Abstract
Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to approximately 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (approximately 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin.ADP and provide evidence of three closely related actin-myosin.ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (approximately 20 s(-1)), and a third complex that releases ADP too slowly (approximately 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin.ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.
Highlights
Mechanochemical properties of each myosin are attributed to a modulation of the rates and equilibrium constants of individual molecular events to match each myosin to its physiological role
We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with myosin heavy chain 2 (MHC-2) and focus on the mechanism of ADP release
Mammalian, muscle tissue the MHC-2 is found as various isoforms (e.g. 2a, 2b, 2x) and it has been established that the essential mechanical properties of the muscle fiber contraction are properties of the MHC present in the tissue, the light chain isoforms play a minor modulatory role (8, 9)
Summary
Mechanochemical properties of each myosin are attributed to a modulation of the rates and equilibrium constants of individual molecular events to match each myosin to its physiological role. We recently undertook a study of mammalian striated muscle myosin isoforms (10, 11) and concluded that the maximum velocity of muscle fiber contraction was limited by the rate of cross-bridge detachment from the actin filament at the end of cross-bridge ATPase cycle. This work demonstrated for the first time that, with a high time resolution optical trap, the mechanical events of myosin II isoforms from fast and slow muscle fibers can be resolved into two distinct steps, as had previously been reported for smooth muscle myosin II and some non-muscle myosins (14 –16) These two steps have been interpreted as a force-generating power stroke that is coupled to Pi release from the cross-bridge followed by a second, smaller mechanical event associated with ADP release. Because this second event is associated with ADP release, the mechanism of ADP release is of particular interest for these muscle myosins
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