To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 μg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (KAD). The apparent second order rate constant of actomyosin dissociation gets faster (K1k+2 = 0.17 -0.26 μM-1.s-1), whereas the affinity for ADP is weakened (250-930 μM) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V 0) of the parent fibers. If the value of KAD is controlled largely by the rate constant of ADP release (k-AD), then the estimated value of k-AD is sufficiently low to limit V 0. In contrast, [ATP]K1k+2 at a physiological concentration of 5 mM ATP would be 2.5-6 times faster than k-AD.
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