Fujita Hideaki, Sasaki Daisuke, Ishiwata Shin'ichi, Kawai Masataka
Department of Anatomy and Cell Biology, College of Medicine, The University of Iowa, Iowa City, Iowa 52242, USA.
Biophys J. 2002 Feb;82(2):915-28. doi: 10.1016/S0006-3495(02)75453-2.
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 < or = 4.66, pH 7.00, 25 degrees C. In control myocardium, the MgATP association constant was 9.1 +/- 1.3 mM(-1), and the Pi association constant 0.14 +/- 0.04 mM(-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 approximately 1.5-1.7x of that in the absence of regulatory proteins. These results further indicate that regulatory proteins promote detachment of cross-bridges.
研究了调节蛋白在牛心肌横桥循环基本步骤中的作用。用凝溶胶蛋白选择性去除细肌丝，并在没有原肌球蛋白或肌钙蛋白的情况下重构肌动蛋白丝。通过添加原肌球蛋白和肌钙蛋白实现进一步重构。在pCa≤4.66、pH 7.00、25℃条件下，研究了MgATP和磷酸盐（Pi）对对照心肌、重构肌动蛋白丝心肌和重构细肌丝心肌中指数过程速率常数的影响。在对照心肌中，MgATP结合常数为9.1±1.3 mM⁻¹，Pi结合常数为0.14±0.04 mM⁻¹。横桥解离步骤的平衡常数为2.6±0.4，力产生步骤的平衡常数为0.59±0.04。在没有调节蛋白的重构肌动蛋白丝心肌中，MgATP结合常数大致相同，Pi结合常数增加到2.8倍。横桥解离的平衡常数降至0.2倍，但力产生步骤的平衡常数增加到4倍。在重构细肌丝后，这些动力学常数恢复到对照值。这些结果表明，存在调节蛋白时的张力/横桥约为不存在调节蛋白时的1.5 - 1.7倍。这些结果进一步表明，调节蛋白促进横桥的解离。
