Elementary steps of the cross-bridge cycle in fast-twitch fiber types from rabbit skeletal muscles

¹ University of Salzburg
² University of IOwa
³ University of Iowa

^* To whom correspondence should be addressed. E-mail: masataka-kawai{at}uiowa.edu.

Submitted on November 19, 2004
Revised on January 15, 2005
Accepted on 19 July 2005

	Abstract

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, Pi) 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: where A=actin, M=myosin, D=MgADP, and S=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, and K4 (=k4/k 4: force generation) was progressively larger in the order IIA, IID, and IIB. K5 (Pi affinity) did not vary much among the fiber types. 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.

Key Words: association constants, isoform, myosin heavy chain, rate constants, sinusoidal analysis, transient analysis

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