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Elementary Steps of the Cross-Bridge Cycle in Fast-Twitch Fiber Types from Rabbit Skeletal Muscles

Stefan Galler ^*, Brant Gang Wang  and Masataka Kawai 

Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242 USA; and ^* Department of Cell Biology, Faculty of Natural Sciences, University of Salzburg, A-5020 Salzburg, Austria

Correspondence: Address reprint requests to Masataka Kawai, Dept. of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242 USA. Tel.: 1-319-335-8101; Fax: 1-319-335-7198; E-mail: masataka-kawai{at}uiowa.edu.

To understand the molecular mechanism underlying the diversity of mammalian skeletal muscle fibers, the elementary steps of the cross-bridge cycle were investigated in three fast-twitch fiber types from rabbit limb muscles. Skinned fibers were maximally Ca²⁺-activated at 20°C and the effects of MgATP, phosphate (P, P_i), and MgADP were studied on three exponential processes by sinusoidal analysis. The fiber types (IIA, IID, and IIB) were determined by analyzing the myosin heavy-chain isoforms after mechanical experiments using high-resolution SDS-PAGE. The results were consistent with the following cross-bridge scheme:

1

where A is actin, M is myosin, D is MgADP, and S is MgATP. All states except for those in brackets are strongly bound states. All rate constants of elementary steps (k₂, 198–526 s^–1; k_–2, 51–328 s^–1; k₄, 13.6–143 s^–1; k_–4, 13.6–81 s^–1) were progressively larger in the order of type IIA, type IID, and type IIB fibers. The rate constants of a transition from a weakly bound state to a strongly bound state (k_–2, k₄) varied more among fiber types than their reversals (k₂, k_–4). The equilibrium constants K₁ (MgATP affinity) and K₂ (=k₂/k_–2, ATP isomerization) were progressively less in the order IIA, IID, and IIB. K₄ (=k₄/k_–4, force generation) and K₅ (P_i affinity) were larger in IIB than IIA and IID fibers. K₁ showed the largest variation indicating that the myosin head binds MgATP more tightly in the order IIA (8.7 mM^–1), IID (4.9 mM^–1), and IIB (0.84 mM^–1). Similarly, the MgADP affinity (K₀) was larger in type IID fibers than in type IIB fibers.

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