; Fax: 36-1-381-2172; E-mail: stoci1{at}gmail.com.

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