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Changes in Orientation of Actin during Contraction of Muscle

J. Borejdo ^*, A. Shepard ^*, D. Dumka ^*, I. Akopova ^*, J. Talent ^*, A. Malka ^* and T. P. Burghardt 

^* Department of Molecular Biology and Immunology, University of North Texas, Fort Worth, Texas and Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota

Correspondence: Address reprint requests to Julian Borejdo, University of North Texas, Dept. of Biochemistry, Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107-2699. Tel.: 817-735-2699; E-mail: jborejdo{at}hsc.unt.edu.

It is well documented that muscle contraction results from cyclic rotations of actin-bound myosin cross-bridges. The role of actin is hypothesized to be limited to accelerating phosphate release from myosin and to serving as a rigid substrate for cross-bridge rotations. To test this hypothesis, we have measured actin rotations during contraction of a skeletal muscle. Actin filaments of rabbit psoas fiber were labeled with rhodamine-phalloidin. Muscle contraction was induced by a pulse of ATP photogenerated from caged precursor. ATP induced a single turnover of cross-bridges. The rotations were measured by anisotropy of fluorescence originating from a small volume defined by a narrow aperture of a confocal microscope. The anisotropy of phalloidin-actin changed rapidly at first and was followed by a slow relaxation to a steady-state value. The kinetics of orientation changes of actin and myosin were the same. Extracting myosin abolished anisotropy changes. To test whether the rotation of actin was imposed by cross-bridges or whether it reflected hydrolytic activity of actin itself, we labeled actin with fluorescent ADP. The time-course of anisotropy change of fluorescent nucleotide was similar to that of phalloidin-actin. These results suggest that orientation changes of actin are caused by dissociation and rebinding of myosin cross-bridges, and that during contraction, nucleotide does not dissociate from actin.

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