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

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 fibbers were maximally Ca2+ -activated at 20 degrees 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: [GRAPHICS] 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 (k(2), 198-526 s(-1); k_(2), 51-328 s(-1); k(4), 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), k(4)) varied more among fiber types than their reversals (k(2), k_(4)). The equilibrium constants K-1 (MgATP affinity) and K-2 (= k(2)/ k_(2), ATP isomerization) were progressively less in the order IIA, IID, and IIB. K-4 (=k(4)/k_(4), force generation) and K-5 (P-i affinity) were larger in IIB than IIA and IID fibers. K-1 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 affnity (K-0) was larger in type IID. bers than in type IIB fibers.