Elementary steps of the cross-bridge cycle in Bovine myocardium with and without regulatory proteins

ABSTRACT The role of regulatory proteins in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed by gelsolin and the actin filament was reconstituted without tropomyosin or troponin. Further reconstitution was achieved by adding tropomyosin and troponin. The effects of MgATP and phosphate (Pi) on the rate constants of exponential processes were studied in control, actin filament-reconstituted, and thin filament-reconstituted myocardium at pCa </=4.66, pH 7.00, 25 deg C. In control myocardium, the MgATP association constant was 9.1 +/- 1.3 mM^sup -1^, and the Pi association constant 0.14 +/- 0.04 mM^sup -1^. The equilibrium constant of the cross-bridge detachment step was 2.6 _ 0.4, and the equilibrium constant of the force generation step was 0.59 +/- 0.04. In actin filament-reconstituted myocardium without regulatory proteins, the MgATP association constant was approximately the same, and the Pi association constant increased to 2.8x. The equilibrium constant of cross-bridge detachment decreased to 0.2x, but the equilibrium constant of the force generation step increased to 4X. These kinetic constants regained control values after reconstitution of the thin filament. These results indicate that tension/cross-bridge in the presence of regulatory proteins is ~1.5-1.7x of that in the absence of regulatory proteins. These results further indicate that regulatory proteins promote detachment of cross-bridges.

The authors thank Dr. Larry S. Tobacman for critical reading of the manuscript and useful suggestions. This work was supported by grant IBN 98-14441 from the National Science Foundation, and the Grant-in-Aid 99-50437N from the American Heart Association National Center. Dr. Fujita is a recipient of a Postdoctoral Fellowship award from Japan Society for Promotion of Science.

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[Author Affiliation]

Hideaki Fujita,* Daisuke Sasaki,^ Shin'ichi Ishiwata,^^^ and Masataka Kawai*

*Department of Anatomy and Cell Biology, College of Medicine, The University of Iowa, Iowa City, Iowa 52242, USA; ^Department of Physics, School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan; and ^^Core Research for Evolutional Science and Technology (CREST) Team-13, Japan.

[Author Affiliation]

Submitted December 11, 2000, and accepted for publication November 7, 2001.

Address reprint request to Dr. Masataka Kawai, Department of Anatomy, College of Medicine, The University of Iowa, Iowa City, IA 52242 USA. Tel.: 319-335-8101; Fax: 319-335-7198; E-mail: masataka-kawai@uiowa.edu.